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WO2007045096A1 - Oligoheteroaromatic luminiscent assemblies as high-affinity dna sequence-directed ligands - Google Patents

Oligoheteroaromatic luminiscent assemblies as high-affinity dna sequence-directed ligands Download PDF

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Publication number
WO2007045096A1
WO2007045096A1 PCT/CA2006/001724 CA2006001724W WO2007045096A1 WO 2007045096 A1 WO2007045096 A1 WO 2007045096A1 CA 2006001724 W CA2006001724 W CA 2006001724W WO 2007045096 A1 WO2007045096 A1 WO 2007045096A1
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Prior art keywords
bzi
alkyl
compound according
dna
compound
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PCT/CA2006/001724
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French (fr)
Inventor
Malvinder Pal Singh
Manashi Nithar Chatterjee
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University Of Saskatchewan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems

Definitions

  • the present invention relates to oligoheteroaromatic assemblies with luminescence characteristics, and in particular novel compounds and compositions based on systematically integrating these assemblies with polyamide oligomers of optimally spaced nitrogen-containing heteroaromatics capable of binding to targeted DNA sequences.
  • DNA sequence targeted chemical agents have been attracting increasing research focus, 1"9 and perhaps the most actively used paradigm with respect to the design of polyamide class of DNA minor groove binding agents is distamycin, a trispyrrolecarboxamide member of the pyrrole amidine class of antibiotics.
  • Other extended oligoheteroaromatic structures such as the bis-benzimidazole class of chromosomal stains (Hoechst 33258), 10'14 diamidine derivatives, 15"17 and designer analogs have also received some attention.
  • the present invention therefore includes a compound of formula I:
  • each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N;
  • R 1 is selected from C 1-6 alkyl, C 3 -i 2 cycloalkyl, aryl, C(O)NHC, -6 alkyl, C(O)NHC 3- ncycloalkyl and C(O)NH(Ci -6 alkylene-N(R 6 ) 2 ), in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N, NR 6 or
  • N(R 6 ) 2 ; R and R >2 ! are independently selected consisting of H and Ci -6 alkyl, or R and R are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 3 is Ci- ⁇ alkylene or C 2 . 6 alkenylene, both of which are optionally substituted with one or more of Ci- ⁇ alkyl, or OH, and both optionally have one or more of the carbons replaced with O, S, NR 6 or N(R 6 ) 2 ;
  • R 4 and R 4 are independently selected from H and or R 4 and R 4 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C 3 _ 6 heterocycle, Ci_ 6 alkyl, OH and OCi -6 alkyl;
  • R 6 is H or C,. 6 alkyl
  • R 7 is Ci_ 4 alkylene or C 2 . 4 alkenylene both of which are optionally substituted with one or more of Ci -6 alkyl, OCi- 6 alkyl or OH and both optionally have one or more carbons replaced with O, S, NR 6 or N(R 6 ) 2 ; m is 1 , 2, 3, 4 or 5, and when m is other than 1, only the terminal monomeric unit represented by m is substituted with R 1 ; n is 1, 2, 3, 4 or 5; o is 0, 1, 2, 3, 4 or 5 p is O, 1, 2, 3, 4 or 5; and q is O, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof, with the proviso that when R 1 is selected from C(O)NHC 1-6 alkyl, C(O)NHC 3 .
  • R 5 is selected from pyrrolyl, imidazolyl, benzimidazolyl, imidazopyridinyl and purinyl.
  • the present invention includes a compound selected from a compound of the formula I:
  • each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N; R 1 is selected from C 3 -i 2 cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N,
  • R 2 and R 2 are independently selected from H and Ci -6 alkyl, or R 2 and R 2 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 3 is Ci. ⁇ alkylene or C 2 . 6 alkenylene, both of which are optionally substituted with one or more of Ci -6 alkyl, or OH, and both optionally have one or more of the carbons optionally replaced with O, S, NR 6 or N(R 6 ) 2 ;
  • R 4 and R 4 are independently selected from H and Ci -6 alkyl, or R 4 and R 4 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3_6heterocycle, Ci -6 alkyl, OH and
  • R 6 is H or Ci -6 alkyl
  • R 7 is Ci- 4 alkylene or C 2-4 alkenylene both of which are optionally substituted with one or more of C ⁇ alkyl, OCi -6 alkyl or OH and both optionally have one or more carbons replaced with O, S, NR 6 or N(R 6 ) 2 ;
  • m is 1, 2, 3, 4 or 5, provided that when m is other than 1, only the terminal monomer unit represented by m is substituted with R 1 ;
  • n is 1, 2, 3, 4 or 5; o is 0, 1, 2, 3, 4 or 5 p is 0, 1 , 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof.
  • the hydrogen bond forming capacity of the Pzi ring system is greater than that of the Bzi system, therefore the assemblies comprising the Pzi system are much more selective for recognition of thymine and cytosine bases in AT and GC base pairs in the configuration that they are used in the compounds of the invention.
  • the Pzi system has 2-3 orders of magnitude greater capacity for such high fidelity binding to the said bases within DNA.
  • a greater selectivity is achieved by the side by side configuration of individual molecules within the groove formed by the pairing of two strands of DNA and it has become possible by the use of the compounds of the present invention to intellectually predict the exact match between the edge-wise shape of the ligand molecules and that formed by the order of the functional groups of DNA bases in the complete length of the target DNA site (regardless of the dimensions of such site).
  • the present invention therefore includes a method for selectively forming a complex between target DNA and a compound of the present invention, the method comprising: contacting the target DNA with the compound of the invention; wherein the compound of the invention is capable of selectively binding to a sequence on the target DNA, for example, under physiological conditions where selective complexes form between the compound of the invention and the target DNA.
  • the sequence is in a minor groove on the target DNA.
  • the compound of the invention and the target DNA are at a binding stoichiometry of about 2:1
  • a method of selectively detecting the presence of a sequence in a sample having DNA is included within the scope of the present invention.
  • complexes By contacting the DNA with a compound of the invention having a detectable label and that is capable of selectively binding to the sequence on the DNA, complexes can be selectively formed between the compound and the target sequence on DNA.
  • the detectable label on the compounds can be observed from the complexes, indicating the presence of a selected sequence in the sample.
  • the detectable label would comprise this moiety.
  • the present invention relates to a method for isolating target DNA from a sample comprising a mixture of DNA.
  • a compound of the invention that is capable of selectively binding to a sequence on the target DNA
  • complexes can be selectively formed between the compound of the invention and a target sequence on the DNA.
  • the target DNA can then be isolated from the mixture of DNA.
  • these ligands have already tested favorably for their ability to obliterate DNA binding proteins like DNAase I, restriction enzymes specific for (A/T)GGCC(T/A) sites, and therefore will be useful in vitro molecular probes in PCR, gene cloning, molecular biology and molecular genetics. Because of their high affinity and specific binding for the said segments, and the occurrence of those nucleotide segments in gene sequences of cancer related proteins, such ligands will also be useful in modulation of such genes to be expressed, both in vitro and in vivo.
  • the present invention relates to a method for modulating transcription of a target gene in a cell comprising contacting the cell with an effective amount of a compound of the invention under conditions sufficient for the formation of complexes selectively between the compound of the invention and the target gene and wherein such complex formation modulates the transcription of the target gene.
  • the target sequence on the gene is a sequence on transcriptional regulatory regions of the gene.
  • the cell may be a bacterial, virus or pathogen cell.
  • the target gene may be any gene implicated in the propagation of the cell or of a disease state.
  • the target gene may be an oncogene.
  • the cell may be in a subject, such as a mammal, as in a human or outside the subject, such as, for example, ex vivo treatment.
  • the present invention also relates to a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof.
  • a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof.
  • complexes can be selectively formed between the compound of the invention and a target oncogene on the DNA.
  • Selective formation of the complexes at sequences on the target oncogene reduces transcription of that oncogene, such as when complexes are formed at transcriptional regulatory regions of the oncogene.
  • the subject is a mammal, such as a human.
  • the present invention further includes a method of treating an infection by a virus, bacterium or pathogen in a subject comprising administering an effective amount of a compound of the invention to a subject in need thereof.
  • the present invention further includes a use of a compound of the invention to treat cancer and a use of a compound of the invention to prepare a medicament to treat cancer. Further, the invention includes a use of a compound of the invention to treat a viral, bacterial or pathogen infection as well as a use of a compound of the invention to prepare a medicament to treat a viral, bacterial or pathogen infection.
  • the alternatively functionalized benzimidazole and pyridoimidazole may also be used for new types of integrated fluorophore-poly amide conjugates for targeting the DNA minor groove in a sequence-selective manner.
  • Advantages of these novel conjugates over the polyamide ligands in the prior art include, for example, the N-methylpiperazine group (when utilized) providing easier trafficking across cellular membranes, and the benzimidazole/pyridoimidazole units, for their useful fluorescence characteristics in diagnostic genomics applications.
  • the reduced number of amide bonds in these conjugates will also make them less susceptible to cellular degradation by peptidase class of enzymes.
  • the fluorogenic rings are photostable and do not suffer from the problems associated with the oxidative degradation as known for electron- rich pyrrole/hydroxypyrrole rings.
  • Figure 1 shows the chemical structures of compounds Fl to F7.
  • the circles with plus sign correspond to the N-methylpiperazine system, as it exists in a protonated positively charged form at neutral pH.
  • Figure 2 shows the chemical structures of compounds Gl to G7.
  • the imidazopyridine (intermittently called pyridoimidazole) fragment is different from the benzimidazole ring in terms of the fluorescence and acid/base characteristics.
  • FIG. 1 shows the chemical structures of compounds Hl to H4. The abbreviated naming of the compounds is the same as that indicated for Figures 1 and 2. Diamonds with a plus sign represent the Dp (dimethylaminopropanamide) end-groups.
  • Figure 4 shows the chemical structures of compounds Il and 12. The abbreviated naming of the compounds is the same as that indicated for Figures 1 and 2.
  • Figure 5 shows the chemical structures of compounds Jl to J4.
  • the abbreviated naming of the compounds is the same that as indicated for Figures 1 and 2.
  • Figure 6 shows the chemical structures of compounds Kl to K6.
  • the imidazole ring substituents on the benzimidazole rings to the left are expected to be protonated at pH ⁇ 7.
  • the Bzi and Bzi are two differently functionalized benzimidazole systems.
  • Figure 7A shows the chemical structures of compounds Ll to L4.
  • the abbreviated naming of the compounds is the same that as indicated for Figures 1 -6.
  • the two benzimidazole-pyrrole building blocks are separated by the imidazole residue alone (Ll), or in combination with aliphatic amino acid spacers, glycine (termed ⁇ ), beta- alanine( ⁇ )and gamma- aminobuty rate ( ⁇ ), in L2, L3, and L4, respectively.
  • Figure 7B shows the chemical structures of compounds Nl to N3.
  • Figure 8 is a graph showing the ⁇ T m values measured for the interaction between distamycin against a panel of AT sites and GC sites.
  • Figure 9 is a graph showing the ⁇ T m values measured for the interaction between actinomycin against a panel of AT sites and GC sites.
  • A ⁇ T m values measured for the interaction between actinomycin and 16 different DNA molecules, containing different permutations of a 4-base pair variation shown in the plots.
  • B ⁇ T m values for actinomycin-stabilized DNA duplexes, containing different permutations of a 4- base pair variation around the G # C base pairs.
  • Actinomycin a known anticancer drug agent is a standard with relatively increased binding preference for GC-rich base sequences in DNA.
  • Figure 10 is a graph showing the ⁇ T m values measured for the interaction between compounds R4, F4 and G5 with the panel of 16 GC-rich sites only (2: 1 binding mode, looking at GC selectivity). Binding to the other 16 AT-rich sites was very poor for each of them.
  • A Compound R4 (an all five-membered ring polyamide),
  • B compound F4 (MP-BziPyPylmlm) and
  • C compound G4 (MP-PziPyPylmlm). Note, binding varies according to their respective module compositions.
  • Figure 11 shows the single mismatch effects on the variation in ⁇ T m values for each of the ligands that contain identical polyamide fragments but with variant modules containing the positively charged end.
  • Q and F refer to the quencher (DABCYL) and fluorescent dye (FITC) covalently attached to the ends of DNA strands.
  • DBCYL quencher
  • FITC fluorescent dye
  • Figure 12 shows the UWVIS absorption spectra for the Bzi ligand (F4) and Bzi:DNA complex.
  • A shows the spectra of the ligand, in absence of DNA, with increasing concentration.
  • B gives the measurement made in a titration experiment with increasing [Bzi] added to a fixed [DNA].
  • C shows difference absorption plots obtained by subtracting the DNA absorption from those in B.
  • D normalized ligand spectra subtracted from those in B. Arrows are drawn immediately to the right of the main absorption bands due to the ligand.
  • DNA 260 ran
  • the E values are expressed in the units M -1 Cm "1 .
  • DNA d(CATGGCCATG) 2 .
  • Figure 13 shows the UV/VIS absorption spectra for the Pzi ligand (G4) and Pzi:DNA complex.
  • A shows the spectra of the ligand, in absence of DNA, with increasing concentration.
  • B gives the measurement made in a titration experiment with increasing [Pzi] added to a fixed [DNA].
  • C shows difference absorption plots obtained by subtracting the DNA absorption from those in B.
  • D normalized ligand spectra subtracted from those in B. Arrows are drawn immediately to the right of the main absorption bands due to the ligand.
  • DNA 260 ran) refers to the band at 260 nm for stacked base pairs in the duplex. The e values are expressed in the units NT 1 Cm "1 .
  • DNA d(CATGGCCATG) 2 .
  • Figure 14 shows a putative 2:1 binding models for 5 different compounds of the present invention on the basis of shape, structure and functional group matched recognition of a specified DNA target sequence.
  • Figure 15 shows a diagrammatic representation of 34 intermolecular ligand-DNA NOE relationships between the ligand F4 and d(C ATGGCC ATG) 2 .
  • a model ball- stick representation of the binding model is shown beneath.
  • Benzimidazole rings are shown as rectangles, pyrrole residues as darker circles, and imidazole systems as lighter circles.
  • Figure 16 shows a diagrammatic representation of 48 intermolecular ligand-DNA NOE relationships between the ligand G4 and d(C ATGGCC ATG) 2 .
  • Pyridoimidazole rings are shown as rectangles, pyrrole residues as darker circles, and imidazole systems as lighter circles.
  • Figure 17 shows a view into the minor groove of the mean structure of the 2:1 Bzi- DNA complex, with each ligand molecule proximal to the strand containing G residues at positions where Im residues reside in the ligand.
  • Figure 18 is a schematic providing a summary of the binding models for the 2:1 Bzi: DNA and Pzi: DNA complexes, showing the intermolecular hydrogen bonds between the ligand molecules and DNA. Distances measured from the model structures are provided beneath the model. In all respects, the Bzi and Pzi ligands bind to the identical core site consisting of G* C base pairs.
  • Ci -y alkyl as used herein means straight and/or branched chain, saturated alkyl groups containing from one to y carbon atoms and includes (depending on the identity of y) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, A- methylpentyl, n-hexyl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
  • Ci -y alkenyl as used herein means straight and/or branched chain, unsaturated alkyl groups containing from one to n carbon atoms and one or two double bonds, and includes (depending on the identity of y) vinyl, allyl, 2- methylprop- 1 -enyl, but-1-enyl, but-2-enyl, but-3-enyl, 2-methylbut-l-enyl, 2- methylpent-1-enyl, 4-methylpent-l-enyl, 4-methylpent-2-enyl, 2-methylpent-2-enyl, 4-methylpenta-l,3-dienyl, hexen-1-yl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
  • Ci- y alkynyl as used herein means straight and/or branched chain, unsaturated alkyl groups containing from one to y carbon atoms and one or two triple bonds, and includes (depending on the identity of y) propargyl, but-1-ynyl, but-2- ynyl, but-3-ynyl, 4-methylpent-l-ynyl, 4-methylpent-2-ynyl, hex-1-ynyl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
  • cyclo(C 3 -C y )alkyl as used herein means saturated cyclic alkyl groups containing from three to y carbon atoms and includes (depending on the identity of y) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexyl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
  • aryl as used herein means a monocyclic or bicyclic ring system containing one or two aromatic rings and from 6 to 14 carbon atoms and includes phenyl, naphthyl, anthraceneyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like.
  • monocyclic or polycyclic heterocycle refers to monocyclic, bicyclic, tricyclic or quadracyclic ring fused systems containing at least one nitrogen atom and 1 to 6 other heteroatoms selected from O, S and N (with the balance of the ring system being C).
  • the ring system may contain from 3 to 20 atoms, suitably 5 to 16 atoms, and may be fully saturated, partially saturated or aromatic.
  • the polycyclic ring system is a fused quadracyclic ring system comprising 4 nitrogen atoms.
  • C 3-6 heterocycle refers monocyclic rings containing 3 to 6 atoms of which 1 to 4 atoms are a heteroatom selected from N, S and O (with the balance of the ring system being carbon).
  • the ring may be fully saturated, partially saturated or aromatic.
  • the heterocyclic rings contain 5 to 6 atoms, of which at least one atom is nitrogen and the rings are aromatic.
  • heterocycles include imidazole, pyrrole, indole, pyridine and the like.
  • alkylene as used herein means bifunctional straight and/or branched alkyl groups containing the specified number of carbon atoms.
  • alkenylene as used herein means bifunctional straight and/or branched alkenyl groups containing the specified number of carbon atoms.
  • pyridoimidazole or "Pzi” as used herein refers to a monomeric unit having the following structure:
  • benzimidazole or "Bzi” as used herein refers to a monomeric unit having the following structure:
  • the compounds of the present invention selectively bind to specific "target DNA” sequences.
  • the target DNA sequences may be single stranded or double stranded DNA (dsDNA).
  • dsDNA double stranded DNA
  • the target DNA sequence is ds DNA.
  • the compounds of the invention can bind in a "sequence specific manner" which means that the compounds of the invention have the ability to form at least one complementary pair with at least one nucleotide base on the DNA target sequence.
  • pharmaceutically acceptable salt means an acid addition salt which is suitable for or compatible with the treatment of patients.
  • pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any base compound of the invention, or any of its intermediates.
  • Basic compounds of the invention that may form an acid addition salt include, for example, where the Ci ⁇ alkyl group of R 1 and/or R 2 is substituted with a group having a basic nitrogen, for example NH 2 and
  • Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form.
  • mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sul
  • the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.
  • the selection of the appropriate salt will be known to one skilled in the art.
  • Other non-pharmaceutically acceptable acid addition salts e.g. oxalates, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
  • solvate means the incorporation of molecules of a suitable solvent into the crystal lattice of a compound of the invention.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate”.
  • solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • prodrug as used herein will be functional derivatives of a compound of the invention which are readily convertible in vivo into the compound from which it is notionally derived.
  • Prodrugs may be conventional esters formed with, for example, available hydroxy, or amino groups.
  • an available OH may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine).
  • Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C 8 -C 24 ) esters, acyloxymethyl esters, carbamates and amino acid esters.
  • the prodrugs are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo.
  • Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985.
  • a “effective amount” as used herein refers to an amount of a compound of the invention that is effective in achieving a desired effect for a particular application. Effective amounts can vary depending on the specific compound of the invention and the accessibility of the target sequence.
  • a "therapeutically effective amount" of a compound of the invention, as used herein is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount” or synonym thereto, when used in therapeutic applications, depends upon the context in which it is being applied.
  • an effective amount of a compound of the invention is that amount sufficient to achieve such a modulation in the transcription of the target gene as compared to the modulation obtained in the absence of the compound of the invention.
  • therapeutically effective amounts of the compounds of the invention are used to treat, modulate, attenuate, reverse, or effect conditions which benefit from modulation of a target gene.
  • An "effective amount" is intended to mean that amount a compound of the invention that is sufficient to treat, prevent or inhibit conditions which benefit from modulation of a target gene or a disease associated with modulation of a target gene.
  • a "therapeutically effective amount" of a compound of the invention is an amount which modulates transcription of a target gene (e.g., as determined by clinical symptoms) in a subject as compared to a control.
  • a therapeutically effective amount of a compound of the invention may be readily determined by one of ordinary skill by routine methods known in the art. Generally ranges of amounts of the compounds of the invention effective for reducing transcription of a target gene in a cell are 1 pM to 50 mM, such as 1 nM to 100 ⁇ M.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • “Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • prevention refers to a reduction in the risk or probability of a subject becoming afflicted with a condition which benefits from modulation of a target gene or manifesting a symptom associated with a condition which benefits from modulation of a target gene.
  • compound(s) of the invention means compound(s) of formula I, pharmaceutically acceptable salts, prodrugs and/or solvates thereof.
  • subject as used herein includes all members of the animal kingdom including human.
  • the animal is preferably a human.
  • a cell as used herein includes a plurality of cells. Administering a compound to a cell includes in vivo, ex vivo and in vitro treatment.
  • a novel class of polyamide oligomers of nitrogen-containing heteroaromatic rings has been prepared.
  • the compounds of the invention are useful as binding agents to targeted DNA sequences in the minor groove.
  • the present invention relates to a compound selected from a compound of formula I:
  • each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N; R 1 is selected from C ⁇ alkyl, C 3 -i 2 cycloalkyl, aryl, C(O)NHC i -6 alkyl, C(O)NHC 3 .
  • R 2 and R 2 are independently selected from H and Ci- 6 alkyl, or R 2 and R 2 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 3 is Ci -6 alkylene or C 2-6 alkenylene, both of which are optionally substituted with one or more of Ci. 6 alkyl, OCi. 6 alkyl or OH, and both optionally have one or more of the carbons replaced with O, S, NR 6 or N(R 6 ) 2 ;
  • R 4 and R 4 are independently selected from H and Ci- 6 alkyl, or R 4 and R 4 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C 3-6 heterocycle, Ci -6 alkyl, OH and 0Ci. 6 alkyl;
  • R 6 is H or C, -6 alkyl
  • R 7 is C ⁇ -4alkylene or C 2-4 alkenylene both of which are optionally substituted with one or more of Ci -6 alkyl, or OH and both optionally have one or more carbons replaced with O, S, NR 6 or N(R 6 ) 2 ; m is 1, 2, 3, 4 or 5, and when m is other than 1, only the terminal monomeric unit represented by m is substituted with R 1 ; n is 1, 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is O, 1, 2, 3, 4 or 5; and q is O, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof, with the proviso that when R 1 is selected from C(O)NHCi -6 alkyl, C(O)NHC 3- , 2 cycloalkyl and C(O)NH(C )-6 alkylene-N(R 6 ) 2 ), R 5 is selected from pyrrolyl, imidazolyl, benzimidazolyl
  • each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N;
  • R 1 is selected from Ci -6 alkyl, C 3 _i 2 cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N,
  • R 2 and R 2 are independently selected from H and Ci -6 alkyl, or R 2 and R 2 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 3 is C ⁇ - 6 alkylene or C 2 - 6 alkenylene, both of which are optionally substituted with one or more of Ci -6 alkyl, or OH, and both optionally have one or more of the carbons optionally replaced with O, S, NR 6 or N(R 6 ) 2 ;
  • R 4 and R 4 are independently selected from H and or R 4 and R 4 are joined by R 7 to form a 5 to 8 membered ring between two adjacent monomeric units;
  • R 5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C 3-6 heterocycle, Ci -6 alkyl, OH and OC]. 6 alkyl;
  • R 6 is H or Ci -6 alkyl
  • R 7 is Ci -4 alkylene or C 2-4 alkenylene both of which are optionally substituted with one or more of or OH and both optionally have one or more carbons replaced with O, S, NR 6 or N(R 6 ) 2 ; m is 1, 2, 3, 4 or 5, provided that when m is other than 1, only the terminal monomer unit represented by m is substituted with R 1 ; n is 1, 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is 0, 1, 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof.
  • m is 1.
  • m is 1 and p is 0.
  • m is 1
  • o 0 and p is 0.
  • n is 1 , 2, 3 or 4 and q is 0 or 1.
  • n and o are both 0.
  • m and p are both 1.
  • m is 1, n is 1 or 2, o is 0, p is 1 and q is 0 or 1.
  • R 1 is selected from
  • R 1 is selected from N-C ⁇ alkyl- piperazinyl, imidazolyl, pyrazolyl, triazolyl, morpholinyl, each of which are connected to the compound of Formula I through the nitrogen atom. More particularly, R 1 is N-methylpiperazinyl. In yet another embodiment of the present invention, R 1 is selected from
  • R 2 and R 2 are, independently, H or
  • Ci- 4 alkyl More particularly, R 2 and R 2 are both methyl. In further embodiments of the present invention, R 2 and R 2 are joined by R 7 to form a 5 to 8 membered ring, suitably a 6 membered ring, between two adjacent monomeric units where R 7 is Ci. 4 alkylene or C 2 ⁇ alkenylene. In embodiments of the present invention, R 3 is Ci ⁇ alkyene. More particularly, R 3 is Ci- 3 alkylene.
  • R 4 and R 4 are independently selected from H and In further embodiments of the present invention, R 4 and R 4 are joined by R 7 to form a 5 to 8 membered ring, suitably a 6 membered ring, between two adjacent monomeric units, where R 7 is Ci ⁇ alkylene or C 2 - 4 alkenylene.
  • R 5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C 5- ⁇ heterocycle, d ⁇ alkyl, OH and OCi -6 alkyl.
  • R 5 is a nitrogen-containing mono- or bicyclic aromatic heterocycle. More particularly, R 5 is selected from optionally substituted derivatives of pyrrole, imidazole, oxazole, thiazole, benzimidazole, imidazopyridine, benzoxazole, benzothiazole, and indole.
  • Optional substituents are one or more OH and OCi ⁇ alkyl, suitably one of more of CH 3 , OH or OCH 3 , more suitably one of CH 3 , OH or OCH 3 .
  • R 5 is unsubstituted.
  • R 5 is Pzi or Bzi, substituted with N-methyl imidazole or N-methylpyrrole at the position between the two nitrogens on the 5 membered ring portion of this group.
  • R 6 is H or Suitably, R 6 is H or CH 3 .
  • the compound of the formula I is selected from:
  • R 4 and R 4 are independently selected from H and Ci- ⁇ alkyl; q is 1, 2, 3, 4 or 5 (suitably q is 2, 3 or 4, most suitably q is 3), and and pharmaceutically acceptable salts, solvates and prodrugs thereof.
  • each X may be the same or different and are independently selected from CH and N; wherein each Y may be the same or different and are independently selected from CH and N, and and pharmaceutically acceptable salts thereof.
  • III. Methods of Detecting and Isolating Target DNA Sequences The formation of complexes between target DNA and the compounds of the present invention may be used for diagnostic, purification or research purposes, and the like. Compounds of the present invention can be used to detect specific dsDNA sequences in a sample without melting the dsDNA. Examples of diagnostic applications for which compounds of the present invention may be used include detection of alleles, identification of mutations, identification of a particular host, e.g.
  • the pathogens may be viruses, bacteria, fungi, protista, chlamydia, or the like.
  • the hosts may be vertebrates or invertebrates, including insects, fish, birds, mammals, and the like or members of the plant kingdom.
  • the present invention therefore includes a method for selectively forming a complex between target DNA and a compound of the present invention, the method comprising: contacting the target DNA with the compound of the invention; wherein the compound of the invention is capable of selectively binding to a sequence on the target DNA.
  • the compound of the invention selectively binds to the target DNA under physiological conditions where complexes form between the compound of the invention and the target DNA.
  • the specific sequence is in a minor groove on the target DNA.
  • the compound of formula I and the target DNA is at a binding stoichiometry of 2:1.
  • the target gene comprises all or a fragment of a sequence selected from AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, suitably 5'-ATGGCCAT and 5'- AAGCGCTT.
  • a method of detecting the presence of a sequence in a sample comprising DNA comprises contacting the sample with a compound of the invention which is capable of selectively binding to sequences on the DNA, and wherein the compound of the invention has at least one detectable label; and monitoring the detectable label in the sample, wherein the presence of the detectable label is indicative of the presence of the selected sequence.
  • the present invention relates to a method for isolating target DNA from a sample comprising a mixture of DNA.
  • the method comprising: contacting the target DNA with a compound of the invention which is capable of selectively binding to a sequence on the target DNA, and wherein complexes selectively form between the compound of the invention and the target DNA; and isolating the selected complexes.
  • Compounds of the present invention are useful for detecting the presence of DNA of a specific sequence for diagnostic or preparative purposes.
  • the sample containing the target DNA sequence can be contacted with a compound of the present invention linked to a solid substrate, thereby isolating DNA comprising a desired sequence.
  • compounds of the invention linked to a suitable detectable marker such as biotin, a hapten, a radioisotope or a dye molecule, can be contacted by a sample containing the target DNA.
  • Detection of complexes of the compounds of the invention and target DNA sequences is facilitated by the presence of luminescent or fluorescent moieties already incorporated with in their molecular structure. These moieties may be utilized by a person skilled in the art using well know methods to detect complexes of the invention with target DNA sequences.
  • detectable labels include additional fluorescers, e.g. dansyl, fluorescein, Texas red, isosulfan blue, ethyl red, and malachite green, chemiluminescers, magnetic particles, colloidal particles, gold particles, light sensitive bond forming compounds, i.e.
  • chelating compounds such as EDTA, NTA, tartaric acid, ascorbic acid, polyhistidines of from 2 to 8 histidines, alkylene polyamines, etc., chelating antibiotics, such as bleomycin, where the chelating compounds may chelate a metal atom, such as iron, cobalt, nickel, technetium, etc., where the metal atom may serve to cleave DNA in the presence of a source of peroxide, intercalating dyes, such as ethidium bromide, thiazole orange, thiazole blue, TOTO, 4',6-diamidino-2-phenylindole (DAPI), etc., enzymes, such as ⁇ -galactosidase, NADH or NADHP dehydrogenase, malate dehydrogenase, lysozyme, peroxida
  • chelating compounds such as EDTA, NTA, tartaric acid, ascorbic acid, polyhistidines of from
  • colloidal particles e.g., gold particles, fluorescent particles, peroxides, DNA cleaving agents, oligonucleotides, oligopeptides, NMR agents, stable free radicals, metal atoms, etc.
  • the compounds of the invention may be combined with other labels, such as haptens for which a convenient receptor exists, e.g. biotin, which may be complexed with avidin or streptavidin and digoxin, which may be complexed with antidigoxin, etc. where the receptor may be conjugated with a wide variety of labels, such as those described above.
  • the compounds may be joined to sulfonated or phosphonated aromatic groups, e.g.
  • naphthalene to enhance inhibition of transcription, particularly of viruses (Clanton et al., Antiviral Res., 27:335-354, 1995).
  • Polymers, particularly water soluble polymers, which may find use are cellulose, poly(vinyl alcohol), poly(vinyl acetate-vinyl alcohol), polyacrylates, and the like.
  • a radioactive moiety may also be employed as a detectable label, such tritium, 14 C, 125 I, or the like.
  • the radiolabel may be a substituent on a carbon or a heteroatom of any atom in any monomer, or the radiolabel may be a substituent at either terminus of the oligomer.
  • the radiolabel may serve numerous purposes in diagnostics, cytohistology, radiotherapy, and the like.
  • the target DNA may be extracellular or intracellular. When extracellular, the DNA may be in solution, in a gel, on a slide, or the like.
  • the DNA may be present as part of a whole chromosome or fragment thereof of one or more centiMorgans.
  • the DNA may be part of an episomal element.
  • the DNA may be present as smaller fragments ranging from about 20, usually at least about 50, to a million base pairs, or more.
  • the DNA may be intracellular, chromosomal, mitochondrial, plastid, kinetoplastid, or the like, part of a lysate, a chromosomal spread, fractionated in gel elecrophoresis, a plasmid, or the like, being an intact or fragmented moiety.
  • the DNA may be combined with the compounds of the inventionin appropriately buffered medium, generally at a concentration in the range of about 0.1 nM to 1 mM.
  • Various buffers may be employed, such as TRIS, HEPES, phosphate, carbonate, or the like, the particular buffer not being critical to this invention.
  • buffer usually in the range of about 10-200 mM.
  • Other additives which may be present in conventional amounts include sodium chloride, generally from about 1-250 mM, dithiothreitol, and the like, the particular nature or quanitity of salt not being critical to this invention.
  • the pH will generally be in the range of about 6.5 to 9, the particular pH not being critical to this invention.
  • the temperature will generally be in a range of 4-45 0 C, the particular temperature not being critical to this invention.
  • the target DNA may be present in from about 0.001 to 100 times the moles of compound of the invention.
  • the present invention also provides a diagnostic system, suitably in kit form, for assaying for the presence of a target DNA sequence bound by compounds of the invention in a body sample, such as brain tissue, cell suspensions or tissue sections, or body fluid samples such as colony stimulating factor (CSF), blood, plasma or serum, where it is desirable to detect the presence, and suitably the amount, of the target DNA sequence bound by the compound of the invention in the sample according to the diagnostic methods described herein.
  • a diagnostic system suitably in kit form, for assaying for the presence of a target DNA sequence bound by compounds of the invention in a body sample, such as brain tissue, cell suspensions or tissue sections, or body fluid samples such as colony stimulating factor (CSF), blood, plasma or serum, where it is desirable to detect the presence, and suitably the amount, of the target DNA sequence bound by the compound of the invention in the sample according to the diagnostic methods described herein.
  • a body sample such as brain tissue, cell suspensions or tissue sections, or body fluid samples such as colony stimulating factor (CSF), blood,
  • the diagnostic system includes, in an amount sufficient to perform at least one assay, a specific compound of the invention as a separately packaged reagent. Instructions for use of the packaged reagent(s) are also typically included.
  • the term "package” refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a compound of the present invention.
  • a package can be a glass vial used to contain milligram quantities of a contemplated compound or it can be a microliter plate well to which microgram quantities of a contemplated compound have been operatively affixed, i.e., linked so as to be capable of being bound by the target DNA sequence.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent or sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system of the present invention might also include a detectable label and a detecting or indicating means capable of signaling the binding of the contemplated compound of the present invention to the target DNA sequence.
  • detectable labels such as biotin, and detecting or indicating means, such as enzyme-linked (direct or indirect) streptavidin, are well known in the art.
  • the detectable label is incorporated into the compound of the invention and the instructions for use will explain to a person skilled in the art how to utilize the compound of the invention to determine the presence and/or amounts of the target DNA sequences using this labeling method.
  • Kits may optionally contain instructions for administering compounds or compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of compounds of the invention by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for compounds of the invention. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies.
  • the kits can include compounds of the invention in the solid phase or in a liquid phase (such as buffers provided) in a package.
  • the kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
  • the present inventors have examined the properties of DNA binding molecules for a non-denaturing fluorescent detection of specific sites in solution within short segments of DNA. Interesting applications such as chromosomal staining and as diagnostic probes are possible.
  • An application for this science may be related to diagnostics and "SNP typing" in particular, in which the identity of the SNP containing sequence is known (and a proper means for SNP detection is the only obstacle).
  • SNPs single nucleotide polymorphisms
  • Most methods of DNA detection involve hybridization of an oligonucleotide probe to its complementary single-strand nucleic acid target leading to signal generation. Detection by hybridization requires DNA denaturation conditions, and it remains a challenge to develop sequence specific fluorescent probes for DNA in the double strand form. IV. Methods of Modulating Transcription of a Target Gene
  • the present invention relates to a method for modulating transcription of a target gene in a cell.
  • the method comprising: contacting the cell with an effective amount of a compound of formula I which is capable of binding to a specific sequence on the target gene under conditions where specific complexes form between the compound of formula I and the target gene, and wherein the level of transcription of the target gene is modulated.
  • exemplary compounds from the library of disclosed assemblies are very selective for binding AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, type self-compementary DNA segments, such compounds that are also light-emissive probes, will also be useful in the detection and quantitative analysis of such sequence contents in diagnostic applications. Likewise, such compounds will also be useful in DNA/gene staining methods for in vitro and in vivo photomicroscopy based on fluorescence capturing detection.
  • these ligands have already tested favorably for their ability to obliterate DNA binding proteins like DNAase I, restriction enzymes specific for (A/T)GGCC(T/A) sites, and therefore will be useful in vitro molecular probes in PCR, gene cloning, molecular biology and molecular genetics. Because of their high affinity and specific binding for the said segments, and the occurrence of those nucleotide segments in gene sequences of cancer related proteins, such ligands will also be useful in modulation of such genes to be expressed, both in vitro and in vivo.
  • the present invention relates to a method for modulating transcription of a target gene in a cell comprising contacting the cell with an effective amount of a compound of the invention under conditions sufficient for the formation of complexes selectively between the compound of the invention and the target gene and wherein such complex formation modulates the transcription of the target gene.
  • the target sequence on the gene is a sequence on transcriptional regulatory regions of the gene. AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, suitably 5'-ATGGCCAT and 5'-AAGCGCTT.
  • the cell may be for example, a mammalian cell, such as a human cell. Alternatively, the cell may be a bacterial, virus or pathogen cell.
  • the target gene may be any gene implicated in the propagation of the cell or of a disease state.
  • the target gene may be an oncogene.
  • the cell may be in a subject, such as a mammal, as in a human or outside the subject, such as, for example, ex vivo treatment.
  • the present invention also relates to a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof.
  • a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof.
  • complexes can be selectively formed between the compound of the invention and a target oncogene on the DNA. Selective formation of the complexes at sequences on the target oncogene reduces transcription of that oncogene, such as when complexes are formed at transcriptional regulatory regions of the oncogene.
  • the subject is a mammal, such as a human.
  • the present invention further includes a method of treating an infection by a virus, bacterium or pathogen in a subject comprising administering an effective amount of a compound of the invention to a subject in need thereof.
  • highly selective representative DNA binding ligands, F4, G4, and F6 were determined to be binding strongly and bindingwith high fidelity to their specific sites, 5'-ATGGCCAT (either of ligands F4 or G4), and 5'-5'- AAGCGCTT (ligand F6). From information on those target protein gene sequences (coding regions of the gene expressing those proteins), the occurrence of the aforementioned targeted octanucleotide sequence (and their frequency of occurrence in the gene) were identified. These potential therapeutic targets are listed below:
  • Glutathione reductase (mitochondrial), 1440 base pairs ; frequency of either
  • Retinoic acid receptor alpha 1389 base pairs; frequency of either AGGCCA,
  • AGGCCT TGGCCA
  • TGGCCT 2 Ribonucleotide reductase, 2379 base pairs, frequency of either AGGCCA, AGGCCT,
  • DNA Topoisomerase II 4596 base pairs; frequency of either AGGCCA, AGGCCT,
  • AAGGCCTA 1. Low density lipoproteins (LDL), 2583 base pairs; frequency of either AGGCCA,
  • PML-RAR alpha protein 200 base pairs, frequency of either AGGCCA, AGGCCT,
  • Apoptosis regulator Bcl-2 720 base pairs [chromosome 18]; frequency of either
  • DNA polymerase 1797 base pairs [chromosome H]; frequency of either AGGCCA,
  • DNA helicase 5130 base pairs [chromosome 5]; frequency of either AGGCCA,
  • COX-2 1815 base pairs [chromosome I]; frequency of either AGGCCA, AGGCCT,
  • Beta tubulin 1356 base pairs [chromosome 20]; frequency of either AGGCCA,
  • AAGGCCAA 1.
  • Matrix metalloprotease 2 (MMP-2), 1983 base pairs [chromosome 16]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT 5; frequency of
  • Estrogen receptor 1593 base pairs [chromosome 14]; frequency of either AGGCCA,
  • Arachi donate 5-lipoxygenase, 1989 base pairs [chromosome 17]; frequency of either
  • Androgen receptor 2760 base pairs [chromosome X]; frequency of either AGGCCA,
  • Alpha- IA adrenergic receptor 1401 base pairs [chromosome 8]; frequency of either
  • Retinoic acid receptor alpha 1389 base pairs; frequency of either AGCGCA,
  • LDL Low density lipoproteins
  • AGCGCT, TGCGCA, or TGCGCT 4.
  • Tubulin, 1335 base pairs [chromosome 6]; frequency of AAGCGCAT 1.
  • Thymidylate synthase 942 base pairs; frequency of either AGCGCA, AGCGCT,
  • Apoptosis regulator Bcl-2 720 base pairs [chromosome 18]; frequency of either
  • AGCGCA, AGCGCT, TGCGCA, or TGCGCT 1.
  • Alpha retinoic acid receptors (RARs), 1389 base pairs [chromosome 17]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT 2.
  • Type III phopshodiesterase 3426 base pairs [chromosome 12]; frequency of either
  • Beta p late let- derived growth factor receptor precursor 3321 base pairs [chromosome
  • Arachnidonate 5-lipoxygenase, 1989 base pairs [chromosome 17]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT 3.
  • Androgen receptor 2760 base pairs [chromosome X]; frequency of either AGCGCA,
  • the compounds of the invention are suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound selected from a compound of the invention and one or more pharmaceutically acceptable carriers or diluents.
  • the described compounds of the invention may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
  • a compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the compound of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • a compound of the invention may also be administered parenterally.
  • Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • a person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
  • compositions suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device.
  • the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
  • the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon.
  • the aerosol dosage forms can also take the form of a pump-atomizer.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine.
  • Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, poly gly colic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, poly gly colic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • the compounds of the invention may be administered to a subject alone or also in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
  • the dosage of the compounds of the invention and/or compositions comprising a compound of the invention and/or a compound of Formula II can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the animal to be treated.
  • One of skill in the art can determine the appropriate dosage based on the above factors.
  • the compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.
  • the compounds of the present invention may be prepared by combining the individual monomer units in a stepwise fashion using methods known in the art.
  • the individual monomer units are either commercially available or may be prepared using methods known in the art.
  • the benzimidazole-pyrrole, benzimidazole- imidazole, imidazopyridine-pyrrole and imidazoimidazole key units may be prepared using a oxidative cyclocondensation reaction as follows:
  • a reagent of formula A wherein R , X, R 2 and R are as defined in formula I is combined with a reagent of formula B, wherein Y, R and R 4' are as defined in formula I and W is either H or NO 2 , in an inert solvent, for example acetonitrile, at a temperature in the range of 50-1 10 0 C, suitably 80 to 100 0 C, to form an intermediate Schiff s base.
  • an inert solvent for example acetonitrile
  • the solvent may then be suitably removed and the crude Schiff s base may be resuspended in a suitable inert solvent (same or different as the first solvent) and reacted with a transition metal salt, most suitably an Fe(III) salt, such as FeCl 3 .6H 2 O, in the presence of oxygen gas at a temperature in the range of 50-110 0 C, suitably 80 to 100 0 C to form the intermediate of formula C.
  • a transition metal salt most suitably an Fe(III) salt, such as FeCl 3 .6H 2 O
  • oxygen gas at a temperature in the range of 50-110 0 C, suitably 80 to 100 0 C to form the intermediate of formula C.
  • Various R groups may be incorporated onto reagents of formula A or C using methods known in the art, for example, using nucleophilic substitution reactions.
  • Imidazole- or pyrrole-containing monomeric units may be added on to intermediate C when W is NO 2 , by first reducing the NO 2 group to an amine using, for example H 2 /Pd-C, and coupling the resulting amine to an imidazole- or pyrrole- containing monomeric unit comprising an activated acid in a suitable location using well known amide bond forming reaction conditions.
  • Other reactions for the preparation and coupling of the monomeric units in the compounds of formula I are well known to those skilled in the art and representative examples of such reactions are described in more detail below.
