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WO2001096313A1 - Analogues de distamycine a - Google Patents

Analogues de distamycine a Download PDF

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WO2001096313A1
WO2001096313A1 PCT/US2001/019404 US0119404W WO0196313A1 WO 2001096313 A1 WO2001096313 A1 WO 2001096313A1 US 0119404 W US0119404 W US 0119404W WO 0196313 A1 WO0196313 A1 WO 0196313A1
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distamycin
subunit
library
binding
analog
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Dale L. Boger
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • 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
    • C07D487/02—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 two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the invention relates to cytotoxic agents. More particularly, the invention relates analogs of distamycin A, to libraries of distamycin A, to their synthesis and screening for DNA binding activity and cytotoxic activies.
  • Solution-phase combinatorial strategies for the synthesis of libraries of distamycin A analogs is described wherein the ⁇ /-methylpyrroIe subunit of distamycin A is systematically replaced with other heterocyclic amino acids.
  • Solution-phase synthesis techniques with reaction workup and purification employing acid/base liquid-liquid extractions were used in the multistep preparation of distamycin A (8 steps, 40% overall yield) and a prototypical library of 2640 analogs providing intermediates and final products that are > 95% pure on conventional reaction scales.
  • This first generation library was further functionalized with a basic side chain to mimic the amidine group of distamycin.
  • One aspect of the invention is directed to an analog of distamycin A represented by the following structure:
  • R- ⁇ R' In the above structure, R is a selected from -C(O)O-(C1-C6 alkyl) and -C(O)CH 2 CH 2 CH 2 NMe 2 ; R' is -O(C1-C6 alkyl), where (C1-C6 alkyl) is any branched or unbranched alkyl having 1 to 6 carbons; and -NH-Subunit A-C(O)- , -NH-Subunit B-C(O)- , and -NH-Subunit C-C(O)- are each a diradical independently selected from the following structures:
  • R is -C(O)O-_Bu.
  • R is -C(0)CH 2 CH 2 CH 2 NMe 2 .
  • R' is selected from the group consisting of -OMe and -OEt.
  • -NH-Subunit A-C(O)- -NH-Subunit B-C(O)-
  • -NH-Subunit C-C(O)- can not all be identical. Examples of this fourth preferred embodiment include the following species:
  • Another aspect of the invention is directed to a positional scanning library comprising a collection of ten or more of the compounds indicated above.
  • Another aspect of the invention is directed to a process for synthesizing a library of amide linked aromatic trimers represented by the following structure:
  • Subunit A is any aromatic radical of a plurality of aromatic radicals
  • Subunit B is a first aromatic radical
  • Subunit C is a second aromatic radical.
  • Subunit B is linked to Subunit C by means of a first amide linkage to form a dimer of the first and second aromatic radicals, the dimer being represented by the following structure:
  • a plurality of the dimers of the first step are linked to a plurality of Subunits A by means of a second amide linkage for forming the library of compounds.
  • Each element of the library is a trimer of aromatic radicals linked by amide linkages.
  • Another aspect of the invention is directed to a process for killing a cancer cell.
  • the process employs the step of contacting the cancer cell with a solution containing a cytotoxic concentration of any of the above compounds.
  • the close distamycin A analogue 17 was identified as the most effective binder to a hairpin oligonucleotide that contains the PSA-ARE-3 consensus sequence and a 5 base-pair AT-rich site.
  • the distinctions in the assay were small and subtle discoveries tucked into the library were not detected, including additional effective binders and those which bound both the PSA-ARE-3 and ARE consensus sequence equally well. Nonetheless, the combined use of solution-phase mixture synthesis and positional scanning is simple and technically nondemanding even for a large library being less demanding than the parallel synthesis of individual compounds or small mixtures, or solid-phase split and mix synthesis (Furka, A.; et al. Abstr. Int.
  • Figure 1 shows the design of the positional scanning library.
  • Figure 2 shows the structures of amino acid monomer units used in the preparation of the libraries.
  • Figure 3 is a scheme illustrating the synthesis of the BOC-trimer positional scanning libraries.
  • Figure 4 is a scheme for the conversion of BOC-trimer into DMABA-trimer libraries.
  • Figure 5 is a table showing the yields of the BOC- and DMABA-trimers.
  • Figure 6 is a bar graph showing the most potent residues that were found using the positional scanning library.
  • Figure 7 is a table showing the cytotoxic activities of the candidate compounds composed of the most potent residues found by the positional scanning library.
  • Figure 8 shows the structure of the two most cytotoxic compounds within the libraries.
  • Figure 9 shows the cytotoxicity (L1210) for DMABA-trimer scanning libraries. Smaller numbers indicate higher cytotoxic activity.
  • Figure 10 shows the structures of 86, 210, 220 and 49.
  • Compounds 86, 210 and 220 were identified as potent DMABA-trimers.
  • Figure 11 shows the results of the ethidium bromide displacement assay for DMABA-trimer libraries (99 ⁇ M). DNA at 0.88 * 10 "5 M, ethidium bromide at 0.44 x 10 "5 M. Smaller numbers indicate higher cytotoxic activity..
  • Figure 12 shows the solution phase strategy for DNA binding agent libraries.
  • Figure 13 illustrates the general procedure for determination of sequence selectivity for a library of DNA binding agents.
  • Figure 14 is a scheme showing the steps in synthesizing distamycin A.
  • Figure 15 shows the reaction sequence for preparation of DNA binding agent libraries.
  • Figure 16 shows the structures of the amino acid subunits used in the preparation of libraries.
  • Figure 17 is a scheme for the EDCI/DMAP coupling of the carboxylic acid and amine.
  • the second reaction was used to couple the sodium salt of carboxylic acids to amines. This method was used when the free carboxylic acids were unstable.
  • Figure 18 is a scheme showing the side reaction and solution to incorporating the indole subunit into analogs.
  • the indole amino acid 13b dimerizes upon attempted coupling with other amino acids.
  • the series of reactions in the second and third row is how 13b was eventually coupled to other amino acids by first protecting the indole nitrogen, coupling and then deprotecting the indole and amine nitrogens simultaneously.
  • Figure 19 shows the formation of the individual trimers from the pyrrole dimer.
  • the lower reaction is the coupling of the dimers to a mixture of free acids to get mixtures of trimers.
  • Figure 20 is a scheme that shows the incorporation of a dimethylaminobutyric acid tail onto the deprotected trimers.
  • Figure 21 is a three-dimensional display of the results of the cytotoxicity assay for the BOC-trimer libraries.
  • Figure 22 shows how the individual trimers were synthesized after finding out which were the best B and C subunits. The activity of each individual trimer is shown in the table.
  • Figure 23 is a three-dimensional display of the results of the cytotoxicity assay of the dimethylaminobutyric acid terminated trimers. A table shows the toxicity of the individual compounds.
  • Figure 24 shows the synthesis of the individual dimethylaminobutyric acid (DMABA) terminated trimers for testing. The most active B and C subunit was determined in the cytotoxicity assay.
  • DMABA dimethylaminobutyric acid
  • Figure 25 shows the general procedure for establishing DNA binding of a library of compounds with a single sequence.
  • Figure 26 shows the ethidium bromide displacement assay for DMABA- trimer libraries with poly[dA]-poly[dT] DNA in graph A and graph B shows the corresponding assay for poly[dG]-poly[dC]. Larger numbers indicate higher affinity for DNA.
  • Figure 27 shows the synthesis of 40 DMABA trimers and the corresponding yields.
  • Figure 28 shows the activity in the L1210 assay and the percent remaining fluorescence with poly[dA]-poly[dT] DNA.
  • Figure 29 shows the graphical results for the ethidium displacement assay for selected DMABA trimers.
  • the hairpin oligonucleotides contain the 14-base pair ARE-consensus and the PSA-ARE-3 sequences.
  • Figure 30 shows the results of the ethidium bromide assay for selected compounds in table form.
  • Figure 31 gives the hairpin structure of the nucleotides representing all possible combinations of five base pairs.
  • Figure 32 shows the graphical results of a screen of distamycin A against the library of DNA hairpin oligonucleotides.
  • Figure 33 is a table with the binding constants of distamycin A with particular short AT-rich sequences.
  • Figure 34 is a table with binding constants of different types of DNA with ethidium bromide.
  • Figure 35 shows the results of a screen of compound 128 with a library of 512 DNA hairpin oligonucleotides. The top 20 sequences are shown.
  • Figure 36 shows the binding constants with two types of DNA, Gibb's free energy of binding to poly[dA]-poly[dT] DNA, and the IC 50 's from the L1210 assay of a selected group of 6 compounds which are analogs of high affinity DNA binding agents.
  • Figure 37 shows the binding constants with two types of DNA, Gibb's free energy of binding to poly[dA]-poly[dT] DNA, and the IC 50 's from the L1210 assay of another group of 6 compounds which are analogs of high affinity DNA binding agents.
  • DNA of interest (homopolymers, heteropolymers, or predefined hairpin oligonucleotides) in 96-well plates is treated with ethidium bromide, yielding a large fluorescence increase upon DNA intercalation. Addition of a nonfluorescent DNA binding agent results in a decrease in fluorescence due to displacement of bound ethidium bromide.
  • the decrease in % fluorescence is directly related to the extent of DNA binding providing relative DNA binding affinities and, through subsequent quantitative titration, is capable of providing accurate absolute binding constants.
  • this technique may be used to screen a library of compounds for DNA binding to a single DNA sequence or for the complementary screening a single compound against a full library of DNA sequences which results in the definition of the sequence specificity of a given agent. Combining these in the assay of a library of compounds against a library of DNA, provides qualitative and/or quantitative information on the binding of all library members against a library of available sequences, allowing complete characterization of the DNA binding profiles of each agent in a single experiment.
  • a general set of coupling conditions that gives high yields of coupled product was developed enlisting the amines and carboxylic acids directly and the water soluble 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) with dimethylaminopyridine (DMAP) as an additive.
  • EDCI water soluble 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
  • DMAP dimethylaminopyridine
  • DMABA N, ⁇ /-dimethylaminobutyric acid
  • heterocyclic amino acids selected for the first prototypical libraries are shown in Figure 16. Included in this set are the pyrrole, imidazole, and thiazole amino acids studied by Dervan and Lown, and the indole and CDPI amino acids studied in our laboratories. While not intended to be a survey of optimal heterocyclic amino acids, this set provides built-in known DNA binding agents and could be expected to address issues of identification, practicality and viability which proved to be especially useful in comparisons with positional scanning libraries.
  • the subunits 1 , 5, 6, 9, 10, and 13 were prepared according to known procedures (Baird, E. E., et al., J. Am. Chem. Soc. 1996, 118, 6141 ; Nishiwaki, E., et al., Heterocycles 1988, 27, 1945; Boger, D. L., et al., J. Org. Chem. 1987, 52, 1521 ; and Boger, D. L., et al., Bioorg. Med. Chem. 1995, 3, 1429).
  • the preparation of the remaining subunits is obtained from readily available materials following established procedures and proceed through intermediates 16-29 (Sprague, J. M., et al., J. Am. Chem. Soc.
  • indole nitrogen was protected with a p-methoxybenzyl group to afford indole 31.
  • Hydrolysis to afford the free acid 32 and coupling to the three individual amines afforded the desired dipeptides in moderate yield.
  • Simultaneous deprotection of both the p-methoxybenzyl and BOC-protecting groups afforded the desired amines.
  • each of the individual dipeptides was converted to a mixture of ten tripeptides (Figure 19).
  • An excess of the amine component was used to ensure complete consumption of the ten acids in the reaction mixture.
  • Full matrix mixtures analyzed by mass spectrometry ensured all expected components were present (see Supporting Information). Since the benzoxazole 14b did not couple efficiently under the standard conditions and required an additional purification step, it was omitted from the first position (subunit A) to ensure library fidelity and purity.
  • each of the BOC-trimer mixtures was converted to the corresponding DMABA-trimer mixture as shown in Figure 20.