  • reaction conditions including reactants, temperature, time and solvent, mentioned below could be varied by a person skilled in the art in order to optimize reaction yields depending on the structure of the starting monomeric units.
  • the structurally analogous diaminopyridine compound (6) was prepared from 2,6-dichloro-3-nitropyridine (Scheme II), 22 using initially a regioselective substitution of one of the chloro-substituents with NH 3 ZEtOH in controlled conditions (amount of condensed NH 3 and low temperature), followed by a similar aromatic substitution reaction with N-methylpiperazine as above. Reduction with Pd/C under high pressure of H 2 provided the diamine (6) in 90% yield; this diamine had to be isolated as its dihydrochloride by treatment of the reaction filtrate with ethanolic HCl due to rapid darkening of the solution on exposure to air.
  • the l-methyl-4-nitro-2-pyrrolecarboxaldehyde (20) was a key intermediate that has previously been reported in a French- language article. 46
  • the procedure in that report is extremely inefficient for preparing the title compound, using ambient temperature nitration of the pyrrole-2-carboxaldehyde precursor (19) with cone HNO 3 .
  • the desired 4-nitro-isomer is only a minor product and the unwanted 5-nitro-isomer is largely formed, making the separation of two regioisomers very difficult.
  • transition metal ions such as Fe(III), Cu(II), and Mn(III) were found to be effective catalysts in mediating oxidative cyclocondensation of a variety of aromatic diamines and aldehydes to furnish benzimidazole-products (Scheme V)
  • iron trichloride hexahydrate FeCh-OH 2 O was chosen to minimize the product loss and catalyst poisoning due to metal complexation by the benzimidazole or imidazopyridine compounds.
  • HCl or the highly nonpolar diisopropyl carbodiimide (DIC) were found to be useful in the reactions employing DMF-H 2 O or anhydrous DMF, respectively, as the solvents.
  • DIC highly nonpolar diisopropyl carbodiimide
  • benzimidazole units may further enhance the DNA binding characteristics of the designer ligands.
  • These benzimidazole units could either be placed in a directly linked form, as found for Hoechst 33258, or could be positioned distal from each other.
  • the placement of two, or more, benzimidazole (or imidazopyridine) units separated from each other in the oligomeric conjugates to determine whether a 2:1 side-by-side complexation mode with the DNA minor groove could still occur with sufficiently high affinity and selectivity for the cognate DNA sequences was investigated.
  • benzimidazole system and related fused heterocycles have a larger aromatic hydrophobic surface with different electronic/steric characteristics and are also subject to tautomerization, compared with the five-membered rings linked via amide groups in the distamycin-like polyamides.
  • Benzimidazole rings in the acid modules were also incorporated using the highly effective catalytic Fe(III)/Fe(II) redox cycling approach.
  • 3,4- diaminobenzoic acid or with its methyl ester two- and three-ring subunits, (64) and (70), were readily obtained using the multistep sequence summarized in Scheme XII.
  • Trichloroacetyl-substituted pyrrole and imidazole derivatives are likely the most efficient class of smallest building blocks for a facile generation of the polyamide assemblies and their conjugates with benzimidazole/pyridoimidazole units as described above.
  • the trichloroacetylimidazole unit has been used as the terminal residue.
  • larger fused heterocyclic fragments, such as an equivalent benzimidazole unit could be incorporated at terminal locations.
  • N-methylbenzimidazole was trichoroacetylated at C2-position using an adaptation of the Nishiwaki method (Scheme XV), previously applied to the pyrrole, imidazole and related five-membered heterocycles. 51"53 Any one-ring or two-ring aminoesters from Scheme VII could thus be treated with the "activated” trichloroacetyl benzimidazole compound (73) in a "haloform-type” condensation reaction to generate a variety of benzimidazole- terminated elongated amides (such as 75).
  • F1-F7 (Figure 1), G1-G7 ( Figure 2), H1-H4 ( Figure 3), Il and 12 ( Figure 4), J1-J4 (Figure 5), Kl -K6 ( Figure 6), and L1-L4 ( Figure 7), 34 compounds overall, were successfully prepared and fully characterized, using a widely diverse set of combinations of different acid and amine modules consisting of benzimidazole, pyridoimidazole, pyrrole, imidazole, and selected aliphatic amino acid residues.
  • the need for chromatography was minimized and liquid-liquid or acid/base extraction procedures were sufficiently successful.
  • the chemistries outlined above may have to be modified, for instance by use of protective groups, to prevent side reactions due to reactive groups, such as reactive groups attached as substituents.
  • This may be achieved by means of conventional protecting groups, for example as described in "Protective Groups in Organic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 rd Edition, 1999.
  • the formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
  • solvates of the compounds of the invention will vary depending on the compound and the solvate.
  • solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent.
  • the solvate is typically dried or azeotroped under ambient conditions.
  • Prodrugs of the compounds of Formula I may be, for example, conventional esters formed with available hydroxy, amino or carboxyl group.
  • available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine).
  • inert solvent e.g. an acid chloride in pyridine.
  • Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C 8 -C 24 ) esters, acyloxymethyl esters, carbamates and amino acid esters.
  • the present invention includes radiolabeled forms of the compounds of the invention, for example, compounds of the invention labeled by incorporation within the structure 3 H, 11 C or 14 C or a radioactive halogen such as 125 I and 18 F.
  • a radiolabeled compound of the invention may be prepared using standard methods known in the art.
  • tritium may be incorporated into a compound of the invention using standard techniques, for example by hydrogenation of a suitable precursor to a compound of the invention using tritium gas and a catalyst.
  • a compound of the invention containing radioactive iodo may be prepared from the corresponding trialkyltin (suitably trimethyltin) derivative using standard iodination conditions, such as [ 125 I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethyl formamide.
  • standard iodination conditions such as [ 125 I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethyl formamide.
  • the trialkyltin compound may be prepared from the corresponding non-radioactive halo, suitably iodo, compound using standard palladium-catalyzed stannylation conditions, for example hexamethylditin in the presence of tetrakis(triphenylphosphine) palladium (0) in an inert solvent, such as dioxane, and at elevated temperatures, suitably 50- 100 0 C.
  • a compound of the invention containing a radioactive fluorine may be prepared, for example, by reaction of K[ I 8 F]/K222 with a suitable precursor compound, such as a compound of Formula I comprising a suitable leaving group, for example a tosyl group, that may be displaced with the 18 F anion.
  • a suitable precursor compound such as a compound of Formula I comprising a suitable leaving group, for example a tosyl group, that may be displaced with the 18 F anion.
  • DNA-Ligand Interactions 1. ⁇ T m Measuements in High-Throughput Screens Using a high throughput assay method based on fluorescence resonance energy transfer (FRET) and a set of 32 duplex DNA molecules (Table I) where the respective complementary strands are alternatively labeled with a quencher (DABCYL) and a fluorescent reporter group (FITC), the influence of compounds designed in the present application on DNA melting was assessed.
  • the prechosen DNA sequences in the set of standard primers are comprised of a central two base pair AT or GC binding site flanked by variable regions whose immediate neighboring sequences are systematically changed to accommodate all nearest neighbor alterations.
  • a direct measure of thermal stabilization of DNA primers in the absence and presence of tested ligands was obtained in experiments conducted under identical conditions and by measuring the difference in fluorescence due to FITC that arises from the separation of strands upon regulated heating.
  • This ultrasensitive high-throughput assay first employed for distamycin (an AT-selective compound) and actinomycin (a GC -binding anticancer drug) provided the necessary benchmarks for comparing multiple sets of compounds of the present invention by measuring the ⁇ T m values (T m for complexed DNA - T m for free DNA). These results are presented in a histogram format to illustrate the AT vs GC selectivity patterns for the two reference compounds ( Figures 8 and 9). All experiments were performed according to a fixed protocol described in the Experimental Section.
  • the binding characteristics of the compounds of the present invention have been examined with regards to the following specific properties: (1) DNA sequence specificity; (2) ligand solubility in physiologically relevant buffer solutions; (3) thermal stability of ligands; (4) assessment of their selectivity for double stranded DNA (versus single-stranded oligomers); (5) relative affinity for DNA as assessed by ⁇ T m changes; and (6) performance in various salt/buffer environments for potential PCR applications.
  • AT-rich segments are preferred binding sites for distamycin, and conversely actinomycin binds to GC-rich segments to a greater extent than AT- sites.
  • the minor groove binding compounds, developed by the present inventors clearly show a higher affinity for the GC segments than a reference polyamide ligand (R4) with equivalent number of DNA interactive modules.
  • Compound F4 of the present invention shows maximum effect of increasing the T m values for a DNA containing the GGCCGGCC and CGCCGGCG segments ( Figure 10).
  • compound G4 (the pyridoimidazole analog) also shows a greater stabilization of GGCCGGCC, CGCCGGCG and CGCT.AGCG segments of duplex DNA.
  • this compound shows a much greater selectivity for these sites compared to F4. From the presence of pyrrole and imidazole units in these candidate ligands, it is likely that 2: 1 complexes are formed with DNA in a sequence selective manner, as shown in model illustrations ( Figure 11).
  • UV-VIS absorption spectra for compound F4 (Bzi analog) in the absence and presence of duplex oligonucleotide, d(CATGGCCATG) 2 , and to the point of identical final ligand concentrations in the two experiments, are shown in Figure 12.
  • the ⁇ increase is quite remarkable compared to previous results with minor groove binding agents, which usually exhibit a small decrease in absorption upon binding DNA. 39 ' 60 It may be that the increased absorption is due to the anticipated 2:1 binding mode on the basis of a correctly matched binding sequence with the linear heterocycle sequence of the Bzi analog.
  • the stacked binding of 2 molecules of the Bzi analog with significant overlap of the heteroaromatic chromophores is expected to show higher absorptivity in the presence of DNA.
  • the UV-VIS absorption spectra for compound G4 (Pzi analog) in the absence and presence of the same DNA molecule as used above, are provided in Figure 13.
  • the two types of difference spectra were deduced by the subtraction of the free ligand and the free DNA spectra from those for the admixtures.
  • Two distinct bands, with significant overlap, centered at 363 and 384 nm are observed.
  • the compounds of the invention having the benzimidazole-pyrrole and imidazopyridine-pyrrole modules, for example, with the N-methylpiperazinyl substituents, in the designed polyamide-fluorophore conjugates have similar (to each other) but unique effect on the ability to bind the DNA minor groove and the mode in which that interaction occurs.
  • the Bzi and Pzi analogs are shown to be well suited for not only ultra-strong binding to the target DNA site, but with two ligand molecules bound simultaneously in the 2: 1 mode.
  • Both the Bzi and Pzi ligands form fluorescent complexes with double-stranded DNA, which facilitates investigation of their binding isotherms in terms of the binding stoichiometry, equilibrium association constants and provides benchmark measures of photophysical parameters.
  • Thermal denaturation experiments were employed as an initial alternative method for the investigation of ligand-DNA complex stabilities and sequence selectivities.
  • the ⁇ T m values for a majority of the 34 ligands prepared in the present invention have been obtained against a panel of 32 different DNA sequences, representing various combinations of the centrally-positioned 4-base pair long site.
  • the terminal units derived from the structure of Hoechst 33258 i.e. the 5-(N- methylpiperazinyl)benzimidazole and 5-(N-methylpiperazinyl)imidazopyridine units in the Bzi and Pzi analogs, respectively, are protonated and positively charged at neutral pH, similar to the tertiary amine (Dp) groups in previously studied polyamides of Py /Im heterocyles. The differences in electrostatic interactions with the negatively charged phosphodiester backbone of DNA are therefore not likely to be significant.
  • the Bzi and Pzi ligands of the present invention are more effective than the polyamide class of molecules since they have a reduced number of amide bonds in integrated combinations of fused heterocycles with five- membered rings (pyrrole) in the Bzi-Py and Pzi-Py modules, relative to the Py- CONH-Py units in the polyamide molecules of the present invention.
  • This reduced number of amide bonds is being compensated by the alternative hydrogen-bond donor functionalities in the benzimidazole or pyridoimidazole rings.
  • the benzimidazole/pyridoimidazole ring NH groups are evidently more acidic than the amide CONH groups.
  • Figure 14 provides a summary of the interactions between 5 closely related ligands selected on the basis of individually matched combinations of the structural models present in the ligand structure with the cognate DNA base pairs in the context of 2:1 ligand-DNA binding models.
  • Analogs F4 and G4 are found to be superior in terms of the propensity to exhibit 2:1 binding stoichiometry and equilibrium association constants.
  • the amide derivatives R4 and H4 are not well suited for the 2:1 interactions and more data will be required on these systems to elucidate the structural or molecular basis for the apparent mismatch due to Dp units in these ligands.
  • Compound K6 was difficult to analyse under the solution conditions due to its insolubility at pH 7. Note that the terminal imidazole ring in this ligand is expected to be protonated at that pH, but the increased lipophilic character of the cation remains. A corresponding methylated derivative is expected to increase solubility in aqueous solutions.
  • the benzimidazole and imidazopyridine are expected to be considerably photostable, relative to free radical and oxidative instability of the hydroxypyrrole rings, as well as more resistant to cellular degradation than the polyamide class of molecules.
  • Cell-permeability of the piperazine-substituted benzimidazole compounds is already well established, judging by the cytological and molecular genetics applications of Hoechst 33258 class of dyes. 10 - 1 1
  • Structures of exemplary ligands when bound to cognate nucleotide sequence in double stranded DNA, were determined by the application of two-dimensional NMR methods to reveal the exact configuration and atomic level details of the respective quaternary complexes. Primarily, very exact closest contact points between hydrogen atoms on the ligand molecules and those in DNA were identified as experimentally observed NOE's in two-dimensional NMR plots.
  • a second exemplary ligand G4 which differs from F4 in the sense that a Pzi unit replaces the Bzi unit
  • the intermolecular contacts observed in its complex with d(CATGGCCATG) 2 are also summarized in Figure 16.
  • These 48 different contact points between the ligand G4 molecules and the DNA also establish the specific shape-based readout of the segment 5'-ATGGCCAT by this specific ligand.
  • this ligand G4 binds 100-1000 fold strongly with the said DNA segment than the preceding ligand F4, revealing the benefit of the Pzi unit in the design of ultra-strong DNA interactive molecular assemblies for targeting specific DNA segments with high fidelity.
  • DNA footprinting is the in-vitro experimental method of choice to determine which specific continuous segment of nucleotides in DNA is protected against enzymatic cleavage - in the presence of added ligand(s).
  • the cleavage of double stranded DNA plasmids for example, by the protein DNase I (an endonucleolytic enzyme) produces a DNA sequence ladder pattern on polyacrylamide gels.
  • sequence pSask-A has 128 possible non-repeating segments that are each 6 base pairs long, and 126 possible segments that are each 8 base pairs long, etc. Similarily, sequence pSask-B has 151 non-repeating 6-basepairs long segments, and 149 such 8-base pairs long segments. Selective binding of any of the test ligands to a specific such segment is easily characterized by the present of obliterated regions on the electrophoresis footprinting gel exposed to autoradiography for generating high resolution images.
  • a pool of 37 different ligands from this invention was placed into 4 different groups for the purposes of evaluating their DNA sequence specific binding characteristics by DNA footprinting method. This division into 4 different groups was on the basis of molecular structures (meaning the configuration order of the individual building blocks in the oligoheteroaromatic assemblies, much like the peptide sequences were defined according to the sequence of the individual amino acids).
  • Series A ligands were the smallest assemblies, consisting of methylpiperazine headgroup, 2 benzimidazole [Bzi] units, and the fourth (variable) heterocyclic or other similar aromatic ring system.
  • Series B ligands contained varying combinations of the methylpiperazinyl head group, Bzi or Pzi units, pyrrole, imidazole, and inverted Bzi ring systems as part of the molecular frameworks.
  • Ligands F4 and G4 were part of this group of compounds.
  • Series C ligands were the various symmetrical dimeric combinations of two "half ligands joined together by variable and novel linkers to separate out the individual "half ligands to the extent that maximum contact is permitted between the ligand edges and such functional groups present on DNA bases that are located in the minor groove of the double stranded DNA. Thus the dimensions of the linkers were a consideration in the way the assemblies were constructed.
  • Series D ligands were a small group of miscellaneous ligands that were selected on the basis of their results from T m data presented earlier in this application. The order in which the several units appear within each ligand was not constant throughout, and yet the original data from T m analysis had identified several of them to be binding to selected DNA segments with very high affinity.
  • Table 2 provides the listing of preferred binding sites for the individual series A ligands, along with their Kd (dissociation constants of DNA complexed ligands) as determined from quantitative footprinting data.
  • the most preferred binding segment in DNA is the 5'-AATT, completely consistent with the structural model of binding which places the methylpiperazine head group to be on the 3 '-side and therefore at the "end" of consecutively placed T nucleotides.
  • 5'- TTAA is the least preferred binding site for any of these series A ligands. This is an relevant aspect of the mode in which such ligands bind to DNA.
  • the footprinting results were necessary to determine whether the DNA sequence selectivity of F4 and G4 ligands is retained in the presence of a pool of multiple but varying octanucleotide segments.
  • the F4 and G4 ligands were both found to be super-selective for the sequence segments identified as 5'-AAGGCCTT and 5'-ATGGCCAT. Binding to any of the other 124 octanucleotide segments was comparatively extremely weak.
  • the functional differences between the Pzi class assemblies and the Bzi class assemblies originate from the different fluorescence characteristics of the Pzi (vs Bzi), difference in pK a values, difference in hydrogen bond forming capacities, and the distinct tautomeric forms of the two types of ring systems - all important features that make the Pzi ligands better than the Bzi ligands; it is important to note that both classes of ligands are in turn far superior than assemblies designed purely on the basis of arranging pyrrole and imidazole ring systems (the so-termed all amide assemblies that had been part of the early attempts made in the 1980s).
  • binding sites and their binding association constants for other series B ligands are all provided in Table 3.
  • a specific additional ligand of interest [F6] is identified to be selective for 5'-AAGCGCTT segments and this specific assembly is also representative of the same structure-based match between the ligand and the DNA site, as determined for ligands F4 and G4 above.
  • the binding association constant for this particular combination of ligand configuration and the targeted DNA sequence was however not as high as picomolar but still falls in the subnanomolar range allowing it to be an effective, ultra-strong and selective ligand.
  • NMR signal assignments where so described, use the following notations for specified ring systems: Bzi, benzimidazole; Dp, (dimethylamino)propyl; Im, imidazole; morph, morpholine; Ph, phenyl; Pip, piperazine; Pri, pyridine; Py, pyrrole; Pyrz, pyrazole; Pzi, pyridoimidazole/imidazopyridine.
  • Mass spectrometry data were obtained through the Analytical Services Division of the Plant Biotechnology Institute, located on our campus.
  • MALDI-TOF-MS was obtained using ⁇ -cyano-4-hydroxycinnamic acid matrix, and accurate mass measurements (HRMS) by secondary ion mass spectrometry were performed using a m-nitrobenzyl alcohol as matrix.
  • the mixture was further stirred for 5-8 h at 90 °C with continuous O 2 bubbling through the mixture, or alternatively allowed to stir exposed to air overnight.
  • the reaction progress was monitored by TLC and NMR analysis of the aliquots, drawn periodically and filtered through a silica gel-filled Pasteur pipette in order to remove iron salts.
  • solvent was removed by evaporation and the residue subjected to silica gel flash chromatography to afford the benzimidazole products in the indicated yields.
  • the hydrochloride salts were prepared by treatment with methanolic HCl, evaporation of the solvent and crystallization from EtOH/Et 2 O.
  • Im-Ph(NO 2 )NH 2 l-(3-Amino-4-nitrophenyl)-lH-imidazole (7) was prepared in 86% isolated yield using imidazole and 5-chloro-2-nitroaniline according to general procedure A described above.
  • Im-Ph(NH 2 ) 2 l-(3,4-Diaminophenyl)-lH-imidazole (8) was prepared in 90% isolated yield from catalytic hydrogenation of 7 as described for 2 above.
  • 1 H-NMR (DMSO-d 6 ) 67.87 (s, IH, Im-CH), 7.39 (s, IH, Im-CH), 7.00 (s, IH, Im-CH), 6.66- 6.58 (m, 3H, Ph-CH), 4.77 (s, 2H, NH 2 ), 4.64 (s, 2H, NH 2 ).
  • Im-Pyr(NO 2 )NH 2 2-Amino-6-(imidazol-l-yl)-3-nitropyridine (9) was prepared in 92% isolated yield from imidazole and compound 3 according to general procedure B described above.
  • FAB-HRMS m/z calcd for C 8 H 8 N 5 O 2 + (MH + ) 206.0678, found 206.0682 (100%).
  • Morph-Pyr(N O 2 )NH 2 2-Amino-6-(morpholin-l-yl)-3-nitropyridine (17) was prepared from morpholine and chloropyridine derivative (3) in 90% isolated yield according to general procedure B described above.
  • MeOH was removed under reduced pressure and the contents were acidified to pH 2 using 2M HCl under cooling, during which acid precipitated out as gelatinous solid.
  • the solid was filtered and the wet cake was taken up in 1 : 1 MeOH/'PrOH when acid precipitated as granular solid, filtered washed with Et20 to furnish 35 (6.48 g, 87 %).
  • Methyl l-methyl-4-nitro-lH-imidazole-2-carboxylate (40) MeOH (40 mL) was added to a sample of the trichloroacetyl compound 25, and the mixture stirred after heating to 40 0 C for 10 min. To ensure complete methanolysis, a small amount (100 mg) of NaH was added to the mixture. After stirring for 2 h, the resultant suspension was filtered to afford the title product (94% yield).
  • 1 H-NMR (CDCl 3 ) 67.84 (s, IH, Im-CH), 4.18 (s, 3H, CH 3 ), 3.93 (s, 3H, CH 3 ).
  • the reaction mixture was filtered through a bed of celite.
  • the celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as black oil.
  • the residue was taken up in benzene and concentrated to furnish the amine as black solid, filtered and washed with ether (2.5 g, 88%).
  • the crude amine was dissolved in CH 3 CN (50 mL) and DIEA (2.3, 18 mmol).
  • a solution of 1- methyl-2-trichloroacetylimidazole 24 (3.6 g, 14.7 mmol) in CH 3 CN (50 mL) was added to the amine solution at 0 0 C.
  • the catalyst was removed by filtration and the filtrate evaporated to afford a dark solid, which was used without further purification. Attempts to crystallize and/or purify by column chromatography led to prolonged exposure to air and degradation to unidentified product mixtures.
  • the title product was alternatively isolated as the hydrochloride salt by treatment with ethanolic HCl, and crystallization (EtOH/Et 2 O).
  • MP-Pzi-Ph(NO 2 )NHAc 2-(4-Acetamido-3-nitrophenyl)-5-(4- methylpiperazin-yl)-lH-imidazo[4,5-b]pyridine (56) was prepared in 70% yield using the diamine derivative 6 and 3-nitro-4-acetamidobenzaldehyde in the general procedure D given above.
  • MP-Pzi-Ph(NH 2 ) 2 2-(3,4-Diaminophenyl)-5-(4-methyl ⁇ iperazinyl)-lH- imidazo[4,5-b]pyridine (60) was prepared in 90% yield from 58 in the hydrogenation protocol described above.
  • EI-HRMS m/z calcd for Ci 2 H 10 N 4 O 2 242.0804, found 242.0897 (14%, M + ), 145.0501 (67%);
  • ES-HRMS m/z calcd for C 12 H n N 4 O 2 + (MH + ) 243.0882, found 243.0876 (100%).
  • MeO-Bzi-Py-NO 2 Methyl 2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H-benzimid- azole-5-carboxylate (65) was prepared in 88% yield using methyl 3,4- diaminobenzoate diamine and aldehyde 20 in the general procedure C given above.
  • MeO-Bzi-Py-NH 2 Methyl 2-(4-amino-l -methyl- lH-pyrrol-2-y I)-I H-benzim- idazole-5-carboxylate (67) Compound 65 was reduced with hydrogen at high pressure (60 psi) using 10% Pd-C and standard practice followed above. The product was not isolated and instead used directly for further reactions below.
  • HO-Bzi-Py-NH 2 2-(4-Amino-l -methyl- lH-pyrrol-2-y I)-I H-benzimidazole-
  • Dp-Bzi-Py-NO2 N,N-Dimethyl-3- ⁇ [2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H- benzimidazo-5-yl]carbonylamino ⁇ propanamine (71).
  • Acid derivative 66 (2 mmol) was dissolved in DMF (20 mL) and a sequence of additions of HOBt (2.4 mmol), DIC (2.3 mmol) and dimethylaminopropylamine (2.5 mmol), in that order, were followed by stirring the suspension at 45 0 C for 1O h The heating was removed and the mixture evaporated to afford a solid residue.
  • Dp-Bzi-Py-NH 2 N,N-Dimethyl-3- ⁇ [2-(4-amino- 1 -methyl- 1 H-pyrrol-2-yl)- lH-benzimidazo-5-yl]carbonylamino ⁇ propanamine (72): Compound 71 was reduced catalytically (10% Pd-C) with H 2 (50 psi pressure) overnight to give the title amine after filtration and evaporation of the filtrate. The red solid was stored under nitrogen before further use.
  • EI-HRMS m/z calcd for C 10 H 7 Cl 3 N 2 O 275.9624; found 275.9669 (M+, 10%), 160.0591 (10%), 159.0556 (M-CHCl 3 , 100%) ⁇ relative isotopic distribution: 275.9669 (100), 276.9707 (13), 277.9640 (95), 278.9685 (13), 279.9633 (30), 281.9635 (4) ⁇ .
  • Im-Bzi-Py-NO 2 5-(Imidazol- 1 -yl)-2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H- benzimidazole (76) was prepared in 78% yield using the diamine derivative 8 and compound 20 in the general procedure C given above.
  • 1 H-NMR 300 MHz, DMSO- d 6 ) 68.94 (s, IH, Im-CH), 7.36 (s, IH, Py-CH), 7.15 (s, IH), 7.15-7.04 (m, 3H), 6.82 (dd, IH, Bzi-CH), 6.32 (s, IH, Im-CH), 3.96 (s, 3H, CH 3 ).
  • FAB-HRMS m/z calcd for Ci 5 H) 3 N 6 O 2 + (MH + ) 309.1 100, found 309.1072 (100%).
  • Im-Bzi-Py-NH 2 2-(4- Amino- 1 -methyl- 1 H-pyrrol-2-yl)-5-(imidazol- 1 -yl)- lH-benz-imidazole (77): catalytic reduction of the nitro compound.
  • Im-Py-Bzi-Gly-OH (81) LiOH mediated hydrolysis, as described for aliphatic linker diesters in Chapter 1, was used for compound 78 to afford the title product after washing the reaction residue with 'PrOH (85% yield).
  • Im-Py-Bzi-Gaba-OH (83) was similarly obtained using LiOH hydrolysis of 80. (75% yield).
  • F1-F4 The following first series of final products (F1-F4) were all prepared from the same amine (49) and different acid derivatives listed individually for each data set below.
  • Mp-Bzi-Py-Im (F-I) was prepared from 2-trichloroacetyl-l-methylimidazole and amine 49 treated together in CH3CN and according to the isolation methodlogy in general procedure F above. Recovery 56%.
  • Mp-Bzi-Py-Im-Py-Im (F-6) was prepared using amine 53, acid 35 and EDCI/HOBt using the general procedure F. Recovery 52%. The alternative combination of amine 49 and acid 47 (with EDCI/HOBt) gave lower recovered yields (10-14%).
  • Mp-Pzi-Py-Py -Im-Im (G-4) was prepared using amine 49, acid 39 and EDCI/HOBt using the general procedure F. Recovery 46%.
  • ES-HRMS m/z calcd for C 32 H 36 Ni 4 O 3 664.3095, found 664.5026 (M+, 38%), 662.51 18 (45%, M-2), 663.5078 (36%, M-I), 665.5318 (100%, M+l), 666.5395 (42%, M+2), 667.5448 (8%, M+3).
  • Mp-Bzi-Bzi-Py-Im (1-1) was prepared using amine 62, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 68%.
  • J1-J4 The following final products (J1-J4) were all prepared from the given combination of amine and acid derivatives listed individually for each data set below.
  • Mp-Bzi-Py-Bzi-Im (J-I) was prepared using amine 49, acid 64 and DIC/HOBt using the general procedure F. Recovery 40%.
  • MALDI-TOF-HRMS m/z calcd for C 29 H 30 Ni 0 O 534.2604, found monoisotonic 535.268; 534.4783 (M+, 100%), 532.4543 (80%, M-2), 533.4643 (48%, M-I), 535.4900 (70%, M+l), 536.4842 (24%, M+2).
  • MALDI-TOF-HRMS m/z calcd for C 35 H 36 Ni 2 O 2 656.3084, found monoisotonic 657.316; 656.4976 (M+, 94%), 654.4490 (65%, M-2), 655.4692 (46%, M-I), 657.4977 (100%, M+l), 658.4888 (36%, M+2).
  • Im-Bzi-Py-Bzim (K-3) was prepared using amine 77, 2-trichloroacetyl-l- methylbenzimidazole (73) and the isolation methodology given in the general procedure F. Recovery 65%.
  • Im-Bzi-Py-Im (K-5) was prepared using amine 77, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 70%.
  • Im-Bzi-Py-Py-Im-Im (K-6) was prepared using amine 77, acid 35 and DIC/HOBt using the general procedure F. Recovery 38%.
  • 1 H-NMR HCl salt in CD 3 OD
  • ES-HRMS m/z calcd for C 40 H 41 N 15 O 3 779.3517, found 779.4863 (M+, 54%), 777.4843 (100%, M-2), 778.5093 (70%, M-I), 780.4712 (40%, M+l), 781.5302 (16%, M+2).
  • ES-HRMS m/z calcd for C 42 H 44 Ni 6 O 4 836.3731, found 836.3855 (M+, 47%), 834.3665 (100%, M-2), 835.3809 (82%, M-I), 837.5764 (58%, M+l), 838.3910 (30%, M+2).
  • ES-HRMS m/z calcd for C 44 H 48 Ni 6 O 4 864.4044, found 864.4870 (M+, 78%), 862.4734 (100%, M-2), 863.4804 (78%, M-I), 865.5003 (92%, M+l), 866.5238 (35%, M+2), 867.5238 (10%, M+3).
  • Reference compounds R1-R7 matching the series F/G in terms of the polyamide composition, can likely be prepared readily on the basis of success with the coupling methods for amide forming reactions. With the detailed studies directed towards a specific ligand composition, R4 was prepared as below.
  • Dp-Py-Py-Py-Im-Im (R4). This compound was prepared from an amine (15b) described and numbered as such in chapter 1, in a coupling reaction with the acid derivative (39) from above and DIC/HOBt combination in the general procedure E. Recovery 55%.
  • ES-HRMS m/z calcd for C 33 H 4 iN ]3 O 5 699.3354, found 700.5727 (63%, M+l), (note: no peaks observed for either deprotonated or further protonated forms than M+l).
  • Biophysical Studies The ligand and DNA stock solutions were prepared in either 10 or 50 mM sodium/potassium phosphate, pH 7.0, buffers containing 50 mM NaCl and 0.1 mM Na 2 EDTA to minimize the effects due to residual metal ions. Quadruply distilled (Millipore) water was employed in all work described below.
  • the method employed the DNA arrays (with high picomolar level sensitivity) where fluorescence dye (FITC) and quencher (DABCYL) labeled oligonucleotides were employed for a fluorescence resonance energy transfer based evaluation of the hybridization of DNA strands in the absence and presence of test ligands as a function of temperature. All experiments were carried out under similar solution conditions relating to the buffer nature and their concentrations. The Tm values were determined using first order differential plots and the experiments were done in triplicate. Averaged values (with ⁇ 5% standard error) were plotted in the histogram format to asses the structure-activity relationships for the various structural subclasses of ligands (series F, G, H etc.)
  • Absorption and Fluorescence Spectroscopy Absorption spectra were measured on a double-beam Varian Cary 500 UV/VIS spectrophotometer with a bandwidth of 1 nm and a scan rate of 0.5 nm/s, with the solutions suspended in quartz cells with a 1 cm pathlength. All measurements were conducted at ambient temperature. Buffer solutions used for titrations were themselves used as reference for background corrections. Absorption spectra were recorded in the range of 200-700 nm. Molar extinction coefficients were determined on solutions with solute concentrations adjusted to provide linear working curves of absorbance at band maxima versus concentration, according to Beer-Lambert relationship.

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Abstract

The present invention provides a novel class of oligoheteroaromatic assemblies with luminescence characteristics and composition based on integrated polyheterocyclic polyamide oligomers of multiple nitrogen-containing heteroaromatic of the general formula (I) This novel class of compounds of the present invention is capable of binding to targeted DNA sequence in the minor groove, and thus is useful for genomics applications. In particular, the compounds of the invention binds to the DNA at a binding stoichiometry of 2: 1 ternary complexation with very high affinity and sequence selectivity.

Description

B&P File No. 13764-14
TITLE: OLIGOHETEROAROMATIC LUMINESCENT ASSEMBLIES AS
HIGH-AFFINITY DNA SEQUENCE-DIRECTED LIGANDS
FIELD OF THE INVENTION
The present invention relates to oligoheteroaromatic assemblies with luminescence characteristics, and in particular novel compounds and compositions based on systematically integrating these assemblies with polyamide oligomers of optimally spaced nitrogen-containing heteroaromatics capable of binding to targeted DNA sequences.
BACKGROUND OF THE INVENTION
DNA sequence targeted chemical agents have been attracting increasing research focus,1"9 and perhaps the most actively used paradigm with respect to the design of polyamide class of DNA minor groove binding agents is distamycin, a trispyrrolecarboxamide member of the pyrrole amidine class of antibiotics. Other extended oligoheteroaromatic structures such as the bis-benzimidazole class of chromosomal stains (Hoechst 33258), 10'14 diamidine derivatives,15"17 and designer analogs have also received some attention.
Figure imgf000003_0001
Distamycin
Figure imgf000003_0002
Despite extensive efforts spanning the past two decades, there are few small molecule inhibitors of transcription events available as drugs or as DNA sequence diagnostic probes. In the case of drug applications, for instance, these limitations exist, in part, due to the criteria required for a therapeutically useful small molecule inhibitor of intranuclear and/or mitochondrial gene expression. Such inhibitors need to be highly potent, have a comparable affinity for DNA to that of proteins, be selective for specific DNA sequences among the transcription factor binding sites, and also possess requisite pharmacodynamic properties for the target of interest. Cell- permeable compounds36'38 comprising structural features for DNA sequences selective binding in the minor groove are thus highly desirable as potential gene regulatory drugs for in vivo activity.
There are relatively few reports describing inhibitors of gene expression. From a design perspective, the bisbenzimidazole (also known as bis-dideazapurine) and amidine class of molecules, for example, are known to be as strong and selective in binding AT-rich segments of DNA as distamycin itself39. Therefore potential design opportunities exist for developing extended polybenzimidazole derivatives as DNA sequence directed compounds that specifically and rationally exploit noncovalent interactions with the DNA base pairs. Some examples of molecules containing more than two benzimidazole units are particularly encouraging, from previously reported results,40"41 suggesting a similar shape and multiple hydrogen bond binding mode for the extended benzimidazole frameworks. Of significant interest are the reported inhibitory effects of the bisbenzimidazole and few tris- benzimidazole compounds on the topoisomerase class of DNA binding enzymes.42"44 The fact that Hoechst 33258 and related compounds have been used widely as chromosomal stains also provides optimism regarding the criterion of desirable cell permeability characteristics for such molecules.
While there has been a steady progress made in the design, synthesis, and discovery of novel DNA minor groove binding polyamides by making use of 4- aminopyrrole- and 4-aminoimidazole-2-carboxamides, the modular assembly approach to Hoechst 33258 analogs has not been exploited fully. In general, the oligobenzimidazole frameworks beyond two consecutive benzimidazole rings are not synthetically accessible due to solubility problems (bricksand-like nature).45 Thus, there remains a need to continually design and develop distamycin and Hoechst 33258-like polyamide analogs to expand the structural repertoire of therapeutically relevant gene-regulatory molecules.
SUMMARY OF THE INVENTION
It has been found that a novel class of combinatorial luminescent assemblages of benzimidazole (or dideazapurine), pyridoimidazole (or deazapurine) and purine fragments, in singular and multiple functionalized units, when integrated with polyamide oligomers of nitrogen-containing heteroaromatics, are capable of binding to targeted DNA sequences in the minor groove.
The present invention therefore includes a compound of formula I:
Figure imgf000005_0001
I wherein each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N;
R1 is selected from C1-6alkyl, C3-i2cycloalkyl, aryl, C(O)NHC, -6alkyl, C(O)NHC3- ncycloalkyl and C(O)NH(Ci-6alkylene-N(R6)2), in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N, NR6 or
N(R6)2; R and R >2! are independently selected consisting of H and Ci -6alkyl, or R and R are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units; R3 is Ci-όalkylene or C2.6alkenylene, both of which are optionally substituted with one or more of Ci-όalkyl,
Figure imgf000006_0001
or OH, and both optionally have one or more of the carbons replaced with O, S, NR6 or N(R6)2;
R4 and R4 are independently selected from H and
Figure imgf000006_0002
or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3_6heterocycle, Ci_6alkyl, OH and OCi-6alkyl;
R6 is H or C,.6alkyl;
R7 is Ci_4alkylene or C2.4alkenylene both of which are optionally substituted with one or more of Ci-6alkyl, OCi-6alkyl or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1 , 2, 3, 4 or 5, and when m is other than 1, only the terminal monomeric unit represented by m is substituted with R1 ; n is 1, 2, 3, 4 or 5; o is 0, 1, 2, 3, 4 or 5 p is O, 1, 2, 3, 4 or 5; and q is O, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof, with the proviso that when R1 is selected from C(O)NHC 1-6alkyl, C(O)NHC3. ,2cycloalkyl and C(O)NH(C ,.6alkylene-N(R6)2), R5 is selected from pyrrolyl, imidazolyl, benzimidazolyl, imidazopyridinyl and purinyl.
In an embodiment, the present invention includes a compound selected from a compound of the formula I:
Figure imgf000006_0003
wherein each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N; R1 is selected from
Figure imgf000007_0001
C3-i2cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N,
NR6 or N(R6)2;
R2 and R2 are independently selected from H and Ci-6alkyl, or R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units; R3 is Ci.όalkylene or C2.6alkenylene, both of which are optionally substituted with one or more of Ci-6alkyl,
Figure imgf000007_0002
or OH, and both optionally have one or more of the carbons optionally replaced with O, S, NR6 or N(R6)2;
R4 and R4 are independently selected from H and Ci-6alkyl, or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units; R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3_6heterocycle, Ci-6alkyl, OH and
Figure imgf000007_0003
R6 is H or Ci-6alkyl;
R7 is Ci-4alkylene or C2-4alkenylene both of which are optionally substituted with one or more of C^alkyl, OCi-6alkyl or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1, 2, 3, 4 or 5, provided that when m is other than 1, only the terminal monomer unit represented by m is substituted with R1 ; n is 1, 2, 3, 4 or 5; o is 0, 1, 2, 3, 4 or 5 p is 0, 1 , 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof.
By virtue of the fact that the NH group of pyridoimidazole (Pzi) system is electronically more polarized than the NH group of benzimidazole (Bzi) system, the hydrogen bond forming capacity of the Pzi ring system is greater than that of the Bzi system, therefore the assemblies comprising the Pzi system are much more selective for recognition of thymine and cytosine bases in AT and GC base pairs in the configuration that they are used in the compounds of the invention. The Pzi system has 2-3 orders of magnitude greater capacity for such high fidelity binding to the said bases within DNA. Since the NH groups in both Pzi and Bzi systems are in turn far more polarized than the amide NH groups in prior art compounds, inclusion of increasing numbers of these units at the expense of amide NH groups increases the affinity and sequence specificity of such compounds over those that are enriched with oligoamide frameworks in the prior art. DNA sequence specificity as well as the association constants in binding of the Pzi/Bzi combinations of the compounds of the present invention is always superior to those containing only amide linkages. It is therefore desirable that the linear sequence of monomers in the compounds of the invention be such that the binding interface brings the Pzi/Bzi NH groups into the same location as where the amide NH groups reside in the prior art compounds. A greater selectivity is achieved by the side by side configuration of individual molecules within the groove formed by the pairing of two strands of DNA and it has become possible by the use of the compounds of the present invention to intellectually predict the exact match between the edge-wise shape of the ligand molecules and that formed by the order of the functional groups of DNA bases in the complete length of the target DNA site (regardless of the dimensions of such site).
The present invention therefore includes a method for selectively forming a complex between target DNA and a compound of the present invention, the method comprising: contacting the target DNA with the compound of the invention; wherein the compound of the invention is capable of selectively binding to a sequence on the target DNA, for example, under physiological conditions where selective complexes form between the compound of the invention and the target DNA. In an embodiment of the invention, the sequence is in a minor groove on the target DNA. In another embodiment of the invention, the compound of the invention and the target DNA are at a binding stoichiometry of about 2:1 Also included within the scope of the present invention is a method of selectively detecting the presence of a sequence in a sample having DNA. By contacting the DNA with a compound of the invention having a detectable label and that is capable of selectively binding to the sequence on the DNA, complexes can be selectively formed between the compound and the target sequence on DNA. The detectable label on the compounds can be observed from the complexes, indicating the presence of a selected sequence in the sample. In the compounds of the invention comprising luminescent moieties, the detectable label would comprise this moiety.
Further, the present invention relates to a method for isolating target DNA from a sample comprising a mixture of DNA. By contacting the DNA with a compound of the invention that is capable of selectively binding to a sequence on the target DNA, complexes can be selectively formed between the compound of the invention and a target sequence on the DNA. The target DNA can then be isolated from the mixture of DNA.
Since exemplary compounds of the present invention are very specific for a given DNA sequence size comprising linear self-complementary sequence types [AAA]x[TTT]x and [AAA]x[TA]y[TTT]x [where x=2 to 6, and y= 1], some of the compounds of the present invention will be useful in specifically targeting gene segments of pathogens like Plasmodium falciparum (and other malaria pathogens] and other microbes that are known to have such long AT base pair stretches. Human genes have negligible frequency of the same types of segments in their genes. Compounds of the invention will also be useful in DNA/gene staining methods for in vitro and in vivo photomicroscopy based on fluorescence capturing detection. Further, due to their DNA binding profiles, these ligands have already tested favorably for their ability to obliterate DNA binding proteins like DNAase I, restriction enzymes specific for (A/T)GGCC(T/A) sites, and therefore will be useful in vitro molecular probes in PCR, gene cloning, molecular biology and molecular genetics. Because of their high affinity and specific binding for the said segments, and the occurrence of those nucleotide segments in gene sequences of cancer related proteins, such ligands will also be useful in modulation of such genes to be expressed, both in vitro and in vivo. Accordingly, the present invention relates to a method for modulating transcription of a target gene in a cell comprising contacting the cell with an effective amount of a compound of the invention under conditions sufficient for the formation of complexes selectively between the compound of the invention and the target gene and wherein such complex formation modulates the transcription of the target gene. In an embodiment of the invention the target sequence on the gene is a sequence on transcriptional regulatory regions of the gene. Alternatively, the cell may be a bacterial, virus or pathogen cell. The target gene may be any gene implicated in the propagation of the cell or of a disease state. For instance, the target gene may be an oncogene. The cell may be in a subject, such as a mammal, as in a human or outside the subject, such as, for example, ex vivo treatment. Furthermore, the present invention also relates to a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof. By contacting the DNA comprising the target oncogene with a compound of the invention that is capable of selectively binding to a target sequence on the oncogene, complexes can be selectively formed between the compound of the invention and a target oncogene on the DNA. Selective formation of the complexes at sequences on the target oncogene reduces transcription of that oncogene, such as when complexes are formed at transcriptional regulatory regions of the oncogene. In an embodiment, the subject is a mammal, such as a human.