  • Full matrix mixtures analyzed by mass spectrometry ensured all components were present (see Supporting Information).
  • 2640 distamycin analogs were available in the format of two small mixture libraries. Cytotoxic Activity.
  • the IC 50 values for the DMABA-trimer mixtures were found to be on the order of 10-100 fold higher than the BOC-trimer libraries ( Figure 23). This may be the result of decreased cell penetration of a charged species.
  • the most active mixture contains the CDPI subunit (10) in the final position (subunit C), and the thiophene subunit (8) at the central position (subunit B). This mixture was deconvoluted by synthesis of the ten components, beginning from the BOCNH-8-CONH-10-OMe dimer ( Figure 24).
  • a second round of screening revealed that the most active component of this library contained the benzothiophene (11 ) at the first position (subunit A), with an IC 50 of 0.46 ⁇ M for 86 and it was >10 times more active than any other compound in the mixture and 100 times more potent than any of the individual components of the 49-58 mixture based on and including the close distamycin analogs (Figure 23).
  • the tripyrrole analog 49 exhibited an IC 50 of 44 ⁇ M indistinguishable from that of distamyin A (42 ⁇ M) and 100 times as less potent than 86.
  • the individual BOC-dimers were also tested in the L1210 functional assay. Nearly all the members were essentially inactive (IC 50 > 1 ⁇ M) with the exception of those containing the B subunit 6 and the bicyclic heterocycles 10-15 in the final position (subunit C), five of which exhibited IC 5Q 's ⁇ 1 ⁇ M.
  • the procedure provides a rapid, flexible, and reliable indication of the relative binding affinities of a wide variety of DNA binding ligands.
  • This technique was first examined as a rapid screen for binding to poly[dA]-poly[dT] and poly[dG]-poly[dC] and subsequently extended to hairpin oligonucleotides containing unique sequences.
  • the relative decrease in % fluorescence is proportional to the affinity of a given mixture or individual compound for a particular DNA sequence.
  • the affinity is much lower than for poly[dA]-poly[dT].
  • the BOC- trimer libraries were also screened for DNA binding to both po!y[dA]-poly[dT] and poly[dG]-poly[dC] (data not shown), and they showed substantially lower affinity as expected.
  • Distamycin A Rank Order Binding to a Library of Hairpin Oligonucleotides. Distamycin A is among the best characterized DNA binding compounds. Its DNA binding properties have been studied in depth through footprinting (Portugal, J., et al., Eur. J. Biochem. 1987, 167, 281 ; Portugal, J., et al., FEBS Lett. 1987, 225, 195; Abu-Daya, A., et al., Nucleic Acids Res. 1995, 23, 3385; Abu-Daya, A., et al., Nucleic Acids Res.
  • the results of screening the 512-membered library of hairpin oligonucleotides using distamycin A is given in Figure 32.
  • affinity increases with increasing AT content.
  • the top sequences include the sites 5'- ATAA, ⁇ '-AATT, 5'-AAAT, and 5'-AAAA and among the twenty hairpins showing the greatest decrease in % fluorescence, three four base-pair sequences occur most often: 5'-AATT, 5'-AAAT, 5'-AATA.
  • 128 bound poly[dA]-poly[dT] with an affinity equal to the distamycin analog 49. However, it also bound poly[dG]-poly[dC] with only a slightly reduced affinity being 25-30 times more effective than 49 and, unlike 49, it bound tightly to both the PSA-ARE-3 and ARE consensus sequences.
  • the sequence selectivity of 128 was established by screening it against the library of 512 hairpin oligonucleotides. Compound 128 was found to clearly bind with a selectivity distinct from that of distamycin A and it appears to exhibit a significant preference for PuPyPy sequences.
  • the primary difference between the two molecules is the presence of an additional potential hydrogen bond donor in distamycin and 130 ( ⁇ /-terminal formamide) that is not present in either 49 (C-terminal ester), 129 ( ⁇ /-terminal BOC-group), or 132 (C-terminal dimethylamide).
  • a potential hydrogen bond donor group is included at the C-terminus (131)
  • the binding affinity does approximate that of distamycin A.
  • the difference in free energy of binding between those molecules containing an additional donor hydrogen bonding group (distamycin A and 130-131) and those which do not (49, 129, 132) is approximately 1 kcal/mol, the value of a single hydrogen bond.
  • reaction mixture was poured into EtOAc (50 mL) and washed with 10% aqueous HCI (3 x 50 mL) and saturated aqueous NaHCO 3 (3 x 50 mL).
  • the organic phase was dried (Na 2 SO 4 ), filtered and concentrated to provide 2 (350 mg, 89%) as a tan foam.
  • the reaction mixture was stirred for 3 h at 25 °C, diluted with EtOAc (10 mL) and washed with 10% aqueous HCI (3 x 10 mL) and saturated aqueous NaHCO 3 (3 x 10 mL).
  • the organic phase was dried (Na 2 SO 4 ), filtered and concentrated to a yellow solid.
  • the solid material was suspended in 1 :1 MeOH/10% NaOH (20 mL) and stirred for 30 min at 25 ° C to decompose small amounts of contaminate symmetrical anhydride. The solution was then poured into EtOAc (20 mL) and washed with NaHCO 3 (3 x 20 mL).
  • Distamycin A Hydrochloride A solution of nitrile 4 (12 mg, 0.022 mmol) in dry EtOH (0.3 mL) was treated with 8.0 N HCI/EtOH (1 mL) at 0 °C for 30 min, then slowly warmed to 25 °C and stirred for 2 h. The solvent was removed under a stream of N 2 and the residue was washed with Et 2 O (3 mL) and dried in vacuo for 30 min. The resulting solid was taken up in EtOH (0.3 mL) and treated with 7% NH 3 /EtOH (1 mL) at 25 °C. After 1 h the reaction was concentrated to a tan solid and dried under reduced pressure for 1 h.
  • the crude amidine was dissolved in MeOH (0.2 mL) and cooled to -40 °C.