The present invention further includes a method of treating an infection by a virus, bacterium or pathogen in a subject comprising administering an effective amount of a compound of the invention to a subject in need thereof.
The present invention further includes a use of a compound of the invention to treat cancer and a use of a compound of the invention to prepare a medicament to treat cancer. Further, the invention includes a use of a compound of the invention to treat a viral, bacterial or pathogen infection as well as a use of a compound of the invention to prepare a medicament to treat a viral, bacterial or pathogen infection.
The alternatively functionalized benzimidazole and pyridoimidazole, with additional ring substituents or N-alkylated ring systems, may also be used for new types of integrated fluorophore-poly amide conjugates for targeting the DNA minor groove in a sequence-selective manner. Advantages of these novel conjugates over the polyamide ligands in the prior art include, for example, the N-methylpiperazine group (when utilized) providing easier trafficking across cellular membranes, and the benzimidazole/pyridoimidazole units, for their useful fluorescence characteristics in diagnostic genomics applications. The reduced number of amide bonds in these conjugates will also make them less susceptible to cellular degradation by peptidase class of enzymes. Further, the fluorogenic rings are photostable and do not suffer from the problems associated with the oxidative degradation as known for electron- rich pyrrole/hydroxypyrrole rings.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in greater detail with reference to the following drawings in which:
Figure 1 shows the chemical structures of compounds Fl to F7. The abbreviated nomenclature scheme of the compounds follows the notation of Bzi=benzimidazole; Py=pyrrole; Im=imidazole; and MP=methylpiperazine. The circles with plus sign correspond to the N-methylpiperazine system, as it exists in a protonated positively charged form at neutral pH.
Figure 2 shows the chemical structures of compounds Gl to G7. The imidazopyridine (intermittently called pyridoimidazole) fragment is different from the benzimidazole ring in terms of the fluorescence and acid/base characteristics. The abbreviated naming of the compounds follows the notation of Pzi=pyridoimidazole;
Py=pyrrole; Im=imidazole; and MP=methylpiperazine. The circles with plus sign correspond to the N-methylpiperazine system, as it exists in a protonated positively charged form at neutral pH. Figure 3 shows the chemical structures of compounds Hl to H4. The abbreviated naming of the compounds is the same as that indicated for Figures 1 and 2. Diamonds with a plus sign represent the Dp (dimethylaminopropanamide) end-groups. Figure 4 shows the chemical structures of compounds Il and 12. The abbreviated naming of the compounds is the same as that indicated for Figures 1 and 2.
Figure 5 shows the chemical structures of compounds Jl to J4. The abbreviated naming of the compounds is the same that as indicated for Figures 1 and 2. Figure 6 shows the chemical structures of compounds Kl to K6. The imidazole ring substituents on the benzimidazole rings to the left are expected to be protonated at pH ~ 7. The shorthand codes are as follows: Bzi=benzimidazole; Py=pyrrole; Im=imidazole; MP=methylpiperazine Im imidazole as head group. The Bzi and Bzi are two differently functionalized benzimidazole systems.
Figure 7A shows the chemical structures of compounds Ll to L4. The abbreviated naming of the compounds is the same that as indicated for Figures 1 -6. The two benzimidazole-pyrrole building blocks are separated by the imidazole residue alone (Ll), or in combination with aliphatic amino acid spacers, glycine (termed α), beta- alanine(β)and gamma- aminobuty rate (γ), in L2, L3, and L4, respectively. Figure 7B shows the chemical structures of compounds Nl to N3. Figure 8 is a graph showing the ΔTm values measured for the interaction between distamycin against a panel of AT sites and GC sites. (A) ΔTm values measured for the interaction between distamycin and 16 different DNA molecules, containing different permutations of a 4-base pair variation shown in the plots. (B) ΔTm values for distamycin-stabilized DNA duplexes, containing different permutations of a 4-base pair variation around the G»C base pairs. For a complete sequence of DNA fragments see Table I. Distamycin, a minor-groove-binding polyamide, is a standard compound selective for 4-5 A1T base pair clusters.
Figure 9 is a graph showing the ΔTm values measured for the interaction between actinomycin against a panel of AT sites and GC sites. (A) ΔTm values measured for the interaction between actinomycin and 16 different DNA molecules, containing different permutations of a 4-base pair variation shown in the plots. (B) ΔTm values for actinomycin-stabilized DNA duplexes, containing different permutations of a 4- base pair variation around the G#C base pairs. For a complete sequence of DNA fragments see Table I. Actinomycin, a known anticancer drug agent is a standard with relatively increased binding preference for GC-rich base sequences in DNA. Figure 10 is a graph showing the ΔTm values measured for the interaction between compounds R4, F4 and G5 with the panel of 16 GC-rich sites only (2: 1 binding mode, looking at GC selectivity). Binding to the other 16 AT-rich sites was very poor for each of them. (A) Compound R4 (an all five-membered ring polyamide), (B) compound F4 (MP-BziPyPylmlm) and (C) compound G4 (MP-PziPyPylmlm). Note, binding varies according to their respective module compositions. Figure 11 shows the single mismatch effects on the variation in ΔTm values for each of the ligands that contain identical polyamide fragments but with variant modules containing the positively charged end. The description also illustrates the principle of FRET-based assay method. Q and F refer to the quencher (DABCYL) and fluorescent dye (FITC) covalently attached to the ends of DNA strands. Upon strand separation (thermal melting) the Q and F are no longer in close proximity and give a measurable increase in the fluorescence signal of F. Ligand- stabilized DNA samples (as shown) melt at higher temperatures than the corresponding controls and the difference in Tm values are shown below each model representation. Of the 16 duplex DNA that contain two G»C base pairs flanked by variant pairs immediately flanking that region, the two "highest" ranking sequences are only shown in the above format. Figure 12 shows the UWVIS absorption spectra for the Bzi ligand (F4) and Bzi:DNA complex. (A) shows the spectra of the ligand, in absence of DNA, with increasing concentration. (B) gives the measurement made in a titration experiment with increasing [Bzi] added to a fixed [DNA]. (C) shows difference absorption plots obtained by subtracting the DNA absorption from those in B. (D) normalized ligand spectra subtracted from those in B. Arrows are drawn immediately to the right of the main absorption bands due to the ligand. DNA (260 ran) refers to the band at 260 nm for stacked base pairs in the duplex. The E values are expressed in the units M-1Cm"1. DNA = d(CATGGCCATG)2. Figure 13 shows the UV/VIS absorption spectra for the Pzi ligand (G4) and Pzi:DNA complex. (A) shows the spectra of the ligand, in absence of DNA, with increasing concentration. (B) gives the measurement made in a titration experiment with increasing [Pzi] added to a fixed [DNA]. (C) shows difference absorption plots obtained by subtracting the DNA absorption from those in B. (D) normalized ligand spectra subtracted from those in B. Arrows are drawn immediately to the right of the main absorption bands due to the ligand. DNA (260 ran) refers to the band at 260 nm for stacked base pairs in the duplex. The e values are expressed in the units NT1Cm"1. DNA = d(CATGGCCATG)2.
Figure 14 shows a putative 2:1 binding models for 5 different compounds of the present invention on the basis of shape, structure and functional group matched recognition of a specified DNA target sequence.
Figure 15 shows a diagrammatic representation of 34 intermolecular ligand-DNA NOE relationships between the ligand F4 and d(C ATGGCC ATG)2. A model ball- stick representation of the binding model is shown beneath. Benzimidazole rings are shown as rectangles, pyrrole residues as darker circles, and imidazole systems as lighter circles.
Figure 16 shows a diagrammatic representation of 48 intermolecular ligand-DNA NOE relationships between the ligand G4 and d(C ATGGCC ATG)2. Pyridoimidazole rings are shown as rectangles, pyrrole residues as darker circles, and imidazole systems as lighter circles. Figure 17 shows a view into the minor groove of the mean structure of the 2:1 Bzi- DNA complex, with each ligand molecule proximal to the strand containing G residues at positions where Im residues reside in the ligand. For hydrogen bonds between Im N3 and guanine NH2, between CONH and cytosine 02, and Bzi NH and adenine N3, to collectively form, the two ligand molecules show a completely overlapped stacked binding mode with only the MP rings staggered off in the outermost base pairs of DNA. Intermolecular hydrogen bond patterns are shown in Figure 18.
Figure 18 is a schematic providing a summary of the binding models for the 2:1 Bzi: DNA and Pzi: DNA complexes, showing the intermolecular hydrogen bonds between the ligand molecules and DNA. Distances measured from the model structures are provided beneath the model. In all respects, the Bzi and Pzi ligands bind to the identical core site consisting of G* C base pairs.
DETAILED DESCRIPTION OF THE INVENTION I. Definitions
The term "Ci-yalkyl" as used herein means straight and/or branched chain, saturated alkyl groups containing from one to y carbon atoms and includes (depending on the identity of y) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, A- methylpentyl, n-hexyl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
The term "Ci-yalkenyl" as used herein means straight and/or branched chain, unsaturated alkyl groups containing from one to n carbon atoms and one or two double bonds, and includes (depending on the identity of y) vinyl, allyl, 2- methylprop- 1 -enyl, but-1-enyl, but-2-enyl, but-3-enyl, 2-methylbut-l-enyl, 2- methylpent-1-enyl, 4-methylpent-l-enyl, 4-methylpent-2-enyl, 2-methylpent-2-enyl, 4-methylpenta-l,3-dienyl, hexen-1-yl and the like, where y is an integer representing the maximum number of carbon atoms in the group. The term "Ci-yalkynyl" as used herein means straight and/or branched chain, unsaturated alkyl groups containing from one to y carbon atoms and one or two triple bonds, and includes (depending on the identity of y) propargyl, but-1-ynyl, but-2- ynyl, but-3-ynyl, 4-methylpent-l-ynyl, 4-methylpent-2-ynyl, hex-1-ynyl and the like, where y is an integer representing the maximum number of carbon atoms in the group.
The term "cyclo(C3-Cy)alkyl" as used herein means saturated cyclic alkyl groups containing from three to y carbon atoms and includes (depending on the identity of y) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexyl and the like, where y is an integer representing the maximum number of carbon atoms in the group. The term "aryl" as used herein means a monocyclic or bicyclic ring system containing one or two aromatic rings and from 6 to 14 carbon atoms and includes phenyl, naphthyl, anthraceneyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. The term "monocyclic or polycyclic heterocycle" refers to monocyclic, bicyclic, tricyclic or quadracyclic ring fused systems containing at least one nitrogen atom and 1 to 6 other heteroatoms selected from O, S and N (with the balance of the ring system being C). The ring system may contain from 3 to 20 atoms, suitably 5 to 16 atoms, and may be fully saturated, partially saturated or aromatic. Suitably the polycyclic ring system is a fused quadracyclic ring system comprising 4 nitrogen atoms.
The term "C3-6heterocycle" as used herein refers monocyclic rings containing 3 to 6 atoms of which 1 to 4 atoms are a heteroatom selected from N, S and O (with the balance of the ring system being carbon). The ring may be fully saturated, partially saturated or aromatic. Suitably the heterocyclic rings contain 5 to 6 atoms, of which at least one atom is nitrogen and the rings are aromatic. Examples of heterocycles include imidazole, pyrrole, indole, pyridine and the like.
The term "alkylene" as used herein means bifunctional straight and/or branched alkyl groups containing the specified number of carbon atoms. The term "alkenylene" as used herein means bifunctional straight and/or branched alkenyl groups containing the specified number of carbon atoms.
The term "optionally substituted" as used herein, when not specified, means that the group is unsubstituted or substituted with one or more substituents independently selected from C,.4alkyl, OCMalkyl, OH, CF3, OCF3, halo, NO2, SH, SCMalkyl, NH2, NHCMalkyl, N(Ci-4alkyl)(Ci.4alkyl), CN, C(O)OH, tetrazolyl, C(O)OCMalkyl, C(O)C1-4alkyl, C(O)NH2, C(O)NHC 1-4alkyl, C(O)N(Ci-4alkyl)(C1. 4alkyl), NHC(O)C Malkyl, OC(O)C Malkyl, SOC1-4alkyl, SO2CMalkyl, SO2NHCi- 4alkyl and SO2NH2.
The term "pyridoimidazole" or "Pzi" as used herein refers to a monomeric unit having the following structure:
Figure imgf000017_0001
The term "benzimidazole" or "Bzi" as used herein refers to a monomeric unit having the following structure:
Figure imgf000017_0002
Unless otherwise noted, all DNA sequences within the present application are written in the 5' - 3' direction.
In one of its aspects, the compounds of the present invention selectively bind to specific "target DNA" sequences. The target DNA sequences may be single stranded or double stranded DNA (dsDNA). Suitably the target DNA sequence is ds DNA. The compounds of the invention can bind in a "sequence specific manner" which means that the compounds of the invention have the ability to form at least one complementary pair with at least one nucleotide base on the DNA target sequence.
The term "pharmaceutically acceptable salt" means an acid addition salt which is suitable for or compatible with the treatment of patients.
The term "pharmaceutically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any base compound of the invention, or any of its intermediates. Basic compounds of the invention that may form an acid addition salt include, for example, where the Ci^alkyl group of R1 and/or R2 is substituted with a group having a basic nitrogen, for example NH2 and
Figure imgf000017_0003
Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable acid addition salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term "solvate" as used herein means the incorporation of molecules of a suitable solvent into the crystal lattice of a compound of the invention. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The term "prodrug" as used herein will be functional derivatives of a compound of the invention which are readily convertible in vivo into the compound from which it is notionally derived. Prodrugs may be conventional esters formed with, for example, available hydroxy, or amino groups. For example, an available OH may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates and amino acid esters. In certain instances, the prodrugs are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.
The term "effective amount" as used herein refers to an amount of a compound of the invention that is effective in achieving a desired effect for a particular application. Effective amounts can vary depending on the specific compound of the invention and the accessibility of the target sequence. The term a "therapeutically effective amount" of a compound of the invention, as used herein is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount" or synonym thereto, when used in therapeutic applications, depends upon the context in which it is being applied. For example, in the context of modulating transcription of a target gene, an effective amount of a compound of the invention is that amount sufficient to achieve such a modulation in the transcription of the target gene as compared to the modulation obtained in the absence of the compound of the invention. In the context of disease, therapeutically effective amounts of the compounds of the invention, are used to treat, modulate, attenuate, reverse, or effect conditions which benefit from modulation of a target gene. An "effective amount" is intended to mean that amount a compound of the invention that is sufficient to treat, prevent or inhibit conditions which benefit from modulation of a target gene or a disease associated with modulation of a target gene. The amount of a compound of the invention that will correspond to such an amount will vary depending upon various factors, such as the given compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a "therapeutically effective amount" of a compound of the invention is an amount which modulates transcription of a target gene (e.g., as determined by clinical symptoms) in a subject as compared to a control. As defined herein, a therapeutically effective amount of a compound of the invention may be readily determined by one of ordinary skill by routine methods known in the art. Generally ranges of amounts of the compounds of the invention effective for reducing transcription of a target gene in a cell are 1 pM to 50 mM, such as 1 nM to 100 μM.
As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.
"Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
The term "prevention" or "prophylaxis", or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a condition which benefits from modulation of a target gene or manifesting a symptom associated with a condition which benefits from modulation of a target gene.
The term "compound(s) of the invention" as used herein means compound(s) of formula I, pharmaceutically acceptable salts, prodrugs and/or solvates thereof.
The term "subject" as used herein includes all members of the animal kingdom including human. The animal is preferably a human.
The term "a cell" as used herein includes a plurality of cells. Administering a compound to a cell includes in vivo, ex vivo and in vitro treatment.
II. Compounds of the Invention
A novel class of polyamide oligomers of nitrogen-containing heteroaromatic rings has been prepared. The compounds of the invention are useful as binding agents to targeted DNA sequences in the minor groove.
Accordingly, the present invention relates to a compound selected from a compound of formula I:
Figure imgf000020_0001
wherein each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N; R1 is selected from C^alkyl, C3-i2cycloalkyl, aryl, C(O)NHC i-6alkyl, C(O)NHC3. i2cycloalkyl and C(O)NH(C i-6alkylene-N(R6)2), in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N, NR6 or
N(R6)2;
R2 and R2 are independently selected from H and Ci-6alkyl, or R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R3 is Ci-6alkylene or C2-6alkenylene, both of which are optionally substituted with one or more of Ci.6alkyl, OCi.6alkyl or OH, and both optionally have one or more of the carbons replaced with O, S, NR6 or N(R6)2;
R4 and R4 are independently selected from H and Ci-6alkyl, or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3-6heterocycle, Ci-6alkyl, OH and 0Ci.6alkyl;
R6 is H or C,-6alkyl;
R7 is Cι-4alkylene or C2-4alkenylene both of which are optionally substituted with one or more of Ci-6alkyl,
Figure imgf000021_0001
or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1, 2, 3, 4 or 5, and when m is other than 1, only the terminal monomeric unit represented by m is substituted with R1 ; n is 1, 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is O, 1, 2, 3, 4 or 5; and q is O, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof, with the proviso that when R1 is selected from C(O)NHCi-6alkyl, C(O)NHC3- ,2cycloalkyl and C(O)NH(C )-6alkylene-N(R6)2), R5 is selected from pyrrolyl, imidazolyl, benzimidazolyl, imidazopyridinyl and purinyl. In an embodiment, the present invention includes a compound selected from a compound of the formula I:
Figure imgf000022_0001
I wherein each X may be the same or different and are independently selected from C and N, and only when X is C does it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; each Y is independently selected from C and N;
R1 is selected from Ci-6alkyl, C3_i2cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N,
NR6 or N(R6)2;
R2 and R2 are independently selected from H and Ci-6alkyl, or R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R3 is Cι-6alkylene or C2-6alkenylene, both of which are optionally substituted with one or more of Ci-6alkyl,
Figure imgf000022_0002
or OH, and both optionally have one or more of the carbons optionally replaced with O, S, NR6 or N(R6)2;
R4 and R4 are independently selected from H and
Figure imgf000022_0003
or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3-6heterocycle, Ci-6alkyl, OH and OC].6alkyl;
R6 is H or Ci-6alkyl;
R7 is Ci-4alkylene or C2-4alkenylene both of which are optionally substituted with one or more of
Figure imgf000022_0004
or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1, 2, 3, 4 or 5, provided that when m is other than 1, only the terminal monomer unit represented by m is substituted with R1; n is 1, 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is 0, 1, 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof. In an embodiment of the invention, m is 1. In another embodiment of the invention, m is 1 and p is 0. In a further embodiment of the invention, m is 1 , o is 0 and p is 0. In this embodiment, it is yet another embodiment that n is 1 , 2, 3 or 4 and q is 0 or 1.
In yet another embodiment of the invention, n and o are both 0. Within this embodiment, it is yet another embodiment that m and p are both 1.
In a still further embodiment of the invention, m is 1, n is 1 or 2, o is 0, p is 1 and q is 0 or 1.
In another embodiment of the present invention, R1 is selected from
Figure imgf000023_0001
C3-i2cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is replaced with N, NR6 or N(R6)2, where R6 is H or Ci-4alkyl. In further embodiments, R1 is selected from N-C^alkyl- piperazinyl, imidazolyl, pyrazolyl, triazolyl, morpholinyl, each of which are connected to the compound of Formula I through the nitrogen atom. More particularly, R1 is N-methylpiperazinyl. In yet another embodiment of the present invention, R1 is selected from
Figure imgf000023_0002
C(O)NHC3-7cycloalkyl and C(O)NH(CMalkylene-N(R6)2), in which one or more of the carbons of the alkyl or cycloalkyl groups is replaced with N, NR6 or N(R6)2, where R6 is H or CMalkyl.
In embodiments of the present invention, R2 and R2 are, independently, H or
Ci-4alkyl. More particularly, R2 and R2 are both methyl. In further embodiments of the present invention, R2 and R2 are joined by R7 to form a 5 to 8 membered ring, suitably a 6 membered ring, between two adjacent monomeric units where R7 is Ci. 4alkylene or C2^alkenylene. In embodiments of the present invention, R3 is Ci^alkyene. More particularly, R3 is Ci-3alkylene.
In embodiments of the invention R4 and R4 are independently selected from H and In further embodiments of the present invention, R4 and R4 are joined by R7 to form a 5 to 8 membered ring, suitably a 6 membered ring, between two adjacent monomeric units, where R7 is Ci^alkylene or C2-4alkenylene.
In embodiments of the present invention, R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C5- όheterocycle, d^alkyl, OH and OCi-6alkyl. In further embodiments of the invention, R5 is a nitrogen-containing mono- or bicyclic aromatic heterocycle. More particularly, R5 is selected from optionally substituted derivatives of pyrrole, imidazole, oxazole, thiazole, benzimidazole, imidazopyridine, benzoxazole, benzothiazole, and indole. Optional substituents are one or more
Figure imgf000024_0001
OH and OCi^alkyl, suitably one of more of CH3, OH or OCH3, more suitably one of CH3, OH or OCH3. In another embodiment R5 is unsubstituted. In another embodiment of the invention R5 is Pzi or Bzi, substituted with N-methyl imidazole or N-methylpyrrole at the position between the two nitrogens on the 5 membered ring portion of this group.
In embodiments of the present invention, R6 is H or
Figure imgf000024_0002
Suitably, R6 is H or CH3. In an embodiment of the invention the compound of the formula I is selected from:
Figure imgf000024_0003
wherein each X may be the same or different and are independently selected from CH and N; each Y may be the same or different and are independently selected from CH and N and when q is greater than 1 , Y in each monomer unit represented by q may be the same or different;
R4 and R4 are independently selected from H and Ci-όalkyl; q is 1, 2, 3, 4 or 5 (suitably q is 2, 3 or 4, most suitably q is 3), and and pharmaceutically acceptable salts, solvates and prodrugs thereof.
Also within the scope of the present invention is a compound of the formula I which is:
Figure imgf000025_0001
and
Figure imgf000025_0002
wherein each X may be the same or different and are independently selected from CH and N; wherein each Y may be the same or different and are independently selected from CH and N, and and pharmaceutically acceptable salts thereof. III. Methods of Detecting and Isolating Target DNA Sequences The formation of complexes between target DNA and the compounds of the present invention may be used for diagnostic, purification or research purposes, and the like. Compounds of the present invention can be used to detect specific dsDNA sequences in a sample without melting the dsDNA. Examples of diagnostic applications for which compounds of the present invention may be used include detection of alleles, identification of mutations, identification of a particular host, e.g. bacterial strain or virus, identification of the presence of a particular DNA rearrangement, identification of the presence of a particular gene, e.g. multiple resistance gene, forensic medicine, or the like. With pathogens, the pathogens may be viruses, bacteria, fungi, protista, chlamydia, or the like. With higher hosts, the hosts may be vertebrates or invertebrates, including insects, fish, birds, mammals, and the like or members of the plant kingdom.
The present invention therefore includes a method for selectively forming a complex between target DNA and a compound of the present invention, the method comprising: contacting the target DNA with the compound of the invention; wherein the compound of the invention is capable of selectively binding to a sequence on the target DNA. In an embodiment of the invention, the compound of the invention selectively binds to the target DNA under physiological conditions where complexes form between the compound of the invention and the target DNA. In an embodiment of the invention, the specific sequence is in a minor groove on the target DNA. In another embodiment of the invention, the compound of formula I and the target DNA is at a binding stoichiometry of 2:1. In a further embodiment of the invention the target gene comprises all or a fragment of a sequence selected from AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, suitably 5'-ATGGCCAT and 5'- AAGCGCTT. Within the scope of the present invention is a method of detecting the presence of a sequence in a sample comprising DNA. The method comprises contacting the sample with a compound of the invention which is capable of selectively binding to sequences on the DNA, and wherein the compound of the invention has at least one detectable label; and monitoring the detectable label in the sample, wherein the presence of the detectable label is indicative of the presence of the selected sequence. Further, the present invention relates to a method for isolating target DNA from a sample comprising a mixture of DNA. The method comprising: contacting the target DNA with a compound of the invention which is capable of selectively binding to a sequence on the target DNA, and wherein complexes selectively form between the compound of the invention and the target DNA; and isolating the selected complexes.
Compounds of the present invention are useful for detecting the presence of DNA of a specific sequence for diagnostic or preparative purposes. For example, the sample containing the target DNA sequence can be contacted with a compound of the present invention linked to a solid substrate, thereby isolating DNA comprising a desired sequence. Alternatively, compounds of the invention linked to a suitable detectable marker, such as biotin, a hapten, a radioisotope or a dye molecule, can be contacted by a sample containing the target DNA.
Detection of complexes of the compounds of the invention and target DNA sequences is facilitated by the presence of luminescent or fluorescent moieties already incorporated with in their molecular structure. These moieties may be utilized by a person skilled in the art using well know methods to detect complexes of the invention with target DNA sequences.
Other detectable labels that may be utilized with the compounds of the present invention include additional fluorescers, e.g. dansyl, fluorescein, Texas red, isosulfan blue, ethyl red, and malachite green, chemiluminescers, magnetic particles, colloidal particles, gold particles, light sensitive bond forming compounds, i.e. psoralens, anthranilic acid, pyrene, anthracene, and acridine, chelating compounds, such as EDTA, NTA, tartaric acid, ascorbic acid, polyhistidines of from 2 to 8 histidines, alkylene polyamines, etc., chelating antibiotics, such as bleomycin, where the chelating compounds may chelate a metal atom, such as iron, cobalt, nickel, technetium, etc., where the metal atom may serve to cleave DNA in the presence of a source of peroxide, intercalating dyes, such as ethidium bromide, thiazole orange, thiazole blue, TOTO, 4',6-diamidino-2-phenylindole (DAPI), etc., enzymes, such as β-galactosidase, NADH or NADHP dehydrogenase, malate dehydrogenase, lysozyme, peroxidase, luciferase, etc., alkylating agents such as haloacetamides, N- ethyl nitrosourea, nitrogen and sulfur mustards, sulfonate esters, etc., and other compounds, such as arylboronic acids, tocopherols, lipoic acid, captothesin, etc. colloidal particles, e.g., gold particles, fluorescent particles, peroxides, DNA cleaving agents, oligonucleotides, oligopeptides, NMR agents, stable free radicals, metal atoms, etc. The compounds of the invention may be combined with other labels, such as haptens for which a convenient receptor exists, e.g. biotin, which may be complexed with avidin or streptavidin and digoxin, which may be complexed with antidigoxin, etc. where the receptor may be conjugated with a wide variety of labels, such as those described above. The compounds may be joined to sulfonated or phosphonated aromatic groups, e.g. naphthalene, to enhance inhibition of transcription, particularly of viruses (Clanton et al., Antiviral Res., 27:335-354, 1995). In some instances, one may bond multiple copies of the subject compounds to polymers, where the subject compounds are pendant from the polymer. Polymers, particularly water soluble polymers, which may find use are cellulose, poly(vinyl alcohol), poly(vinyl acetate-vinyl alcohol), polyacrylates, and the like. A radioactive moiety may also be employed as a detectable label, such tritium, 14C, 125I, or the like. The radiolabel may be a substituent on a carbon or a heteroatom of any atom in any monomer, or the radiolabel may be a substituent at either terminus of the oligomer. The radiolabel may serve numerous purposes in diagnostics, cytohistology, radiotherapy, and the like. For detecting the presence of a target sequence, the target DNA may be extracellular or intracellular. When extracellular, the DNA may be in solution, in a gel, on a slide, or the like. The DNA may be present as part of a whole chromosome or fragment thereof of one or more centiMorgans. The DNA may be part of an episomal element. The DNA may be present as smaller fragments ranging from about 20, usually at least about 50, to a million base pairs, or more. The DNA may be intracellular, chromosomal, mitochondrial, plastid, kinetoplastid, or the like, part of a lysate, a chromosomal spread, fractionated in gel elecrophoresis, a plasmid, or the like, being an intact or fragmented moiety. When involved in vitro or ex vivo, the DNA may be combined with the compounds of the inventionin appropriately buffered medium, generally at a concentration in the range of about 0.1 nM to 1 mM. Various buffers may be employed, such as TRIS, HEPES, phosphate, carbonate, or the like, the particular buffer not being critical to this invention. Generally, conventional concentrations of buffer will be employed, usually in the range of about 10-200 mM. Other additives which may be present in conventional amounts include sodium chloride, generally from about 1-250 mM, dithiothreitol, and the like, the particular nature or quanitity of salt not being critical to this invention. The pH will generally be in the range of about 6.5 to 9, the particular pH not being critical to this invention. The temperature will generally be in a range of 4-45 0C, the particular temperature not being critical to this invention. The target DNA may be present in from about 0.001 to 100 times the moles of compound of the invention. The present invention also provides a diagnostic system, suitably in kit form, for assaying for the presence of a target DNA sequence bound by compounds of the invention in a body sample, such as brain tissue, cell suspensions or tissue sections, or body fluid samples such as colony stimulating factor (CSF), blood, plasma or serum, where it is desirable to detect the presence, and suitably the amount, of the target DNA sequence bound by the compound of the invention in the sample according to the diagnostic methods described herein.
The diagnostic system includes, in an amount sufficient to perform at least one assay, a specific compound of the invention as a separately packaged reagent. Instructions for use of the packaged reagent(s) are also typically included. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a compound of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated compound or it can be a microliter plate well to which microgram quantities of a contemplated compound have been operatively affixed, i.e., linked so as to be capable of being bound by the target DNA sequence. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent or sample admixtures, temperature, buffer conditions and the like. A diagnostic system of the present invention might also include a detectable label and a detecting or indicating means capable of signaling the binding of the contemplated compound of the present invention to the target DNA sequence. As noted above, numerous detectable labels, such as biotin, and detecting or indicating means, such as enzyme-linked (direct or indirect) streptavidin, are well known in the art. Alternatively, the detectable label is incorporated into the compound of the invention and the instructions for use will explain to a person skilled in the art how to utilize the compound of the invention to determine the presence and/or amounts of the target DNA sequences using this labeling method.
Kits may optionally contain instructions for administering compounds or compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of compounds of the invention by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for compounds of the invention. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compounds of the invention in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
The present inventors have examined the properties of DNA binding molecules for a non-denaturing fluorescent detection of specific sites in solution within short segments of DNA. Interesting applications such as chromosomal staining and as diagnostic probes are possible. An application for this science may be related to diagnostics and "SNP typing" in particular, in which the identity of the SNP containing sequence is known (and a proper means for SNP detection is the only obstacle). For example, single nucleotide polymorphisms (SNPs) are the most common variation in the human genome and can be diagnostic of particular genetic predisposition to disease. Most methods of DNA detection involve hybridization of an oligonucleotide probe to its complementary single-strand nucleic acid target leading to signal generation. Detection by hybridization requires DNA denaturation conditions, and it remains a challenge to develop sequence specific fluorescent probes for DNA in the double strand form. IV. Methods of Modulating Transcription of a Target Gene
Still further, the present invention relates to a method for modulating transcription of a target gene in a cell. The method comprising: contacting the cell with an effective amount of a compound of formula I which is capable of binding to a specific sequence on the target gene under conditions where specific complexes form between the compound of formula I and the target gene, and wherein the level of transcription of the target gene is modulated.
Since exemplary compounds of the present invention are very specific for a given DNA sequence size comprising linear self-complementary sequence types [AAA]x[TTT]x and [AAA]x[TA]y[TTT]x [where x=2 to 6, and y= 1], some of the compounds of the present invention will be useful in specifically targeting gene segments of pathogens like Plasmodium falciparum (and other malaria pathogens] and other microbes that are known to have such long AT base pair stretches. Human genes have negligible frequency of the same types of segments in their genes. Since exemplary compounds from the library of disclosed assemblies are very selective for binding AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, type self-compementary DNA segments, such compounds that are also light-emissive probes, will also be useful in the detection and quantitative analysis of such sequence contents in diagnostic applications. Likewise, such compounds will also be useful in DNA/gene staining methods for in vitro and in vivo photomicroscopy based on fluorescence capturing detection. Further, due to their DNA binding profiles, these ligands have already tested favorably for their ability to obliterate DNA binding proteins like DNAase I, restriction enzymes specific for (A/T)GGCC(T/A) sites, and therefore will be useful in vitro molecular probes in PCR, gene cloning, molecular biology and molecular genetics. Because of their high affinity and specific binding for the said segments, and the occurrence of those nucleotide segments in gene sequences of cancer related proteins, such ligands will also be useful in modulation of such genes to be expressed, both in vitro and in vivo.
Accordingly, the present invention relates to a method for modulating transcription of a target gene in a cell comprising contacting the cell with an effective amount of a compound of the invention under conditions sufficient for the formation of complexes selectively between the compound of the invention and the target gene and wherein such complex formation modulates the transcription of the target gene. In an embodiment of the invention the target sequence on the gene is a sequence on transcriptional regulatory regions of the gene. AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT, suitably 5'-ATGGCCAT and 5'-AAGCGCTT. The cell may be for example, a mammalian cell, such as a human cell. Alternatively, the cell may be a bacterial, virus or pathogen cell. The target gene may be any gene implicated in the propagation of the cell or of a disease state. For instance, the target gene may be an oncogene. The cell may be in a subject, such as a mammal, as in a human or outside the subject, such as, for example, ex vivo treatment.
Furthermore, the present invention also relates to a method for treating cancer comprising administering an effective amount of the compound of the invention to a subject in need thereof. By contacting the DNA comprising the target oncogene with a compound of the invention that is capable of selectively binding to a target sequence on the oncogene, complexes can be selectively formed between the compound of the invention and a target oncogene on the DNA. Selective formation of the complexes at sequences on the target oncogene reduces transcription of that oncogene, such as when complexes are formed at transcriptional regulatory regions of the oncogene. In an embodiment, the subject is a mammal, such as a human. The present invention further includes a method of treating an infection by a virus, bacterium or pathogen in a subject comprising administering an effective amount of a compound of the invention to a subject in need thereof.
In one aspect of the invention, highly selective representative DNA binding ligands, F4, G4, and F6 were determined to be binding strongly and bindingwith high fidelity to their specific sites, 5'-ATGGCCAT (either of ligands F4 or G4), and 5'-5'- AAGCGCTT (ligand F6). From information on those target protein gene sequences (coding regions of the gene expressing those proteins), the occurrence of the aforementioned targeted octanucleotide sequence (and their frequency of occurrence in the gene) were identified. These potential therapeutic targets are listed below:
Listing of antineoplasmic targets containing the truncated or complete length variants of 5'-ATGGCCAT (the preferred binding site of ligands F4 and G4):
Glutathione reductase (mitochondrial), 1440 base pairs ; frequency of either
AGGCCA, AGGCCT, TGGCCA, orTGGCCT -4
Retinoic acid receptor alpha, 1389 base pairs; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 2 Ribonucleotide reductase, 2379 base pairs, frequency of either AGGCCA, AGGCCT,
TGGCCA, or TGGCCT = 3; frequency of ATGGCCTT = 1
DNA Topoisomerase II, 4596 base pairs; frequency of either AGGCCA, AGGCCT,
TGGCCA, or TGGCCT - 6; frequency of AAGGCCAA = 1; frequency of
AAGGCCTA = 1. Low density lipoproteins (LDL), 2583 base pairs; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 7; frequency of ATGGCCAA = 1.
Tubulin, 1335 base pairs; frequency of either AGGCCA, AGGCCT, TGGCCA, or
TGGCCT = 7; frequency of AAGGCCTT = 1
Retinoid receptor beta, 1602 base pairs; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 3 ; frequency of TTGGCCTT = 1.
PML-RAR alpha protein, 200 base pairs, frequency of either AGGCCA, AGGCCT,
TGGCCA, or TGGCCT - 0.
Ribonucleoside-diphosphate reductase large subunit, 2379 base pairs; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 3; frequency of ATGGCCTT = 1.
Apoptosis regulator Bcl-2, 720 base pairs [chromosome 18]; frequency of either
AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 1.
Purine nucleoside phosphorylase, 870 base pairs [chromosome 14]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 2; frequency of AAGGCCAA = I . Hypoxanthine-guanine phosphoribosyltransferase, 657 base pairs [chromosome X]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 1.
DNA polymerase, 1797 base pairs [chromosome H]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 2; frequency of TTGGCCTT = 1 ; frequency of ATGGCCAT = I .
DNA helicase, 5130 base pairs [chromosome 5]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 7; frequency of AAGGCCAA = 2; frequency of
TTGGCCAA = 1 ; frequency of AAGGCCTT = 1.
Alpha retinoic acid receptors (RARs), 1389 base pairs [chromosome 17]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 2.
COX-2, 1815 base pairs [chromosome I]; frequency of either AGGCCA, AGGCCT,
TGGCCA, or TGGCCT = 0.
Beta tubulin, 1356 base pairs [chromosome 20]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 3. DNA ligase III, 2769 base pairs [chromosome 17]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 5.
Cytochrome P450 CYPl IBl (steroid hydroxylase), 1512 base pairs [chromosome 8]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 6; frequency of
AAGGCCAA = 1. Matrix metalloprotease 2 (MMP-2), 1983 base pairs [chromosome 16]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 5; frequency of
AAGGCCAA = 1.
Estrogen receptor, 1593 base pairs [chromosome 14]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 6; frequency of AAGGCCTT = 1 ; frequency of AAGGCCAA = 1 ; frequency of AAGGCCAT = 1.
26S proteosome non-ATPase regulatory subunit I, 2862 base pairs [chromosome 2]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 4; frequency of
ATGGCCTT = 1 ; frequency of ATGGCCAA = 1 ; frequency of TTGGCCTT = 1.
Beta platelet-derived growth factor receptor precursor, 3321 base pairs [chromosome 5]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 11 ; frequency of ATGGCCAA = 1 ; frequency of AAGGCCAT = 1. Arachi donate 5-lipoxygenase, 1989 base pairs [chromosome 17]; frequency of either
AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 7; frequency of AAGGCCAA = 1.
Androgen receptor, 2760 base pairs [chromosome X]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 3; frequency of AAGGCCTT = 1. Estrogen receptor, 1788 base pairs [chromosome 6]; frequency of either AGGCCA,
AGGCCT, TGGCCA, or TGGCCT = 7; frequency of AAGGCCTT = 1; frequency of
TTGGCCAA = 1 ; frequency of ATGGCCAA = 1.
Alpha- IA adrenergic receptor, 1401 base pairs [chromosome 8]; frequency of either
AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 6; frequency of AAGGCCAT = 1; frequency of AAGGCCTT = 1.
FKBPl 2-rapamycin complex-associated protein, 7650 base pairs [chromosome I]; frequency of either AGGCCA, AGGCCT, TGGCCA, or TGGCCT = 38; frequency of
TTGGCCAT = 1 ; frequency of AAGGCCAT = 2; frequency of TTGGCCAA = 2.
Listing of specific antineoplasmic targets from above and this time containing the acceptable variants of AAGCGCTT (the preferred binding site of ligand F6):
Retinoic acid receptor alpha, 1389 base pairs; frequency of either AGCGCA,
AGCGCT, TGCGCA, or TGCGCT = 2.
Low density lipoproteins (LDL), 2583 base pairs; frequency of either AGCGCA,
AGCGCT, TGCGCA, or TGCGCT = 4. Tubulin, 1335 base pairs [chromosome 6]; frequency of AAGCGCAT = 1.
Thymidylate synthase, 942 base pairs; frequency of either AGCGCA, AGCGCT,
TGCGCA, or TGCGCT = 2.
Apoptosis regulator Bcl-2, 720 base pairs [chromosome 18]; frequency of either
AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1. Alpha retinoic acid receptors (RARs), 1389 base pairs [chromosome 17]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 2.
Cytochrome P450 CYPI lBl (steroid hydroxylase), 1512 base pairs [chromosome 8]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1.
Eestrogen receptor, 1593 base pairs [chromosome 14]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1. Type III phopshodiesterase, 3426 base pairs [chromosome 12]; frequency of either
AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1.
26S proteasome non-ATPase regulatory subunit I, 2862 base pairs [chromosome 2]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1. Epidermal growth factor receptor precursor, 3633 base pairs [chromosome 7]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 4.
Beta p late let- derived growth factor receptor precursor, 3321 base pairs [chromosome
5]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 3.
Arachnidonate 5-lipoxygenase, 1989 base pairs [chromosome 17]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 3.
Aromatase, 1512 base pairs [chromosome 15]; frequency of either AGCGCA,
AGCGCT, TGCGCA, or TGCGCT = 1; frequency of ATGCGCAA = 1.
Androgen receptor, 2760 base pairs [chromosome X]; frequency of either AGCGCA,
AGCGCT, TGCGCA, or TGCGCT = 3. Alpha- IA adrenergic receptor, 1401 base pairs [chromosome 8]; frequency of either
AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 3.
FKBP 12-rapamycin complex-associated protein, 7650 base pairs [chromosome I]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 7.
Dihydroorotate dehydrogenase, mitochondrial (precursor), 1188 base pairs [chromosome 16] ; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT
= 1.
Inosine-5' -monophosphate dehydrogenase 2, 1545 base pairs [chromosome 3]; frequency of either AGCGCA, AGCGCT, TGCGCA, or TGCGCT = 1.
The compounds of the invention are suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound selected from a compound of the invention and one or more pharmaceutically acceptable carriers or diluents. In accordance with the methods of the invention, the described compounds of the invention may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
A compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compound of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.
Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, poly gly colic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
The compounds of the invention may be administered to a subject alone or also in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
The dosage of the compounds of the invention and/or compositions comprising a compound of the invention and/or a compound of Formula II, can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.
V. Chemical Synthesis
The compounds of the present invention may be prepared by combining the individual monomer units in a stepwise fashion using methods known in the art. The individual monomer units are either commercially available or may be prepared using methods known in the art. For example, the benzimidazole-pyrrole, benzimidazole- imidazole, imidazopyridine-pyrrole and imidazoimidazole key units may be prepared using a oxidative cyclocondensation reaction as follows:
Figure imgf000039_0001
A B C
wherein a reagent of formula A, wherein R , X, R 2 and R are as defined in formula I is combined with a reagent of formula B, wherein Y, R and R 4' are as defined in formula I and W is either H or NO2, in an inert solvent, for example acetonitrile, at a temperature in the range of 50-1 10 0C, suitably 80 to 100 0C, to form an intermediate Schiff s base. The solvent may then be suitably removed and the crude Schiff s base may be resuspended in a suitable inert solvent (same or different as the first solvent) and reacted with a transition metal salt, most suitably an Fe(III) salt, such as FeCl3.6H2O, in the presence of oxygen gas at a temperature in the range of 50-110 0C, suitably 80 to 100 0C to form the intermediate of formula C. Various R groups may be incorporated onto reagents of formula A or C using methods known in the art, for example, using nucleophilic substitution reactions. Imidazole- or pyrrole-containing monomeric units may be added on to intermediate C when W is NO2, by first reducing the NO2 group to an amine using, for example H2/Pd-C, and coupling the resulting amine to an imidazole- or pyrrole- containing monomeric unit comprising an activated acid in a suitable location using well known amide bond forming reaction conditions. Other reactions for the preparation and coupling of the monomeric units in the compounds of formula I are well known to those skilled in the art and representative examples of such reactions are described in more detail below. A person skilled in the art would appreciate that the reaction conditions, including reactants, temperature, time and solvent, mentioned below could be varied by a person skilled in the art in order to optimize reaction yields depending on the structure of the starting monomeric units.