  • the solution was treated with a solution containing N- formylimidazole, prepared by treating carbonyldiimidazole (18 mg, 0.11 mmol) in THF (0.4 mL) with a solution of formic acid (4.3 mL, 0.11 mmol) in THF (0.4 mL) at 25 °C for 15 min.
  • the reaction mixture was stirred at -40 ° C for 1 h, then concentrated to a volume of 0.2 mL.
  • the product was precipitated with EtOAc (1 mL), and collected by filtration.
  • the crude product was dissolved in cold /-PrOH (2 mL) containing decolorizing carbon (100 mg), stirred at 0 ° C for 30 min, filtered and concentrated to a light yellow solid.
  • the solid material was taken up EtOAc/acetone/MeOH/0.01 N HCI (5:3:1 :1 , 2 mL) and stirred with SiO 2 for 30 min, then filtered through Ceiite to remove traces of NH 4 CI and afford pure distamycin A (4.9 mg, 45%) identical in all respects with authentic material: mp 186-188 ° C.
  • DNA hairpin oligonucleotides were purchased from Genbase Inc. (San Diego) as 880 ⁇ M (base pairs) solutions in water and stored as stock solutions at -80 °C. Prior to use, each oligonucleotide was diluted to 88 ⁇ M in water and stored at 0 °C for no longer than two days. Each well of a 96-well plate was loaded with Tris buffer containing ethidium bromide (0.1 M Tris, 0.1 M NaCl, pH 8, 0.44 x 10 "5 M ethidium bromide final concentration, 88 ⁇ L). To each well was added one hairpin oligonucleotide (10 ⁇ L, 0.88 x 10 ⁇ 5 M in DNA base pairs final concentration).
  • a 3 mL quartz cuvette was loaded with Tris buffer (0.1 M Tris, 0.1 M NaCl, pH 8) and ethidium bromide (0.44 x 10 ⁇ 5 M final concentration).
  • Tris buffer 0.1 M Tris, 0.1 M NaCl, pH 8
  • ethidium bromide 0.44 x 10 ⁇ 5 M final concentration.
  • the fluorescence was measured (ex. 545 nm, em. 595 nm) and normalized to 0% relative fluorescence (free ethidium bromide is only weakly fluorescent).
  • the DNA hairpin oligonucleotide of interest was added (0.88 x 10 "5 M in DNA base pairs final concentration), the fluorescence was measured again and normalized to 100% relative fluorescence.
  • either the substrate or the reacting attachment group may be limiting in solution-phase chemistry. This dictates the use of mix and split synthesis for the solid-phase in order to accommodate differential reaction rates, whereas the simpler protocol of mixture synthesis with limiting reagent stoichiometry may be used in solution to ensure all library members are generated (The exception for solid-phase synthesis enlists an excess of the reacting monomers in adjusted concentrations to accommodate the different reaction rates and requires that this relative rate information be available at the onset of the mixture synthesis. Houghten, R. A.; et al. Nature 1991 , 354, 84; Houghten, R. A.; et al.
  • Each positional scanning library consists of 30 sublibraries that can be divided into three sets. These sets differ in the fixed positions of a monomer subunit within the tripeptide ( Figure 1).
  • the library was prepared by substitution of the same ten subunits for each of the three 4-aminopyrrole-2-carboxylic acid subunits of distamycin A ( Figure 2).
  • a mixture of 100 dimers was synthesized on a 144 ⁇ mol scale by coupling the set of ten amino acid esters 1a and 5a-13a with the corresponding set of ten BOC-amino acids 1b and 5b-13b using 1-[3-(dimethylamino)propyl]- 3-ethylcarbodiimide hydrochloride (EDCI) and dimethylaminopyridine (DMAP) as an additive.
  • EDCI 1-[3-(dimethylamino)propyl]- 3-ethylcarbodiimide hydrochloride
  • DMAP dimethylaminopyridine
  • sublibraries I For the preparation of sublibraries I, where the first position within the trimer is fixed with a single A residue, ten portions of the dimer mixture were deprotected with HCI/EtOAc and coupled to ten individual BOC-amino acids providing ten sublibraries each containing a different and single A residue.
  • the set of ten sublibraries II was assembled by coupling ten individual BOC-amino acids to a mixture of amino acid esters. Subsequent deprotection of the BOC-group and coupling to a mixture of BOC-amino acids yielded the set of ten trimer sublibraries each containing a single and different B residue.
  • dimer mixture of 100 compounds was saponified with LiOH, divided into ten portions, and coupled with ten individual amino acid esters (C residue) to give the third set of sublibraries III.
  • the entire library containing 1000 compounds was synthesized conducting 43 reactions.
  • the most potent residues identified in the scanning library were A 9 , B 9 and B 1 ( and C 10 , C 2 , C 5 and C 6 .
  • the combination of the most potent residues, BOC-A 9 -B 9 -C 10 -OEt was not a compound that exhibited potent cytotoxic activity.
  • none of the alternative 14 possible combinations exhibited cytotoxic activity that approached that of 11 and 12.
  • the screening of the library which entails measurement of the loss of fluorescence derived from compound binding and displacement of prebound ethidium bromide, identified A 6 , B 6 , and C 6 as the most effective residues for binding to the PSA-ARE-3 hairpin containing the 5 base-pair AT-rich site as well as the ARE-consensus hairpin.
  • binding to the latter sequence was less effective ( Figure 11). This constitutes the identification of DMABA-A 6 -B 6 -C 6 -OMe (49), the direct distamycin A analogue, in the 1000-member library as the most effective agent.
  • BOCNH-X-CONH-B ⁇ o-CONH-X-OR Ten single BOC-amino acids 1b and 5-13b (160 ⁇ mol, 11 equiv) and a mixture of ten amino acid esters 1a and 5-13a (each 14.4 ⁇ mol, 1 equiv) were dissolved in DMF (1.5 mL) and treated with EDCI (75 mg, 390 ⁇ mol, 24.4 equiv) and DMAP (49 mg, 400 ⁇ mol, 25 equiv). The solutions were stirred for 16 h at 25 °C.
  • BOCNH-X-CONH-X-CONH-C ⁇ o-OR The mixture of dimers BOCNH-X-CONH-X-OR (190 ⁇ mol, 1.0 equiv) were dissolved in THF/MeOH/H 2 O (20 mL, 2:1 :1), LiOH (760 ⁇ mol, 4 equiv) was added and the mixture was stirred at 25 °C for 18 h. The solvent was removed under reduced pressure and the residue was acidified with 10% aqueous HCI. The dipeptides acids were extracted with EtOAc (3 * 20 mL), the combined organic layers were washed with 10% aqueous HCI and water, dried (Na 2 SO 4 ), filtered, and concentrated in vacuo.
  • Hairpin oligonucleotides (0.887 10 ⁇ 5 M bp) were mixed with ethidium bromide (0.444 10 ⁇ 5 M) in a 2:1 ratio of base-pair:ethidium bromide in a 0.1 M Tris-HCI, 0.1 M NaCl, pH 8 buffer.
  • the fluorescence measurements were conducted at 545 nm excitation and 595 nm emission.
  • 96-well plates (Costar: black, 360 ⁇ L, flat-bottom) were loaded with the premixed ethidium bromide/DNA solution (100 ⁇ L) and single aliquots of each library (1 ⁇ L of 10 mM solutions in DMSO, 99 ⁇ M final concentration) were added. Each plate was incubated at 25 °C for 30 min before reading on a fluorescence plate reader (Molecular Devices SpectraMax Gemini) using 545 nm excitation and 595 nm emission.
  • FIG. 1 shows how the positional scanning libraries were designed.
  • Each positional scanning library consists of 30 sublibraries that can be divided into three sets. These sets differ in the fixed positions of a monomer subunit within the tripeptide. Some of the same compounds which were assembled in a prior study were in the two 1000-member libraries. This was to insure that the quality of information derived from the library assessment could be confirmed.
  • Figure 2 shows the structures of amino acid monomer units used in the preparation of the libraries.
  • the library was prepared by substitution of the same 10 subunits for each of the three 4-aminopyrrole-2-carboxylic acid subunits of distamycin A. Included in this set was the authentic 4-aminopyrrole-2-carboxylic acid subunit of distamycin A so that the natural product analogue was also among the library members.
  • the C-terminus of the library compounds was capped as methyl or ethyl esters and the N-terminus was acylated with 4- dimethylaminobutyric acid (DMABA), a basic side chain that mimics the distamycin A amidine, providing analogues which bear functionalization and a substitution pattern established to provide DNA affinities comparable to that of the natural product.
  • DMABA 4- dimethylaminobutyric acid
  • Another set of library compounds had the N-terminus capped with a terf-butyloxycarbonyl group which renders it neutral and non-nucleophilic.
  • Figure 3 is a scheme illustrating how the positional scanning libraries were synthesized. The synthesis of the library was divided into four parts.
  • a mixture of 100 dimers was synthesized on a 144 mmol scale by coupling the set of ten amino acid esters 1a, 5a-13a with the corresponding set of ten BOC-amino acids 1b, 5b-13b using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDCI) and dimethylaminopyridine (DMAP) as an additive.
  • EDCI 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride
  • DMAP dimethylaminopyridine
  • sublibraries I For the preparation of sublibraries I, where the first position within the trimer is fixed with a single A residue, ten portions of the dimer mixture were deprotected with HCI/EtOAc and coupled to ten individual BOC-amino acids providing 10 sublibraries each containing a different and single A residue.
  • the set of ten sublibraries II was assembled by coupling ten individual BOC-amino acids to a mixture of amino acid esters. Subsequent deprotection of the BOC-group and coupling to a mixture of BOC-amino acids yielded the set of 10 trimer sublibraries each containing a single and different B residue. Finally, the dimer mixture of 100 compounds was saponified with LiOH, divided in ten portions, and coupled with ten individual amino acid esters (C residue) to give the third set of sublibraries III.
  • Figure 4 is a scheme showing how the three sets of ten libraries were functionalized on the N-terminus.
  • the 30 positional scanning libraries were also converted into their corresponding dimethylaminobutyric acid (DMABA) derivatives as shown in Scheme 2.
  • DMABA dimethylaminobutyric acid
  • Figure 5 is a table showing the yields of the BOC- and DMABA-trimers.
  • Figure 6 is a bar graph showing the most potent residues that were found using the positional scanning library.
  • the most potent residues identified in the scanning library were A north and A 12 , B 12 and B 5 , and C 13 , C 6 , C 9 and C,.
  • the combination of the most potent residues, BOC-A 12 -B 12 -C 13 -OEt was not a compound that exhibited potent cytotoxic activity. Moreover, none of the remaining 14 possible combinations exhibited cytotoxic activity that approached that of 67 and 66.
  • FIG. 7 is a table showing the cytotoxic activities of the candidate compounds composed of the most potent residues found by the positional scanning library. Only two compounds, 67 and 66, exhibited potent cytotoxic activity while the third most potent compound was 15-fold less active than 67.
  • Figure 8 shows the structures of the two most potent compounds found in the BOC-trimer library. Both of these compounds have the B 12 and the C 9 residues in common.
  • Figure 9 shows the bar graph of the results of the positional scanning DMABA-trimer library.
  • the DMABA-trimers were less active than the corresponding BOC-trimers.
  • the most potent residues identified were A 12 and A 9 , B ⁇ and C 13 .
  • the preparation and testing of two candidate structures DMABA-A 12 - B ⁇ r C 13 -OEt (210) and DMABA-A g -B ⁇ r C 13 -OEt (220) revealed IC 50 's of 0.32 mM and 3.2 mM, respectively.
  • Figure 10 shows the structures of 86, 210, 220 and 49. Compounds 86, 210 and 220 were identified as potent DMABA-trimers.
  • Compound 49 is a close analogue of distamycin A.
  • Compound 49 and distamycin A have IC 50 's of 42 mM and 44 mM respectively.
  • Figure 11 shows the results of the positional scanning library for the ethidium bromide displacement assay with two hairpin nucleotides. The two hairpin nucleotides are part of the dimer androgen receptor binding consensus sequences, ARE-consensus and PSA-ARE-3.