1. Synthesis of Intermediates and Monomer Residues
For the synthesis of 4-(N-methylpiperazinyl)-l,2-phenylenediamine (2), the commercially available 5-chloro-2-nitroaniline as starting material was used in a direct nucleophilic aromatic substitution reaction protocol (Scheme I). The nitroaniline derivative (1) was readily converted into the diamine (2, 93%) via catalytic hydrogenation, and isolated in its free base form. At times, when large-scale reactions were formed, the diamine (2) was stored in the form of its dihydrochloride salt to avoid aerobic degradation.
Figure imgf000041_0001
Scheme I. Reagent/conditions: (i) N-methylpiperazine/K2CO3ZDMSO; (ii) H2/Pd-C.
The structurally analogous diaminopyridine compound (6) was prepared from 2,6-dichloro-3-nitropyridine (Scheme II),22 using initially a regioselective substitution of one of the chloro-substituents with NH3ZEtOH in controlled conditions (amount of condensed NH3 and low temperature), followed by a similar aromatic substitution reaction with N-methylpiperazine as above. Reduction with Pd/C under high pressure of H2 provided the diamine (6) in 90% yield; this diamine had to be isolated as its dihydrochloride by treatment of the reaction filtrate with ethanolic HCl due to rapid darkening of the solution on exposure to air.
Figure imgf000041_0002
3 major fraction minor product separated off
(II)
Figure imgf000041_0003
Scheme II. Reagents/conditions: (i) dry NH3/EtOH; (ii) /V-methylpiperazine, K2CO1ZDMF; (iii) H2/Pd-C, followed by HCl treatment in large-scale preparation
Figure imgf000042_0001
Scheme III. Reagents/conditions: (i) 5-chloro-2-nitroaniline or (3), K2CO3, DMF; (ii) H2/Pd-C.
Using the same overall strategy as above, several additional diamines were prepared as shown in Scheme III, such as the N-pyrazolyl- (8, 10), N-imidazolyl- (12, 14), and N-morpholinyl- (16, 18) derivatives of phenylene- (8, 12 and 16) and pyridine-diamines (10, 14, and 18).
The l-methyl-4-nitro-2-pyrrolecarboxaldehyde (20) was a key intermediate that has previously been reported in a French- language article.46 The procedure in that report is extremely inefficient for preparing the title compound, using ambient temperature nitration of the pyrrole-2-carboxaldehyde precursor (19) with cone HNO3. Under those conditions, the desired 4-nitro-isomer is only a minor product and the unwanted 5-nitro-isomer is largely formed, making the separation of two regioisomers very difficult. Since bulk amounts of this compound is required, a low temperature (at -70 0C) nitration of l-methyl-2-pyrrolecarboxaldehyde with excess fuming HNO3 and very watchfully raising the temperature to -30 0C over 60-90 min, and under no circumstances allowing it to warm to > -20 0C, provided exclusively the desired isomer in 65-70% yield (Scheme IV).
The commercial samples of precursor l-methyl-2-pyrrolecarboxaldehyde (19) are usually contaminated with the oxidized acid impurities and in fact the nitration reaction under present conditions would invariably cause unwanted explosions of the reaction contents, presumably due to rapid and exothermic decarboxylation of the acid. Compound (19) was therefore alternatively prepared by a Vilsmeier route (POCI3/DMF) and freshly distilled batches of the aldehyde were more amenable to the nitration conditions for conversion to (20). The imidazole compound (22) was prepared in moderate yields as shown in Scheme IV, using a two-step procedure from N-methylimidazole. The lower reactivity of the electron-deficient imidazole unit, relative to the pyrrole, in the electrophilic nitration reaction was compensated by the relatively high-temperature conditions.
Figure imgf000043_0001
19 20
Figure imgf000043_0002
21 22
Scheme IV. Reagents/conditions: (i) POCl3-DMF, 0 C then reflux; (ii) 90% HNO3, must be kept < - 20 °C; (iii) nBuLi/THF, -40 °C, followed by DMF; (iv) 90% HNO3, 40 °C.
Multi-gram amounts of the building blocks 1 -methyl-4-nitro-2- trichloroacetylpyrrole (23), l-methyl-2-trichloroacetylimidazole (24), and l-methyl-4- nitro-2-trichloroacetylimidazole (25), all equivalent to "active ester" synthons in haloform-type acylation reactions, were prepared.
COCCI3
Figure imgf000043_0003
Figure imgf000043_0004
23 24 25
Chart I. "Active" ester equivalent synthons for haloform-type acylation reactions. 2. Novel Benzimidazole-Pyrrole Type Building Blocks.
The key step that is common to the synthesis of the benzimidazole- and imidazopyridine-pyrrole units is a highly efficient and versatile oxidative cyclocondensation reaction, for which a mechanistic rationale allowed the implentation of a catalytic redox-cycling approach.31 Not only were the two desired intermediates (26/27) (Scheme V) accessible in high yields using the procedure developed, but many such benzimidazole or imidazopyridine derivatives used in the present invention were prepared in an analogous set of reaction conditions. While several transition metal ions such as Fe(III), Cu(II), and Mn(III) were found to be effective catalysts in mediating oxidative cyclocondensation of a variety of aromatic diamines and aldehydes to furnish benzimidazole-products (Scheme V), iron trichloride hexahydrate (FeCh-OH2O) was chosen to minimize the product loss and catalyst poisoning due to metal complexation by the benzimidazole or imidazopyridine compounds.
Figure imgf000044_0001
2 X = CH 26 X X == CH ) 6 X = N 27 X = N j
Scheme V. Oxidative cyclocondensation route to the key building blocks, (26) and (27).
An alternative route to imidazopyridine derivatives (Scheme VI) was also explored, using a strategy based on initial selective amidation of diaminopyridine (6) with trichloroacyl derivatives (23/25).27 Mixtures of isomers (28a/b and 29a/b) were usually formed in the first step, but subsequent acid-catalyzed cyclizations to imidazopyridine derivatives (27/30) were fraught due to the presence of unreacted amides (28a/29a), when the batches of 28(a+b) or 29(a+b) were used without prior separation. Appropriate conditions for an efficient chromatography based separation of the regioisomeric amides (28a/b and 29a/b) were not found, rendering this alternative more tedious than the Fe(III)/Fe(II)-cycled oxidative cyclocondensation reactions with aldehydic precursors.
Figure imgf000045_0001
Scheme Vl. Alternative acid-catalyzed cyclization route to pyridoimidazole derivatives. Reagents/conditions: (i) 'Pr2N(Et) (DIPEA), THF-DMF; (ii) 6 M HCl, or glacial AcOH, 80 °C.
3. Polyamide Fragments: Im-X-Y-CO2R Intermediates
For the preparation of acid intermediates comprising the oligomeric two- and three-ring polyamides, where the ring combinations comprise the N-methylpyrrole and N-methylimidazole systems, a highly convergent synthesis plan was successfully worked out (Schemes VII/VIII).47 This composite scheme starts with the alkyl esters of either the nitropyrrole (31) or a nitroimidazole (40) analog, each obtained by NaH- catalyzed methanolysis of the corresponding trichloroacetylated ring derivative (23/25), and builds the peptide chain using repetitive reduction and coupling reaction protocol as shown in Schemes VII/VIII.
Figure imgf000046_0001
34
32 R = OCH3 33
(V) C 35 R = OH
Figure imgf000046_0002
36 R = OCH3
(V) CL 38 R = OH (V) CL 39 R = OH
Scheme VII. Two- and three-ring residues with pyrrole and imidazole termini, (i) CH3OH/NaH (cat); (ii) H2/Pd-C, 24/CH3CN; (iii) H2/Pd-C, 23/CH3CN; (iv) H2/Pd-C, 25/CH3CN; (v) NaOH, H2O-CH3OH; (vi) H2/Pd-C, 24/CH3CN.
The electrophilic monomers trichloroacetylpyrrole (23) and trichloroacetylimidazole (25), used as starting points as well as for repetitive use in multiple reactions shown in Schemes VII & VIII, were also interchangeable across the two partial schemes, and the terminal coupling for each of the desired acid synthons was performed with N-methyl-2-trichloroacetylimidazole (24) to afford a total of six esters, (32), (36), (37), (41), (45) and (46), in a combinatorial/assortment approach (Schemes VII/VIII). The corresponding carboxylic acids, (35), (38), (39), (44), (47), and (48), were obtained in each instance by hydrolysis of the ester compounds with LiOH. In several cases, and for those with the COOH on imidazole ring in particular (44, 47, 48), the reaction mixtures were acidified under cold conditions to avoid decarboxylation, but quite often the products formed gel-like precipitates that were lyophilized before further use. All intermediates and the products were purified to > 95% purity (by NMR), characterized by 1H/13C NMR and mass spectrometry, and the chemical shift assignments were made from two- dimensional HMBC/HMQC NMR spectra for an unambiguous confirmation of product identity.
Figure imgf000047_0001
42 43 p 41 R = OCH3 L-" 44 R = OH
Figure imgf000047_0002
45 R = OCH3 r— 46 R = OCH3
[Z 47 R = OH U- 48 R = OH
Scheme VIII. Two- and three-ring residues with imidazole and imidazole termini.
4. Final Target Products (Series F/G Molecules)
With the advent of a wide variety of amine-acid coupling procedures from the field of peptide synthesis (Chart II),48'49 a significantly expanded set of structurally diverse final products that consist of a range of modular permutations can be generated. However, the dicyclohexyl carbodiimide (DCC) method was clearly not going to be useful and was avoided because of inherent practical difficulties associated with the generation of dicyclohexyl urea as the by-product in the coupling reactions. Instead, either the water-soluble l-ethyl-3-(N,N-dimethylaminopropyl) carbodiimide (EDCI. HCl) or the highly nonpolar diisopropyl carbodiimide (DIC) were found to be useful in the reactions employing DMF-H2O or anhydrous DMF, respectively, as the solvents. In all these reactions, 1 -hydroxybenzotriazole (HOBt) or 1 -hydroxysuccinimide (HOSu) was used as an additive to promote the coupling efficiencies.
/
V- N=C=N- < CH 3CH2-N=C=N-CH2CH2CH2N(CH3);, HCI
DCC DIC EDCI
cr ~N
OH HOBt OH HOSu
Chart II. Coupling reagents (DCC, DIC and EDCI), and auxiliary nucleophilic promoters (HOBt, HOSu) used for peptide (amide) bond forming reactions.
The DIC/HOBt method was particularly useful when the acid components in the reaction were soluble in straight DMF only or when susceptible to decarboxylation in DMF/H20 mixtures. The series wise description of the synthesis of the first-generation various target products (F1-F7 and G1-G7) is summarized in Schemes IX and X and Figures 1 and 2, and illustrates the versatility due to common intermediates for the preparation of a structurally diverse set of oligomeric modular assemblies combining the benzimidazole, imidazopyridine, pyrrole and imidazole building blocks.
Figure imgf000049_0001
26 X = CH 49 X = CH
27 X = N 50 X = N
CH
Figure imgf000049_0002
N
Scheme IX. Preparation of initial subset of target compounds (F1-F4 and G1 -G4) from benzimidazole- pyrrole and pyridoimidazole-pyrrole amine derivatives , (49) and (50), respectively.
Figure imgf000050_0001
CH
Figure imgf000050_0002
N
Scheme X. Preparation of F5-F7 and G5-G7. By using amines (53) and (54), the respective coupling reactions were more efficient than if (49/50) were used in combinations with (44), (46), and (48) in place of (24), (35), and (44), respectively, (i) (24)/CH3CN-DMF; (ii) H2/Pd-C, DMF-CH3OH.
In the case of aforementioned compounds, F1-F7, G1-G7, and in general for all the target derivatives (Series H-L), the products isolated by silica gel flash chromatography were first characterized in their free-base forms by high-field IH- NMR (solutions in DMSOd6) and mass spectrometry (FAB and/or EIMS), and again after conversion to their hydrochloride salt forms by RP-HPLC, Η/13C-NMR, and MALDI-TOF mass spectrometry. 5. Design and Synthesis of Additional Ligands (Series H - L Molecules)
The synthetic schemes made available in the preceding section hallmarks the development of a versatile and facile method to a variety of benzimidazole- and imidazopyridine-tagged polyamide conjugates for targeting DNA. More so, the variations of the individual modules, in their numbers and identity, provides for a facile access to a fairly large repertoire of a promising class of compounds in binding specified DNA sequences. It has been found that the DNA binding affinity of such multipurpose polyamide conjugates with N-methylpiperazinyl-benzimidazole and - imidazopyridine groups rivals and surpasses, in selected combinations, those observed with polyamides alone.
Multiple copies of benzimidazole units may further enhance the DNA binding characteristics of the designer ligands. These benzimidazole units could either be placed in a directly linked form, as found for Hoechst 33258, or could be positioned distal from each other. The placement of two, or more, benzimidazole (or imidazopyridine) units separated from each other in the oligomeric conjugates to determine whether a 2:1 side-by-side complexation mode with the DNA minor groove could still occur with sufficiently high affinity and selectivity for the cognate DNA sequences was investigated. Note that the benzimidazole system and related fused heterocycles have a larger aromatic hydrophobic surface with different electronic/steric characteristics and are also subject to tautomerization, compared with the five-membered rings linked via amide groups in the distamycin-like polyamides.
Figure imgf000052_0001
Figure imgf000052_0002
Scheme XI. The higher-generation, three-residue benzimidazole-benzimidazole-pyrrole building block, in amine form (62) as employed for the preparation of target molecules Il and 12. (i) FeCl3.6H2O (cat), O2, CH3CN/DMF; (ii) NaOH, H2O-CH3OH; (iii) H2/Pd-C, CH30H/DMF, HCl; (iv) (20), FeCl3.6H2O (cat), O2, DMF; (v) H2/Pd-C, CH3OH, HCl.
Starting with 4-(N-methylpiperazinyl)- 1 ,2-diaminobenzene (2) or 6-(N- methylpiperazinyl)-l,2-diaminopyridine (6) that were prepared earlier (see Scheme XXV) as starting materials, condensation reactions with 4-acetamido-3- nitrobenzaldehyde50 using FeCl3/O2/DMF provided the three-ring modules (55/56), respectively, in high yields. Removal of the acetyl protecting group with NaOH followed by acidification and catalytic hydrogenation for each of them furnished the diamine derivatives (59) and (60). The FeCl3ZO2 mediated cyclocondensation of these diamines with l-methyl-4-nitro-2-pyrrolecarboxaldehyde (20) affords the desirable four-ring modules (61/62) according to the general Fe(III)/Fe(II) redox-cycling protocol discussed above. Coupling of the amine (62) with (24), and with (44), affords two desirable targeted products (Il and 12) (Figure 4). Importantly, the oxidative cyclocondensation reactions of diamines (59) and (60) were also used to obtain various novel Hoechst 33258 (itself equivalent to MP-Bzi-Bzi-PhOH) analogs of the general sequence MP-Xzi-Bzi-Ar, where MP represents the N- methylpiperazine ring, Xzi being either benzimidazole (Bzi) or imidazopyridine (Pzi), Bzi and Ar describes any aromatic or heteroaromatic ring system.
Benzimidazole rings in the acid modules were also incorporated using the highly effective catalytic Fe(III)/Fe(II) redox cycling approach. Starting with 3,4- diaminobenzoic acid or with its methyl ester, two- and three-ring subunits, (64) and (70), were readily obtained using the multistep sequence summarized in Scheme XII. Coupling reactions of the active esters generated in situ from (64) and (70), with amines (49), (53a) or (53b), afford the additional final products J1-J4 (Figure 5), each consisting of two benzimidazole ring residues separated by the pyrrole and/or imidazole carboxamide units.
Figure imgf000053_0001
Scheme XII. Ester, acid and amide forms of the benzimidazole-imidazole modules.
Figure imgf000054_0001
Scheme XIII. Preparation of series J (J1-J4) molecules that contain two benzimidazole residues separated from each other by intervening five-membered ring heterocycles.
An additional coupling reaction of the intermediate (66), with 3- (dimethylamino)propylamine (Dp) provided (71), and (72) for further additional structural diversity, since the use of 72 in amido-coupling with the same three acid derivatives (24, 35, 39) as used in the preparation of Series-F and -G sets of analogs (Scheme XIV), affords an equivalent Series-H (Figure 3); replacing the N- methylpiperazine head-groups by the Dp-groups.
Figure imgf000055_0001
Scheme XIV. Preparation of the H1 -H4 subset, where the terminal Dp residues replace the N- methylpiperazine groups of series F and G products. H4 sequence is analogous to F4 and G4.
Trichloroacetyl-substituted pyrrole and imidazole derivatives are likely the most efficient class of smallest building blocks for a facile generation of the polyamide assemblies and their conjugates with benzimidazole/pyridoimidazole units as described above. In all of the ligands prepared to this point, the trichloroacetylimidazole unit has been used as the terminal residue. As a replacement for that fragment, larger fused heterocyclic fragments, such as an equivalent benzimidazole unit, could be incorporated at terminal locations. Thus, to structurally diversified ligands, commercially available N-methylbenzimidazole was trichoroacetylated at C2-position using an adaptation of the Nishiwaki method (Scheme XV), previously applied to the pyrrole, imidazole and related five-membered heterocycles.51"53 Any one-ring or two-ring aminoesters from Scheme VII could thus be treated with the "activated" trichloroacetyl benzimidazole compound (73) in a "haloform-type" condensation reaction to generate a variety of benzimidazole- terminated elongated amides (such as 75). Alternatively the trichloroacetylbenzimidazole was also used directly in coupling reactions with the amines (49/77) that were prepared (Schemes XVI and XVII for the preparation of (Kl -K4) (Figure 6). Products (K5) and (K6) are similarly derived from amine (77), and (K6) matches in the sequence content (BziPyPylmlm) with analogs F4, G4 and H4.
Figure imgf000056_0001
73 ,— 74 R = OCH3 NaOH (aq) Q 75 R = 0H 3
Scheme XV. Preparation of a benzimidazole "active ester" synthon (73), and its further conversion to a three-ring acid derivative (75).
Figure imgf000056_0002
Scheme XVI. Preparation of Kl and K2, as "increased aromatic surface" surrogates of Fl and F4, respectively.
Figure imgf000057_0001
Scheme XVIII. An imidazole (Im') substituted benzimidazole-pyrrole module (77) and its use in the preparation of analogs K3-K6, which all provide a new head group as a replacement for the N- methylpiperazinyl unit that was retained throughout F1 -F7, G1 -G7 and K1 -K2.
Lastly, the Series-L molecules (Figure 7) are derived from acid fragments containing natural aliphatic amino acids, glycine, β-alanine and γ-aminobutyric acid, precoupled to the C-terminus of the benzimidazole-pyrrole-imidazole system. Scheme XVIII provides for the reactions used in the preparation of L1-L4.
Figure imgf000058_0001
Scheme XVIII. Preparation of L1 -L4, from a common single amine (53b) and a common acid intermediate, 70, and from which three other acid derivatives were obtained prior to ultimate couplings.
Preparation of these compounds (L1-L4) involved initial coupling of the aliphatic amino acids, in ester forms, to a common benzimidazole-pyrrole-imidazole 'activated' acid (70, acid group on benzimidazole), followed by alkali hydrolysis of each ester derivative (78-80) to the acids 81-83 and their subsequent coupling with another common amine derived from (piperazinyl)benzimidazole-pyrrole-imidazole (53b, amino group on imidazole). Note that model compound Ll is a contiguous arrangement of the 6 heteroaromatic residues (MP-BziPyϊm-X-BziPylm; X = none), whereas in L2-L4, the X = glycine, β-alanine and γ-aminobutyrate linkers would allow conformational flexibility and a systematic extension for potentially improved DNA affinity by relaxing the overall curvature of the ligand edges containing all the hydrogen bond donor/acceptor groups.
In summary, F1-F7 (Figure 1), G1-G7 (Figure 2), H1-H4 (Figure 3), Il and 12 (Figure 4), J1-J4 (Figure 5), Kl -K6 (Figure 6), and L1-L4 (Figure 7), 34 compounds overall, were successfully prepared and fully characterized, using a widely diverse set of combinations of different acid and amine modules consisting of benzimidazole, pyridoimidazole, pyrrole, imidazole, and selected aliphatic amino acid residues. Along many of the reaction pathways, the need for chromatography was minimized and liquid-liquid or acid/base extraction procedures were sufficiently successful. It is evident that the generality and versatility of the solution-phase reaction protocols, employing aromatic substitutions, nitro group reductions, amide bond formation, and the newly applied FeCl3-catalyzed oxidative cyclocondensation reactions, could enable any specific combination of a multi-residue oligomer at will. The bis- heteroaryl modules, benzimidazole-pyrrole and pyridoimidazole-pyrrole have been prepared. A similar synthesis strategy is applicable to the potential utility of benzimidazole-imidazole and pyridoimidazole-imidazole building blocks. It will also serve well in solid-phase synthesis protocols for a versatile high-throughput approach.
In some cases the chemistries outlined above may have to be modified, for instance by use of protective groups, to prevent side reactions due to reactive groups, such as reactive groups attached as substituents. This may be achieved by means of conventional protecting groups, for example as described in "Protective Groups in Organic Chemistry" McOmie, J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd Edition, 1999. The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
The formation of solvates of the compounds of the invention will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
Prodrugs of the compounds of Formula I may be, for example, conventional esters formed with available hydroxy, amino or carboxyl group. For example, available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates and amino acid esters.
The present invention includes radiolabeled forms of the compounds of the invention, for example, compounds of the invention labeled by incorporation within the structure 3H, 11C or 14C or a radioactive halogen such as 125I and 18F. A radiolabeled compound of the invention may be prepared using standard methods known in the art. For example, tritium may be incorporated into a compound of the invention using standard techniques, for example by hydrogenation of a suitable precursor to a compound of the invention using tritium gas and a catalyst. Alternatively, a compound of the invention containing radioactive iodo may be prepared from the corresponding trialkyltin (suitably trimethyltin) derivative using standard iodination conditions, such as [125I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethyl formamide. The trialkyltin compound may be prepared from the corresponding non-radioactive halo, suitably iodo, compound using standard palladium-catalyzed stannylation conditions, for example hexamethylditin in the presence of tetrakis(triphenylphosphine) palladium (0) in an inert solvent, such as dioxane, and at elevated temperatures, suitably 50- 1000C. Further, a compound of the invention containing a radioactive fluorine may be prepared, for example, by reaction of K[I 8F]/K222 with a suitable precursor compound, such as a compound of Formula I comprising a suitable leaving group, for example a tosyl group, that may be displaced with the 18F anion.
VI. DNA-Ligand Interactions 1. ΔTm Measuements in High-Throughput Screens Using a high throughput assay method based on fluorescence resonance energy transfer (FRET) and a set of 32 duplex DNA molecules (Table I) where the respective complementary strands are alternatively labeled with a quencher (DABCYL) and a fluorescent reporter group (FITC), the influence of compounds designed in the present application on DNA melting was assessed. The prechosen DNA sequences in the set of standard primers are comprised of a central two base pair AT or GC binding site flanked by variable regions whose immediate neighboring sequences are systematically changed to accommodate all nearest neighbor alterations. A direct measure of thermal stabilization of DNA primers in the absence and presence of tested ligands was obtained in experiments conducted under identical conditions and by measuring the difference in fluorescence due to FITC that arises from the separation of strands upon regulated heating.
This ultrasensitive high-throughput assay, first employed for distamycin (an AT-selective compound) and actinomycin (a GC -binding anticancer drug) provided the necessary benchmarks for comparing multiple sets of compounds of the present invention by measuring the ΔTm values (Tm for complexed DNA - Tm for free DNA). These results are presented in a histogram format to illustrate the AT vs GC selectivity patterns for the two reference compounds (Figures 8 and 9). All experiments were performed according to a fixed protocol described in the Experimental Section.
The binding characteristics of the compounds of the present invention have been examined with regards to the following specific properties: (1) DNA sequence specificity; (2) ligand solubility in physiologically relevant buffer solutions; (3) thermal stability of ligands; (4) assessment of their selectivity for double stranded DNA (versus single-stranded oligomers); (5) relative affinity for DNA as assessed by ΔTm changes; and (6) performance in various salt/buffer environments for potential PCR applications.
It is known that AT-rich segments are preferred binding sites for distamycin, and conversely actinomycin binds to GC-rich segments to a greater extent than AT- sites. Within the panel of 16 GC sites, there is rather poor selectivity observed for actinomycin, consistent with its intercalative interaction with DNA. The minor groove binding compounds, developed by the present inventors clearly show a higher affinity for the GC segments than a reference polyamide ligand (R4) with equivalent number of DNA interactive modules.
Compound F4 of the present invention shows maximum effect of increasing the Tm values for a DNA containing the GGCCGGCC and CGCCGGCG segments (Figure 10). Likewise, compound G4 (the pyridoimidazole analog) also shows a greater stabilization of GGCCGGCC, CGCCGGCG and CGCT.AGCG segments of duplex DNA. In fact, this compound shows a much greater selectivity for these sites compared to F4. From the presence of pyrrole and imidazole units in these candidate ligands, it is likely that 2: 1 complexes are formed with DNA in a sequence selective manner, as shown in model illustrations (Figure 11). Considering the cofacial pairing of pyrrole with imidazole in the 2: 1 side-by-side binding in the DNA minor groove, selectivity for GC base pairs at locations matching with the pyrrole/imidazole pairs is observed in a way that is similar to the polyamide-DNA interactions derived from 2: 1 binding models for ditamycin and related analogs. The single base-pair mismatched models (the next higher affinity sites to GGCC) are shown for relative comparisons for each of the test analogs, F4, G4, and R4.
2. DNA-Ligand Interactions by UV/VIS Spectrophotometry Five compounds (F4, G4, H4, K6 and R4) were studied by three different spectroscopy methods since they contain a similar oligomeric arrangement of the different modules, with variant fluorescent moieties at one end of the molecule and an identical polyamide composition (-PyImIm) forming the other end. Note that compound R4 is a control polyamide with the equivalent number of hydrogen-bond donor and acceptor groups as the other analogs, and is also a completely non- fluorescent material. A rather poor affinity for DNA is observed in the Tm assay for this 5 -ring polyamide in accord with a conclusion drawn by Dervan and co-workers from the generalized binding site size limitations for oligomeric polyamide ligands due to curvature defects.54*55 Subsequent to determination of fundamental light absorption and emission parameters for these compounds, titration experiments with selected model DNA under different conditions were conducted wherein optical absorption, fluorescence and circular dichroism measurements were used to further establish the isothermal binding curves, binding stoichiometry and equilibrium association constants. A significant enhancement of the benzimidazole and pyridoimidazole emission intensities is observed upon binding of the Bzi (F4) and Pzi (G4) analogs to the matched sequence DNA predicted by the ΔTm results described above and the pairing rules for 2:1 binding of polyamide ligands.56"59 The induced CD as well as changes in the steady-state absorption spectra were significantly large for the Bzi and Pzi-analogs in the presence of matched DNA. These two compounds behave in a similar manner with regards to their ability to bind the matched DNA in a 2:1 binding mode, and the changes in spectral profiles provide unambiguous proof-of-concept data.
UV-VIS absorption spectra for compound F4 (Bzi analog) in the absence and presence of duplex oligonucleotide, d(CATGGCCATG)2, and to the point of identical final ligand concentrations in the two experiments, are shown in Figure 12. Two types of difference spectra, obtained by subtracting the free ligand from complexed- ligand, and the free DNA from ligand-DNA complex, are also provided in Figure 12. These provide a direct measure and visual display of the ~ 5 nm bathochromic shift (red-shifted λmax), and an enhanced hyperchromic molar absorption coefficient (Δε = 2,600 M"1 cm'1) upon binding of the ligand to DNA. The ε increase is quite remarkable compared to previous results with minor groove binding agents, which usually exhibit a small decrease in absorption upon binding DNA.39'60 It may be that the increased absorption is due to the anticipated 2:1 binding mode on the basis of a correctly matched binding sequence with the linear heterocycle sequence of the Bzi analog. The stacked binding of 2 molecules of the Bzi analog with significant overlap of the heteroaromatic chromophores is expected to show higher absorptivity in the presence of DNA.
The UV-VIS absorption spectra for compound G4 (Pzi analog) in the absence and presence of the same DNA molecule as used above, are provided in Figure 13. The two types of difference spectra were deduced by the subtraction of the free ligand and the free DNA spectra from those for the admixtures. In this case, the bathochromic shift is phenomenally large (Δα = 43 nm) and the ε increase corresponding to the two absorption maxima (in the free and complexed states of the ligand) is of the same order of magnitude as exhibited by the preceding Bzi analog. Two distinct bands, with significant overlap, centered at 363 and 384 nm are observed. Even for the free ligand, there appear to be two closely positioned bands and the Δαmax difference is difficult to precisely be assigned to a particular type of So- Si transition. Lastly, for this specific analog (Pzi), the spectral profile for the DNA bound form (Figure 13D) is also visually very similar to the complexation-induced spectral band in CD titrations. In qualitative terms, this feature that identifies an induced CD band (due to the asymmetric environment of DNA) directly in difference absorption spectra by UV-VIS spectrophotometry has not been used, or reported in the literature. Also, most studies with ligands that bind in the 1 :1 mode with DNA have shown that induced CD bands for the bound ligand have both a positive and negative Cotton effect;61 a positive Cotton effect is observed herein to a large extent, and it is likely that such identification and comparison with the UV absorption spectra could be a diagnostic signature profile for the 2:1 binding modes for DNA minor groove binding ligands.
The compounds of the invention having the benzimidazole-pyrrole and imidazopyridine-pyrrole modules, for example, with the N-methylpiperazinyl substituents, in the designed polyamide-fluorophore conjugates have similar (to each other) but unique effect on the ability to bind the DNA minor groove and the mode in which that interaction occurs. Unlike the all-five-membered heterocycle frameworks (the model polyamides), containing five such consecutive ring residues, that show rather poor affinity for the DNA minor groove, and presumably a lack of effective side-by-side 2:1 mode of binding, the Bzi and Pzi analogs are shown to be well suited for not only ultra-strong binding to the target DNA site, but with two ligand molecules bound simultaneously in the 2: 1 mode. Both the Bzi and Pzi ligands form fluorescent complexes with double-stranded DNA, which facilitates investigation of their binding isotherms in terms of the binding stoichiometry, equilibrium association constants and provides benchmark measures of photophysical parameters. Thermal denaturation experiments were employed as an initial alternative method for the investigation of ligand-DNA complex stabilities and sequence selectivities. The ΔTm values for a majority of the 34 ligands prepared in the present invention have been obtained against a panel of 32 different DNA sequences, representing various combinations of the centrally-positioned 4-base pair long site. The interactions between selected compounds and d(CATGGCCATG)2, using spectroscopy methods, has further enabled the determination of key features of ligand-DNA interactions for compounds in which benzimidazole-pyrrole (Bzi-Py), imidazopyridine-pyrrole (Pzi-Py), benzimidazole-benzimidazole-pyrrole (Bzi-Bzi- Py), and benzimidazole-pyrrole (Bzi-Py) in combination with imidazole- benzimidazole (Bzi-Im and Im-Bzi) building blocks substitute for the pyrrole-pyrrole (Py-CONH-Py) and imidazole-imidazole (Im-CONH-Im) segments in polyamide class of agents.
The terminal units derived from the structure of Hoechst 33258, i.e. the 5-(N- methylpiperazinyl)benzimidazole and 5-(N-methylpiperazinyl)imidazopyridine units in the Bzi and Pzi analogs, respectively, are protonated and positively charged at neutral pH, similar to the tertiary amine (Dp) groups in previously studied polyamides of Py /Im heterocyles. The differences in electrostatic interactions with the negatively charged phosphodiester backbone of DNA are therefore not likely to be significant.
It has been found that the Bzi and Pzi ligands of the present invention are more effective than the polyamide class of molecules since they have a reduced number of amide bonds in integrated combinations of fused heterocycles with five- membered rings (pyrrole) in the Bzi-Py and Pzi-Py modules, relative to the Py- CONH-Py units in the polyamide molecules of the present invention. This reduced number of amide bonds is being compensated by the alternative hydrogen-bond donor functionalities in the benzimidazole or pyridoimidazole rings. The benzimidazole/pyridoimidazole ring NH groups are evidently more acidic than the amide CONH groups. This lowering of pKa values is explained by increased number of resonance-delocalized forms of the corresponding anions (Scheme XIX). Another chemical analogy to support the hypothesis is the fact that N-alkylation reactions of benzimidazole and imidazopyridine derivatives are more facile, feasible even with weak bases such as K2CO3ZEtJN,62 than the corresponding reactions with amides that are usually possible only with strong organometallic bases such as LDA.63 The superior acidic character of the heteroaromatic benzimidazole and pyridoimidazole rings is consistent with the expectation that the ring NH groups form stronger hydrogen-bonds to the acceptor sites on DNA, compared to the CONH functional groups present exclusively in the minor-groove-binding polyamide ligands.
Figure imgf000066_0001
Scheme XIX. (A) The hydrogen-bond donor groups in the Bzi, Pzi and Pyrrole-amide modules. (B) Anionic deprotonated forms, as a close analogy to the increased capacity of Bzi system to donate a proton towards hydrogen-bonding. (C) Corresponding resonance forms for the anion from Pzi, that is presumably the strongest hydrogen-bond forming unit of the three modules.
Figure 14 provides a summary of the interactions between 5 closely related ligands selected on the basis of individually matched combinations of the structural models present in the ligand structure with the cognate DNA base pairs in the context of 2:1 ligand-DNA binding models. Analogs F4 and G4 are found to be superior in terms of the propensity to exhibit 2:1 binding stoichiometry and equilibrium association constants. The amide derivatives R4 and H4 are not well suited for the 2:1 interactions and more data will be required on these systems to elucidate the structural or molecular basis for the apparent mismatch due to Dp units in these ligands. Compound K6 was difficult to analyse under the solution conditions due to its insolubility at pH 7. Note that the terminal imidazole ring in this ligand is expected to be protonated at that pH, but the increased lipophilic character of the cation remains. A corresponding methylated derivative is expected to increase solubility in aqueous solutions.
The observed 2: 1 ligand-DNA complex stoichiometry for the various analogs, on the basis of systematically introduced modules mentioned above argues strongly for a significant expansion of the repertoire of ligands with high-affinity sequence specific recognition of DNA. Until now, Dervan's work has mainly focused on the polyamide model and much of the expansion in the digital readout of DNA is reported on the hundreds of sequences that can be targeted in a specified manner using a rather limited structural diversity, overall length (size) and composition of the polyamide ligands.7"9 The present invention potentially quadruple the number of various permutations of the fused heterocycles and their linear conjugation with either the pyrrole or other five-membered ring residues.
To highlight this specific aspect of increased structural diversity, it may be possible that a total of 55 (= 3125) molecules are available for a ligand assembly that would be five residues in length, arising from variable combinations of benzimidazole, imidazopyridine, pyrrole, imidazole, and hydroxypyrrole ring systems; currently prevalent polyamide methodology in the area thrives largely on a combination of pyrrole and imidazole only, with limited use of hydroxypyrrole and aliphatic amino acid residues. Importantly, the benzimidazole and imidazopyridine are expected to be considerably photostable, relative to free radical and oxidative instability of the hydroxypyrrole rings, as well as more resistant to cellular degradation than the polyamide class of molecules. Cell-permeability of the piperazine-substituted benzimidazole compounds is already well established, judging by the cytological and molecular genetics applications of Hoechst 33258 class of dyes.10-1 1
3. Structure of the quaternary complexes consisting of two ligand molecules and two target DNA strangs by NMR
Structures of exemplary ligands, when bound to cognate nucleotide sequence in double stranded DNA, were determined by the application of two-dimensional NMR methods to reveal the exact configuration and atomic level details of the respective quaternary complexes. Primarily, very exact closest contact points between hydrogen atoms on the ligand molecules and those in DNA were identified as experimentally observed NOE's in two-dimensional NMR plots.
In the first case of evaluating the binding of one representative ligand (F 4) to double stranded DNA oligomer d(CATGGCCATG)2, 34 such intermolecular contacts are summarized in Figure 15. The complete analysis of the NMR experiments, in conjunction with quantitative computer-driven molecular model building, provided a comprehensive picture of the atomic level details of the exact configuration in which two molecules of the ligand F4 are bound to the two DNA strands in a very specific static orientation. The specifics of how the ligand structure matches with the order in which the nucleotide sequence of the binding site appear is revealed. This particular binding site is the sequence 5'-ATGGCCAT and it represents one of the possible 48 [=65536] octanucleotide segments.
For a second exemplary ligand G4, which differs from F4 in the sense that a Pzi unit replaces the Bzi unit, the intermolecular contacts observed in its complex with d(CATGGCCATG)2 are also summarized in Figure 16. These 48 different contact points between the ligand G4 molecules and the DNA also establish the specific shape-based readout of the segment 5'-ATGGCCAT by this specific ligand. Furthermore, this ligand G4 binds 100-1000 fold strongly with the said DNA segment than the preceding ligand F4, revealing the benefit of the Pzi unit in the design of ultra-strong DNA interactive molecular assemblies for targeting specific DNA segments with high fidelity.
The structures generated for the quaternary complexes between d(CATGGCCATG)2 and either ligand F4 or G4 molecules are collectively represented in Figures 17 and 18. Any changes made to either of the 8 locations within the binding site (5'-ATGGCCAT,) were determined to reveal a loss in binding of either F4 or G4 ligands, as established by NMR titration experiments conducted for the said altered DNA segments. Specifically, these changes were made to the centrally located GGCC segment in a stepwise manner, and thus results obtained with five other oligomers d(CATGCGCATG)2, d(CATAGCTATG)2, d(CATCGCGATG)2, d(CATGATC ATG)2 and d(CATAATTATG)2 were compared with the original designed experiments with d(C ATGGCC ATG)2. The above data collectively demonstrates that to have an effective high- affinity binding of such representative ligands as F4 and G4 to a defined (and specific) DNA segment, it is desirable to optimize the spatial configuration of the ligand assemblies for their ability to match with the contours of the specific DNA sequence to which they can effectively bind, and that any defects at the ligand-DNA interface should be minimized. The role of Pzi unit (compared to Bzi) is particularly noteworthy and is attributed to the difference in electron density of the pyridoimidazole attached to methylpiperazine (Pzi system) from that of the benzimidazole (Bzi) system, thereby making the NH group of Pzi more polarized than the NH group of Bzi. The resulting impact on the difference in acidity constant (pKa value) makes the Pzi ring NH group more capable of hydrogen bonding than that of Bzi. Both types of NH groups are stronger hydrogen bond donors than the amide class of NH groups, and they are primarily responsible for the recognition of carbonyl groups of DNA bases. This ranking of hydrogen bond donation capacity in the order of Pzi > Bzi » amides, is a feature that allows for the exact configuration in which the molecular assemblies of the type described in the present application match with the exact desirable order in which a selected nucleotide sequence of DNA may be targeted with high fidelity and to allow binding to all other competing mutated sites be minimized. 4. Analysis of ligand-DNA binding: Target sequence specificity and affinity
DNA footprinting is the in-vitro experimental method of choice to determine which specific continuous segment of nucleotides in DNA is protected against enzymatic cleavage - in the presence of added ligand(s). The cleavage of double stranded DNA plasmids, for example, by the protein DNase I (an endonucleolytic enzyme) produces a DNA sequence ladder pattern on polyacrylamide gels. Radioactively labeled double stranded plasmid sequences pSask-1 (133 basepairs) and pSask-2 (156 basepairs), were judiciously designed and used for the determination of exact segments where the ligands under investigation would bind. The sequence pSask-A has 128 possible non-repeating segments that are each 6 base pairs long, and 126 possible segments that are each 8 base pairs long, etc. Similarily, sequence pSask-B has 151 non-repeating 6-basepairs long segments, and 149 such 8-base pairs long segments. Selective binding of any of the test ligands to a specific such segment is easily characterized by the present of obliterated regions on the electrophoresis footprinting gel exposed to autoradiography for generating high resolution images. Standardized protocols were used to conduct such experiments, including those for quantitative assessment of binding affinity wherein the concentration of the test ligands was gradually incremented in individual lanes running parallel in a single gel used for the electrophoresis experiment, thereby minimizing any errors in conclusively determining the DNA segment specificity and relative affinity constants for the ligands under investigation. Almost all of the ligands described in the present application were first evaluated for their DNA sequence specificity and relative affinity against pSask-A and pSask-B. For some of the ligands, additional radioactively labeled plasmids called pSask-C (133 base pairs length; 128 unique 6 base pairs long segments; 126 unique 8 base pairs long segments) were designed and used. This plasmid pSask-C sequence is:
5'-GGTGGATCCA GCAAGCGCGC TTGCAACCCT ATAGGGTTGC AAGTCTTGCA AATATATATA TTTGCAAGAT CTTGCAAGGC TTGCGCCAAG CTTGCAAGGG TATACCCTTG CAAGCTAGCT TGCAAGCGCT TGC-3' and its corresponding complementary second strand
5'-GCA AGCGCTTGCA AGCTAGCTTG CAAGGGTATA CCCTTGCAAG CTTGGCGCAA GCCTTGCAAG ATCTTGCAAA TATATATATT TGCAAGACTT GCAACCCTAT AGGGTTGCAA GCGCGCTTGC TGGATCCACC-3' A pool of 37 different ligands from this invention was placed into 4 different groups for the purposes of evaluating their DNA sequence specific binding characteristics by DNA footprinting method. This division into 4 different groups was on the basis of molecular structures (meaning the configuration order of the individual building blocks in the oligoheteroaromatic assemblies, much like the peptide sequences were defined according to the sequence of the individual amino acids). Series A ligands were the smallest assemblies, consisting of methylpiperazine headgroup, 2 benzimidazole [Bzi] units, and the fourth (variable) heterocyclic or other similar aromatic ring system. Series B ligands contained varying combinations of the methylpiperazinyl head group, Bzi or Pzi units, pyrrole, imidazole, and inverted Bzi ring systems as part of the molecular frameworks. Ligands F4 and G4 were part of this group of compounds.
Series C ligands were the various symmetrical dimeric combinations of two "half ligands joined together by variable and novel linkers to separate out the individual "half ligands to the extent that maximum contact is permitted between the ligand edges and such functional groups present on DNA bases that are located in the minor groove of the double stranded DNA. Thus the dimensions of the linkers were a consideration in the way the assemblies were constructed. Series D ligands were a small group of miscellaneous ligands that were selected on the basis of their results from Tm data presented earlier in this application. The order in which the several units appear within each ligand was not constant throughout, and yet the original data from Tm analysis had identified several of them to be binding to selected DNA segments with very high affinity. On the aspect of their sequence specificity from Tm and flurosecence methods [vide infra], it was not totally clear whether these ligands were exhibiting any preference for a given set of nucleotide sequence in DNA. Therefore additional screening by DNA footprinting was carried out.
Series A ligands were determined to be selective for segments containing AT base pairs only. The overall DNA segment length that these ligands span was consistently between 4-5 base pairs for any one of these series A ligands. However, within such DNA length made of only AT base pairs, there were the following possible DNA sequences that were incorporated in the test DNA template used to test for binding of series A ligands: 5 ' -AAAA (and its complement 5 ' -TTTT) ; 5'-AAAT (and its complement 5'-ATTT); 5'-AATA (and its complement 5'-TATT); 5'-ATAA (and its complement 5'-TTAT); 5'-TAAA (and its complement 5'-TTTA) 5'-AATT (and its complement 5'-AATT); 5'-ATAT (and its complement 5'-ATAT; 5'-TAAT (and its complement 5'-ATTA; 5'-ATTA (and its complement 5'-TAAT; 5'-TATA (and its complement 5'-TATA); and 5'-TTAA (and its complement 5'-TTAA). These were all the 16 possible 4-bp long AT rich segments present together within the DNA template to allow for even small changes in DNA sequence content to be distinguishable by ligand binding. Table 2 provides the listing of preferred binding sites for the individual series A ligands, along with their Kd (dissociation constants of DNA complexed ligands) as determined from quantitative footprinting data. For all of these ligands, the most preferred binding segment in DNA is the 5'-AATT, completely consistent with the structural model of binding which places the methylpiperazine head group to be on the 3 '-side and therefore at the "end" of consecutively placed T nucleotides. It is important to note that in comparison, 5'- TTAA is the least preferred binding site for any of these series A ligands. This is an relevant aspect of the mode in which such ligands bind to DNA.
Series B ligands were tested thoroughly on pSask-A, the 133 base pairs long double stranded DA fragment that contains in particular the several variations of the aforementioned binding site for F4 and G4 ligands - i.e., the 5'-ATGGCCAT segment. These specific variations include, in addition to the said segment, the following octanucleotide sets 5'-AAGCGCTT, 5'-TAGGCCAT , 5'-AAGCCCTT, 5'-AAGTCCTT, 5'-TTGGCCAA, 5'-AAGATCTT and 5'-AACCGGTT, all of which are a representation of one or more changes to the consensus cognate binding site. The footprinting results were necessary to determine whether the DNA sequence selectivity of F4 and G4 ligands is retained in the presence of a pool of multiple but varying octanucleotide segments. Remarkably, the F4 and G4 ligands were both found to be super-selective for the sequence segments identified as 5'-AAGGCCTT and 5'-ATGGCCAT. Binding to any of the other 124 octanucleotide segments was comparatively extremely weak. These results establish the ultra-selective targeting of 5'-AAGGCCTT and 5'-ATGGCCAT sequence contexts within a large pool of other 8 base pairs long sites, and the results complement those discussed above. The binding association constants for F4 [Ka = 1012] and G4 [K8 = 1014] determined from the discussed footprinting results were also consistent with the above conclusions derived from NMR, fluorescence and Tm results, presented above. The Pzi-containing assemblies [equivalent to G4] exceed the picomolar association constants of their corresponding Bzi-containing ligand [F4] assemblies. The functional differences between the Pzi class assemblies and the Bzi class assemblies originate from the different fluorescence characteristics of the Pzi (vs Bzi), difference in pKa values, difference in hydrogen bond forming capacities, and the distinct tautomeric forms of the two types of ring systems - all important features that make the Pzi ligands better than the Bzi ligands; it is important to note that both classes of ligands are in turn far superior than assemblies designed purely on the basis of arranging pyrrole and imidazole ring systems (the so-termed all amide assemblies that had been part of the early attempts made in the 1980s).
The identity of binding sites and their binding association constants for other series B ligands are all provided in Table 3. A specific additional ligand of interest [F6] is identified to be selective for 5'-AAGCGCTT segments and this specific assembly is also representative of the same structure-based match between the ligand and the DNA site, as determined for ligands F4 and G4 above. The binding association constant for this particular combination of ligand configuration and the targeted DNA sequence was however not as high as picomolar but still falls in the subnanomolar range allowing it to be an effective, ultra-strong and selective ligand.
These following examples are intended only to be illustrative, and not limiting. Persons skilled in the art will readily understand and appreciate a wide range of useful applications for the silicone polymer of the present invention. The following non-limiting examples are illustrative of the invention: VII. EXAMPLES:
Materials and Methods All commercially available starting materials, l-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride (EDCI), diiosopropylcarbodiimide (DIC), N- hydroxy-succinimide, 1 -hydroxybenzotriazole hydrate (HOBt), N ,N- diisopropylethylamine (DIPEA), POCl3, FeCl3-OH2O, fuming nitric acid, 1- methylimidazole, 1 -methylpyrrole, were used as received. Solvents were distilled prior to use. Anhydrous DMF, CH3CN, CH2Cl2, CH3OH were obtained according to standard purification methods for purification and drying. All products were characterized by 1H-NMR, recorded on Bruker AMX-300 spectrometer unless stated otherwise. 13C-NMR spectra were also recorded in selected cases. Deuterated solvents, D2O, CDCl3, DMSO-d6, were used as received. The NMR signal assignments, where so described, use the following notations for specified ring systems: Bzi, benzimidazole; Dp, (dimethylamino)propyl; Im, imidazole; morph, morpholine; Ph, phenyl; Pip, piperazine; Pri, pyridine; Py, pyrrole; Pyrz, pyrazole; Pzi, pyridoimidazole/imidazopyridine. Mass spectrometry data were obtained through the Analytical Services Division of the Plant Biotechnology Institute, located on our campus. MALDI-TOF-MS was obtained using α-cyano-4-hydroxycinnamic acid matrix, and accurate mass measurements (HRMS) by secondary ion mass spectrometry were performed using a m-nitrobenzyl alcohol as matrix.
General Procedure A: The nucleophilic aromatic substitution reaction of 5-chloro-2- nitroaniline with amines.
To a mixture of 10 mmol of 5-chloro-2-nitroaniline and 30 mmol (3 mol equiv) of anhydrous K2CO3 in 20 mL of dimethylsulfoxide (DMSO), was added 20 mmol (2 mol equiv) of the amine (e.g., 1 -methylpiperazine, imidazole, morpholine or pyrazole), and the resultant mixture was stirred at 150 0C overnight. The reaction mixture was poured into iced water, and the solid material was filtered and washed with water. Crystallization with 'PrOH or CH3CN afforded pure 5-substituted nitroaniline derivatives. Note: these reactions were slower in low-boiling solvents (CH3CN, alcohols, THF etc.); DMF was unsuitable because of high-temperature requirement and base-catalyzed decomposition of solvent to generate dimethylamine and subsequent formation of a side-product, 5-(dimethylamino)-2-nitroaniline. General Procedure B: The nucleophilic aromatic substitution reaction of 2- amino-6-chloro-3-nitropyridine with amines. Typically, a mixture of 10 mmol of 2- amino-6-chloro-3-nitropyridine, 30 mmol (3 mol equiv) of anhydrous K2CO3, and 20 mmol (2 mol equiv) of the requisite amine reactant in 50 mL of dimethylformamide (DMF) was stirred at 40-50 °C for 5-8 h. The reaction mixture was poured into iced water, and extracted with EtOAc or CHCl3. The organic extracts, after drying (over anhydrous Na2SO4) and evaporation afford the respective 6-substituted-3- nitropyridin-2-amine derivatives which were usually purified by silica gel flash chromatography. General Procedure C: Redox-cycling-based oxidative cyclocondensation reaction of phenylenediamines and aldehydes to benzimidazole products. Typically, to a stirred solution of the diamine (3.2 mmol) in CH3CN (25 mL) was added a solution of the aldehyde (3 mmol) in CH3CN, and the resultant solution was stirred for 1 h at 90 ° C. The mixture was then evaporated to dryness and the resultant Schiff s base was resuspended in CH3CN (30 mL), followed by addition of solid FeCl3.6H2O (9 mg, 0.03 mmol; 0.01 mol equiv). The mixture was further stirred for 5-8 h at 90 °C with continuous O2 bubbling through the mixture, or alternatively allowed to stir exposed to air overnight. The reaction progress was monitored by TLC and NMR analysis of the aliquots, drawn periodically and filtered through a silica gel-filled Pasteur pipette in order to remove iron salts. At the completion of reaction, solvent was removed by evaporation and the residue subjected to silica gel flash chromatography to afford the benzimidazole products in the indicated yields. The hydrochloride salts were prepared by treatment with methanolic HCl, evaporation of the solvent and crystallization from EtOH/Et2O. General Procedure D: Redox-cycling-based oxidative cyclocondensation reaction of pyridinediamines and aldehydes to imidazopyridine products. A procedure that is particularly applicable to diaminopyridine reactants or when reactions according to general procedure A were sluggish. Typically, to a stirred solution of the diamine (3.2 mmol) in DMF (15 mL) was added a solution of the aldehyde (3 mmol) in DMF, and the solution was stirred for 1 h at 80 °C. Solid FeCl3.6H2O (24 mg, 0.09 mmol; 0.03 mol equiv) was then added and the mixture stirred at 120 "C with continuous O2 bubbling through the solution for 10 h. The reaction progress was monitored by TLC analysis of the aliquots. DMF was removed by evaporation and products isolated in pure form by silica gel flash chromatography. Hydrochloride salts were prepared by treatment with methanolic HCl, followed by evaporation of the solvent and crystallization from EtOHZEt2O.
General Procedure E: In-situ generation of activated -OBt esters from acid derivatives, using EDCI or other carbodiimide reagent (e.g. DIC), and subsequent amide formation with amine subtrates. Typically the appropriate acid derivative (0.5 - 1 mmol) of heterocyclic systems was suspended in DMF (5 - 10 niL) and 1.2 mol equiv of 1 -hydroxybenzotriazole (HOBt) was added to the mixture. After 5-10 mins, a batch of 1.1 mol equiv of the chosen carbodiimide (EDCI or DIC) was added, followed by a several-fold excess of diisopropyl ethyl amine (DIPEA, usually 1 mL). The resulting mixture was stirred at ambient temperature for 4-5 h. Without isolation of the materials, the requisite amine derivative (1 mol equiv) was then added and the mixture further allowed to stir at 40-60 0C overnight. Reaction progress was monitored by TLC. At completion, the reaction mixture was evaporated to dryness and treated with isopropanol to afford solid. Filtration, washing with acetone and CHC13 was found sufficient for obtaining the first crude crop of the products. Further purification of the materials was by column chromatography, using a gradient polarity from 4: 1 CHCl3:MeOH solvent mixture up to purely MeOH and/or containing 2-4% NH4OH to ensure complete elution of polar materials. The product fractions from chromatography were evaporated and usually first characterized in their free base forms. The corresponding hydrochloride salt forms were obtained by treatment with methanolic HCl, followed by recrystallization from alcohol/ether mixtures. MP-Ph(NO2)NH2: l-(3-Amino-4-nitrophenyl)-4-methylpiperazine (1) was prepared in 80% isolated yield according to general procedure A described above. 1H-NMR (DMSO-d6) 67.80 (d, IH, J = 8.5 Hz, Ph-CH), 7.27 (s, exch, 2H, NH2), 6.35 (dd, IH, J = 8.5 & 2.3 Hz, Ph-CH), 6.20 (d, IH, J = 2.3 Hz, Ph-CH), 3.29 (m, 4H, CH2), 2.38 (m, 4H, CH2), 2.21 (s, 3H, CH3). EI-HRMS: m/z calcd for CnH16N4O2 236.1273, found 236.1271 (100%). MP-Ph(NH2)2: l-(3,4-Diaminophenyl)-4-methyliperazine (2). To a solution of 1 (12 g, 50 mmol) in EtOAc (40 mL) was added 10% Pd-C (1 g) and the mixture was shaken under 45 psi H2 at normal temperature. The catalyst was filtered through a bed of Celite and the filtrate evaporated to dryness to afford light purple solid. The material was recrytallized from benzene/'PrOH to yield the title product (94% yield). 1H-NMR (DMSOd6) 66.47 (d, IH, J = 8.2 Hz, Ph-CH), 6.22 (s, IH, Ph-CH), 6.04 (d, IH, J = 8.2 Hz, Ph-CH), 4.41 (s, exch, 2H, NH2), 3.90 (s, exch, 2H, NH2), 2.89 (m, 4H, CH2), 2.40 (m, 4H, CH2), 2.18 (s, 3H, CH3). 13C-NMR (125 MHz, D2O) 6136.9 (+, C), 133.4 (+, C), 132.6 (+, C), 107.9 (-, CH), 106.2 (-, CH), 104.7 (-, CH), 53.8 (+, CH2), 45.3 (+, CH2), 42.6 (-, CH3). EI-HRMS: m/z calcd for CnH18N4 206.1531, found 206.1538 (100%).
Cl-Pyr(NO2)NH2: 2-Amino-6-chloro-3-nitropyridine (3). Gaseous ammonia was distilled through a cold (dry-ice) trap into a solution of 2,6-dichloro-3- nitropyridine (9.65 g, 50 mmol) in anhydrous EtOH (100 mL), until the dichloro derivative was consumed (by TLC). The resultant solution was evaporated to dryness to afford a solid containing the title product and compound 4. Crystallization from iPrOH afforded the title product in pure form (65% yield). 1H-NMR (CDCl3) 68.36 (d, IH, J = 8.7 Hz, Pri-CH), 6.73 (d, IH, J = 8.7 Hz, Pri-CH), 5.2 (s, exch, 2H, NH2). EI-HRMS: m/z calcd for C5H4ClN3O2 172.9992, found 172.9987 (100%), 174.0023 (6%), 174.9961 (32%), 175.9992 (2%).
NH2-Pyr(NO2)NH2: 2,6-Diamino-3-nitropyridine (4) was formed in the reaction leading to product 3 above. Isolation of this material was by column chromatography (silica gel; eluent CHCl3). 1H-NMR (CDCl3) 68.19 (d, IH, J = 8.7 Hz, Pri-CH), 6.71 (s, exch, 2H, NH2), 6.43 (d, IH, J = 8.7 Hz, Pri-CH). EI-HRMS: m/z calcd for C5H6N4O2 154.0491, found 154.0496 (100%).
MP-Pyr(NO2)NH2: 2-Amino-6-(4-methylpiperazinyl)-3-nitropyridine (5) was prepared in 88% isolated yield according to general procedure B described above. 1H- NMR (DMSO-d6) 68.05 (d, IH, J = 9 Hz, Pri-CH), 7.73 (s, exch, 2H, NH2), 6.32 (d, IH, J = 9 Hz, Pri-CH), 3.35 (m, 4H, CH2), 2.37 (m, 4H, CH2), 2.19 (s, 3H, CH3). 13C- NMR (125 MHz, CDCl3) 6159.9 (+, C), 154.8 (+, C), 136.7 (+, C), 119.6 (-, CH), 99.4 (-, CH), 55.08 (+, CH2), 46.3 (+, CH2), 44.9 (-, CH3). EI-HRMS: m/z calcd for Ci0Hi5N5O2 237.1226, found 237.1223 (25%, M+).
MP-Pyr(NH2)2: 2,3-Diamino-6-(4-methylpiperazinyl)pyridine (6). To a solution of 1 (12 g, 50 mmol) in MeOH (40 niL) was added 10% Pd-C (1 g) and the mixture was shaken under 45 psi H2 at normal temperature. The catalyst was filtered through a bed of Celite into a methanolic HCl solution. The filtrate was evaporated to dryness to afford deep green solid which was recrystallized from EtOH/Et2O to yield the title product (90% yield). 1H-NMR (CDCl3) δ6.86 (d, IH, J = 9.1 Hz, Pri-CH), 5.95 (d, IH, J = 9.1 Hz, Pri-CH), 4.19 (s, exch, 2H, NH2), 3.47 (m, 4H, CH2), 2.73 (s, exch, 2H, NH2), 2.50 (m, 4H, CH2), 2.32 (s, 3H, CH3). 13C-NMR (125 MHz, D2O) 6155.1 (+, C), 153.2 (+, C), 132.4 (+, C), 11 1.8 (-, CH), 101.4 (-, CH), 54.7 (+, CH2), 46.8 (+, CH2), 44.3 (-, CH3). EI-HRMS: m/z calcd for C0Hi7N5 207.1484, found 207.1490 (60%), 192.1248 (20%), 137.0827 (100%).
Im-Ph(NO2)NH2: l-(3-Amino-4-nitrophenyl)-lH-imidazole (7) was prepared in 86% isolated yield using imidazole and 5-chloro-2-nitroaniline according to general procedure A described above. 1H-NMR (DMSO-d6) 68.27 (s, IH, Im-CH), 8.1 1 (d, IH, J = 8.1 Hz, Ph-CH), 7.69 (s, IH, Im-CH), 7.55 (s, exch, 2H, NH2), 7.19 (d, IH, J = 1.8 Hz, Ph-CH), 7.15 (s, IH, Im-CH), 6.96 (dd, IH, J = 8.1 Hz, Ph-CH). '3C-NMR (125 MHz, CDCl3 + DMSO-d6) 6147.6, 142.6, 135.7, 131.1, 130.4, 128.8, 1 17.9, 109.4, 108.5. EI-HRMS: m/z calcd for C9H8N4O2 204.0647, found 204.0650 (100%), 158.0702 (10%), 131.0606 (14%).
Im-Ph(NH2)2: l-(3,4-Diaminophenyl)-lH-imidazole (8) was prepared in 90% isolated yield from catalytic hydrogenation of 7 as described for 2 above. 1H-NMR (DMSO-d6) 67.87 (s, IH, Im-CH), 7.39 (s, IH, Im-CH), 7.00 (s, IH, Im-CH), 6.66- 6.58 (m, 3H, Ph-CH), 4.77 (s, 2H, NH2), 4.64 (s, 2H, NH2). 13C-NMR (125 MHz, DMSO-d6) 6136.0, 135.3, 134.3, 129.0, 127.8, 118.5, 114.3, 109.8, 107.2. ES- HRMS: m/z calcd for C9HnN4 (MH+) 175.0984, found 175.0936 (100%).
Im-Pyr(NO2)NH2: 2-Amino-6-(imidazol-l-yl)-3-nitropyridine (9) was prepared in 92% isolated yield from imidazole and compound 3 according to general procedure B described above. 1H-NMR (500 MHz, CDCl3) 68.48 (s, IH, Im-CH), 8.41 (d, IH, J = 9.5 Hz, Pri-CH), 7.68 (s, IH, Im-CH), 7.29 (d, IH, J = 9.5 Hz, Pri- CH), 7.12 (s, IH, Im-CH), 6.99 (s, exch, 2H, NH2). FAB-HRMS: m/z calcd for C8H8N5O2 + (MH+) 206.0678, found 206.0682 (100%).
Im-Pyr(NH2)2: 2,3-Diamino-6-(imidazol-l-yl)pyridine (10) was prepared in
80% isolated yield from catalytic hydrogenation of 9 as described for 6 above. 1H- NMR (500 MHz, D2O) 68.24 (s, IH, Im-CH), 7.61 (s, IH, Im-CH), 7.52 (d, IH, J = 9
Hz, Pri-CH), 7.09 (d, IH, J = 9 Hz, Pri-CH), 7.04 (s, IH, Im-CH). FAB-HRMS: m/z calcd for C8Hi0N5 + (MH+) 176.0936, found 176.0948 (100%).
Pyraz-Ph(NO2)NH2: l-(3-Amino-4-nitrophenyl)-lH-pyrazole (11) was prepared in 84% isolated yield from pyrazole and 5-chloro-2-nitroaniline according to general procedure A described above. 1H-NMR (CDCl3) 68.07 (d, IH, J = 9 Hz, Ph- CH), 7.91 (d, IH, J = 2.4 Hz, Pyrz-CH), 7.62 (d, IH, J = 1.5 Hz, Pyrz-CH), 7.14 (d, IH, J = 2.4 Hz, Ph-CH), 6.82 (dd, IH, J = 9 & 2.4 Hz, Ph-CH), 6.40 (t, IH, J = 1.8 Hz, Pyrz-CH); 13C-NMR (125 MHz, CDCl3 + DMSO-d6) 6144.6 (+, C), 140.1 (-, CH), 138.7 (+, C), 130.7 (+, C), 129.2 (-, CH), 128.8 (-, CH), 117.2 (-, CH), 109.8 (- CH), 108.2 (-CH). FAB-HRMS: m/z calcd for C9H9N4O2 + (MH+) 205.0726, found 205.0728 (100%).
Pyraz-Ph(NH2)2: l-(3,4-Diaminophenyl)-lH-pyrazole (12) was prepared in 86% isolated yield from catalytic hydrogenation of 11 as described for 2 above. 1H- NMR (DMSO-d6) 68.36 (d, IH, J = 2.7 Hz, Pyrz-CH), 7.84 (s, IH, Pyrz-CH), 6.72 (dd, IH, Ph-CH), 6.64-6.59 (m, 2H, Ph-CH), 6.52 (m, IH, Pyrz-CH), 4.75 (s, exch, 2H, NH2), 4.60 (s, exch, 2H, NH2); 13C-NMR (75MHz, D2O) 6139.5, 135.9, 133.8, 131.8, 126.7, 114.5, 107.6, 106.5, 105.9. FAB-HRMS: m/z calcd for C9HnN4 + (MH+) 175.0984, found 175.0988 (100%).
Pyraz-Pyr(NO2)NH2: 2-Amino-6-(pyrazol-l-yl)-3-nitropyridine (13) K2CO3 (41.6 mmol), pyrazole (33.8 mmol) and pyridine (26.8 mmol) were dissolved in acetonitrile separately. The resulting solutions were combined and heated at 80 0C for approximately 24 hours with mixing. Product isolation was achieved via evaporation and reconstitution in water, followed by vacuum filtration; yield 57 % (15.4 mmol). 1H-NMR (CDCl3) 68.48 (d, IH, J = 9 Hz, Pri-CH), 8.42 (d, IH, J = 2.7 Hz, Pyrz-CH), 7.73 (s, IH, Pyrz-CH), 7.32 (d, IH, J = 9 Hz, Pri-CH), 6.44 (m, IH, Pyrz-CH); 13C- NMR (75MHz, CDCl3) 6154.0 (s, C), 153.4 (s, C), 144.0 (s, CH), 138.6 (s, CH), 128.3 (s, CH), 125.7 (s, C), 109.4 (s, CH), 102.4 (s, CH); FAB-HRMS: m/z calcd for C8H8N5O2 + (MH+) 206.0678, found 206.0657 (100%).
Pyraz-Pyr(NH2)2: 2,3-Diamino-6-(pyrazol-l-yl)pyridine (14) was prepared in 92% isolated yield from catalytic hydrogenation of 13 as described for 6 above. 1H- NMR (D2O) 68.34 (d, IH, J = 2.7 Hz, Pyrz-CH), 7.82 (s, IH, Pyrz-CH), 7.59 (d, IH, J = 8.4 Hz, Pri-CH), 7.07 (d, IH, J = 8.4 Hz, Pri-CH), 6.58 (m, IH, Pyrz-CH). 13C- NMR (75MHz, CDCl3) 6155.0 (s, C), 153.1 (s, C), 139.2 (-, CH), 131.9 (s, C), 127.8 (-, CH), 1 12.9 (-, CH), 109.3 (-CH), 101.8 (-CH). EI-HRMS: m/z calcd for C8H9N5 175.0858, found 175.0858 (100%, M+). Morph-Ph(NO2)NH2: l-(3-Amino-4-nitrophenyl)morpholine (15) was prepared in 92% isolated yield using morpholine and 5-chloro-2-nitroaniline, according to general procedure A described above. 1H-NMR (CDCl3) 67.92 (d, IH, J = 8.1 Hz, Ph-CH), 6.52 (dd, IH, J = 2 & 8.1 Hz, Ph-CH), 6.36 (d, IH, J = 2 Hz, Ph- CH), 3.76 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2), 3.65 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2). EI-HRMS: m/z calcd for Ci0Hi3N3O3 223.0957, found 223.0958 (100%).
Morph-Ph(NH2)2: l-(3,4-diaminophenyl)morpholine (16) was prepared in 91% isolated yield from catalytic hydrogenation of 15 as described for 2 above, 1H- NMR (DMSO-d6) 66.48 (d, IH, J = 8.1 Hz, Ph-CH), 6.24 (s, IH, Ph-CH), 6.13 (d, IH, J = 8.1 Hz, Ph-CH), 4.38 (br s, exch, 2H, NH2), 3.92 (br s, exch, 2H, NH2), 3.74 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2), 3.66 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2). EI-HRMS: m/z calcd for C10H15N3O 193.1215, found 193.1222 (100%).
Morph-Pyr(N O2)NH2: 2-Amino-6-(morpholin-l-yl)-3-nitropyridine (17) was prepared from morpholine and chloropyridine derivative (3) in 90% isolated yield according to general procedure B described above. 1H-NMR (CDCl3) 68.18 (d, IH, J = 9.3 Hz, Pyr-CH), 6.03 (d, IH, J = 9.3 Hz, Pyr-CH), 3.74 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2), 3.67 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2). 13C-NMR (75 MHz, CDCl3) 6157.3 (+), 151.9 (+), 134.1 (-), 117.1 (+), 95.7 (-), 64.1 (+), 42.5 (+). EI- HRMS: m/z calcd for C9Hi2N4O3 224.0909, found 224.0909 (100%), 193.0724 (40%), 167.0571 (35%), 139.0381 (45%). Morph-Pyr(NH2)2: 2,3-Diamino-6-(morpholin-l-yl)pyridine (18) was prepared in 94% isolated yield from catalytic hydrogenation of 17 as described for 6 above. 1H-NMR (CDCl3) 66.74 (d, IH, J = 9.3 Hz, Pri-CH), 6.03 (d, IH, J = 9.3 Hz, Pri-CH), 3.74 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CH2), 3.67 (dd, 4H, J = 3.6 & 8.7 Hz, morph-CHz). FAB-HRMS: m/z calcd for C9H15N4O+ (MH+) 195.1246, found 195.1249 (100%).
1 -Methyl- 1 H-pyrrole-2-carboxaldehyde (19) was prepared according to standard method for Villsmeier reaction, using POCI3, DMF and 1-methylpyrrole. The product was obtained in pure form by repeated fractional distillation, and stored over Zn powder and KOH pellets. 1H-NMR (CDCl3) 69.50 (s, IH, CHO), 6.87-6.84 (2 x d, 2H, Py-CH), 6.16 (d, IH, J = 3.9 Hz, Py-CH), 3.90 (s, 3H, CH3). EI-HRMS: m/z calcd for C6H7NO 109.0528, found 109.0531 (100%), 81.0540 (40%). l-Methyl-4-nitro-lH-ρyrrole-2-carboxaldehyde (20). To 100 mL of 90% HNO3 cooled to -70 0C for 30 min, was added dropwise neat 1 -methylpyrrole-2- carboxaldehyde (25 g, 250 mmol). The temperature inside the reaction flask was carefully monitored and maintained between -50 and -30 0C during addition. After complete addition, the reaction mixture was allowed to warm up to -20 0C for 30 min, followed by pouring the materials onto ice. The precipitated product was collected by filtration and recrystallized from 1PrOH to afford 27 g of the title product (70% yield). 1H-NMR (CDCl3) 69.61 (s, IH, CHO), 7.70 (d, IH, J = 1.8 Hz, Py-CH), 7.41 (d, IH, J = 1.8 Hz, Py-CH), 4.01 (s, 3H, CH3). 13C-NMR (125 MHz, CDCl3) 6180.5 (+, C), 131.3 (+, C), 129.7 (-, CH), 126.5 (+, C), 117.8 (-, CH), 38.1 (-, CH3). EI-HRMS: m/z calcd for C6H6N2O3 154.0378, found 154.0380 (100%), 126.0377 (25%).
1 -Methyl- lH-imidazole-2-carboxaldehyde (21). To a solution of 1- methylimidazole (8.2 g, 100 mmol) in dry THF (100 mL) cooled to -70 0C, was added dropwise a solution of nBuLi in THF. After stirring for 30 min at the same temperature, the solution was treated with freshly distilled DMF (7.3 g, 100 mmol). The resultant yellow suspension was stirred at 0 0C for 2 h, and quenched with ice- water. The mixture was extracted with EtOAc, and the organic extracts dried (Na2SO4) before evaporation to afford an oily residue, which was distilled under reduced pressure to afford the title product (68% yield). 1H-NMR (CDCl3) 69.76 (s, IH, CHO), 7.22 (s, IH, Im-CH), 7.07 (s, IH, Im-CH), 3.96 (s, 3H, CH3). EI-HRMS: m/z calcd for C5H6N2O 110.0480, found 110.0481 (100%), 82.0493 (60%). l-Methyl-4-nitro-lH-imidazole-2-carboxaldehyde (22). Compound 21 was treated with 90% HNO3 at 0 0C and the resulting mixture further stirred at 45 0C for 2 h. The mixture was poured on to ice and the precipitate collected by filtration. The solid was recrystallized from acetone to afford the title compound (50% yield). 1H- NMR (CDCl3) 69.70 (s, IH, CHO), 8.34 (s, IH, Im-CH), 4.03 (s, 3H, CH3). 13C- NMR (125 MHz, CDCl3) 6182.2 (+, C), 140.9 (+, C), 132.5 (+, C), 127.2 (-, CH), 36.5 (-, CH3). EI-HRMS: m/z calcd for C5H5N3O3 155.0331, found 155.0328 (100%), 127.0386 (38%).
MP-Bzi-Py-NO2: 2-(l-Methyl-4-nitro-lH-pyrrol-2-yl)-5-(4-methylpiperazi- nyl)-lH-benzimidazole (26) was prepared in 74% yield using the diamine derivative 2 and compound 20 in the general procedure C given above. 1H-NMR (DMSO-d6) 612.57 (s, exch, Bzi-NH), 8.21 (d, J = 1.8, IH, Py-CH), 7.43 (d + m, J = 1.8 for d, 2H, Py-CH & Bzi-CH overlapped), 6.96 (2 overlapping d, J = 8.4, 2H, Bzi-CH + Bzi- CH), 4.14 (s, 3H, Py-NCH3), 3.12 (t, 4H, J = 4.5, CH2), 2.48 (t, 4H, J = 4.5, CH2), 2.23 (s, 3H, NCH3). 13C-NMR (DMSO-d6) 6 151.4 (+), 142.5 (+), 138.4 (+), 135.6 (+), 132.6 (-), 129.8 (+), 120.9 (-), 117.6 (-), 113.8 (-), 102.9 (-), 56.2 (+), 49.7 (+), 45.4 (-), 39.6 (-). EI-HRMS: m/z calcd for Ci7H20N6O2 340.1648, found 340.1648 (100%), 270.0983 (15%), 222.0905 (10%); ES-HRMS: m/z calcd for C17H2IN6O2 + (MH+) 341.1720, found 341.1732 (100%).
MP-Pzi-Py-NO2: 2-(l-Methyl-4-nitro-lH-pyrrol-2-yl)-5-(4-methylpiperazi- nyl)-lH-imidazo[4,5-b]pyridine (27) was prepared in 64% yield using the diamine derivative 6 and compound 20 in the general procedure D given above, m.pt. 284 0C; 1H-NMR (DMSO-d6) 08.12 (s, IH, Py-CH), 7.73 (d, IH, J = 8.8 Hz, Pzi-CH), 7.45 (s, IH, Py-CH), 6.70 (d, IH, J = 8.8 Hz, Pzi-CH), 4.11 (s, 3H, Py-NCH3), 3.46 (t, 4H, CH2), 2.39 (t, 4H, CH2), 2.18 (s, 3H, NCH3). 13C-NMR (DMSO-d6) 6156.6 (+), 142.5 (+), 134.9 (+), 127.4 (-), 126.6 (-), 124.6 (+), 105.5 (-), 103.6 (-), 54.4 (+), 45.8 (+), 45.6 (-), 38.0 (-) (Note 2 C nulled with long T). FAB-HRMS: m/z calcd for Ci6H20N7O2 + (MH+) 342.1679, found 342.1671 (100%). 2-Amino-3-[(l -methyl-4-nitro- lH-pyrrole-2-carbonyl)-amino]-6-(4-methyl-l- piperazinyl)pyridine and 3-amino-2-[( 1 -methyl-4-nitro- 1 H-pyrrole-2-carbonyl)- amino]-6-(4-methyl-l-piperazinyl)pyridine (28a/28b) Diamine 6 (3.1 g, 15 mmol) was dissolved in CH3CN (60 mL) and the solution added to a stirred mixture of 1- methyl-4-nitro-2-trichloroacetylpyrrole (4.07 g, 15 mmol) and DIPEA (2 g, 16 mmol) in CH3CN. The mixture was stirred at 40 0C for 16 h, and then poured into ice. The precipitate formed was collected by filtration, washed with acetone and dried to provide 3.8 g (70% yield) as a mixture of two regioisomeric amides 28a and 28b in nearly 3: 1 ratio (as determined by 1H-NMR) as a light brown solid which was used for acid-promoted cyclization to compound 27 as described in the following paragraph. 1H NMR (DMSO-d6) δ 9.21 (br s, 0.75H), 8.96 (br s, 0.25H), 7.82-7.94 (m, 3H), 6.81-6.93 (m, IH), 6.32 (br s, 0.25 H), 6.1 1 (br s, 0.75H), 3.92, 3.88 (2s, 3H, Py-NCH3), 3.15 (m, 4H, CH2), 2.48 (m, 4H, CH2), 2.23, 2.21 (2s, 3H, NCH3).
2-Amino-3-[(l -methyl-4-nitro- 1 H-imidazole-2-carbonyl)-amino]-6-(4-methyl- 1 -piperazinyl)pyridine and 3-amino-2-[(l-methyl-4-nitro-lH-imidazole-2-carbonyl)- amino]-6-(4-methyl-l-piperazinyl)pyridine (29a/29b) Diamine 6 (3.1 g, 15 mmol) was dissolved in CH3CN (60 mL) and the solution added to a stirred mixture of 1- methyl-4-nitro-2-trichloroacetylimidazole (4.1 g, 15 mmol) and DIPEA (2 g, 16 mmol) in CH3CN. The mixture was stirred at 40 0C for 16 h, and then poured into ice. The precipitate formed was filtered, washed with dichloromethane and dried to provide 4.1 g (75% yield) as a mixture of two regioisomeric amides 29a and 29b in nearly 2:1 ratio (as determined by 1H-NMR) as a light yellow solid which was used without further separation. 1H NMR (DMSO-d6) δ 9.49 (br s, 0.68H), 9.21 (br s, 0.32H), 7.82-7.94 (m, 3H), 6.81-6.93 (m, IH), 3.94, 3.91 (2s, 3H, Im-NCH3), 3.15 (m, 4H, CH2), 2.48 (m, 4H, CH2), 2.23, 2.21 (2s, 3H, NCH3).
MP-Pzi-Im-NO2: 2-(l -Methyl-4-nitro- 1 H-imidazol-2-yl)-5-(4-methy lpipe- razin-yl)-lH-imidazo[4,5-b]pyridine (30) was prepared in 62% yield using the diamine derivative 6 and imidazole derivative (22) in the general procedure D given above. 1H-NMR (DMSOd6) 68.68 (s, IH, Im-CH), 7.79 (d, IH, J = 8.8 Hz, Pzi-CH), 6.74 (d, IH, J = 8.8 Hz, Pzi-CH), 4.13 (s, 3H, Im-NCH3), 3.48 (t, 4H, CH2), 2.41 (t, 4H, CH2), 2.20 (s, 3H, NCH3). Methyl l-methyl-4-nitro-lH-pyrrole-2-carboxylate (31) Compound 23 (2.7 g,
10 mmol) was dissolved in dry MeOH and a catalytic amount of NaH (100 mg) was added to the solution. After stirring the mixture for 30 min at ambient temperature, the solid present in the suspension was collected by filtration and recrystallized (1PrOH) to afford the title product (80% yield). HRMS: C7H8N2O4 184.0484
[ImPy-COOMe] : Methyl 1 -methy 1-4- { [( 1 -methyl- 1 H-imidazol-2-y l)-carbon- yl]amino}-lH-pyrrole-2-carboxylate (32). Methyl 1 -methyl-4-nitropyrrole-2- carboxylate 31 (6.0 g, 32.6 mmol) was dissolved in EtOAc (100 mL). A slurry of moist 10% Pd/C (600 mg) in EtOAc was added and the contents were stirred vigorously under H2 (70 psi) for 16 h. The reaction mixture was filtered through a bed of Celite. The Celite was washed with MeOH and the combined filtrate was evaporated under reduced pressure to furnish crude amine as dark oil. The crude oil, without any further purification, was taken up in CH3CN (60 mL). Contents were cooled to 0 °C and DIPEA (12 mL, 62.4 mmol) was added. A solution of l-methyl-2- trichloroacetylimidazole 24 (8.5 g, 31 mmol) in CH3CN (10 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h. Solvents were removed under reduced pressure and the solid obtained was washed with hexanes and filtered to afford 32. (8.0 g, 94 %). 1H-NMR (CDCl3) 09.08 (s, exch, IH, NH), 7.41 (s, IH, Py-C5H), 7.03 (s, IH, Im-C5H), 6.98 (s, IH, Im-C4H), 6.80 (s, IH, Py-C3H), 4.08 (s, 3H, CH3), 3.90 (s, 3H, CH3), 3.78 (s, 3H, OCH3). 13C-NMR (75 MHz, DMSO-d6) 6161.8 (+, s, CO), 157.1 (+, s, CO), 139.1 (+, s, Im-C2), 128.3 (-, d, Im- C4), 127.1 (-, d, Im-C5), 122.6 (+, s, Py-C2), 121.7 (-, d, Py-C5), 119.7 (+, s, Py-C4), 109.5 (-, d, Py-C3), 51.2 (-, q, OCH3), 37.9 (-, q, CH3), 35.5 (-, q, CH3). EI-HRMS: m/z calcd for C12H14N4O3 262.1066, found 262.1056 (M+, 100%), 174.0661 (20%), 109.0407 (40%).
[NO2-PyPy-COOCH3] : Methyl 1 -methyl-4- { [( 1 -methyl-4-nitro- 1 H-pyrrol-2- yl)-carbonyl]amino}-lH-pyrrole-2-carboxylate (33) was prepared in a similar manner as the preceding compound, by using l-methyl-3-nitro-2-trichloroacetylpyrrole instead. Isolated yield of 33 was 92%. 1H-NMR (DMSO-d6) δlθ.27 (s, IH, exch NH), 8.19 (d, IH, J = 1.2 Hz, Py-CH), 7.55 (d, IH, J = 1.2 Hz, Py-CH), 7.46 (d, IH, J = 1.2 Hz, Py-CH), 6.89 (d, IH, J = 1.2 Hz, Py-CH), 3.95 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.74 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6160.9 (+, s, CO), 157.1 (+, s, CO), 134.0 (+, s, Py-C4), 128.5 (-, d, Py-C5), 126.3 (+, s, Py-C2), 122.3 (+, s, Py- C2), 121.0 (-, d, Py-C5), 1 19.0 (+, s, Py-C4), 108.5 (-, d, Py-C3), 107.8 (-, d, Py-C3), 51.2 (-, q, OCH3), 37.6 (-, q, CH3), 36.4 (-, q, CH3). EI-HRMS: m/z calcd for C13Hi4N4O5 306.0964; found 306.0962 (M+, 100%), 153.0690 (84%), 107.0374 (32%).
[NO2-ImPy-COOCH3] : Methyl 1 -methyl-4- { [( 1 -methyl-4-nitro- 1 H-imidazol- 2-yl)carbonyl]amino}-lH-pyrrole-2-carboxylate (34). Methyl 4-nitro-pyrrole-2- carboxylate 31 (3.8 g, 20.6 mmol) was dissolved in a mixture of MeOH-EtOAc (1 : 1, 150 mL). A slurry of moist 10% Pd/C in EtOAc was added and the contents were stirred vigorously under hydrogen (70 psi) for 14 h. The reaction mixture was filtered through a bed of Celite. The Celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as oil. The crude amine without any further purification was taken up in CH3CN (150 mL) and DIEA (5 mL). A solution of l-methyl-4-nitro-2-trichloroacetylimidazole 25 (5.4 g, 19.8 mmol) in CH3CN (20 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h, during which solid precipitated out. The reaction mixture was filtered to furnish 34 as yellow solid (4.8 g, 70.5 %). 1H-NMR (CDCl3) 68.96 (s, exch, IH, NH), 7.82 (s, IH, Im-CH), 7.37 (d, IH, J = 1.9 Hz, Py-CH), 6.86 (d, IH, J = 1.9 Hz, Py-CH), 4.19 (s, 3H, CH3), 3.92 (s, 3H, CH3), 3.82 (s, 3H, CH3), 13C-NMR (75 MHz, DMSO-d6) 6160.9 (+, s, CO), 154.9 (+, s, CO), 144.5 (+, s, Im-C4), 137.8 (+, s, Im-C2), 126.9 (-, d, Im-C5), 121.9 (+, s, Py-C2), 121.6 (-, d, Py-C5), 119.1 (+, s, Py-C4), 109.3 (-, d, Py-C3), 51.2 (-, q, OCH3), 36.8 (-, q, CH3), 36.5 (-, q, CH3). EI- HRMS: m/z calcd for Ci2H13N5O5 307.0917; found 307.0920 (M+, 100%), 180.0538 (88%), 149.0358 (42%).