  • the screening of the library which entails measurement of the loss of fluorescence derived from compound binding and displacement of prebound ethidium bromide, identified A.,, B 1 and C 1 as the most effective residues for binding to the PSA-ARE-3 hairpin containing the 5 base-pair AT-rich site as well as the ARE-consensus hairpin. Binding to the latter sequence was less effective. This constitutes the identification of DMABA-A B C OMe (49), the direct distamycin A analogue, in the 1000 member library as the most effective binding agent.
  • Figure 12 illustrates the solution-phase strategy for developing libraries of new DNA binding agents.
  • the strategy involves systematically replacing the N- methylpyrrole subunit with other heterocyclic amino acids to give a first generation library in a small mixture format. This is done by using liquid-liquid purification protocols. Then a basic side chain is added to expand the possible number of compounds and to mimic the amidine side chain of distamycin A.
  • Figure 13 is a simplified illustration of the general procedure for the rapid DNA binding screen.
  • the chosen DNA as homopolymers, heteropolymers or hairpin oligonucleotides is placed in 96 well plates.
  • Upon treatment with ethidium bromide there is a large increase in fluorescence as ethidium bromide intercalates with the DNA.
  • a non-fluorescent DNA binding agent is added there is a percentage decrease in the fluorescence due to binding.
  • the percentage decrease in fluorescence is proportional to the extent of DNA binding. This provides the relative DNA binding affinities and through quantitative titration that may be carried out later, an accurate, absolute binding constant is obtained.
  • Figure 14 is a scheme showing the synthesis of distamycin A.
  • pyrrole carboxylic acid 1a (Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 778, 6141.) coupling with aminopyrrole 1b (Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 778, 6141.) using EDCI/DMAP afforded 2 in high yield (97%).
  • Removal of the BOC protecting group with HCI/EtOAc followed by coupling to pyrrole 1a afforded the tripeptide 3 in good yield (96%).
  • Figure 15 shows how two prototypical libraries of potential DNA binding agents were prepared in a small mixture format.
  • eleven ⁇ /-BOC heterocyclic amino acids and twelve amino esters the individual subunits were coupled using EDCI/DMAP to provide all possible 132 individual dipeptides in parallel.
  • the use of EDCI and DMAP allows for the removal of excess coupling agents and their reaction by-products along with unreacted starting materials by acid/base liquid-liquid extraction.
  • These individual dimers were deprotected and coupled to a mixture of ten ⁇ /-BOC carboxylic acids to give 132 mixtures of ten N- BOC-trimers where only the last position (subunit A) is undefined (1320 compounds).
  • FIG. 16 shows the heterocyclic amino acids selected for the first prototypical libraries. This set includes the pyrrole, imidazole, and thiazole amino acids which were studied by Dervan and Lown and the indole and CDPI amino acids previously studied by the inventor.
  • Figure 17 shows the preparation of the dimers using 1 b and 5b-13c as the acid component and 1a and 5a-15a as the amine component, 120 individual dimers were prepared. Each dimer was prepared in 70-80 mg quantities in parallel, using only acid/base liquid-liquid extraction purification to afford products in typically >80% yield and >95% purity.
  • Figure 18 shows the three instances when using the indole subunit 13b as the acid component in couplings with the three unreactive amines (5a, 7a, and 14a), the diketopiperazine 30 was isolated due to indole dimerization. To circumvent this problem, the indole nitrogen was protected with a p- methoxybenzyl group to afford indole 31.
  • Figure 19 shows how the preparation of the trimer libraries was investigated initially by preparing several sets of individual trimers to ensure the reaction conditions were appropriate. Deprotection of the dimer with HCI/EtOAc followed by coupling with 1b, 5b-13b afforded the ten BOC-trimers in high yield and with >90% purity using only acid/base liquid-liquid extraction purification.
  • This set based on the dipyrrole dimer is of special interest because it contains a close analog of distamycin, the tripyrrole 39 (BOCNH-1-CONH-1-CONH-1-OMe).
  • Figure 20 shows the reaction conditions for adding the dimethylaminobutyric acid side chain (DMABA) to the individual trimers. The yields were much more variable than the previous steps since some of the derivatives were appreciably soluble. The typical acid/base liquid-liquid purification protocol was modified because of this. The solvent was removed from the reactions, the products were suspended in water and extraction with EtOAc gave the desired products.
  • Figure 21 shows the graphical results of the cytotoxicity assay (L1210) for the BOC-trimer libraries. Thirteen of the libraries showed activity at less than 1 ⁇ M, one showed activity at 100 nM. The most active library contained the benzofuran subunit (12) at the central position (subunit B) and the imidazole subunit (9) at the final position (subunit C).
  • Figure 22 shows the deconvolution of the mixture by the resynthesis of the
  • Figure 23 shows the graphical results of the cytotoxicity assay (L1210) for the DMABA-trimer libraries.
  • the IC 50 values for the DMABA-trimer libraries are on the order of 10-100 fold higher than the BOC-trimer libraries ( Figure 10).
  • the most active mixture contains the CDPI subunit (10) in the final position (subunit C), and the thiophene subunit (8) at the central position (subunit B).
  • Figure 24 illustrates the synthesis of the individual members of the library for deconvolution of the mixture.
  • a second round of screening revealed that the most active component of this library contained the benzothiophene (11 ) at the first position (subunit A), with an IC 50 of 0.46 ⁇ M for 86 and it was >10 times more active than any other compound in the mixture and 100 times more potent than any of the individual components of the 49-58 mixture based on and including the close distamycin analogues ( Figure 12).
  • the corresponding BOC-trimers were tested as well and these show a 10-100 fold greater activity than the DMABA- trimers.
  • Figure 25 shows the general procedure for establishing DNA binding of a library of compounds with a single sequence.
  • Figure 26 shows the binding results for the DMABA-trimer library with poly[dA]-poly[dT].
  • 1 all the DMABA-trimers induce some decrease in fluorescence, indicating the libraries have an overall AT affinity; (2) high affinity libraries contain one of the larger subunits at the second (B) position (monomers 10-14); and (3) the smaller subunits at the third (C) position (monomers 1, 5-9) appear to be more active.