[ImPy-COOH] : 1 -Methyl-4- { [( 1 -methyl- 1 H-imidazol-2-yl)carbonyl]amino} - lH-pyrrole-2-carboxylic acid (35). A solution of methyl 1 -methyl- [4-(l-methyl- imidazole-2-carboxamido)]-pyrrole-2-carboxylate 32 (7.8 g, 30 mmol) in MeOH (400 mL), NaOH (6 g, 154 mmol) and H2O (200 mL) was allowed to stir at 60 °C for 12 h. MeOH was removed under reduced pressure and the contents were acidified to pH 2 using 2M HCl under cooling, during which acid precipitated out as gelatinous solid. The solid was filtered and the wet cake was taken up in 1 : 1 MeOH/'PrOH when acid precipitated as granular solid, filtered washed with Et20 to furnish 35 (6.48 g, 87 %). 1H-NMR (DMSO-d6) 612.23 (bs, COOH), 10.48 (s, exch, IH, NH), 7.47 (s, IH, Im- C5H), 7.38 (s, IH, Py-C5H), 7.03 (s, IH, Im-C4H), 6.97 (s, IH, Py-C3H), 3.97 (s, 3H, CH3), 3.81 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6162.2 (+, s, CO), 156.2 (+, s, CO), 138.8 (+, s, Im-C2), 127.1 (-, d, Im-C4), 126.6 (-, d, Im-C5), 122.1 (+, s, Py- C2), 120.7 (-, d, Py-C5), 119.9 (+, s, Py-C4), 109.0 (-, d, Py-C3), 36.4 (-, q, CH3), 35.3 (-, q, CH3). EI-HRMS: m/z calcd for CnH)2N4O3 248.0909; found 248.0912 (M+, 100%), 203.0944 (M-CO2, 15%), 174.0658 (17%), 109.0401 (32%), 82.0526 (37%). [ImPyPy-COOMe]: Methyl l-methyl-4-{[(l-methyl-4-{[(l-methyl-lH- imidaz-ol-2-yl)carbonyl]amino}-lH-pyrrol-2-yl)carbonyl]amino}-lH-pyrrole-2- carboxylate (36). A solution of 33 (4.6 g, 15 mmol) in 1 : 1 MeOH:DMF (100 mL) was stirred with hydrogen at 70 psi in the presence of Pd/C (400 mg) for 14 h. The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as oil. The crude amine without any further purification was taken up in DMF (50 mL) and DIEA (4 mL). A solution of 24 (3.4 g, 15 mmol) in DMF (20 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h. Solvents were removed under reduced pressure and the contents were taken up in H2O (100 mL), upon which the product 36 precipitated out, filtered and washed with Et2O (5.2 g, 91%). 1H-NMR (DMSO-d6) 610.47 (s, exch, IH, NH), 9.96 (s, exch, IH, NH), 7.47 (s, IH, Im-C5H), 7.39 (s, IH, Py-C5H), 7.28 (s, IH, Py-C5H), 7.19 (s, IH, Py-C3H), 7.04 (s, IH, Im-C4H), 6.92 (s, IH, Py-C3H), 3.99 (s, 3H, CH3), 3.84 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.73 (s, 3H, OCH3). 13C-NMR (75 MHz, DMSO-d6) 6161.0 (+, s, CO), 158.7 (+, s, CO), 156.3 (+, s, CO), 139.0 (+, s, Im-C2), 127.2 (-, d, Im-C4), 126.6 (-, d, Im-C5), 123.2 (+, s, Py-C2), 123.0 (+, s, Py-C2), 121.7 (+, s, Py-C4), 121.0 (+, s, Py-C4), 119.0 (-, d, Py-C5), 118.7 (-, d, Py-C5), 108.6 (-, d, Py-C3), 105.2 (-, d, Py-C3), 51.1 (-, q, -OCH3), 36.4 (-, q, CH3), 36.3 (-, q, CH3), 35.3 (-, q, CH3). EI- HRMS: m/z calcd for C18H20N6O4 384.1546; found 384.1549 (M+, 100%), 302.0999 (8%), 231.0881 (86%), 149.0355 (55%), 109.0402 (44%). [ImImPy-COOMe]: Methyl 1 -methy 1-4- { [(I -methyl-4-{ [(I -methyl- IH- imidaz-ol-2-yl)carboxnyl]amino}-lH-imidazol-2-yl)carbonyl]amino}-lH-pyrrole-2- carboxy-late (37). A solution of 34 (4.6 g, 15 mmol) in 1 :1 MeOH:DMF (100 niL) was stirred with hydrogen at 70 psi in the presence of Pd/C (400 mg) for 14 h. The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as oil. The crude amine without any further purification was taken up in CH3CN (50 rnL) and DIEA (4 mL). A solution of 24 (3.4 g, 15 mmol) in DMF (20 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h during which solid precipitated out. Reaction mixture was filtered and washed with Et2O to furnish 34 (3.6 g). The filtrate was chromatographed to yield additional 1 g of product, (yield 80.7 %). 1H-NMR (CDCl3) 59.53 (s, exch, IH, NH), 8.88 (s, exch, IH), 7.46 (s, IH, Im'-C5H), 7.41 (s, IH, Py-C5H), 7.06 (s, IH, Im-C5H), 7.00 (s, IH, Im-C4H), 6.80 (s, IH, Py-C3H), 4.09 (s, 3H), 4.07 (s, 3H), 3.91 (s, 3H), 3.81 (s, 3H, OCH3). 1H-NMR (DMSO-d6) δlθ.43 (s, exch, IH, NH), 9.72 (s, exch, IH), 7.66 (s, IH, Im'-C5H), 7.55 (s, IH, Py-C5H), 7.43 (s, IH, Im-C5H), 7.06 (s, IH, Im-C4H), 7.01 (s, IH, Py-C3H), 4.00 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.84 (s, 3H, CH3), 3.73 (s, 3H, OCH3). '3C-NMR (75 MHz, DMSO-d6) 6161.0 (+, s, CO), 155.9 (+, s, CO), 155.8 (+, s, CO), 138.0 (+, s, Im-C2), 134.9 (+, s, Im-C2), 134.4 (+, s, Im- C4), 127.9 (-, d, Im-C4), 127.3 (-, d, Im-C5), 122.3 (+, s, Py-C2), 121.1 (+, s, Py-C4), 1 18.9 (-, d, Py-C5), 1 14.3 (-, d, Im-C5), 108.9 (-, d, Py-C3), 51.2 (-, q, -OCH3), 36.5 (-, q, CH3), 35.4 (-, q, CH3), 35.3 (-, q, CH3). EI-HRMS: m/z calcd for CnH19N7O4 385.1499; found 385.1493 (M+, 100%), 303.0975 (39%), 244.0838 (16%), 205.0961 (6%), 180.0525 (8%), 150.0301 (6%), 109.0396 (15%). [ImPyPy-COOH] : 1 -Methy 1-4- { [ 1 -methy l-4-{ [( 1 -methyl- 1 H-imidazol-2- yl)car-bonyl]amino}-lH-pyrrol-2-yl)carbonyl]amino}-lH-pyrrole-2-carboxylic acid (38). To a solution of 36 (5.1 g, 13.3 mmol) in MeOH (100 mL) was added a solution of NaOH (1 g, 26 mmol) in H2O (100 mL). The reaction mixture was allowed to stir for 12 h at 60 °C during which the contents became homogeneous. The reaction mixture was worked up, as reported for 35, to furnish 38 (4.8 g, 98 %). 1H-NMR (DMSO-d6) δlθ.96 (s, exch, IH, NH), 10.01 (s, exch, IH, NH), 7.58 (s, IH, Im-C5H), 7.43 (s, IH, Py-C5H), 7.33 (s, IH, Im-C4H), 7.18 (s, IH, Py-C3H), 6.86 (s, IH, Py- C3H), 4.03 (s, 6H, 2 x CH3), 3.85 (s, 3H, CH3), 3.82 (s, 3H, CH3), 13C-NMR (75 MHz, DMSO-d6) 6162.2 (+, s, CO), 158.5 (+, s, CO), 154.1 (+, s, CO), 137.7 (+, s, Im-C2), 126.7 (-, d, Im-C4), 124.3 (-, d, Im-C5), 123.2 (+, s, Py-C2), 122.8 (+, s, Py- C2), 121.2 (+, s, Py-C4), 120.6 (-, d, Py-C5), 119.7 (+, s, Py-C4), 119.1 (-, d, Py-C5), 108.7 (-, d, Py-C3), 105.0 (-, d, Py-C3), 36.4 (-, q, CH3), 36.3 (-, q, CH3), 36.0 (-, q, CH3). EI-HRMS: m/z calcd for CnHi8N6O4 370.1390; found 326.1489 (M-CO2, 100%), 231.0879 (71%), 149.0355 (58%), 109.0397 (55%).
[ImImPy-COOH] : 1 -Methyl-4- { [( 1 -methyl-4- { [( 1 -methyl- 1 H-imidazol-2-yl)- carbony 1] amino } - 1 H-pyrrol-2-y l)carbony 1] amino } - 1 H-pyrrole-2-carboxy licacid (39). To a solution of 37 (3.2 g, 8 mmol) in MeOH (100 mL) was added a solution of NaOH (640 mg, 16 mmol) in H2O (100 mL). The reaction mixture was allowed to stir for 12 h at 60 °C during which the contents became homogeneous. The reaction mixture was worked up, as reported for 3, to furnish 37. (2.8 g, 87 %). 1H-NMR (DMSO-d6) 612.24 (bs, IH), 10.42 (s, exch, IH, NH), 9.74 (s, exch, IH, NH), 7.56 (s, IH, Im'-C5H), 7.47 (s, IH, Py-C5H), 7.44 (s, IH, Im-C5H), 7.07 (s, IH, Im-C4H), 6.96 (s, IH, Py-C3H), 4.00 (2 x s, 6H, 2 x CH3), 3.82 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6162.1 (+, s, CO), 155.9 (+, s, CO), 155.7 (+, s, CO), 138.0 (+, s, Im-C2), 134.9 (+, s, Im-C2), 134.5 (+, s, Im-C4), 127.9 (-, d, Im-C4), 127.3 (-, d, Im- C5), 122.0 (+, s, Py-C2), 120.7 (-, d, Py-C5), 120.0 (+, s, Py-C4), 114.3 (-, d, Im-C5), 109.0 (-, d, Py-C3), 36.4 (-, q, CH3), 35.4 (-, q, CH3), 35.3 (-, q, CH3). EI-HRMS: m/z calcd for C16H17N7O4 371.1342; found 371.1343 (M+, 100%), 327.1436 (M-CO2, 61%), 289.0821 (28%), 245.0921 (35%), 109.0401 (32%).
Methyl l-methyl-4-nitro-lH-imidazole-2-carboxylate (40): MeOH (40 mL) was added to a sample of the trichloroacetyl compound 25, and the mixture stirred after heating to 40 0C for 10 min. To ensure complete methanolysis, a small amount (100 mg) of NaH was added to the mixture. After stirring for 2 h, the resultant suspension was filtered to afford the title product (94% yield). 1H-NMR (CDCl3) 67.84 (s, IH, Im-CH), 4.18 (s, 3H, CH3), 3.93 (s, 3H, CH3). EI-HRMS: m/z calcd for C6H7N3O4 185.0437, found 185.0476 (100%). [ImIm-COOMe]: Methyl 1 -methy 1-4- { [(I -methyl- lH-imidazol-2- yl)carbonyl]-amino}-lH-imidazole-2-carboxylate (41). Methyl l-methyl-4- nitroimidazole-2-carboxylate 40 (3.3 g, 17.8 mmol), dissolved in 1 : 1 MeOH:EtOAc (100 niL), was shaken with hydrogen at 70 psi, for 14 h in presence of Pd/C (400 mg). The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as black oil. The residue was taken up in benzene and concentrated to furnish the amine as black solid, filtered and washed with ether (2.5 g, 88%). The crude amine was dissolved in CH3CN (50 mL) and DIEA (2.3, 18 mmol). A solution of 1- methyl-2-trichloroacetylimidazole 24 (3.6 g, 14.7 mmol) in CH3CN (50 mL) was added to the amine solution at 0 0C. The reaction mixture was allowed to stir for 10 h, solvent was removed and the residue was taken up in MeOH and the product was precipitated by addition Of Et2O to furnish desired compound 41 (2.13 g, 68%). 1H- NMR (DMSO-d6) δlθ.1 1 (s, exch, IH, NH), 7.69 (s, IH, Im'-C5H), 7.43 (s, IH, Im- C5H), 7.07 (s, IH, Im-C4H), 3.99 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.82 (s, 3H, OCH3). 13C-NMR (75 MHz, DMSO-d6) 6159.4 (+, s, CO), 157.2 (+, s, CO), 138.9 (+, s, Im- C2), 137.2 (+, s, Im-C2), 132.4 (+, s, Im-C4), 128.9 (-, d, Im-C4), 127.7 (-, d, Im-C5), 117.3 (-, d, Im-C5), 61.9 (+, t, -OCH2-), 37.6 (-, q, CH3), 35.8 (-, q, CH3), 14.2 (-, q, CH3). EI-HRMS: m/z calcd for C1 1H13N5O3 263.1018; found 263.1016 (M+, 100%), 204.0888 (27%), 182.0573 (27%), 150.0301 (30%), 109.0393 (26%).
[NO2-PyIm-COOMe] : Methyl 1 -methy 1-4- { [( 1 -methyl-4-nitro- 1 H-pyrrol-2- yl)-carbonyl] amino}- 1 H-imidazole-2-carboxylate (42). Methyl-4-nitro-imidazole-2- carboxylate 40 (5 g, 27 mmol) was dissolved in 1 : 1 mixture of MeOH and EtOAc (150 mL). A slurry of moist 10% PdVC in EtOAc was added and the contents were stirred vigorously under hydrogen (70 psi) for 14 h. The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as green solid. The crude amine without any further purification was taken up in CH3CN (150 mL). Contents were cooled to 0 0C and DIEA (6.9 mL, 37.7 mmol) was added. A solution of 1- methyl-4-nitro-2-trichloroacetylpyrrole 23 (7.6 g, 28.2 mmol) in CH3CN (20 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h, during which solid precipitated out. The reaction mixture was filtered to furnish 42 as yellow solid (5 g, 60%). The filtrate was concentrated and the residue was taken up in MeOH. Et2O was added to furnish a second crop of 42 (1 g, 12%). 1H-NMR (DMSO-d6) 611.14 (s, exch, IH, NH), 8.19 (d, IH, J = 1.2 Hz, Py-CH), 7.80 (d, IH, J = 1.2 Hz, Py-CH), 7.68 (s, IH, Im-CH), 3.96 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.82 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6158.9 (+, s, CO), 157.4 (+, s, CO), 137.1 (+, s, Im-C2), 134.1 (+, s, Py-C4), 131.2 (+, s, Im-C2), 128.2 (-, d, Py-C5), 125.4 (+, s, Py-C2), 1 15.2 (-, d, Im-C5), 1 10.2 (-, d, Py-C3), 52.0 (-, q, OCH3), 37.9 (-, q, CH3), 35.2 (-, q, CH3). EI-HRMS: m/z calcd for Ci3Hi5N5O5 321.1073; found 321.1094 (M+, 20%), 279.0997 (100%), 194.0830 (72%), 153.0317 (35%).
[NO2-ImIm-COOMe] : Methyl 1 -methyl-4- { [( 1 -methyl-4-nitro- 1 H-imidazol- 2-yl)-carbonyl]amino}-lH-imidazole-2-carboxylate (43). Methyl- l-methyl-4- nitroimidazole-2-carboxylate 40 (4.62 g, 25 mmol) was dissolved in 1 :1 mixture of MeOH and EtOAc (150 mL). ). A slurry of moist 10% Pd/C in EtOAc was added and the contents were stirred vigorously under hydrogen (70 psi) for 14 h. The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as green solid (3.6 g, 23.2 mmol). The crude amine without any further purification was taken up in CH3CN (150 mL). Contents were cooled to 0 "C and DIEA (2.6 mL, 25 mmol) was added. A solution of l-methyl-4-nitro-2-trichloroacetylimidazole 25 (6.8 g, 25 mmol) in CH3CN (20 mL) was added and the reaction mixture was allowed to stir at ambient temperature for 14 h, during which solid precipitated out. The reaction mixture was filtered to furnish 43 as yellow solid (4 g). The filtrate was concentrated and residue was chromatographed using EtOAc to furnish 2 g of 43 (combined yield 78%). 1H-NMR (CDCl3) 69.65 (s, exch, IH, NH) 7.80 (s, IH, Im-CH), 7.53 (s, IH, Im-CH), 4.18 (s, 3H, CH3), 4.03 (s, 3H, CH3), 3.95 (s, 3H, CH3). 1H-NMR (DMSO- d6) 610.88 (s, exch, IH, NH), 8.61 (s, IH, Im-CH), 7.71 (s, IH, Im-CH), 4.02 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.81 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6160.9 (+, s, CO), 157.1 (+, s, CO), 144.6 (+, s, Im-C4), 137.3 (+, s, Im-C2), 136.1 (+, s, Im- C2), 131.8 (+, s, Im-C4), 126.9 (-, d, Im-C5), 1 16.5 (-, d, Im-C5), 52.1 (-, q, OCH3), 36.5 (-, q, CH3), 35.9 (-, q, CH3). EI-HRMS: m/z calcd for CnHi2N6O5 308.0869; found 308.0881 (M+, 100%), 265.0870 (79%), 182.0617 (56%), 180.0699 (68%), 154.0197 (17%), 123.0440 (17%).
[ImIm-COOH] : 1 -Methyl-4- { [( 1 -methyl- 1 H-imidazol-2-yl)carbonyl]amino } - lH-imidazole-2-carboxylic acid (44). Methyl l-methyl-[4-(l-methyl-imidazole-2- carboxamido)]-imidazole-2-carboxylate 41 (3 g, 11.4 mmol) was dissolved in MeOH (75 mL). A solution of NaOH (2.3 g, 57 mmol) in H2O (75 mL) was added and the reaction mixture was allowed to stir at 50 °C for 12 h. Solvents were removed under reduced pressure and the residue was dissolved in H2O (25 mL). The solution was carefully acidified by dropwise addition of 2M HCl to pH 2 at 0 °C during which product precipitated. The solid was filtered washed with Et2O to furnish compound 44 (2.3 g, 82%). 1H-NMR (DMSO-d6) 610.13 (s, exch, IH, NH), 7.64 (s, IH, Im'- C5H), 7.49 (s, IH, Im-C5H), 7.09 (s, IH, Im-C3H), 3.99 (s, 3H, CH3), 3.93 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-de) 6159.9 (+, s, CO), 156.2 (+, s, CO), 137.8 (+, s, Im-C2), 135.9 (+, s, Im-C2), 132.6 (+, s, Im-C4), 127.8 (-, d, Im-C4), 127.1 (-, d, Im- C5), 115.2 (-, d, Im-C5), 35.9 (-, q, CH3), 35.3 (-, q, CH3). EI-HRMS: m/z calcd for Ci0HnN5O3 249.0862; found 205.0962 (M-CO2, 100%), 163.0965 (11%), 124.0511 (42%), 109.0401 (27%), 82.0524 (28%).
[NH2-ImIm-COOCH3]: Methyl 1 -methyl-4- {[(4-amino-l -methyl- IH- imidazol-2-yl)-carbonyl]amino}-lH-imidazole-2-carboxylate was isolated for characterization, during the hydrogenation of 43. 1H-NMR (DMSO-d6) 69.50 (s, exch, IH, NH), 7.64 (s, IH, Im-CH), 6.45 (s, IH, Im-CH), 4.56 (s, exch 2H, NH2), 3.93 (s, 3H, CH3), 3.87 (s, 3H, CH3), 3.81 (s, 3H, CH3).
[ImPyIm-COOMe]: Methyl 1 -methyl-4- { [(I -methyl-4- { [(I -methyl- 1 H- imidazol-2-yl)carbonyl]amino}-lH-pyrrol-2-yl)carbonyl]amino}-lH-imidazole-2- carboxylate (45). Methyl l-methyl-[4-(l-methyl-4-nitro-pyrrole-2-carboxamido)]- imidazole-2-carboxylate 42 (6 g, 19.5 mmol) was dissolved in 1 :1 DMF/MeOH. A slurry of moist 10% Pd/C in DMF was added and the contents were stirred vigorously under hydrogen (70 psi) for 14 h. The reaction mixture was filtered through a bed of celite. The celite was washed with MeOH and the filtrate was concentrated under reduced pressure to furnish crude amine as an oil. The oil was taken up in benzene and solvents were removed under reduced pressure. The residue was taken up in CH3CN and the solvents were removed to furnish amine as brown solid, filtered washed with Et2O and set for coupling reaction. To a solution of amine (5.1 g, 95%) in CH3CN (150 mL) and DIEA (5 mL, 27 mmol) was added a solution of l-methyl-2- trichloroacetylimidazole 24 (4.5 g, 20 mmol) in CH3CN (40 mL). The reaction mixture was allowed to stir at ambient temperature for 14 h. Solvents were removed under reduced pressure and the residue was treated with 1 : 1 CH3CN/Et20 during which 45 precipitated out as brown solid (5 g, 70%). 1H-NMR (DMSO-d6) δlθ.71 (s, exch, IH, NH), 10.36 (s, exch, IH, NH), 7.67 (s, IH, Im'-C5H), 7.39 (2 x overlapping s, IH each, Im-C5H + Py-C5H), 7.22 (s, IH, Py-C3H), 7.04 (s, IH, Im-C4H), 3.98 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.82 (s, 3H, OCH3). 13C-NMR (75 MHz, DMSO-d6) 6159.1 (+, s, CO), 158.9 (+, s, CO), 156.3 (+, s, CO), 139.0 (+, s, Im-C2), 138.0 (+, s, Im-C2), 130.9 (+, s, Im-C4), 127.2 (-, d, Im-C4), 126.5 (-, d, Im- C5), 122.2 (+, s, Py-C2), 121.6 (+, s, Py-C4), 1 19.8 (-, d, Py-C5), 1 15.7 (-, d, Im-C5), 106.3 (-, d, Py-C3), 51.9 (-, q, -OCH3), 36.5 (-, q, CH3), 35.7 (-, q, CH3), 35.3 (-, q, CH3). EI-HRMS: m/z calcd for C17H19N7O4 385.1499; found 385.1506 (M+, 100%), 327.1450 (7%), 303.1014 (4%), 231.0907 (81%), 203.0860 (10%), 149.0426 (40%), 109.0342 (59%).
[ImImIm-COOMe]: Methyl l-methyl-4-{[(l-methyl-4-{[(l-methyl-lH- imidazol-2-yl)carbonyl]amino}-lH-imidazol-2-yl)carbonyl]amino}-lH-imidazole-2- carboxylate (46). A solution of methyl l-methyl-4-[(l-methyl-4-nitro-imidazole-2- carboxamido)]-imidazole-2-carboxylate 43 (4 g, 13 mmol) in 1 :1 MeOH/EtOAc (200 mL) was hydrogenated using 5% Pd/C (400 mg) at 70 psi for 18 h. The green reaction mixture was filtered through a bed of celite. The celite bed was washed with MeOH and the filtrate was concentrated to furnish crude amine as an oil. The oil obtained was taken up in benzene, solvents were removed to furnish amine as a green solid (2 g, 60%). To a solution of amine (1.8 g, 6.47 mmol) in CH3CN (50 mL), DMF (10 mL) and DIEA (5 mL), was added a solution of l-methyl-2- trichloroacetylimidazole 24 (1.47 g, 6.47 mmol) in CH3CN (20 mL). The reaction mixture was allowed to stir at ambient temperature for 14 h. Solvents were removed and the residue was taken up in 1PrOH during which solid product precipitated out, filtered washed with ether to furnish crude product 46, which was purified by column chromatography using 1 : 1 MeOH:EtOAc (800 mg, 31%). 1H-NMR (CDCl3) 69.45 (s, exch, IH, NH), 9.41 (s, exch, IH, NH), 7.56 (s, IH, Im'-C5H), 7.49 (s, IH, InV-C5H), 7.09 (s, IH, Im-C5H), 7.01 (s, IH, Im-C4H), 4.08 (s, 3H, CH3), 4.06 (s, 3H, CH3), 4.01 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.92 (s, 3H, OCH3). 13C-NMR (75 MHz, DMSO-d6) 6160.4 (+, s, CO), 159.2 (+, s, CO), 157.1 (+, s, CO), 138.8 (+, s, Im-C2), 137.0 (+, s, Im-C2), 134.7 (+, s, Im-C2), 134.4 (+, s, Im-C4), 132.2 (+, s, Im-C4), 129.4 (-, d, Im-C4), 127.5 (-, d, Im-C5), 127.1 (-, d, Im-C5), 1 17.3 (-, d, Im-C5), 51.3 (-, q, - OCH3), 37.6 (-, q, CH3), 36.1 (-, q, CH3), 35.8 (-, q, CH3). EI-HRMS: m/z calcd for C16H18N8O4 386.1451; found 385.1456 (M+, 100%), 328.1402 (18%), 304.0920 (10%), 205.0962 (20%), 182.0562 (22%), 150.0308 (24%), 109.0399 (30%).
[ImPyIm-COOH] : 1 -Methy 1-4- { [( 1 -methy 1-4- { [( 1 -methyl- 1 H-imidazol-2-yl)- carbonyl]amino}-lH-pyrrol-2-yl)carbonyl]amino}-lH-imidazole-2-carboxylic acid (47). To a solution of methyl l-methyl-4-{l-methyl-4-[(l-methyl-imidazole-2- carboxamido)]-pyrrole-2-carboxamido}imidazole-2-carboxylate 45 (500 mg, 1.3 mmol) in MeOH (20 mL) was added a solution of NaOH (57 mg, 1.4 mmol) in H2O (20 mL). The reaction mixture was allowed to stir at 60 °C for 12 h. MeOH was removed under reduced pressure and the contents were acidified to pH 2 using 1 M HCl under cooling, during which acid precipitated out as gelatinous solid. The solid was filtered and the wet cake was taken up in benzene. Solvents were removed and the residue was treated with 1 :1 MeOH/'PrOH when acid precipitated as granular solid, filtered washed with Et20 to furnish 47. (400 mg, 83%). 1H-NMR (DMSO-d6) δl l .14 (s, exch, IH, NH), 10.76 (s, exch, IH, NH), 7.68 (s, IH, Im'-C5H), 7.63 (s, IH, Im-C5H), 7.46 (s, IH, Py-C5H), 7.42 (s, IH, Im-C4H), 7.26 (s, IH, Py-C3H), 4.04 (s, 3H, CH3), 3.92 (s, 3H, CH3), 3.87 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6) 6160.1 (+, s, CO), 158.7 (+, s, CO), 156.0 (+, s, CO), 138.7 (+, s, Im-C2), 137.3 (+, s, Im-C2), 131.5 (+, s, Im-C4), 126.7 (-, d, Im-C4), 126.6 (-, d, Im-C5), 121.9 (+, s, Py- C2), 121.4 (+, s, Py-C4), 120.9 (-, d, Py-C5), 1 15.9 (-, d, Im-C5), 106.9 (-, d, Py-C3), 36.5 (-, q, CH3), 35.5 (-, q, CH3), 35.4 (-, q, CH3). EI-HRMS: m/z calcd for C16Hi7N7O4 371.1342; found 327.1446 (M-CO2, 100%), 231.0876 (63%), 149.0350 (56%) 109.0401 (55%). [ImImIm-COOH] : 1 -Methyl-4- { [( 1 -methyl-4- { [( 1 -methyl- 1 H-imidazol-2-y I)- carbonyl]amino}-lH-imidazol-2-yl)carbonyl]amino}-lH-imidazole-2-carboxylic acid (48). To a solution of methyl 1 -methyl-4- {l-methyl-4-[(l -methyl-imidazole-2- carboxamido)]-pyrrole-2-carboxamido}imidazole-2-carboxylate 46 (500 mg, 1.3 mmol) in MeOH (20 mL) was added a solution of NaOH (57 mg, 1.4 mmol) in H2O (10 mL). The reaction mixture was carefully acidified using 2 M HCl to pH 2, during which jelly like precipitate was obtained. The reaction mixture was concentrated under reduced pressure and the residue was taken up in MeOH (50 mL). Insoluble salts were filtered and the filtrate was concentrated under reduced pressure. The residue obtained was dissolved in MeOH and the product was precipitated by adding Et2O to furnish 48 as grey solid (620 mg, 83 %). 1H-NMR (DMSO-d6) δ9.55 (s, IH, NH), 9.53 (s, IH, NH), 7.38 (s, IH), 7.36 (s, IH), 7.13 (s, IH), 7.09 (s, IH), 3.98 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.82 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-da) 0160.6 (+, s, CO), 157.8 (+, s, CO), 156.8 (+, s, CO), 137.1 (+, s, Im-C2), 135.8 (+, s, Im-C2), 132.9 (+, s, Im-C2), 134.3 (+, s, Im-C4), 132.0 (+, s, Im-C4), 128.4 (-, d, Im- C4), 127.0 (-, d, Im-C5), 126.5 (-, d, Im-C5), 1 15.4 (-, d, Im-C5), 37.2 (-, q, CH3), 36.7 (-, q, CH3), 36.3 (-, q, CH3). EI-HRMS: m/z calcd for C15H16N8O4 372.1295; found 328.1404 (M-CO2, 100%), 246.0854 (14%), 220.1060 (23%), 124.0474 (61%), 109.0394 (27%). MP-Bzi-Py-NH2: 2-(4-Amino-l -methyl- lH-pyrrol-2-yl)-5-(4-methylpiperaz- inyl)-lH-benzimidazole (49). A mixture of the nitro derivative 26 and 10% Pd-C in 50 mL MeOH was hydrogenated in a Paar shaker for 8 h. The catalyst was removed by filtration and the filtrate evaporated to afford a dark solid, which was used without further purification. Attempts to crystallize and/or purify by column chromatography led to prolonged exposure to air and degradation to unidentified product mixtures. The title product was alternatively isolated as the hydrochloride salt by treatment with ethanolic HCl, and crystallization (EtOH/Et2O). 1H-NMR (HCl salt, DMSO-d6) δl l .28 (s, exch, IH, Bzi-NH), 10.52 (s, exch, 2H, NH2), 7.66 (d, J = 9 Hz, IH, Bzi- CH), 7.45 (d, IH, J = 1.4 Hz, Bzi-CH), 7.30 (dd, IH, J = 9 & 1.4 Hz, Bzi-CH), 7.20 (s, 2H, 2 x Py-CH), 4.09 (s, 3H, Py-NCH3), 3.83 (m, 2H, CH2), 3.50 (m, 2H, CH2), 3.20 (m, 4H, CH2), 2.83 (s, 3H, NCH3). 13C-NMR (125 MHz, D2O) 6148.8 (+), 140.7 (+), 132.3 (+), 126.2 (+), 124.2 (-), 118.6 (-), 1 16.6 (+), 114.7 (-), 1 14.6 (+), 111.3 (-), 100.7 (-), 53.4 (+), 47.7 (+), 43.2 (-), 36.3 (-). FAB-HRMS: m/z calcd for C7H23N6 + (MH+) 310.1984, found 31 1.1983.
MP-Pzi-Py-NH2: 2-(4- Amino- 1 -methyl- lH-pyrrol-2-yl)-5-(4-methylpiperaz- inyl)-lH-imidazo[4,5-b]pyridine (50). Preparation of this compound posed similar problems as for 49. With a modified procedure, entailing hydrogenation of a mixture of the nitropyrrole derivative 27 and 10% Pd-C with 4 mol equiv of HCl added to it, the title product was obtained in its hydrochloride salt form (85% yield). 1H-NMR
(HCl salt, D2O) 08.03 (d, IH, J = 9 Hz, Pzi-C7H), 7.35 (d, IH, J = 1.2 Hz, Py-C5H), 7.14 (d, IH, J = 9 Hz, Pzi-C6H), 7.04 (d, IH, J = 1.2 Hz, Py-C3H), 4.53 (d, 2H, J =
14.4 Hz, Pip-CH2), 3.95 (s, 3H, Py-CH3), 3.66 (d, 2H, J = 12 Hz, Pip-CH2), 3.36-3.45
(m, 2H, Pip-CH2), 3.20-3.27 (m, 2H, PJp-CH2), 2.98 (s, 3H, Pip-CH3). 13C-NMR
(125 MHz, D2O) 5157.0 (+, C), 125.6 (-), 124.4 (-), 118.2 (+), 1 14.5 (+), 110.8 (-),
108.2 (-), 52.9 (+), 43.3 (+), 43.1 (-), 35.9 (-) (Note 2 C with long Tl). ES-HRMS: m/z calcd for Ci6H22N7 + (MH+) 312.1931, found 312.1936 (90%, MH+), 623.3883
(4%, M2H+), 255.1334 (100%), 156.6008 (48%), 128.0716 (20%).
MP-Bzi-Ph(NO2)NHAc: 2-(4-Acetamido-3-nitrophenyl)-5-(4- methylpiperazin-yl)-lH-benzimidazole (55) was prepared in 80% yield using the diamine derivative 2 and 3-nitro-4-acetamidobenzaldehyde in the general procedure C given above. M.pt. > 300 0C; 1H-NMR (DMSO-d6) 610.96 (s, IH), 10.71 (s, IH, exch), 8.78 (s, IH, Ph-C3H), 8.53 (d, IH, J = 8.7 Hz, Bzi-C7H), 7.87 (d, IH, J = 8.7 Hz, Ph-C5H), 7.61 (d, IH, J = 8.7 Hz, Bzi-C4H), 7.17 (d, IH, J = 8.7 Hz, Ph-C6H), 7.14 (s, IH, BZi-C6H), 3.62-3.76 (m, 4H, CH2), 3.18 (m, 4H, CH2), 2.89 (s, 3H, CH3), 2.16 (s, 3H, COCH3); 13C-NMR (75 Hz, DMSO-d6 at 318 K) 6173.7 (s), 150.9 (s), 150.7 (s), 147.8 (s), 143.8 (s), 133.7 (s), 130.1 (d), 129.2 (d), 126.4 (s), 123.1 (d),
122.3 (s), 1 19.6 (d), 1 15.7 (d), 102.6 (d), 55.6 (t), 49.1 (t), 45.7 (q), 26.2 (q); FAB- HRMS: m/z calcd for C20H23N6O3 + (MH+) 395.1823; found 395.1828 (100%).
MP-Pzi-Ph(NO2)NHAc: 2-(4-Acetamido-3-nitrophenyl)-5-(4- methylpiperazin-yl)-lH-imidazo[4,5-b]pyridine (56) was prepared in 70% yield using the diamine derivative 6 and 3-nitro-4-acetamidobenzaldehyde in the general procedure D given above. 1H-NMR (DMSO-d6) 613.20 (s, exch, IH, NH), 10.56 (s, exch, IH, NH), 8.67 (s, IH, Ph-C3H), 8.39 (d, IH, J = 8.5 Hz, Ph-C5H), 7.90 (d, IH, J - 8.5 Hz, Ph-C6H), 7.79 (d, IH, J = 9 Hz, PzI-C7H), 6.93 (d, IH, J - 9 Hz, PzI-C5H), 3.30-3.50 (m, 8H, CH2), 2.79 (s, 3H, CH3), 2.1 1 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 169.5, 156.3, 143.2, 132.8, 131.6, 127.5, 126.3, 123.0, 1 13.0, 109.9, 105.6, 103.7, 52.6, 43.8, 42.9. FAB-HRMS: m/z calcd for Ci8H21N8O3 + (MH+) 396.1713, found 396.1836 (100%).
MP-BzI-Ph(NO2)NH2: 2-(4-Amino-3-nitrophenyl)-5-(4-methyrpiperazinyl)- 1 H-benzimidazole (57). A suspension of 55 (770 mg, 2 mmol) and NaOH (100 mg, 2.5 mmol) in a mixture of MeOH (20 mL) and H2O (5 mL) was stirred at 70 0C for 4 h. Acidification with 0.1 N HCl to pH 3 gave a precipitate that was collected by filtration and washed with MeOH to afford the title product (95% yield). M.pt. 165- 168 X; 1H-NMR (DMSO-d6) 612.59 (s, exch, IH, NH), 8.75 (s, IH, Ph-C3H), 8.13 (d, IH, J = 8.7 Hz, BZi-C7H), 7.76 (s, IH, Bzi-C4H), 7.38 (d, IH, J = 8.6 Hz, Ph-C5H), 7.13 (d, IH, J = 8.7 Hz, Bzi-C6H), 6.93 (d, IH, J = 8.6 Hz, Ph-C6H), 3.17-3.10 (m, 4H, CH2), 2.42-2.49 (m, 4H, CH2), 2.24 (s, 3H, CH3). 1H-NMR (DMSO-d6) 68.76 (s, IH), 8.18 (d, IH, J = 8.4 Hz), 7.79 (s, 2H, NH2), 7.38 (d, IH, J = 8.7 Hz), 7.13 (d, IH, J - 8.9 Hz), 6.97 (s, IH), 6.89 (d, IH, J = 8.9 Hz), 3.10 (m, 4H, CH2), 2.53-2.49 (m, 4H, CH2), 2.23 (s, 3H, CH3); 13C-NMR (125 MHz, DMSO-d6) δ 148.5, 147.9, 146.8, 133.4, 130.1, 122.9, 119.9, 118.1, 113.8, 54.9, 50.0, 45.9 (note: not all signals observed due to Tl effects). EI-HRMS: m/z calcd for C18H20N6O2 352.1648, found 352.1644 (M+, 100%);
MP-PZi-Ph(NO2)NH2: 2-(4-Amino-3-nitrophenyl)-5-(4-methylpiperazinyl)- lH-imidazo[4,5-b]pyridine (58): A suspension of 56 (770 mg, 2 mmol) and NaOH (100 mg, 2.5 mmol) in a mixture of MeOH (20 mL) and H2O (5 mL) was stirred at 70 0C for 4 h. Acidification with 0.1 N HCl to pH 6 gave a precipitate that was collected by filtration and washed with acetone to afford the title product (90% yield). 1H- NMR (DMSO-d6) 613.11 (s, exch, IH, NH), 8.68 (s, IH, Ph-C3H), 7.62 (d, IH, J = 9 Hz, Pzi-C7H), 7.38 (d, IH, J = 8.6 Hz, Ph-C5H), 7.02 (d, IH, J = 9 Hz, Pzi-C6H), 6.93 (d, IH, J = 8.6 Hz, Ph-C6H), 3.47 (m, 4H, CH2), 3.32 (m, 4H, CH2), 2.76 (s, 3H, CH3); 13C-NMR (125 MHz, DMSO-d6) 6 156.1, 142.9, 133.4, 132.8, 130.8, 127.3, 126.1, 122.4, 112.9, 109.1, 105.0, 103.8, 53.5, 43.4, 42.6. FAB-HRMS: m/z calcd for CnH20N7O2 + (MH+) 354.1678, found 354.1646 (100%).
MP-Bzi-Ph(NH2)2: 2-(3,4-Diaminophenyl)-5-(4-methylpiperazinyl)-lH-benz- imidazole (59). A mixture of compound 57 and 10% Pd-C in MeOH was hydrogenated at high pressure (70 psi) in a Paar shaker for 14 h. Evaporation of the filtrate obtained by removing the catalyst through a bed of Celite afforded the title compound (88% yield). M.pt. 265-267 °C; 1H-NMR (DMSO-d6) 612.17 (s, exch, IH, NH), 7.36 (s+d overlapping, 2H), 7.19 (d, IH, J = 8.2 Hz, Ph-C5H), 6.90 (s, IH, Ph- C3H), 6.83 (d, IH, J = 8.4 Hz, Bzi-C6H), 6.60 (d, IH, J = 8.2 Hz, Ph-C6H), 4.91 (s, 2H, NH2), 4.65 (s, 2H, NH2), 3.08 (m, 4H), 2.47 (m, 4H), 2.21 (s, 3H); 1H-NMR (500 MHz, HCl salt, D2O) δ7.24 (d, IH, J = 9 Hz), 6.99 (d, IH, J = 8.5 Hz), 6.95 (s, IH), 6.89 (d, IH, J = 8.5 Hz), 6.79 (s, IH), 6.64 (d, IH, J = 9 Hz), 3.52-3.48 (m, 4H, CH2), 2.98 (m, 2H, CH2), 2.89 (m, 2H, CH2), 2.85 (s, 3H, CH3). 13C-NMR (125 MHz, CD3OD) δ 155.2 (+), 150.1 (+), 139.9 (+), 137.5 (+), 122.1 (+), 118.9 (-), 116.5 (-), 1 15.7 (-), 115.3 (-), 57.7 (+), 52.9 (+), 48.5 (-) (note: not all signals observed due to Tl effects). EI-HRMS: m/z calcd for Ci8H22N6 322.1906; found 322.1903 (100%), 252.1243 (24%), 251.1 173 (29%);
MP-Pzi-Ph(NH2)2: 2-(3,4-Diaminophenyl)-5-(4-methylρiperazinyl)-lH- imidazo[4,5-b]pyridine (60) was prepared in 90% yield from 58 in the hydrogenation protocol described above. 1H-NMR (HCl salt, D2O) 67.22 (d, IH, J = 5.2 Hz, Pzi- CH), 6.53 (d+s, Pzi-CH + Ph-CH), 6.53-6.37 (m, 2H, Ph-CH), 3.98 (m, 2H), 3.43 (m, 2H), 2.95 (m, 4H), 2.82 (s, 3H, CH3); 13C-NMR (125 MHz, HCl salt in D2O) δ 156.3, 146.1, 142.7, 142.1, 128.9, 123.6, 122.0, 116.6, 115.8, 115.7, 109.7, 106.6, 53.0, 43.2, 42.6. EI-HRMS: m/z calcd for Ci7H2[N7 323.1858; found 323.1857 (M+, 73%, 308.1653 (23%), 253.1194 (100%), 108.0687 (22%).
MP-Bzi-Bzi-Py-NO2: 2- {2-( 1 -Methy 1-4-nitro- 1 H-pyrrol-2-y I)- 1 H- benzimidaz-ol-5-yl}-5-(4-methylpiρerazinyl)-lH-benzimidazole (61) was prepared in 82% yield using the diamine derivative 59 and pyrrole derivative (20) in the general procedure D given above. m.pt. 235-238 °C. 1H-NMR (500 MHz, DMSO-d6) 612.61 (s, exch, IH, NH), 8.33 (s, IH, Py-CH), 8.24 (s, IH, Bzi-CH), 8.03 (d, IH, J = 8.4 Hz, Bzi-CH), 7.68 (d, IH, J = 8.4 Hz, Bzi-CH), 7.58 (s, IH, Py-CH), 7.43 (d, IH, J - 8.7 Hz, Bzi-CH), 6.99 (s, IH, Bzi-CH), 6.92 (d, IH, J = 8.7 Hz, Bzi-CH), 4.21 (s, 3H, Py-NCH3), 3.11 (m, 4H, CH2), 2.51 (m, 4H, CH2), 2.28 (s, 3H, CH3). 1H-NMR (CD3OD) 58.06 (s, IH, Bzi-CH), 7.79 (d, IH, J = 8.4 Hz, Bzi-CH), 7.74 (s, IH, Py- CH), 7.53 (d, IH, J = 8.4 Hz, Bzi-CH), 7.37 (d, IH, J = 8.4 Hz, Bzi-CH), 7.20 (s, IH, Py-CH), 6.99 (s, IH, Bzi-CH), 6.91 (d, IH, J = 8.4 Hz, Bzi-CH), 4.03 (s, 3H, Py- NCH3), 3.11 (t, 4H, CH2), 2.56 (t, 4H, CH2), 2.27 (s, 3H, NCH3). FAB-HRMS: m/z calcd for C24H25N8O2 + (MH+) 457.2101 ; found 457.2097.
MP-Bzi-Bzi-Py-NH2: 2- {2-(4- Amino- 1 -methyl- 1 H-pyrrol-2-yl)- 1 H- benzimid-azol-5-yl}-5-(4-methylpiperazinyl)-lH-benzimidazole (62). A mixture of compound 61 and 10% Pd-C in MeOH was hydrogenated at high pressure (70 psi) in a Paar shaker for 14 h. The catalyst was filtered through a bed of Celite and the filtrate was evaporated to afford the title compound (80% yield). 1H-NMR (CD3OD) 68.15 (s, IH, Bzi-CH), 7.87 (d, IH, J = 8.4 Hz, Bzi-CH), 7.60 (d, IH, J = 8.4 Hz, Bzi- CH), 7.46 (d, IH, J = 8.4 Hz, Bzi-CH), 7.10 (s, IH, Py-CH), 6.97 (d, IH, J = 8.4 Hz, Bzi-CH), 6.47 (s, IH, Bzi-CH), 6.43 (s, IH, Py-CH), 3.93 (s, 3H, Py-NCH3), 3.18 (t, 4H, CH2), 2.64 (t, 4H, CH2), 2.33 (s, 3H, NCH3). FAB-HRMS: m/z calcd for C24H27N8 + (MH+) 427.2358 found 427.2369 (100%).