  • four of the 10 compound mixtures showed a higher affinity than the pyrrole sublibrary containing 49, the tripyrrole analog of distamycin.
  • the highest affinity mixture contains the CDPI subunit (10) at the second position and the imidazole subunit (9) at the third position.
  • the second most effective mixture contains the benzothiophene (11) at the second position and the pyrrole (1) at the third position.
  • Figure 27 shows the synthesis of mixtures that were deconvoluted, both the BOC- and DMABA trimers.
  • Figure 28 tabulates the results of the L1210 assay on individual compounds synthesized for deconvolution. The DNA binding properties of three sets of individual compounds are also shown in the table. The activity of the mixtures in the cell-based assay approximated that of the individual components and established the reliability of testing in the small mixture format for libraries.
  • Figure 29 is a bar graph illustrating the binding affinity to two related sequences of the androgen response element, the 14-base pair ARE-consensus (Cato, A. C. B.; Hernerson, D.; Ponta, H. EMBO J. 1987, 33, 545) and the PSA- ARE-3 (Cleutjens, D. B. J. M.; et al., Mol. Endocrinol. 1993, 7, 23) sequences in the ethidium bromide displacement assay.
  • the 14-base pair ARE-consensus Cato, A. C. B.; Hernerson, D.; Ponta, H. EMBO J. 1987, 33
  • Figure 30 displays the same results of the ethidium bromide assay in table form. Screening the individual components of this mixture afforded the direct distamycin A analog 49 as having the highest affinity, followed closely by 53 containing the thiophene subunit at the first position (A). The same overall pattern was observed with poly[dA]-poly[dT], where 49 and 53 also showed the highest affinity (Table 3). Both these agents exhibited diminished affinity for the ARE-consensus sequence presumably resulting from the intervening GC base pair.
  • Figure 31 shows the hairpin DNA oligomer used in the binding affinity assay.
  • a survey of distamycin A binding to all possible 5 base pair DNA sequences was conducted using a library of 512 of these hairpin DNA oligonucleotides containing all possible five base pair sequences of the general format 5'-GCXXXXXC-3' with a 5-A loop.
  • two complementary sequences are contained in each hairpin differing only in their location relative to the position of the adenine loop making, for example, the sequence 5'-ATGCA equivalent to the sequence 5'- TGCAT as shown in the lower portion of the figure.
  • Figure 32 shows the results of screening the 512-membered library of hairpin oligo-nucleotides using distamycin A. As expected, affinity increases with increasing AT content.
  • the top sequences include the sites 5'-ATAA, 5'-AATT, 5'-AAAT, and 5'-AAAA and among the twenty hairpins showing the greatest decrease in % fluorescence, three four base-pair sequences occur most often: 5'- AATT, 5'-AAAT, 5'-AATA.
  • Figure 33 is a table showing the few absolute binding constants for distamycin A to short AT-rich sequences that have been published. The comparison of all those disclosed show the relative trend 5'- AATTT>AAAAA>AATAA>ATTAA (Rentzeperis, D.; et al. Biochemistry 1995, 34, 2937 and Wade, W. S.; Mrksich, M.; Dervan, P. B. Biochemistry 1993, 32,
  • the ethidium bromide displacement assay revealed the same general trend and a quantitative titration measurement of binding constants with the hairpin oligo-nucleotides containing these sequences afforded binding constants that are not only consistent with the relative trend ( Figure 21), but also within a factor of 2-3 of all the absolute binding constants previously determined through calorimetry and footprinting (Table 4). Given that the DNA upon which the measurements were made is different, that the buffer conditions are not identical, and that entries 2-4 were derived from a close analog of distamycin A, all which may contribute to small discrepancies in the absolute binding constants, the ethidium bromide displacement titration assay appears to be remarkably accurate at reproducing absolute binding constants.
  • Figure 34 shows the ethidium bromide binding constants obtained from Baguley, B.C.; Falkenhaug, E.-M. Nucleic Acid Res. 1978, 5, 161.
  • the trend displayed is that the ethidium bromide binding constant varies considerably and the displacement does not follow a 1 :1 stoichiometry. Both factors complicate the use of a competitive binding model for establishing binding constants.
  • Figure 35 is a bar chart showing the 20 highest affinity sequences.
  • the sequence selectivity of compound 128 was established by screening it against the library of 512 hairpin oligonucleotides. It was found to clearly bind with a selectivity distinct from that of distamycin A and it appears to exhibit a significant preference for PuPyPy (purine-pyrimidine-pyrimidine) sequences.
  • 16 contain the PuPyPy motif (80%), where statistically 37.5% of the sequences would be expected to contain this motif in a random sample.
  • One of the four exceptions contained a five-base-pair AT-rich site. Within both of the androgen response elements used to identify 128, the PuPyPy motif is repeated three times.
  • Figure 36 shows a number of derivatives of distamycin A where the side chains were altered in a systematic manner. Analysis of these derivatives using the quantitative titration with displacement of prebound ethidium bromide showed that there is very little difference between an amidine as the basic side chain and the dimethylamino group (Distamycin vs. 130) or between placing the basic side chain at the C- or /V-terminal end of the trimer (49 vs. 129).
  • Amine 129 shows slightly lower binding affinity to poly[dA]-poly[dT] than 49 which may arise from the incorporation of a bulky f-butyl group present at the /V-terminus. Interestingly, there is approximately a three-fold difference in binding between distamycin A or 130 and the tripyrrole 49. The primary difference between the two molecules is the presence of an additional potential hydrogen bond donor in distamycin and
  • Figure 37 shows a number of derivatives of the trimer core of 128 where the side chains were altered in a systematic manner. Analysis of these derivatives using the quantitative titration with displacement of prebound ethidium bromide showed that there is very little difference between an amidine as the basic side chain and the dimethylamino group (136 vs. 138) or between placing the basic side chain at the C- or /V-terminal end of the trimer (138 vs. 128).
  • Amine 128 shows slightly lower binding affinity to poly[dA]-poly[dT] than 138. Interestingly, there is approximately a three-fold difference in binding between 136 and 138.