[Im-Bzi-COOMe]: Methyl 2-(l -methyl- lH-imidazol-2-y I)- IH- benzimidazole-5-carboxylate (63) was prepared in 80% yield using methyl 3,4- diaminobenzoate diamine and aldehyde 21 in the general procedure D given above. 1H-NMR (DMSO-d6) 512.78 (s, exch, IH, NH), 8.19 (s, IH, Bzi-CH), 7.85 (d, IH, J = 8.6 Hz, Bzi-CH), 7.69 (d, IH, J = 8.6 Hz, Bzi-CH), 7.46 (s, IH, Im-CH), 7.17 (s, IH, Im-CH), 4.19 (s, 3H, NCH3), 3.87 (s, 3H, OCH3); 13C-NMR (DMSO-d6 at 318 K) 6171.1 (+, s, CO), 147.2 (+, s, Bzi-C2), 145.5 (+, s, Im-C2), 140.3 (+, s, Bzi-C3a), 130.6 (-, d, Im-C5), 129.5 (+, s, Bzi-C5), 129.1 (-, d, Im-C4), 127.7 (-, d, Bzi-C6), 127.3 (+, s, Bzi-C7a) 120.8 (-, d, Bzi-C4), 118.8 (-, d, Bzi-C7), 52.6 (-, q, OCH3), 38.0 (-, q, CH3). EI-HRMS: m/z calcd for Ci3Hi2N4O2 256.0960; found 256.0956 (M+, 100%), 225.0777 (34%), 197.0816 (21%).
[Im-Bzi-COOH] : 2-( 1 -Methyl- 1 H-imidazol-2-yl)- 1 H-benzimidazole-5- carbox-ylic acid (64): was prepared in 76% yield using 3,4-diaminobenzoic acid and aldehyde 21 in the general procedure D given above. 1H-NMR (DMSO-dό) 512.82 (s, exch, IH, NH), 8.18 (s, IH, Bzi-CH), 7.84 (d, IH, J = 8.7 Hz, Bzi-CH), 7.65 (d, IH, J = 8.7 Hz, Bzi-CH), 7.49 (s, IH, Im-CH), 7.20 (s, IH, Im-CH), 4.19 (s, 3H, NCH3); 13C-NMR (DMSOd6 at 318 K) 6170.2 (+, s, CO), 148.2 (+, s, Bzi-C2), 143.2 (+, s, Im-C5), 138.9 (+, s, Bzi-C3a), 131.3 (-, d, Im-C5), 130.8 (+, s, Bzi-C5), 129.5 (-, d, Im- C4), 128.1 (-, d, Bzi-C6), 127.6 (+, s, Bzi-C7a) 122.1 (-, d, Bzi-C4), 119.6 (-, d, Bzi- C7), 37.8 (-, q, CH3). EI-HRMS: m/z calcd for Ci2H10N4O2 242.0804, found 242.0897 (14%, M+), 145.0501 (67%); ES-HRMS: m/z calcd for C12HnN4O2 + (MH+) 243.0882, found 243.0876 (100%).
MeO-Bzi-Py-NO2 : Methyl 2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H-benzimid- azole-5-carboxylate (65) was prepared in 88% yield using methyl 3,4- diaminobenzoate diamine and aldehyde 20 in the general procedure C given above. 1H-NMR (DMSO-d6) 512.59 (s, exch, IH, NH), 8.22 (d, IH, J = 1.5 Hz, Py-CH), 8.14 (s, IH, Bzi-CH), 7.85 (d, IH, J = 8.6 Hz, Bzi-CH), 7.63 (d, IH, J = 8.6 Hz, Bzi-CH), 7.51 (d, IH, J = 1.5 Hz, Py-CH), 4.10 (s, 3H, NCH3), 3.85 (s, 3H, OCH3). 13C-NMR (DMSO-d6, 330 K) 6166.7 (s, C), 142.3 (s, C), 139.8 (s, C), 136.7 (s, C), 136.2 (s, C),
128.5 (d, CH), 124.7 (s, C), 124.4 (d, CH), 122.1 (s, C), 116.5 (d, CH), 114.5 (d, CH),
108.6 (d, CH), 52.2 (q, CH3), 37.7 (q, CH3). FAB-HRMS: m/z calcd for C14H12N4O4 + (MH+) 301.0937, found 301.0932 (100%).
HO-Bzi-Py-NO2: 2-(l -Methyl-4-nitro- 1 H-pyrrol-2-yl)- lH-benzimidazole-5- carboxylic acid (66). To a solution of preceding ester derivative (65) in 4:1 MeOH-
H2O (30 mL/mmol) was added NaOH (2 mol equiv) and the mixture stirred at 60 0C for 2 h. After cooling, the mixture was acidified with 1 N HCl to pH 2 and the precipitate collected by filtration. Crystallization from acetone gave the title product
(90% yield). 1H-NMR (DMSO-d6) 612.90 (s, exch, IH, Bzi-NH), 8.18 (s, IH, Py- CH), 8.13 (s, IH, Bzi-CH), 7.82 (d, IH, J = 8.4 Hz, Bzi-CH), 7.59 (d, IH, J = 8.4 Hz,
Bzi-CH), 7.49 (s IH, Py-CH), 4.14 (s, 3H, NCH3); 13C-NMR (DMSO-d6) 6167.8 (s,
C), 146.6, 135.0, 128.1, 124.9, 123.8, 106.9, 38.0. (5 aromatics nulled due to high Tl relaxation). EI-HRMS: m/z calcd for C13H10N4O4 286.0702, found 286.0699 (100%,
M+), 239.0689 (65%); ES-HRMS: m/z calcd for Ci3H1 1N4O4 + (MH+) 287.0780, found 287.0762 (80%). MeO-Bzi-Py-NH2: Methyl 2-(4-amino-l -methyl- lH-pyrrol-2-y I)-I H-benzim- idazole-5-carboxylate (67) Compound 65 was reduced with hydrogen at high pressure (60 psi) using 10% Pd-C and standard practice followed above. The product was not isolated and instead used directly for further reactions below. HO-Bzi-Py-NH2: 2-(4-Amino-l -methyl- lH-pyrrol-2-y I)-I H-benzimidazole-
5-carboxylic acid (68) Compound 66 was hydrogenated as in the case of preceding compound and the material obtained after filtration was used directly without purification in the steps given below.
MeO-Bzi-Py-Im (69) The amine derivative 67 was treated with a 1.5-fold excess of 2-trichloroacetyl-l-methylimidazole dissolved in CH3CN. The mixture was stirred at 30 0C for 2 h, and the resultant suspension filtered to afford a yellow solid which was recrystallized (MeOH) to give the title product (80% yield based on imidazole). 1H-NMR (500 MHz, DMSO-d6) (Mix of tautomers) 612.89 (s, exch, IH, NH), 10-56 (s, exch, IH, NH), 8.19 + 8.01 (split s, IH, Bzi-C4H), 7.83 + 7.78 (split d, IH, Bzi-C6H), 7.68 + 7.52 (split d, IH, Bzi-C7H), 7.43 (d, IH, J = 1.8 Hz, Py-C5H), 7.38 (s, IH, Im-C5H), 7.27 + 7.24 (split d, IH, Py-C3H), 7.07 (s, IH, Im-C4H), 4.07 (s, 3H, CH3), 4.03 (s, 3H, CH3), 3.86 (s, 3H, CH3); 13C-NMR (125 MHz, DMSO-d6) 6167.7, 156.9, 149.1, 139.6, 127.8 (CH), 127.2 (CH), 123.7 (CH), 120.6 (CH), 119.3 (CH), 1 12.9, 1 1 1.4 (CH), 105.4 (CH), 52.8 (CH3), 37.4 (CH3), 35.9 (CH3) (assigned using HMBC); ES-HRMS: m/z calcd for Ci9Hi9N6O3 + (MH+) 379.1513, found 379.1523 (100%), 757.3095 (4%, M2H+), 271.1233 (15%), 190.0797 (17%),
Im-Py-Bzi-COOH (70): Amine derivative 68 was coupled with 2- trichloroacetyl-1-methylimidazole as described for the preceding product. The title compound was purified by crystallization (acetone; 84% yield). In an alternative procedure, this compound was also obtained when 69 was hydrolysed under basic conditions as described for the preparation of 66. 1H-NMR (DMSO-d6) 610.66 (s, exch, IH, NH), 8.14 (s, IH, Bzi-CH), 7.85 (d, IH, J = 8.1, Bzi-CH), 7.62 (d, IH, J = 8.1, Bzi-CH), 7.47 (d, IH, J = 1.8, Py-CH), 7.43 (s, IH, Im-CH), 7.26 (d, IH, J = 1.8, Py-CH), 7.13 (s, IH, Im-CH), 4.09 (s, 3H, Im-CH3), 4.02 (s, 3H, Py-CH3); 13C-NMR (125 MHz, DMSO-d6) 6168.1, 156.2, 138.9, 127.1, 126.5, 124.2, 122.9, 120.0, 118.4, 104.5, 36.7, 35.2. (3 aromatics nulled due to high Tl relaxation); ES-HRMS: m/z calcd for Ci8H17N6O3 + (MH+) 365.1362, found 365.1371 (100%), 366.1435 (14%, M+2), 283.0880 (10%),
Dp-Bzi-Py-NO2 : N,N-Dimethyl-3- { [2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H- benzimidazo-5-yl]carbonylamino}propanamine (71). Acid derivative 66 (2 mmol) was dissolved in DMF (20 mL) and a sequence of additions of HOBt (2.4 mmol), DIC (2.3 mmol) and dimethylaminopropylamine (2.5 mmol), in that order, were followed by stirring the suspension at 45 0C for 1O h The heating was removed and the mixture evaporated to afford a solid residue. This material was triturated with hot CHCl3 (to remove the urea product), followed by washings with acetone and 'PrOH. The resultant yellow solid was crystallized (MeOH) to afford the title product (64% yield). 1H-NMR (HCl salt, D2O) 68.01 (s, IH, Bzi-CH), 7.91 (s, IH, Py-C5H), 7.78 (d, IH, J = 6.9 Hz, Bzi-CH), 7.71 (d, J = 6.9 Hz, Bzi-CH), 7.23 (s, IH, Py-C3H), 3.98 (s, 3H, NCH3), 3.55 (t, 2H, CH2), 3.34 (t, 2H, CH2), 3.05 (s, 6H, N(CH3)2), 2.17 (qnt, 2H, CH2); 13C-NMR (75 MHz, HCl salt, D2O, 318 K) 6169.5, 143.0, 135.9, 135.5, 133.6, 130.9, 103.3, 124.7, 119.0, 114.5, 114.0, 111.3, 56.1, 43.4, 37.4, 24.6. ES- HRMS: m/z calcd for C18H23N6O3 + (MH+) 371.1832, found 371.1813 (100%), 185.1 169 (15%).
Dp-Bzi-Py-NH2: N,N-Dimethyl-3- { [2-(4-amino- 1 -methyl- 1 H-pyrrol-2-yl)- lH-benzimidazo-5-yl]carbonylamino}propanamine (72): Compound 71 was reduced catalytically (10% Pd-C) with H2 (50 psi pressure) overnight to give the title amine after filtration and evaporation of the filtrate. The red solid was stored under nitrogen before further use. 1H-NMR (HCl salt, D2O) 67.99 (s, IH, Bzi-CH), 7.77 (d, IH, J = 8.4 Hz, Bzi-CH), 7.66 (dd, J = 2.7 & 8.4 Hz, Bzi-CH), 7.38 (s, IH, Py-CH), 7.12 (s, IH, Py-CH), 3.94 (s, 3H, NCH3), 3.42 (t, 2H, CH2), 3.20 (t, 2H, CH2), 2.87 (s, 6H, N(CH3)2), 2.03 (qnt, 2H, CH2); 13C-NMR (75 MHz, HCl salt, D2O) 6166.5 (s, C), 140.1 (s, C), 130.6 (s, C), 128.5 (s, C), 128.3 (s, C), 122.8 (d, CH), 122.3 (d, CH), 1 13.4 (s, C), 1 1 1.9 (s, C), 1 1 1.2 (d, CH), 1 10.4 (d, CH), 109.3 (d, CH), 52.8 (t, CH2), 40.2 (q, CH3), 34.2 (t, CH2), 33.6 (q, CH3), 21.6 (t, CH2); ES-HRMS: m/z calcd for C18H25N6O+ (MH+) 341.2090, found 341.2082 (100%), 185.1166 (20%), 171.1067 (45%). [BZi-COCCl3]: l-Methyl-2-trichloroacetyl-lH-benzimidazole (73). Freshly distilled trichloroacetyl chloride (50 mmol) dissolved in dry CH2C12 was added dropwsie to a CH2Cl2 solution of 1-methylbenzimidazole. The resultant mixture was stirred at room temperature for 16 h. The solution was passed through a bed of silica gel and eluted with CHCl3. The filtrate was evaporated to dryness to afford a light yellow solid. Recrystallization (1PrOH) provided the title product in 82% yield. 13C- NMR (75 MHz, DMSO-d6) 6164.5 (+, s, CO), 144.8 (+, s, Bzi-C2), 139.4 (+, s, Bzi- C3a), 138.7 (+, s, Bzi-C7a), 121.7 (-, d, Bzi-C6), 120.5 (-, d, Bzi-C5), 118.3 (-, d, Bzi- C4), 110.8 (-, d, Bzi-C7), 31.9 (-, q, Bzi-CH3). EI-HRMS: m/z calcd for C10H7Cl3N2O 275.9624; found 275.9669 (M+, 10%), 160.0591 (10%), 159.0556 (M-CHCl3, 100%) {relative isotopic distribution: 275.9669 (100), 276.9707 (13), 277.9640 (95), 278.9685 (13), 279.9633 (30), 281.9635 (4)}.
[Bzi-ImPy-COOMe] (74). The nitroimidazole derivative 34 was hydrogenated (as described in the preparation of 37) and treated with 73 dissolved in CH3CN to afford a suspension which was stirred further at 45 0C for 3 h. The solid was collected by filtration and recrystallized from MeOH to afford the title product in 76% yield. 1H-NMR (DMSO-d6) 610.48 (s, exch, IH, NH), 10.29 (s, exch, IH, NH), 7.79 (d, IH, J = 7.5 Hz, Bzi-CH), 7.72 (d, IH, J = 7.5 Hz, Bzi-CH), 7.65 (s, IH, Im- CH), 7.54 (d, IH, J = 1.8 Hz, Py-CH), 7.46 (t, IH, J = 7.2 Hz, Bzi-CH), 7.38 (t, IH, J = 7.2 Hz, Bzi-CH), 7.02 (d, IH, J = 1.8 Hz, Py-CH), 4.19 (s, 3H, NCH3), 4.02 (s, 3H, NCH3), 3.85 (s, 3H, NCH3), 3.74 (s, 3H, OCH3); 13C-NMR (75 MHz, DMSO-d6) 0160.9 (+, s, CO), 156.5 (+, s, CO), 155.7 (+, s, CO), 143.1 (+, s, Bzi-C2), 140.8 (+, s, Bzi-C3a), 140.7 (+, s, Bzi-C7a), 137.2 (+, s, Im-C2), 134.6 (+, s, Im-C4), 124.7 (-, d, Bzi-C6), 123.5 (-, d, Bzi-C5), 122.3 (+, s, Py-C2), 121.2 (-, d, Py-C5), 120.4 (+, s, Py- C4), 118.9 (-, d, Bzi-C4), 114.8 (-, d, Im-C5), 111.6 (-, d, Bzi-C7), 108.8 (-, d, Py-C3), 51.1 (-, q, OCH3), 36.4 (-, q, CH3), 35.4 (-, q, CH3), 31.9 (-, q, Bzi-CH3). ES-HRMS: m/z calcd for C20H20N7O4 + (MH+) 422.1577, found 422.1556 (100%), 423.1574 (14%, M+2); EI-HRMS: m/z calcd for C20H19N7O4 (M+) 421.1499. found 421.1504 (10%), 377.1587 (100%), 245.0919 (70%), 133.0766 (18%). [Bzi-ImPy-COOH] (75). The preceding ester derivative 74 was hydrolysed with NaOH in the same manner as described for 39. The final product obtained as a white solid was washed with acetone and dried (yield = 68%). 1H-NMR (DMSOd6) δlθ.46 (s, exch, IH, NH), 10.32 (s, exch, IH, NH), 7.79 (d, IH, J = 7.8 Hz, Bzi-CH), 7.73 (d, IH, J = 7.8 Hz, Bzi-CH), 7.65 (s, IH, Im-CH), 7.48 (d, IH, J = 1.5 Hz, Py- CH), 7.43 (t, IH, J = 7.2 Hz, Bzi-CH), 7.36 (t, IH, J = 7.2 Hz, Bzi-CH), 6.98 (d, IH, J = 1.5 Hz, Py-CH), 4.19 (s, 3H, NCH3), 4.03 (s, 3H, NCH3), 3.83 (s, 3H, NCH3); 13C- NMR (75 MHz, DMSOd6) 6161.9 (+, s, CO), 156.3 (+, s, CO), 155.5 (+, s, CO), 142.9 (+, s, Bzi-C2), 140.6 (+, s, Bzi-C3a), 140.5 (+, s, Bzi-C7a), 136.9 (+, s, Im-C2), 134.5 (+, s, Im-C4), 124.5 (-, d, Bzi-C6), 123.3 (-, d, Bzi-C5), 121.8 (+, s, Py-C2), 120.5 (-, d, Py-C5), 120.3 (+, s, Py-C4), 119.8 (-, d, Bzi-C4), 114.6 (-, d, Im-C5), 111.4 (-, d, Bzi-C7), 108.8 (-, d, Py-C3), 36.3 (-, q, CH3), 35.2 (-, q, CH3), 31.8 (-, q, Bzi- CH3). EI-HRMS: m/z calcd for C20Hi9N7O4 421.1499, found 421.1493 (M+, 100%), 377.1446 (M-CO2, 80%), 339.0833 (20%), 295.0919 (30%).
Im-Bzi-Py-NO2: 5-(Imidazol- 1 -yl)-2-( 1 -methyl-4-nitro- 1 H-pyrrol-2-yl)- 1 H- benzimidazole (76) was prepared in 78% yield using the diamine derivative 8 and compound 20 in the general procedure C given above. 1H-NMR (300 MHz, DMSO- d6) 68.94 (s, IH, Im-CH), 7.36 (s, IH, Py-CH), 7.15 (s, IH), 7.15-7.04 (m, 3H), 6.82 (dd, IH, Bzi-CH), 6.32 (s, IH, Im-CH), 3.96 (s, 3H, CH3). FAB-HRMS: m/z calcd for Ci5H)3N6O2 + (MH+) 309.1 100, found 309.1072 (100%).
Im-Bzi-Py-NH2: 2-(4- Amino- 1 -methyl- 1 H-pyrrol-2-yl)-5-(imidazol- 1 -yl)- lH-benz-imidazole (77): catalytic reduction of the nitro compound. 1H-NMR (HCl salt, D2O) 69.1 1 (s, IH, Im-CH), 7.84 (s, IH, Im-CH), 7.81 (s, IH, Im-CH), 7.73 (d, IH, J = 8.7 Hz, Bzi-CH), 7.61 (s, IH, Py-CH), 7.49 (dd, IH, J = 2.1 & 8.7 Hz, Bzi- CH), 7.16 (d, IH, J = 2.1 Hz, Bzi-CH), 6.85 (s, IH, Py-CH), 3.94 (s, 3H, NCH3); EI- HRMS: m/z calcd for Ci5H14N6278.1280, found 278.1249 (100%, M+). Im-Py-Bzi-Gly-OEt (78): Glycine ethyl ester (2 mmol) was added to a mixture of acid derivative 70 (1.5 mmol), HOBt (1.7 mmol), EDCI (1.6 mmol) and DIPEA (4 mmol), all added neat in the order as written to 10 mL DMF. The resultant mixture was stirred at 45 0C for 16 h, and allowed to cool afterwards. The mixture was evaporated to dryness and treated (for washings) with H2O, followed by acetone and CHCl3. The remaining residue was purified by silica gel column chromatography (4: 1 CHCl3 :MeOH to MeOH gradient elution) to afford the title product (68% yield). 1H-NMR (CDCl3) 69.32 (s, exch, IH, NH), 8.01 (s, IH, Bzi-CH), 7.57 (d, IH, J = 7.8 Hz, Bzi-CH), 7.46 (d, IH, J = 7.8 Hz, Bzi-CH), 7.40 (t, exch, IH, NH), 7.24 (d, IH, J = 1.8 Hz, Py-CH), 6.99 (s, IH, Im-CH), 6.95 (s, IH, Im-CH), 6.81 (d, IH, J = 1.8 Hz, Py-CH), 4.24 (q, 2H, J = 7.2 Hz, OCH2), 4.19 (d, 2H, gly-CH2), 4.02 (s, 3H, Im-CH3), 3.97 (s, 3H, Py-CH3), 1.26 (t, 3H, J = 7.2 Hz, CH3).
Im-Py-Bzi-Beta-OEt (79): Beta-alanine ethyl ester hydrochloride was used instead of glycine in the procedure given for the preceding compound. Titled product was obtained similarly (72% yield). 1H-NMR (CDCl3) 69.30 (s, exch, IH, NH), 7.97 (s, IH, Bzi-CH), 7.55 (d, IH, J = 7.8 Hz, Bzi-CH), 7.47 (d, IH, J = 7.8 Hz, Bzi- CH), 7.41 (t, exch, IH, NH), 7.22 (d, IH, J = 1.8, Py-CH), 6.96 (s, IH, Im-CH), 6.91 (s, IH, Im-CH), 6.83 (d, IH, J = 1.8 Hz, Py-CH), 4.15 (q, 2H, J = 7.2 Hz, OCH2),
4.01 (s, 3H, Im-CH3), 3.95 (s, 3H, Py-CH3), 3.71 (d, 2H, β-CH2), 2.65 (t, 2H, β-CH2), 1.31 (t, 3H, J = 7.2 Hz, CH3). ES-HRMS: m/z calcd for C23H26N7O4 + (MH+) 464.2046, found 464.2045 (100%), 927.4165 (6%, M2H+); EI-HRMS: m/z calcd for C23H25N7O4 (M+) 463.1968, found 463.1976 (100%), 381.1439 (40%), 265.0728 (40%), 145.0400 (17%).
Im-Py-Bzi-Gaba-OEt (80): Ethyl 4-aminobutyrate was used as the amine substrate for the coupling reaction protocol described above for 78 and 79. The product was isolated as in those cases (58% yield). 1H-NMR (CDCl3) 69.37 (s, exch, IH, NH), 7.97 (s, IH, Bzi-C4H), 7.54 (d, IH, J = 7.8 Hz, Bzi-C6H), 7.45 (d, IH, J = 7.8 Hz, Bzi-C7H), 7.29 (d, IH, J = 1.8 Hz, Py-CH), 7.13 (t, exch, IH, NH), 6.97 (s, IH, Im-CH), 6.92 (s, IH, Im-CH), 6.80 (d, IH, J = 1.8 Hz, Py-CH), 4.09 (q, 2H, J =
7.2 Hz, OCH2), 4.01 (s, 3H, Im-CH3), 3.96 (s, 3H, Py-CH3), 3.47 (q, 2H, γ-NCH2), 2.42 (t, 2H, J = 7.5 Hz, γ-COCH2), 1.95 (qnt, 2H, J = 7.5 Hz, γ-CH2), 1.21 (t, 3H, J = 7.2, CH3). 13C-NMR (75 MHz, CD3OD) 6174.1, 169.3, 169.2, 156.1, 147.9, 140.8, 138.6, 128.4, 127.3, 126.5, 126.1, 121.9, 120.8, 120.7, 118.3, 114.0, 103.8, 60.7, 39.4, 36.4, 35.5, 31.7, 24.6, 13.8. ES-HRMS: m/z calcd for C24H28N7O4 + (MH+) 478.2197, found 478.2199 (100%), 347.1287 (12%), 174.0662 (35%).
Im-Py-Bzi-Gly-OH (81): LiOH mediated hydrolysis, as described for aliphatic linker diesters in Chapter 1, was used for compound 78 to afford the title product after washing the reaction residue with 'PrOH (85% yield). 1H-NMR (DMSO-d6) 610.63 (s, exch, IH, NH), 8.84 (t, exch, IH, NH), 8.09 (s, IH, Bzi-CH), 7.75 (d, IH, J = 8.1 Hz, Bzi-CH), 7.57 (d, IH, J = 8.1 Hz, Bzi-CH), 7.44 (d, IH, J = 1.8 Hz, Py-CH), 7.43 (s, IH, Im-CH), 7.25 (d, IH, J = 1.8 Hz, Py-CH), 7.09 (s, IH, Im-CH), 4.08 (s, 3H, Im-CH3), 4.01 (s, 3H, Py-CH3), 3.94 (q, 2H, G-NCH2). ES-HRMS: m/z calcd for C20H20N7O4 + (MH+) 422.1577, found 422.1566 (100%), 843.3197 (5%, M2H+), 314.1257 (10%).
Im-Py-Bzi-Beta-OH (82). This was obtained from 79 as in the preceding example (80% yield). 1H-NMR (DMSOd6) δlθ.64 (s, exch, IH, NH), 8.53 (t, exch, IH, NH), 8.06 (s, IH, Bzi-CH), 7.69 (d, IH, J = 8.1 Hz, Bzi-CH), 7.52 (d, IH, J = 8.1 Hz, Bzi-CH), 7.44 (d, IH, J = 1.8 Hz, Py-CH), 7.43 (s, IH, Im-CH), 7.25 (d, IH, J = 1.8 Hz, Py-CH), 7.09 (s, IH, Im-CH), 4.07 (s, 3H, Im-CH3), 4.01 (s, 3H, Py-CH3), 3.48 (q, 2H, β-NCH2), 2.55 (t, 2H, β-COCH2). ES-HRMS: m/z calcd for C21H22N7O4 + (MH+) 436.1733, found 436.1713 (100%), 239.0965 (15%).
Im-Py-Bzi-Gaba-OH (83) was similarly obtained using LiOH hydrolysis of 80. (75% yield). 1H-NMR (DMSO-d6) δlθ.57 (s, exch, IH, NH), 8.45 (t, exch, IH, NH), 8.06 (s, IH, Bzi-CH), 7.71 (d, IH, J = 8.1 Hz, Bzi-CH), 7.53 (d, IH, J = 8.1 Hz, Bzi-CH), 7.42 (d, IH, J = 1.8 Hz, Py-CH), 7.41 (s, IH, Im-CH), 7.22 (d, IH, J = 1.8 Hz, Py-CH), 7.06 (s, IH, Im-CH), 4.08 (s, 3H, Im-CH3), 4.01 (s, 3H, Py-CH3), 3.29 (q, 2H, Y-NCH2), 2.29 (t, 2H, γ-COCH2), 1.77 (qnt, 2H, γ-CH2). General Procedure F. Typically, an acid derivative (1 mmol) was added to DMF (5 mL), to which the following were added in the order as written: HOBt (1.2 mmol), a carbodiimide (either EDCI or DIC, 1.1 mmol), and DIPEA (2 mmol). The resultant suspension was stirred at 45 0C for 3-4 h. After that period, a solution of the amine derivative (1 mmol) in 5 mL DMF was added and the reaction mixture was stirred at 60 0C for an additional 10-16 h. At the end of that period, the mixture was concentrated or evaporated to dryness to remove most of DMF, followed by addition of CH3CN to the contents that resulted in a suspension. Filtration of the material afforded solid residue that was subjected to silica gel column chromatography (eluting the material with a gradient system from 4:1 CHCl3 :MeOH to neat MeOH and finally with 2-4% NH4OH in MeOH). The final products are quite polar and the initial fractions from the column (that were discarded) contained the urea and HOBt. Fractions containing the products were pooled together and evaporated. After an initial characterization and confirming the presence of products, the material was extensively lyophilized to remove traces Of NH3. Final purification of the products entailed conversion to the hydrochloride salt forms by treatment with methanolic HCl and crystallization (MeOH/Et2O or MeOH/acetone binary solvent mixtures). Final product yields are unoptimized since our foremost requirement was recovery of as much material of high purity (by 1H-NMR) as possible. Note: Free base forms were usually dissolved in DMSOd6 or CD3OD for characterization; the former generally gave better resolution spectra but the attendant problems due to mixtures of tautomers for the benzimidazole ring were overcome with fast-exchanging solvent (CD3OD) or by addition of small aliquots of either acid (CF3COOD) or base (1,4- dimethylpiperazine). The C-13 NMR and HMBC/QC experiments were performed only on representative members of each family of compounds due to limited solubility of these compounds and impossible recovery from solution states when dissolved in DMSO-d6. The HCl salt forms were readily soluble in D2O or CD3OD for ease in analysis.
The following first series of final products (F1-F4) were all prepared from the same amine (49) and different acid derivatives listed individually for each data set below. Mp-Bzi-Py-Im (F-I) was prepared from 2-trichloroacetyl-l-methylimidazole and amine 49 treated together in CH3CN and according to the isolation methodlogy in general procedure F above. Recovery 56%. 1H-NMR (500 MHz, HCl salt in CD3OD) 67.61 (d, IH, Bzi-C7H), 7.56 (s, 2H, 2 x Im-CH), 7.48 (s, IH, Py-C5H), 7.25 (d, IH, BzI-C6H), 7.23 (s, IH, Bzi-C4H), 7.1 1 (s, IH, Py-C3H), 4.03 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.82 (m, 2H, CH2), 3.61 (m, 2H, CH2), 3.2 (m, 4H, CH2), 2.93 (s, 3H, CH3). (note the assignment of singlets for Py/Im-CH may be ambiguous). 13C- NMR (125 MHz, HCl salt in CD3OD) 6153.0 (+), 150.3 (+), 142.0 (+), 137.3 (+), 133.6 (+), 127.9, 127.1 (+), 124.0, 123.7 (+), 122.3, 119.2, 115.9 (+), 115.4, 110.0, 101.0, 54.5 (+), 43.9 (-), 37.4 (-), 37.0 (-). ES-HRMS: m/z calcd for C22H26N8O 418.2230, found 418.2981 (M+, 100%), 416.2827 (54%, M-2), 417.2919 (40%, M-I), 419.2964 (50%, M+l), 420.3125 (14%, M+2). Mp-Bzi-Py-Py-Im (F-2) was prepared using amine 49, acid 35 and EDCI/HOBt using the general procedure F. Recovery 44%. 1H-NMR HCl salt (500 MHz, CD3OD) 67.79 (s, IH, Im-C5H), 7.72 (s, IH, Im-C4H), 7.68 (d, IH, J = 8.5 Hz, BZi-C7H), 7.62 (s, IH, Py-C5H), 7.43 (s, IH, Py-C5H), 7.35 (d, IH, J = 8.5 Hz, Bzi- C6H), 7.30 (s, IH, Bzi-C4H), 7.29 (d, IH, J = 1.8 Hz, Py-C3H), 7.15 (d, IH, J = 1.8 Hz, Py-C3H). 13C-NMR (HCl salt in D2O) 6157.2, 151.0, 147.9, 139.4, 135.0, 131.2, 127.0, 124.5, 123.8, 123.4, 121.2, 119.2, 118.8, 115.6, 1 13.3, 112.2, 107.3, 103.5, 98.1, 53.3, 46.4, 43.1, 36.9, 36.7, 36.5. ES-HRMS: m/z calcd for C28H32Ni0O3 540.2710, found 540.4717 (M+, 100%), 538.4405 (84%, M-2), 539.4510 (58%, M-I), 541.4715 (70%, M+l), 542.4916 (18%, M+2).
Mp-Bzi-Py-Py-Py-Im (F-3) was prepared using amine 49, acid 38 and DIC/HOBt using the general procedure F. Recovery 38%. 1H-NMR (500 MHz, DMSO-de) 612.27 (s, exch, IH, NH), 10.45 (s, exch, IH, NH), 10.01 (s, exch, IH, NH), 9.99 (s, exch, IH, NH), 7.42 (d, IH, BzJ-C7H), 7.40 (s, IH, Im-C5H), 7.31 (m, 2H, Im-C4H + Py-C5H), 7.27 (s, IH, Py-C5H), 7.19 (s, IH, Py-C5H), 7.09 (s, IH, Py- C3H), 7.05 (s, IH, Py-C3H), 6.96 (s, IH, Py-C3H), 6.90 (d, IH, BzI-C6H), 6.87 (s, IH, Bzi-C4H), 4.1 1 (s, 3H), 4.04 (s, 6H, 2 x CH3), 3.88 (s, 3H, CH3), 3.81 (s, 3H, CH3), 2.87 (m, 4H, CH2), 2.60-2.37 (m, 4H, CH2), 2.24 (s, 3H, CH3). 1H-NMR (HCl salt in CD3OD) 67.70 (s, IH, Im-CH), 7.64 (m, 2H, Bzi-C7H + Py-C5H), 7.60 (s, IH, Im- CH), 7.54 (d, IH, J = 1.8 Hz, Py-C5H), 7.36 (d, IH, J = 1.8 Hz, Py-C5H), 7.29 (d, IH, J = 9 Hz, Bzi-C6H), 7.22 (2H, Py-C3H + Bzi-C4H), 7.03 (d, IH, J = 1.8 Hz, Py-C3H), 7.01 (d, IH, J = 1.8 Hz, Py-C3H). FAB-HRMS: m/z calcd for C34H39Ni2O3 + (MH+) 663.3268, found 663.3284 (48%);
Mp-Bzi-Py-Py-Im-Im (F-4) was prepared using amine 49, acid 39 and EDCI/HOBt using the general procedure F. Recovery 54%. 1H-NMR (CD3OD/D2O) 67.69 (d, IH, J = 8.7, Bzi-C7H), 7.57 (s, IH, Im-C5H), 7.54 (s, IH, Im-C4H), 7.45 (s, IH, Py-C5H), 7.41 (s, IH, Im-C5H), 7.37 (d, IH, Bzi-C6H), 7.31 (s, IH, Bzi-C4H), 7.28 (s, IH, Py-C3H), 7.07 (s, IH, Py-C3H), 4.03 (s, 3H, CH3), 4.01 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.72 (t, 4H, CH2), 2.92 (t, 4H, CH2), 2.71 (s, 3H, CH3). ES-HRMS: m/z calcd for C33H37NnO3 663.3142, found 663.5617 (M+, 100%), 662.5340 (12%, M-I), 664.5888 (72%, M+l), 665.5561 (40%, M+2). The following series of final products (F5-F7) were all prepared from the same amine (53) and different acid derivatives listed individually for each data set below.
Mp-Bzi-Py-Im-Im (F-5) was prepared using amine 53, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 62%. IH-NMR (300 MHz, DMSO-d6) D 12.29 (s, exch, IH, NH), 9.62 (s, exch, IH, NH), 9.04 (s, each, IH, NH), 7.48 (s, IH, Im-C5H), 7.29 (d, IH, J = 8.1 Hz, Bzi-C7H), 7.25 (s, IH, Im-C4H), 7.03 (s, IH, Py-C5H), 6.97 (2 x s, IH each, Bzi- C4H + Im-C4H), 6.86 (d, IH, J = 8.1 Hz, Bzi-C6H), 6.68 (s, IH, Py-C3H), 4.06 (s, 3H, CH3), 4.03 (s, 3H, CH3), 4.02 (s, 3H, CH3), 3.22 (m, 4H, CH2), 2.77 (m, 4H, CH2), 2.46 (s, 3H, CH3). ES-HRMS: m/z calcd for C27H31N11O2 541.2662, found 541.2991 (M+, 100%), 542.3008 (40%, M+l), 543.3020 (14%, M+2).
Mp-Bzi-Py-Im-Py-Im (F-6) was prepared using amine 53, acid 35 and EDCI/HOBt using the general procedure F. Recovery 52%. The alternative combination of amine 49 and acid 47 (with EDCI/HOBt) gave lower recovered yields (10-14%). 1H-NMR (300 MHz, CD3OD) 67.40 (d, IH, J = 8.7 Hz, Bzi-C7H), 7.32 (s, IH, Im-C5H), 7.24 (d, IH, J = 1.6 Hz, Py-C5H), 7.23 (d, IH, J = 1.6 Hz, Py-C5H), 7.15 (s, IH, Im-C5H), 7.01 (s, IH, Bzi-C4H), 6.96 (s, IH, Im-C4H), 6.94 (d, IH, J = 8.7 Hz, Bzi-C6H), 6.92 (d, IH, J = 1.6 Hz, Py-C3H), 6.76 (d, IH, J = 1.6 Hz, Py- C3H), 3.97 (2 x s, 3H each, 2 x CH3), 3.91 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.14 (m, 4H, CH2), 2.65 (m, 4H, CH2), 2.34 (s, 3H, CH3). ES-HRMS: m/z calcd for C33H37N13O3 663.3142, found 663.4462 (M+, 100%), 661.4217 (48%, M-2), 662.4875 (28%, M-I), 664.4494 (62%, M+l), 665.4199 (12%, M+2).
Mp-Bzi-Py-Im-Im-Im (F-7) was prepared using amine 53, acid 44 and DIC/HOBt using the general procedure F. Recovery 22%. 1H-NMR (HCl salt, CD3OD/D2O) 67.70 (d, IH, J = 8.7, Bzi-C7H), 7.61 (s, IH, Im-C5H), 7.56 (s, IH, Im- C4H), 7.50 (s, IH, Im-C5H), 7.45 (s, IH, Im-C5H), 7.40 (d, IH, J = 1.6 Hz, Py-C5H), 7.31 (d, IH, Bzi-C6H), 7.27 (s, IH, Bzi-C4H), 7.09 (s, IH, Py-C3H), 4.04 (s, 3H, CH3), 4.00 (2 x s, 6H, 2 x CH3), 3.91 (s, 3H, CH3), 3.72 (t, 4H, CH2), 2.94 (t, 4H, CH2), 2.89 (s, 3H, CH3). ES-HRMS: m/z calcd for C32H36N14O3 664.3095, found 664.3101 (M+, 100%), 665.3135 (30%, M+l), 666.3166 (10%, M+2). The following first series of final products (G1-G4) were all prepared from the same amine (50) and different acid derivatives listed individually for each data set below.
Mp-Pzi-Py-Im (G-I) was prepared using amine 50, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 70%. 1H-NMR (HCl salt in CD3OD) 67.95 (d, IH, J = 9, Pzi-C7H), 7.67 (d, IH, J = 1.2, Py- C5H), 7.61 (s, IH, Im-C5H), 7.51 (s, IH, Im-C4H), 7.22 (d, IH, J = 1.2, Py-C3H), 7.15 (d, IH, J = 9, Pzi-C6H), 4.57 (d, 2H, CH2), 4.09 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.59 (d, 2H, CH2), 3.40-3.15 (m, 4H), 2.92 (s, 3H, CH3). ES-HRMS: m/z calcd for C21H25N9O 419.2182, found 419.2187 (M+, 100%), 420.2245 (44%, M+l), 421.2279 (18%, M+2).
Mp-Pzi-Py-Py-Im (G-2) was prepared using amine 50, acid 35 and EDCI/HOBt using the general procedure F. Recovery 56%. 1H-NMR (500 MHz, CD3OD) 67.68 (d, IH, J = 8.7, Pzi-C7H), 7.24 (d, IH, Py-C5H), 7.19 (d, IH, Py- C5H), 7.17 (s, IH, Im-C5H), 6.98 (s, IH, Im-C4H), 6.88 (d, IH, Py-C3H), 6.79 (d, IH, Py-C5H), 6.74 (d, IH, J = 8.7, Pzi-C6H), 3.98 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.86 (s, 3H, CH3), 3.64 (m, 4H, CH2), 2.88 (m, 4H, CH2), 2.52 (s, 3H, CH3). 1H- NMR (500 MHz, HCl salt in CD3OD) 68.02 (d, IH, J = 9, Pzi-C7H), 7.74 (s, IH, Im- C5H), 7.72 (s, IH, Im-C4H), 7.63 (s, IH, Py-C5H), 7.42 (s, IH, Py-C5H), 7.25 (s, IH, Py-C3H), 7.18 (d, IH, J = 9, Pzi-C6H), 7.12 (s, IH, Py-C3H), 4.60 (d, 2H, CH2), 4.18 (s, 3H, CH3), 4.06 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.65 (d, 2H, CH2), 3.41 (m, 2H, CH2), 3.25 (m, 2H, CH2), 2.98 (s, 3H, CH3). ES-HRMS: m/z calcd for C27H3INnO2 541.2662, found 541.4320 (M+, 100%), 539.4316 (26%, M-2), 540.4044 (15%, M-I), 542.4469 (52%, M+l), 543.5817 (14%, M+2) Mp-Pzi-Py-Py -Py-Im (G-3) was prepared using amine 50, acid 38 and
DIC/HOBt using the general procedure F. Recovery 52%. 1H-NMR (500 MHz, DMSO-d6) 612.81 (s, exch, IH, NH), 10.43 (s, exch, IH, NH), 10.04 (s, exch, IH, NH), 10.02 (s, exch, IH, NH), 8.04 (d, IH, J = 9 Hz, Pzi-C7H), 7.74 (s, IH, Im-C5H), 7.66 (s, IH, Im-C4H), 7.60 (s, IH, Py-C5H), 7.46 (s, IH, Py-C5H), 7.31 (s, IH, Py- C3H), 7.18 (s, IH, Py-C5H), 7.12 (s, IH, Py-C3H), 7.04 (s, IH, Py-C3H), 6.94 (d, IH, J = 9 Hz, Pzi-C6H), 4.02 (s, 3H, CH3), 3.95 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.81 (s, 3H, CH3), 3.21 (m, 4H, CH2), 2.57 (m, 4H, CH2), 2.21 (s, 3H, CH3). ES-HRMS: m/z calcd for C33H37N13O3 663.3142, found 663.3195 (M+, 100%), 664.3229 (52%, M+l), 664.3268 (10%, M+2).
Mp-Pzi-Py-Py -Im-Im (G-4) was prepared using amine 49, acid 39 and EDCI/HOBt using the general procedure F. Recovery 46%. 1H-NMR (DMSOd6) 67.74 (d, IH, J = 8.7, Pzi-CH), 7.58 (s, IH, Im-CH), 7.46 (s, IH, Im-CH), 7.35 (s, IH, Py-CH), 7.30 (s, IH, Py-CH), 7.19 (s, IH, Py-CH), 7.03 (s, IH, Im-CH), 6.93 (s, IH, Py-CH), 6.72 (d, IH, J = 8.7, Pzi-CH), 4.05 (s, 3H, CH3), 4.02 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.87 (s, 3H, CH3), 3.73 (t, 4H, CH2), 2.46 (t, 4H, CH2), 2.25 (s, 3H, CH3). 1H-NMR (500 MHz, HCl salt in CD3OD) 68.03 (d, IH, J = 9 Hz, Pzi-C7H), 7.76 (s, IH, Im-C5H), 7.64 (s, IH, Im-C4H), 7.53 (d, IH, Py-C5H), 7.48 (s, IH, Im-C5H), 7.37 (d, IH, Py-C3H), 7.21 (d, IH, Py-C5H), 7.11 (d, IH, Py-C3H), 6.90 (d, IH, J = 9 Hz, Pzi-C6H), 4.60 (d, 2H, CH2), 4.18 (s, 3H, CH3), 4.06 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.65 (d, 2H, CH2), 3.41 (m, 2H, CH2), 3.25 (m, 2H, CH2), 2.98 (s, 3H, CH3). ES-HRMS: m/z calcd for C32H36N14O3 664.3095, found 664.3142 (M+, 100%), 665.3178 (30%, M+l), 666.3212 (18%, M+2).
The following final products (G5-G7) were all prepared from the same amine (54) and different acid derivatives listed individually for each data set below.
Mp-Pzi-Py-Im-Im (G-5) was prepared using amine 54, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 66%. 1H-NMR (300 MHz, DMSO-d6) 612.29 (s, exch, IH, NH), 9.62 (s, exch, IH, NH), 9.04 (s, each, IH, NH), 7.71 (d, IH, J = 8.8 Hz, Pzi-C7H), 7.59 (s, IH, Im-C5H), 7.48 (s, IH, Im-C4H), 7.18 (d, IH, J = 1.8 Hz, Py-C5H), 7.03 (s, IH, Im-C5H), 6.96 (d, IH, J = 1.8 Hz, Py-C3H), 6.70 (d, IH, J = 8.8 Hz, Pzi-C6H), 4.03 (s, 3H, CH3), 4.01 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.28 (m, 4H, CH2), 2.68 (m, 4H, CH2), 2.29 (s, 3H, CH3). ES-HRMS: m/z calcd for C26H30N12O2 542.2615, found 542.2998 (M+, 100%), 543.3014 (40%, M+l), 544.3028 (8%, M+2).
Mp-Pzi-Py-Im- Py-Im (G-6) was prepared using amine 54, acid 35 and EDCI/HOBt using the general procedure F. Recovery 45%. 1H-NMR (CD3OD) 67.68 (d, IH, J = 9 Hz, Pzi-C7H), 7.30 (d, IH, J = 1.5 Hz, Py-C5H), 7.26 (d, IH, J = 1.5 Hz, Py-C5H), 7.23 (s, IH, Im-C5H), 7.19 (s, IH, Im-C5H), 7.14 (s, IH, Im-C4H), 6.88 (d, IH, J = 1.5 Hz, Py-C3H), 6.76 (d, IH, J = 9 Hz, Pzi-CH), 6.72 (d, IH, J = 1.6 Hz, Py-C3H), 3.97 (2 x s, 3H each, 2 x CH3), 3.91 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.14 (m, 4H, CH2), 2.65 (m, 4H, CH2), 2.34 (s, 3H, CH3). ES-HRMS: m/z calcd for C32H36Ni4O3 664.3095, found 664.5026 (M+, 38%), 662.51 18 (45%, M-2), 663.5078 (36%, M-I), 665.5318 (100%, M+l), 666.5395 (42%, M+2), 667.5448 (8%, M+3).
Mp-Pzi-Py-Im-Im-Im (G-7) was prepared using amine 53, acid 44 and DIC/HOBt using the general procedure F. Recovery 18%. 1H-NMR (CD3OD) 67.72 (d, IH, J = 9 Hz, Pzi-C7H), 7.38 (s, IH, Im-C5H), 7.23 (d, IH, J = 1.5 Hz, Py- C5H), 7.18 (s, IH, Im-C5H), 7.09 (s, IH, Im-C5H), 6.96 (s, IH, Im-C4H), 6.77 (d, IH, J = 9 Hz, Pzi-CH), 6.71 (d, IH, J = 1.6 Hz, Py-C3H), 3.99 (2 x s, 3H each, 2 x CH3), 3.96 (s, 3H, CH3), 3.91 (s, 3H, CH3), 3.18 (m, 4H, CH2), 2.68 (m, 4H, CH2), 2.31 (s, 3H, CH3). ES-HRMS: m/z calcd for C3iH35Ni5O3 665.3047, found 665.3991 (M+, 100%), 666.4008 (41%, M+l), 667.4020 (6%, M+2).
The following final products (H1-H4) were all prepared from the same amine (72) and different acid derivatives listed individually for each data set below.
Dp-Bzi-Py-Im (H-I) was prepared using amine 72, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 58%. 1H-NMR (HCl salt, CD3OD) 68.39 (s, IH, Bzi-C4H), 8.03 (d, IH, J = 8.6, Bzi-C6H), 7.88 (d, IH, J = 8.6, Bzi-C7H), 7.63 (d, IH, J = 1.7 Hz, Py-C5H), 7.58 (s, IH, Im- C5H), 7.44 (d, IH, J = 1.7 Hz, Py-C3H), 7.21 (s, IH, Im-C4H), 4.13 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.57 (t, 2H, J = 6.5 Hz, C CH2), 3.27 (t, 2H, J = 7.5 Hz, CH2), 2.96 (s, 6H, 2 x CH3), 2.10 (qnt, 2H, CH2). ES-HRMS: m/z calcd for C23H28N8O2 448.2335, found 448.3588 (M+, 10%), 447.3455 (12%, M-I), 449.3738 (100%, M+l), 450.3937 (44%, M+2). Dp-Bzi-Py-Py-Im (H-2) was prepared using amine 72, acid 35 and DIC/HOBt using the general procedure F. Recovery 36%. 1H-NMR (D2O, note all signals were broadened) 67.54 (s, IH, Bzi-C4H), 7.24 (m, 2H, 2 x Bzi-CH), 7.03 (s, IH, Im-C5H), 6.93 (s, IH, Im-C4H), 6.83 (s, IH, Py-C5H), 6.66 (d, IH, Py-C5H), 6.60 (s, IH, Py- C3H), 6.18 (d, IH, Py-C3H), 3.58-3.53 (3 x s, 3H each, 3 x CH3), 3.18-3.13 (m, 4H, CH2), 2.90 (s, 6H, CH3), 1.94 (m, 2H, CH2). ES-HRMS: m/z calcd for C29H34Ni0O3 570.2815, found 570.4726 (M+, 14%), 569.4786 (22%, M-I), 571.5069 (100%, M+l), 572.5124 (35%, M+2).
Dp-Bzi-Py-Im-Im (H-3) was prepared using amine 72, acid 47 and DIC/HOSu using the general procedure F. Recovery 25%. 1H-NMR (500 MHz, CD3OD) 68.12 (s, IH, Bzi-CH), 7.74 (d, IH, J = 8.2, Bzi-CH), 7.58 (br d, IH, Bzi-CH), 7.48 (s, IH), 7.37 (s, IH), 7.27 (s, IH), 7.08 (s, IH), 6.96 (s, IH), 4.08 (s, 3H, CH3), 4.06 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.53 (t, 2H, CH2), 3.03 (t, 2H, CH2) 2.73 (s, 6H, 2 x CH3), 2.06 (qnt, 2H, CH2). 1H-NMR (500 MHz, HCl salt, CD3OD) 08.33 (s, IH, Bzi-C4H), 8.10 (dd, IH, J = 8.6 & 1.5 Hz, Bzi-C6H), 7.86 (d, IH, J = 8.6, Bzi-C7H), 7.78 (s, IH, Im-C5H), 7.76 (s, IH, Im-C5H), 7.71 (s, IH, Im-C4H), 7.68 (s, IH, Py-C5H), 7.46 (d, IH, J = 1.7 Hz, Py-C3H), 4.20 (s, 3H, CH3), 4.13 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.57 (t, 2H, J = 6.5 Hz, CH2), 3.27 (t, 2H, J = 7.5 Hz, CH2), 2.96 (s, 6H, 2 x CH3), 2.10 (qnt, 2H, CH2). ES-HRMS: m/z calcd for C28H33NnO3 571.2768, found 571.2994 (M+, 100%), 572.3024 (54%, M+l), 573.3062 (17%, M+2). Dp-Bzi-Py-Py -Im-Im (H-4) was prepared using amine 72, acid 39 and
EDCI/HOBt using the general procedure F. Recovery 46%. 1H-NMR (500 MHz, CD3OD) 68.31 (s, IH, Bzi-C4H), 8.13 (dd, IH, J = 8.6 Hz, Bzi-C6H), 7.89 (d, IH, J = 8.6, Bzi-C7H), 7.74 (s, IH, Im-C5H), 7.68 (s, IH, Im-C4H), 7.60 (d, IH, J = 1.2 Hz, Py-C5H), 7.54 (s, IH, Im-C5H), 7.41 (d, IH, J = 1.1 Hz, Py-C5H), 7.36 (d, IH, J = 1.2 Hz, Py-C3H), 7.22 (d, IH, J = 1.1 Hz, Py-C3H), 4.18 (s, 3H, CH3), 4.13 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.87 (s, 3H, CH3), 3.55 (t, 2H, J = 6.5 Hz, CH2), 3.24 (t, 2H, J = 7.5 Hz, CH2), 2.97 (s, 6H, 2 x CH3), 2.08 (qnt, 2H, CH2). ES-HRMS: m/z calcd for C34H39NnO4 693.3248, found 693.3257 (M+, 100%), 694.3288 (44%, M+l), 695.3328 (10%, M+2). The following two products (Il and 12) were all prepared from the same amine
(72) and different acid derivatives listed individually for each data set below.
Mp-Bzi-Bzi-Py-Im (1-1) was prepared using amine 62, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 68%. 1H-NMR (CD3OD) 68.12 (s, IH, Bzi-CH), 7.89 (d, IH, J = 7.8, Bzi- CH), 7.68 (s, IH, Im-C5H), 7.62 (d, IH, J = 7.8, Bzi-CH), 7.43 (d, IH, J = 8.4, Bzi'- CH), 7.32 (s, IH, Py-C5H), 7.15 (s, IH, Im-C4H), 7.11 (s, IH, Bzi'-CH), 6.98 (d, IH, J = 8 Hz, Bzi-CH), 6.85 (s, IH, Py-C3H), 4.03 (s, 3H, CH3), 3.96 (s, 3H, CH3), 3.18 (t, 4H, CH2), 2.68 (t, 4H, CH2), 2.32 (s, 3H, CH3). FAB-HRMS: m/z calcd for C29H31N10O + (MH+) 535.2682, found 535.2668.
Mp-Bzi-Bzi-Py-Im-Im (1-2) was prepared using amine 62, acid 44 and DIC/HOBt using the general procedure F. Recovery 26%. 1H-NMR (CD3OD) 68.13 (s, IH, Bzi-CH), 7.83 (d, IH, J = 7.8, Bzi-CH), 7.57 (d, IH, J = 7.8, Bzi-CH), 7.42 (d, IH, J = 8.4, Bzi'-CH), 7.39 (s, IH, Im'-CH), 7.28 (s, IH, Py-CH), 7.18 (s, IH, Im- CH), 7.04 (s, IH, Bzi'-CH), 6.99 (s, IH, Im-CH), 6.95 (dd, IH, J = 8.4 & 1.2, Bzi'- CH), 6.87 (s, IH, Py-CH), 3.99 (s, 3H, Im-NCH3), 3.97 (s, 3H, InT-NCH3), 3.92 (s, 3H, Py-NCH3), 3.14 (t, 4H, CH2), 2.59 (t, 4H, CH2), 2.29 (s, 3H, NCH3). 13C-NMR (free base, CD3OD) 6156.4 (+), 156.3 (+), 152.8 (+), 148.4 (+), 148.1 (+), 138.6 (+), 135.8 (+), 134.6 (+), 127.8 (-), 126.7 (-), 124.3 (+), 122.7 (+), 121.2 (-), 120.9 (+), 1 18.4 (-), 1 15.2 (-), 114.4 (-), 1 12.7 (-), 109.9 (-), 104.3 (-), 101.0 (-), 55.2 (+), 50.8 (+), 45.2 (-), 36.1 (-), 35.2 (-), 35.0 (-). FAB-HRMS: m/z calcd for C34H36N13O2 + (MH+) 658.3115, found 658.3106 (100%).
The following final products (J1-J4) were all prepared from the given combination of amine and acid derivatives listed individually for each data set below. Mp-Bzi-Py-Bzi-Im (J-I) was prepared using amine 49, acid 64 and DIC/HOBt using the general procedure F. Recovery 40%. 1H-NMR (CD3OD) 68.18 (s, IH, Bzi- CH), 7.73 (d, IH, J = 8.7, Bzi-CH), 7.61 (d, br, IH, Bzi-CH), 7.40 (d, IH, J = 8.1, Bzi-CH), 7.32 (s, IH, Py-CH), 7.19 (s, IH, Im-CH), 7.04 (s, IH, Im-CH), 7.03 (d, IH, Bzi-CH), 6.94 (s, IH, Bzi-CH), 6.85 (s, IH, Py-CH), 4.03 (s, 3H, CH3), 3.95 (s, 3H, CH3), 3.14 (t, 4H, CH2), 2.59 (t, 4H, CH2), 2.29 (s, 3H, CH3). MALDI-TOF-HRMS: m/z calcd for C29H30Ni0O 534.2604, found monoisotonic 535.268; 534.4783 (M+, 100%), 532.4543 (80%, M-2), 533.4643 (48%, M-I), 535.4900 (70%, M+l), 536.4842 (24%, M+2).
Mp-Bzi-Py-Bzi-Py-Im (J-2) was prepared using amine 49, acid 70 and DIC/HOBt using the general procedure F. Recovery 32%. 1H-NMR (CD3OD) 68.08 (s, IH, Bzi-CH), 7.74 (d, IH, J = 8.7, Bzi-CH), 7.56 (d, br, IH, Bzi-CH), 7.39 (d, IH, J = 8.1, Bzi-CH), 7.32 (s, IH, Py-CH), 7.29 (s, IH, Py-CH), 7.16 (s, IH, Im-CH), 6.97 (s, IH, Im-CH), 7.02 (s, IH, Bzi-CH), 6.94 (d, IH, J = 8.7, Bzi-CH), 6.88 (s, IH, Py-CH), 6.84 (s, IH, Py-CH), 4.01 (s, 3H, CH3), 3.98 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.13 (t, 4H, CH2), 2.59 (t, 4H, CH2), 2.29 (s, 3H, CH3). MALDI-TOF-HRMS: m/z calcd for C35H36Ni2O2 656.3084, found monoisotonic 657.316; 656.4976 (M+, 94%), 654.4490 (65%, M-2), 655.4692 (46%, M-I), 657.4977 (100%, M+l), 658.4888 (36%, M+2).
Mp-Bzi-Py-Py-Bzi-Py-Im (J-3) was prepared using amine 53a, acid 64 and DIC/HOBt using the general procedure F. Recovery 36%. 1H-NMR (CD3OD) 68.08 (s, IH, Bzi-CH), 7.73 (d, IH, J = 8.1, Bzi-CH), 7.54 (d, IH, J = 8.7, Bzi-CH), 7.38 (d, IH, J = 8.7, Bzi-CH), 7.33 (s, IH, Py-CH), 7.26 (s, IH, Py-CH), 7.21 (s, IH, Py-CH), 7.18 (s, IH, Im-CH), 7.03 (s, IH, Bzi-CH), 6.98 (s, IH, Im-CH), 6.96 (s, IH, Py-CH), 6.93 (d, IH, J = 8.7, Bzi-CH), 6.89 (s, IH, Py-CH), 6.76 (s, IH, Py-CH), 4.01 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.87 (s, 3H, CH3), 3.14 (t, 4H, CH2), 2.59 (t, 4H, CH2), 2.29 (s, 3H, CH3). ES-HRMS: m/z calcd for C4IH42N14O3 778.3564, found 778.3862 (M+, 100%), 779.3898 (24%, M+l), 780.3941 (10%, M+2). Mp-Bzi-Py-Im-Bzi-Im (J-4) was prepared using amine 53b, acid 64 and
DIC/HOBt using the general procedure F. Recovery 28%. 1H-NMR (CD3OD) 68.18 (s, IH, Bzi-CH), 7.80 (d, IH, J = 8.7, Bzi-CH), 7.63 (d, br, IH, Bzi-CH), 7.46 (s, IH), 7.34 (d, IH, J = 8.1 , Bzi-CH), 7.22 (s, IH, Py-CH), 7.22 (s, IH, Im-CH), 7.07 (s, IH, Im-CH), 7.00 (s, IH, Bzi-CH), 6.91 (d, IH, J = 8.7, Bzi-CH), 6.75 (s, IH, Py-CH), 4.09 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.89 (s, 3H, CH3), 3.12 (t, 4H, CH2), 2.61 (t, 4H, CH2), 2.30 (s, 3H, CH3). ES-HRMS: m/z calcd for C34H35N13O2 657.3037, found 657.3430 (M+, 100%), 658.3471 (55%, M+l), 659.3418 (9%, M+2).
The following final products (Kl -K6) were all prepared from the given combination of amine and acid derivatives listed individually for each data set below. Mp-Bzi-Py-Bzim (K-I) was prepared using amine 49, 2-trichloroacetyl-l- methylbenzimidazole 73 and the isolation methodology given in the general procedure E. Recovery 70%. 1H-NMR (HCl salt, CD3OD) 67.89 (d, IH), 7.84 (d, IH), 7.73 (s, IH), 7.65-7.61 (m, 3H), 7.31-7.25 (m, 3H), 4.26 (s, 3H, CH3), 4.01 (s, 3H, CH3), 3.87 (d, 2H, CH2), 3.60 (d, 2H, CH2), 3.26-3.13 (m, 4H, CH2), 2.92 (s, 3H, CH3). 13C-NMR (TFA + DMSO-d6) 6156.6, 148.6, 143.6, 141.2, 140.5, 136.8, 132.7, 125.7, 124.5, 123.4, 122.1, 119.9, 117.1, 1 14.3, 111.5, 108.8, 99.3, 52.3, 46.5, 35.9, 31.9, 31.8. (1 other aromatic C nulled due to long Tl relaxation time). ES-HRMS: m/z calcd for C26H28N8O 468.2386, found 468.2768 (M+, 100%), 469.2800 (60%, M+l), 470.2839 (18%, M+2).
Mp-Bzi-Py-Py-Im-Bzim (K-2) was prepared using amine 49, acid 75 and EDCI/HOBt using the general procedure F. Recovery 40%. 1H-NMR (DMSOd6)
612.38 ( br s, exch, IH, Bzi-NH), 10.41 (s, exch, IH, NH), 10.39 (s, exch, IH, NH),
10.09 (s, exch, IH, NH), 7.83 (d, IH), 7.72 (d, IH), 7.66 (s, IH), 7.46-7.32 (m, 5H), 7.29 (s, IH), 6.99 (s, IH), 6.87 (m, 2H), 4.19 (s, 3H, CH3), 4.04 (s, 6H, 2 x CH3), 3.87 (s, 3H, CH3), 3.17 (t, 4H, CH2), 2.72 (t, 4H, CH2), 2.38 (s, 3H, CH3). ES-HRMS: m/z calcd for C36H38Ni4O3 714.3251, found 714.3564 (M+, 100%), 715.3600 (30%, M+l), 716.3641 (7%, M+2).
Im-Bzi-Py-Bzim (K-3) was prepared using amine 77, 2-trichloroacetyl-l- methylbenzimidazole (73) and the isolation methodology given in the general procedure F. Recovery 65%. 1H-NMR (DMSO-d6) 68.23 (s, IH, Im-CH), 7.88 (s, IH, Bzi-CH), 7.80 (d, IH, J = 8.1, Bzi-CH), 7.43 (s, IH, Im-CH), 7.41 (d, IH, J = 8.1, Bzi-CH), 7.51 (s, IH, Im-CH), 7.41-7.33 (m, 4H, Bzi'-CH), 7.29 (s, IH, Py-CH), 7.10 (s, IH Py-CH), 4.20 (s, 3H, CH3), 4.12 (s, 3H, CH3). 1H-NMR (HCl salt, CD3OD) 69.62 (s, IH, Im-CH), 8.22-8.19 (d+s, 2H, 2 x Bzi-CH), 8.02 (d, IH, J = 8.4, Bzi- CH), 7.93-7.84 (m, 5H), 7.66-7.54 (m, 2H), 7.54 (d, IH, J = 1.8, Py-CH), 4.32 (s, 3H, Bzi-NCH3), 4.09 (s, 3H, Py-NCH3). 13C-NMR (ImBziPyBzi.s in CD3OD) 6158.1 (+), 143.4 (+)135.9 (+), 134.7 (+), 134.3 (+), 134.2 (+), 134.0 (+), 128.3 (-), 128.2 (-), 124.9 (+), 123.5 (-), 122.2 (-), 1 17.8 (-), 116.5 (-), 1 15.8 (+), 113.9 (-), 111.2 (-), 1 10.1 (-), 37.1 (-), 33.4 (-). ES-HRMS: m/z calcd for C24H20N8O 436.1760, found 436.1864 (M+, 100%), 437.1902 (38%, M+l), 438.1975 (12%, M+2). Im-Bzi-Py-Py-Im-Bzim (K-4) was prepared using amine 77, acid 75 and
EDCI/HOBt using the general procedure F. Recovery 52%. 1H-NMR (DMSO-d6) 612.73 (s, exch, IH, Bzi-NH), 10.44 (s, exch, IH, NH), 10.39 (s, exch, IH, NH),
10.10 (s, exch, IH, NH), 8.22 (s, IH, Im-C2H), 7.83 (d, IH), 7.72 (m, 2H), 7.66 (s, IH), 7.65 (s, IH, Im-C5H), 7.47-7.34 (m, 4H, Bzi-CH), 7.32 (s, IH, Im-C5H), 7.26 (d, IH, J = 1.5 Hz, Py-C3H), 7.21 (d, IH, J = 1.5 Hz, Py-C3H), 7.11 (s, IH, Im-C4H), 7.10 (s, IH, Bzi-C4H), 4.18 (s, 3H, CH3), 4.03 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.87 (s, 3H, CH3). FAB-HRMS: m/z calcd for C35H32N13O3 + (MH+) 682.2751, found 682.2749 (100%).
Im-Bzi-Py-Im (K-5) was prepared using amine 77, trichloroacetyl derivative 24 and the isolation methodology given in the general procedure F. Recovery 70%. 1H-NMR (HCl salt in CD3OD) 69.18 (s, IH, Im-C2H), 7.68 (d, IH, J = 8.7 Hz, Bzi- C7H), 7.62 (s, IH, Im-C4H), 7.58 (s, IH, Im-C5H), 7.53 (s, IH, Im-C4H), 7.48 (d, IH, J = 1.8 Hz, Py-C5H), 7.28 (d, IH, J = 8.7 Hz, Bzi-C6H), 7.24 (s, IH, Bzi-C4H), 7.17 (d, IH, J = 1.8 Hz, Py-C3H), 6.83 (s, IH, Im-C5H), 4.09 (s, 3H, CH3), 3.96 (s, 3H, CH3). ES-HRMS: m/z calcd for C20H18N8O 386.1604, found 386.1750 (M+, 100%), 387.1784 (46%, M+l), 388.1821 (8%, M+2).
Im-Bzi-Py-Py-Im-Im (K-6) was prepared using amine 77, acid 35 and DIC/HOBt using the general procedure F. Recovery 38%. 1H-NMR (HCl salt in CD3OD) 09.22 (s, IH, Im-C2H), 7.75 (s, IH, Im-C4H), 7.66 (s, IH, Im-C5H), 7.62 (d, IH, J = 8.5 Hz, Bzi-C7H), 7.59 (s, IH, Im-C4H), 7.54 (d, IH, J = 1.4 Hz, Py- C5H), 7.45 (s, IH, Im-C5H), 7.38-7.34 (m, 2H, 2 x Py-CH), 7.28 (d, IH, J = 8.5 Hz, Bzi-C6H), 7.23 (s, IH, Bzi-C4H), 7.09 (d, IH, J = 1.4 Hz, Py-C3H), 6.84 (s, IH, Im- C5H), 4.01 (s, 3H, CH3), 3.99 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.83 (s, 3H, CH3). ES- HRMS: m/z calcd for C31H29N13O3 631.2516, found 631.2602 (M+, 100%), 632.2640 (46%, M+l), 633.2683 (16%, M+2). The following final products (L1-L4) were all prepared from the same amine
(53b) and different acid derivatives listed individually for each data set below.
Mp-Bzi-Py-Im-Bzi-Py-Im (L-I) was prepared using acid 70 and DIC/HOBt using the general procedure F. Recovery 48%. 1H-NMR (free base in CD3OD) 68.04 (s, IH, Bzi'-C4H), 7.69 (d, IH, J = 8.7 Hz, Bzi'-C6H), 7.46 (d, IH, J = 8.5 Hz, Bzi- CH), 7.28-7.38 (m, 2H), 7.24 (s, IH), 7.13-7.17 (m, 2H), 6.82-7.00 (m, 4H), 6.74 (s, IH), 3.98 (s, 3H, ring-CH3), 3.95 (s, 3H, ring-CH3), 3.93 (s, 3H, ring-CH3), 3.87 (s, 3H, ring-CH3), 3.10 (m, 4H, Pip-CH2), 2.57 (m, 4H, Pip-CH2), 2.29 (s, 3H, Pip-CH3). (Note: only selected signals assignable in the aromatic region in the NMR; structure assignment supported by data from MS and aliphatic proton resonances in NMR and the relative integration of signals). ES-HRMS: m/z calcd for C40H41N15O3 779.3517, found 779.4863 (M+, 54%), 777.4843 (100%, M-2), 778.5093 (70%, M-I), 780.4712 (40%, M+l), 781.5302 (16%, M+2).
Mp-Bzi-Py-Im-α-Bzi-Py-Im (L-2) was prepared using acid 81 and DIC/HOBt using the general procedure F. Recovery 30%. 1H-NMR (free base in CD3OD) 67.40-7.43 (m, 2H), 7.34 (br d, IH), 7.29 (br s, IH), 7.26 (br s, IH), 7.19 (s, IH), 7.08 (m, 2H), 6.96-6.99 (m, 3H), 6.85-6.87 (m, 2H), 4.39 (s, 3H, ring-CH3), 4.01 (s, 6H, 2 x ring-CH3), 3.97 (s, 3H, ring-CH3), 3.93 (m, 2H, gly-CH2), 3.32 (m, 4H, Pip-CH2), 3.24 (m, 4H, Pip-CH2), 2.79 (s, 3H, Pip-CH3). (Note: the aromatic region in the NMR being complex, structure was confirmed using data from MS and aliphatic proton resonances in NMR and the relative integration of signals). ES-HRMS: m/z calcd for C42H44Ni6O4 836.3731, found 836.3855 (M+, 47%), 834.3665 (100%, M-2), 835.3809 (82%, M-I), 837.5764 (58%, M+l), 838.3910 (30%, M+2).
Mp-Bzi-Py-Im-β-Bzi-Py-Im (L-3) was prepared using acid 82 and DIC/HOBt using the general procedure F. Recovery 28%. 1H-NMR (free base in CD3OD) 67.40-7.43 (m, 3H), 7.34 (br d, IH), 7.29 (br s, IH), 7.10-7.19 (m, 3H), 7.00-7.03 (m, 3H), 6.88-6.90 (m, 2H), 4.26 (s, 3H, ring-CH3), 4.01 (s, 9H, 3 x ring-CH3), 3.97 (m, 2H, β-CH2), 3.43-3.33 (m, 6H, β-CH2 + Pip-CH2), 3.19 (m, 4H, Pip-CH2), 2.76 (s, 3H, Pip-CH3). (Note: the aromatic region in the NMR being complex, structure was confirmed using data from MS and aliphatic proton resonances in NMR and the relative integration of signals). ES-HRMS: m/z calcd for C43H46Ni6O4 850.3888, found 850.3776 (M+, 62%), 848.3402 (28%, M-2), 849.3868 (46%, M-I), 851.4337 (100%, M+l), 852.4489 (88%, M+2).
Mp-Bzi-Py-Im-γ-Bzi-Py-Im (L-4) was prepared using acid 83 and DIC/HOBt using the general procedure F. Recovery 36%. 1H-NMR (HCl salt in CD3OD) 67.71- 7.76 (m, 3H), 7.57-7.64 (m, 3H), 7.38 (br d, IH), 7.29-7.33 (m, 2H), 7.21-7.26 (m, 4H), 4.34 (s, 3H, ring-CH3), 4.1 1 (s, 3H, ring-CH3), 4.04 (m, 2H, γ-CH2), 4.01 (s, 6H, 2 x ring-CH3), 3.95 (m, 2H, γ-CH2), 3.86 (m, 2H, Pip-CH2), 3.59 (m, 2H, Pip-CH2), 3.15-3.28 (m, 4H, Pip-CH2), 2.93 (s, 3H, Pip-CH3), 2.87 (m, 2H, γ-CH2). (Note: the aromatic region in the NMR being complex, structure was confirmed using data from MS and aliphatic proton resonances in NMR and the relative integration of signals). ES-HRMS: m/z calcd for C44H48Ni6O4 864.4044, found 864.4870 (M+, 78%), 862.4734 (100%, M-2), 863.4804 (78%, M-I), 865.5003 (92%, M+l), 866.5238 (35%, M+2), 867.5238 (10%, M+3).
Reference compounds R1-R7, matching the series F/G in terms of the polyamide composition, can likely be prepared readily on the basis of success with the coupling methods for amide forming reactions. With the detailed studies directed towards a specific ligand composition, R4 was prepared as below.
Dp-Py-Py-Py-Im-Im (R4). This compound was prepared from an amine (15b) described and numbered as such in chapter 1, in a coupling reaction with the acid derivative (39) from above and DIC/HOBt combination in the general procedure E. Recovery 55%. 1H-NMR (DMSOd6) δlθ.45 (s, exch, IH, NH), 10.28 (s, exch, IH, NH), 9.98 (s, exch, IH, NH), 9.93 (s, exch, IH, NH), 8.18 (t, exch, IH, NH), 7.63- 7.61 (m, 4H, Py/Im-CH), 7.36 (s, IH), 7.30 (d, IH, J = 1.8 Hz, Py-CH), 7.24 (d, IH, J = 1.8 Hz, Py-CH), 7.19-7.18 (2 x d, 2H, Py/Im-CH), 7.07 (d, IH, J = 1.8 Hz, Py-CH), 6.93 (d, IH, J = 1.8 Hz, Py-CH), 4.03, 4.01, 3.86, 3.84, 3.83 (5 x s, 3H each, 5 x CH3), 3.26 (q, 2H, NH CH2), 3.06 (q, 2H, CH2), 2.74, 2.73 (2 x s, 3H each, 2 x CH3), 1.85 (t, 2H, CH2). ES-HRMS: m/z calcd for C33H4iN]3O5 699.3354, found 700.5727 (63%, M+l), (note: no peaks observed for either deprotonated or further protonated forms than M+l). Biophysical Studies The ligand and DNA stock solutions were prepared in either 10 or 50 mM sodium/potassium phosphate, pH 7.0, buffers containing 50 mM NaCl and 0.1 mM Na2EDTA to minimize the effects due to residual metal ions. Quadruply distilled (Millipore) water was employed in all work described below. Concentrations were determined spectrophotometrically; nucleic acid concentrations were expressed in terms of base pairs with average ε values (M"1 cm" 1) as indicated. In some cases (Job plots), DNA concentrations were used in terms of phosphate contents. The extinction coefficients determined for the various ligands were: Hoechst 33258 (Aldrich, batch 05305BP), ε345 = 41,000; ethidium bromide (Aldrich, batch 1239-45-8), ε48o = 5800, ligand F4, ε323 = 22,500, ligand G4, ε320 = 19,100, and ligand R4, ε246 = 37,000. DNA Thermal Melting (Tm) Measurements. The method employed the DNA arrays (with high picomolar level sensitivity) where fluorescence dye (FITC) and quencher (DABCYL) labeled oligonucleotides were employed for a fluorescence resonance energy transfer based evaluation of the hybridization of DNA strands in the absence and presence of test ligands as a function of temperature. All experiments were carried out under similar solution conditions relating to the buffer nature and their concentrations. The Tm values were determined using first order differential plots and the experiments were done in triplicate. Averaged values (with <5% standard error) were plotted in the histogram format to asses the structure-activity relationships for the various structural subclasses of ligands (series F, G, H etc.)
Absorption and Fluorescence Spectroscopy: Absorption spectra were measured on a double-beam Varian Cary 500 UV/VIS spectrophotometer with a bandwidth of 1 nm and a scan rate of 0.5 nm/s, with the solutions suspended in quartz cells with a 1 cm pathlength. All measurements were conducted at ambient temperature. Buffer solutions used for titrations were themselves used as reference for background corrections. Absorption spectra were recorded in the range of 200-700 nm. Molar extinction coefficients were determined on solutions with solute concentrations adjusted to provide linear working curves of absorbance at band maxima versus concentration, according to Beer-Lambert relationship. While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term. Table I
ΔTm values for DNA = 5' - AAATA - X1X2X3X4X5X6X7X8 - AA - 3'
16 AT Sites (X1-8) DST Act R4 F4 G4
TAAATATA 6 4 2 4 1 TACATATA 1 6 3 5 1 TAGATATA 2 3 2 5 2 TATATATA 6 4 2 6 3
TAAATCTA 1 3 1 6 5 TACATCTA 0 4 1 6 6 TAGATCTA 0 2 0 6 5
TATATCTA 3 1 0 7 6
TAAATGTA 1 5 4 10 5 TACATGTA 1 3 2 10 5
TAGATGTA 0 2 2 9 5 TATATGTA 0 4 3 11 8
TA AATTTA 8 3 1 5 4
TACATTTA 3 1 1 4 3 TAGATTTA 5 3 2 5 5 TATATTTA 8 3 1 6 4
16 GC Sites (X1-8)
TAAGCATA 1 6 0 0 0 TACGCATA 0 5 0 2 0 TAGGCATA 0 4 1 2 0 TAT Ω CATA 0 6 1 1 1
TAAGCCTA 0 3 1 1 1 TACGCCTA 0 1 0 3 5 TAGGCCTA 0 3 1 6 6
TATGCCTA 0 4 0 2 1
TAAGCGTA 0 5 1 1 0 TAC Ω CGTA 0 4 0 3 0
TAGGCGTA 0 2 0 2 0 TATGCGTA 0 6 1 3 1
TAA Ω CTTA 1 5 0 1 0
TACGCTTA 0 4 1 4 5 TAGGCTTA 0 2 0 1 1 TATGCTTA I 6 0 1 0 FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
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Claims

WE CLAIM:
A compound of formula I:
Figure imgf000126_0001
wherein
X is independently CH or N, and when X is C, it serves as the point of attachment for an adjacent group and only one X or 2 non-adjacent X groups are N; Y is independently CH or N;
R1 is selected from Chalky!, C3.i2cycloalkyl, aryl, C(O)NHC 1-6alkyl, C(O)NHC3. πcycloalkyl and C(O)NH(C i_6alkylene-N(R6)2), in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N, NR6 or N(R6)2;
R2 and R2 are independently selected from H and Ci-6alkyl, or R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units; R3 is Ci-6alkylene or C2-6alkenylene, both of which are optionally substituted with one or more of Ci-6alkyl, 0Ci-6alkyl or OH, and both optionally have one or more of the carbons replaced with O, S, NR6 or N(R6)2;
R4 and R4 are independently selected from H and Ci.6alkyl, or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units; R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3_6heterocycle,
Figure imgf000126_0002
R6 is H or C,-6alkyl; R7 is Ci-4alkylene or C2-4alkenylene both of which are optionally substituted with one or more of Ci-6alkyl, OCi-6alkyl or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1, 2, 3, 4 or 5, and when m is other than 1, only the terminal monomeric unit represented by m is substituted with R1; n is 1, 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is O, 1, 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof, with the proviso that when R1 is selected from C(O)NHC i.6alkyl, C(O)NHC3. ,2cycloalkyl and C(O)NH(C i-6alkylene-N(R6)2), R5 is selected from pyrrolyl, imidazolyl, benzimidazolyl, imidazopyridinyl and purinyl.
2. The compound according to claim 1 , wherein m is 1.
3. The compound according to claim 1 , wherein m is 1 and p is 0.
4. The compound according to claim 1 , wherein m is 1 , o is 0 and p is 0.
5. The compound according to claim 4, wherein n is 1 , 2, 3 or 4 and q is 0 or 1.
6. The compound according to claim 1, wherein n and o are both 0.
7. The compound according to claim 6, wherein m and p are both 1.
8. The compound according to claim 1, wherein m is 1 , n is 1 or 2, o is 0, p is 1 and q is 0 or 1.
9. The compound according to any one of claims 1-8, wherein R1 is selected from Ci-6alkyl, C3_i2cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is replaced with N, NR6 or N(R6)2, where R6 is H or Ci- 4alkyl.
10. The compound according to claim 9, wherein R1 is selected from
Figure imgf000128_0001
piperazinyl, imidazolyl, pyrazolyl, triazolyl, morpholinyl, each of which are connected to the compound of Formula I through the nitrogen atom.
11. The compound according to claim 10, wherein R1 is N-methylpiperazinyl.
12. The compound according to any one of claims 1-8, wherein R1 is selected from C(O)NHC i-4alkyl, C(O)NHC3.7cycloalkyl and C(O)NH(C Malkylene-N(R6)2), in which one or more of the carbons of the alkyl or cycloalkyl groups is replaced with N, NR6 or N(R6)2, where R6 is H or CMalkyl.
13. The compound according to any one of claims 1-12, wherein R2 and R2 are, independently, H or Ci-4alkyl.
14. The compound according to claim 13, wherein R2 and R2 are both methyl.
15. The compound according to any one of claims 1-12, wherein R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units.
16. The compound according to claim 15, wherein R2 and R2 are joined by R7 to form a 6 membered ring between two adjacent monomeric units.
17. The compound according to claim 15 or 16, wherein R7 is Ci-4alkylene or C2. 4alkenylene.
18. The compound according to any one of claims 1-17, wherein R3 is Ci- 6alkyene.
19. The compound according to claim 18, wherein R is Ci-3alkylene.
20. The compound according to any one of claims 1-19, wherein R4 and R4 are independently selected from H and Ci-4alkyl.
21. The compound according to claim 20, wherein R4 and R4 are both methyl.
22. The compound according to any one of claims 1-19, wherein R4 and R4 are joined by R7 to form a 5 to 8 membered ring, between two adjacent monomeric units.
23. The compound according to claim 22, wherein wherein R4 and R4 are joined by R7 to form a 6 membered ring, between two adjacent monomeric units.
24. The compound according to claim 22 or 23 wherein R7 is Cι-4alkylene or C2- 4alkenylene.
25. The compound according to any one of claims 1-24, wherein R5 is a nitrogen- containing mono- or bicyclic aromatic heterocycle.
26. The compound according to claim 25, wherein R5 is selected from pyrrole, imidazole, oxazole, thiazole, benzimidazole, imidazopyridine, benzoxazole, benzothiazole, and indole all of which are optionally substituted with one or more Ci- 4alkyl, OH and OC,-4alkyl.
27. The compound according to claim 26, wherein R5 is optionally substituted with one of more of CH3, OH or OCH3.
28. The compound according to claim 27, wherein R5 is optionally substituted with one of CH3, OH or OCH3.
29. The compound according to any one of claims 25-28 wherein R5 is unsubstituted.
30. The compound according to any one of claims 1-29, wherein R6 is H or Ci- 4alkyl.
31. The compound according to claim 30, wherein R6 is H or CH3.
32. The compound according to claim 1 selected from:
Figure imgf000130_0001
wherein each X may be the same or different and are independently selected from CH and N; each Y may be the same or different and are independently selected from CH and N and when q is greater than 1 , Y in each monomer unit represented by q may be the same or different;
R4 and R4 are independently selected from H and Ci -6alkyl; and q is 1, 2, 3, 4 or 5.
33. The compound according to claim 1 selected from:
Figure imgf000131_0001
and
Figure imgf000131_0002
wherein each X may be the same or different and are independently selected from CH and N; and wherein each Y may be the same or different and are independently selected from CH and N.
34. The compound of formula I according to claim 1 :
Figure imgf000132_0001
I wherein
X is independently CH or N, and when X is C, it serves as the point of attachment for the adjacent group and only one X or 2 non-adjacent X groups are N;
Y is independently CH or N;
R1 is selected from Ci-6alkyl, C3_i2cycloalkyl, aryl, in which one or more of the carbons of the alkyl, cycloalkyl or aryl groups is optionally replaced with O, S, N,
NR6 or N(R6)2; R2 and R2 are independently selected from H and Ci-δalkyl, or R2 and R2 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R3 is Ci-όalkylene or C2.6alkenylene, both of which are optionally substituted with one or more of Ci-6alkyl, OCi_6alkyl or OH, and both optionally have one or more of the carbons optionally replaced with O, S, NR6 or N(R6)2; R4 and R4 are independently selected from H and Ci-6alkyl, or R4 and R4 are joined by R7 to form a 5 to 8 membered ring between two adjacent monomeric units;
R5 is a nitrogen-containing monocyclic or polycyclic heterocycle optionally substituted with one or more C3-6heterocycle,
Figure imgf000132_0002
OH and OCi-6alkyl;
R6 is H or C,.6alkyl; R7 is
Figure imgf000132_0003
or C2-4alkenylene both of which are optionally substituted with one or more of Ci-6alkyl, OCi-όalkyi or OH and both optionally have one or more carbons replaced with O, S, NR6 or N(R6)2; m is 1 , 2, 3, 4 or 5, provided that when m is other than 1, only the terminal monomer unit represented by m is substituted with R1 ; n is 1 , 2, 3, 4 or 5; o is O, 1, 2, 3, 4 or 5 p is 0, 1, 2, 3, 4 or 5; and q is 0, 1, 2, 3, 4 or 5, and pharmaceutically acceptable salts, solvates and prodrugs thereof.
35. A method for selectively forming a complex between target DNA and a compound according to any one of claims 1-34, comprising: contacting the target
DNA with the compound according to any one of claims 1-34; wherein the compound is capable of selectively binding to a sequence on the target DNA.
36. The method according to claim 35, wherein the compound selectively binds to the target DNA under physiological conditions where complexes form between the compound and the target DNA.
37. The method according to claim 35 or 36, wherein the specific sequence is in a minor groove on the target DNA.
38. The method according to any one of claims 35-37, wherein the compound and the target DNA is at a binding stoichiometry of 2: 1.
39. The method according to any one of claims 35-38, wherein the target gene comprises all or a fragment of a sequence selected from AAGGCCTT, ATGGCCAT,
AAGCGCTT and ATGCGCAT.
40. The method according to claim 39, wherein the target gene comprises all or a fragment of a sequence selected from 5'-ATGGCCAT and 5'-AAGCGCTT.
41. A method of detecting the presence of a sequence in a sample comprising DNA comprising contacting the sample with a compound according to any one of claims 1 -34 which is capable of selectively binding to sequences on the DNA, and wherein the compound has at least one detectable label; and monitoring the detectable label in the sample, wherein the presence of the detectable label is indicative of the presence of the selected sequence.
42. A method for isolating target DNA from a sample comprising a mixture of DNA comprising contacting the target DNA with a compound according to any one of claims 1-34 which is capable of selectively binding to a sequence on the target DNA, and wherein complexes selectively form between the compound of the invention and the target DNA; isolating the selected complexes.
43. A method for modulating transcription of a target gene in a cell comprising contacting the cell with an effective amount of a compound according to any one of claims 1-34 under conditions sufficient for the formation of complexes selectively between the compound of the invention and the target gene and wherein such complex formation modulates the transcription of the target gene.
44. The method according to claim 43, wherein the target sequence on the gene is a sequence on transcriptional regulatory regions of the gene.
45. The method according to claim 43 or 44, wherein the target gene comprises all or a fragment of a sequence selected from AAGGCCTT, ATGGCCAT, AAGCGCTT and ATGCGCAT.
46. The method according to claim 45, wherein the target gene comprises all or a fragment of a sequence selected from 5'-ATGGCCAT and 5'-AAGCGCTT.
47. The method according to any one of claims 43-46, wherein the target gene is any gene implicated in the propagation of the cell or of a disease state.
48. A method for treating cancer comprising administering an effective amount of the compound according to any one of claims 1-34 to a subject in need thereof.
49. A method of treating an infection by a virus, bacterium or pathogen in a subject comprising administering an effective amount of a compound according to any one of claims 1-34 to a subject in need thereof.
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