  • the primary difference between the two molecules is the presence of an additional potential hydrogen bond donor in 136 ( ⁇ /-terminal formamide) that is not present in either 128 (C-terminal ester), 138 ( V-terminal BOC-group), or 135 (C-terminal dimethylamide).
  • the difference in free energy of binding between those molecules containing an additional donor hydrogen bonding group (136, 134) and those which do not (138, 135) is approximately 0.7 kcal/mol, which is 70% of the value of a single hydrogen bond.
  • Adding a second substituent containing an additional basic, protonated amine (128 vs 137) does not further increase the DNA binding affinity.

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Abstract

L'invention se rapporte à la mise en oeuvre d'une synthèse en phase soluble de distamycine A et à son extension à la préparation de 2640 analogues. On a ainsi mis en oeuvre des techniques de synthèse en phase soluble avec préparation à la réaction et purification utilisant des extractions liquide-liquide, acide-base, dans la préparation à étapes multiples de la distamycine A (8 étapes, 40 % de rendement global) ainsi qu'une banque prototypique de 2640 analogues fournissant des intermédiaires et des produits finis qui sont purs à plus de 95 % sur l'échelle des réactions classiques. La recherche dans la banque prototypique a fourni des composés qui sont 1000 fois plus puissants que la distamycine A dans les analyses cytotoxiques (67, IC50 = 29 nM, L1210), qui se lient à poly[dA]-poly[dT] avec une affinité comparable, et qui présentent une sélectivité de séquence de liaison à l'ADN modifiée. On a identifié plusieurs candidats qui se lient au site riche de cinq paires de base de la séquence PSA-ARE-3, et l'on a conservé (128, K = 3.2 x 106 M-1) la forte liaison par affinité (K = 4.5 x 106 M-1) à la séquence de consensus-ARE contenant un site riche de cinq paires de base AT, interrompu par une paire de base GC, adapté à l'inhibition de la transcription génique initialisée par dimérisation des récepteurs androgènes insensibles aux hormones et liaison à l'ADN caractéristique du cancer de la prostate résistant aux traitements.
PCT/US2001/019404 2000-06-14 2001-06-14 Analogues de distamycine a Ceased WO2001096313A1 (fr)

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WO2004101554A1 (fr) * 2003-05-19 2004-11-25 Sanofi-Aventis Deutschland Gmbh Nouveaux dérivés d'indole en tant qu'inhibiteurs du facteur xa
US6825228B2 (en) 2001-06-13 2004-11-30 Genesoft Pharmaceuticals, Inc. Benzothiophene compounds having antiinfective activity
US7129214B2 (en) 2002-12-10 2006-10-31 Oscient Pharmaceuticals Corporation Antibacterial compounds having a (pyrrole carboxamide)-(benzamide)-(imidazole carboxamide) motif
US7141680B2 (en) 2002-09-20 2006-11-28 Genelabs Technologies, Inc. Aromatic compounds possessing antifungal or antibacterial activity
US7265129B2 (en) 2002-10-25 2007-09-04 Genesoft Pharmaceuticals, Inc. Anti-infective biaryl compounds
US7498349B2 (en) 2002-08-02 2009-03-03 Genesoft Pharmaceuticals, Inc. Biaryl compounds having anti-infective activity
US8012967B2 (en) 2006-09-30 2011-09-06 University Of Strathclyde Minor groove binders
US10562912B2 (en) 2013-06-05 2020-02-18 C&C Research Laboratories Heterocyclic derivatives and use thereof

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US5401851A (en) * 1992-06-03 1995-03-28 Eli Lilly And Company Angiotensin II antagonists
US5475092A (en) * 1992-03-25 1995-12-12 Immunogen Inc. Cell binding agent conjugates of analogues and derivatives of CC-1065
US5786377A (en) * 1993-11-19 1998-07-28 Universidad De Santiago De Compostela Pyrrolo 3,2-E!indol derivatives, process for the preparation thereof and applications

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US5475092A (en) * 1992-03-25 1995-12-12 Immunogen Inc. Cell binding agent conjugates of analogues and derivatives of CC-1065
US5401851A (en) * 1992-06-03 1995-03-28 Eli Lilly And Company Angiotensin II antagonists
US5786377A (en) * 1993-11-19 1998-07-28 Universidad De Santiago De Compostela Pyrrolo 3,2-E!indol derivatives, process for the preparation thereof and applications

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6825228B2 (en) 2001-06-13 2004-11-30 Genesoft Pharmaceuticals, Inc. Benzothiophene compounds having antiinfective activity
US7329765B2 (en) 2001-06-13 2008-02-12 Genesoft Pharmaceuticals, Inc. Benzothiophene compounds having antiinfective activity
US7498349B2 (en) 2002-08-02 2009-03-03 Genesoft Pharmaceuticals, Inc. Biaryl compounds having anti-infective activity
US7141680B2 (en) 2002-09-20 2006-11-28 Genelabs Technologies, Inc. Aromatic compounds possessing antifungal or antibacterial activity
US7265129B2 (en) 2002-10-25 2007-09-04 Genesoft Pharmaceuticals, Inc. Anti-infective biaryl compounds
US7129214B2 (en) 2002-12-10 2006-10-31 Oscient Pharmaceuticals Corporation Antibacterial compounds having a (pyrrole carboxamide)-(benzamide)-(imidazole carboxamide) motif
US7642245B2 (en) 2002-12-10 2010-01-05 Oscient Pharmaceuticals Corporation Antibacterial compounds having a (pyrrole carboxamide)-(benzamide)-(imidazole carboxamide) motif
WO2004101554A1 (fr) * 2003-05-19 2004-11-25 Sanofi-Aventis Deutschland Gmbh Nouveaux dérivés d'indole en tant qu'inhibiteurs du facteur xa
US8012967B2 (en) 2006-09-30 2011-09-06 University Of Strathclyde Minor groove binders
US10562912B2 (en) 2013-06-05 2020-02-18 C&C Research Laboratories Heterocyclic derivatives and use thereof

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