- Email: [email protected]
Combinatorial Chemistry Online Volume 11, Issue 2, February 2009
Combinatorial Chemistry - An Online Journal 11 (2009) 4–7
Contents lists available at ScienceDirect
Combinatorial Chemistry - An Online Journal journal homepage: www.elsevier.com/locate/comche
Combinatorial Chemistry Online Volume 11, Issue 2, February 2009 N. K. Terrett Ensemble Discovery Corp., Cambridge, MA 02139, USA
O
1. Current literature highlights R1
1.1. Delivery of RNAi therapeutics from a combinatorial library of lipid-like materials
R1
1) R2NH2 2) MeI
O O
N+ R2
O 1 3 R O
1
Gene expression can be modulated through the application of RNA interference (RNAi) technology. Such modulation offers the potential of a new class of therapeutics that addresses previously untreatable diseases. However, in vivo delivery of these putative RNAi therapeutics is challenging. Certain viruses, such as respiratory syncytial virus, can be inhibited locally after administration of naked small interfering RNA (siRNA), and the systemic delivery of siRNA to the liver of non-human primates has been demonstrated using a lipid formulation. Despite these examples, there are relatively few reports demonstrating efficacy of systemically delivered siRNA in primates. Research to date has focused on cationic lipid delivery systems, but the development of safe and effective methods, both in vitro and in vivo, has proven problematic. The delivery of siRNA has been mediated, for example, by direct conjugation of delivery agents to the RNA moiety or peptide-based delivery systems. A barrier to the exploration of new delivery materials is the synthesis of the delivery material itself. Conventional lipid synthesis typically requires individually optimised, multiple-step synthesis, including protection and deprotection stratagems, use and removal of catalysts, solvent exchanges and purification. The optimisation of each (multiple) synthetic step therefore has limited throughput and so limits the ability to generate libraries of substantial size and diversity. Recent work has attempted to develop chemical methods capable of the rapid, parallel generation of lipid-like molecules.1 Library compounds were synthesised through a conjugate addition of primary or secondary amines to alkylacrylates (1) or alkylacrylamides (2) to generate compounds of general structure 3 and 4. The advantage of this type of chemistry is that, unlike many traditional lipid synthesis chemistries, it allows for reaction in the absence of solvent or catalysts and results only in lipidoid product, thereby eliminating the need for protection and deprotection steps and purification.
O
H N
O
R1
O O R1
N H
NH2
H2N
R1
O
2
R1
H N
N
N H
R1
O
N
4
N H
Using this methodology, several libraries totalling more than 1200 lipidoid members were synthesised. Library members were then screened for their ability to deliver siRNA to a HeLa cell line that stably expresses both firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase. Efficacy of siRNA delivery by lipidoids was determined by treating cells with these complexes, prepared using a firefly luciferase-targeting siRNA, and then measuring the ratio of firefly to Renilla luciferase expression. A number of compounds were found to enhance the delivery of siRNA to HeLa cells. In a further round of experiments, Factor VII, a blood clotting factor, was chosen to assess targeted siRNA delivery to the liver. At least seven lipidoid formulations were identified that mediated significant reduction of serum Factor VII protein levels, with the largest reduction observed for compound 5. The development of a library of lipid-like materials represents an expansion in the diversity and range of intracellular delivery materials, and has clearly demonstrated potential for in vivo utility. (CH2)11CH3 HN
O
O
H N
(CH2)11CH3
O H3C(CH2)11
N
N H O
H3C(CH2)11
N H
N H
N
(CH2)11CH3 NH
N 5
O
1.2. New thrombin binding aptamer analogues containing an acyclic nucleotide
E-mail: [email protected] doi:10.1016/j.comche.2009.01.001
The serine protease thrombin plays a central role in blood clotting, converting soluble fibrinogen to fibrin, activating the factors
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7
V, VIII, and XI, and stimulating platelet aggregation. There are many examples of endogenous molecules that bind to thrombin allowing regulation of the clotting process, and so preventing any loss of blood and inappropriate clotting in healthy individuals. The development of anticoagulant strategies to inhibit thrombogenesis in patients is of great interest and among these the most important are focused on direct or indirect inhibition of thrombin. One such inhibitor that specifically binds to human thrombin (with an EC50 of 20 nM) is a 15-oligomeric aptamer. The solution-state three-dimensional structure of the thrombin binding aptamer has been determined by NMR methods. On the basis of previous NMR results produced by studying the folding of modified aptamers, it can be deduced that the tertiary structure of the thrombin binding aptamer adopts a chair-like conformation. Recent work has attempted to obtain new insights into the recognition event between thrombin and the thrombin binding aptamer.2 This work has undertaken to study the synthesis and the structural and biological characterisation of a new series of modified thrombin binding aptamers in which the thymidines have been replaced, sequentially, by the acyclic nucleoside N1-(3-hydroxy-2hydroxymethyl-2-methylpropyl)-thymine to give 6. Oligonucleotides incorporating acyclic nucleotides are of interest, in part, because they are less susceptible to nuclease action than their natural counterparts. The structural changes between the acyclic 6 and the unmodified T residue 7 result in a one bond shortening between the T base and one of the phosphate groups. Additionally the replacement results in the introduction of a hydrophobic methyl group, and the presence of a prochiral carbon in the linker that develops into a chiral center when the modified nucleotide is inserted into the DNA sequence.
H N
O
O
O
P
CH 3
H 3C O
O-
O O
6
P
O
O P
O-
O
H N
O N
O
O
CH 3
O O
O7
O
P
an aqueous ammonia solution (33%) at 55 °C overnight. The oligonucleotides were evaluated for any effect, following incubation with human plasma, on prothrombin time. The concentration of oligonucleotide required to double the fibrinogen clotting time was also determined. From this library, a number of active oligonucleotides were obtained. One of the most active gave a prothrombin time of 24 s at a concentration of 2 M, and a concentration of 70 nM of the oligonucleotide was found to double time to clot in the fibrogen clotting assay. 2. A summary of the papers in this month’s issue 2.1. Solid-phase synthesis A general solid-phase synthesis of 1,2,5-trisubstituted imidazolidin-4-ones has been described. The key synthetic transformation incorporates a microwave-assisted condensation of an aamino amide on solid support with an aldehyde in solution to give the corresponding resin-bound imidazolidin-4-one in a simple one-pot procedure.3 A new straightforward solid-phase synthesis of perfluoroalkylated dimerisable detergents used as gene delivery systems has been presented. The route used Fmoc-Cys(SASRINTM–Ò)-OH resin as solid support, affording the target products in almost quantitative yields and high purity.4 An efficient method for the solid-phase synthesis of aryl amines, heteroaryl amines, and sterically hindered alkyl amines has been developed. The key step in this process was the formation of resin-bound carbamates by the Curtius rearrangement of aryl carboxylic acids with Wang resin providing the trapping hydroxyl group. The developed methods can be applied to the preparation of libraries containing aryl, heteroaryl, and sterically hindered alkyl amine structures as the pharmacophores.5 2.2. Solution-phase synthesis
N O
5
O-
O
The ability of the synthesised sequences to assume a quadruplex conformation has been evaluated by NMR, UV, and CD spectroscopies, and thrombin inhibitory activity was determined in prothrombin time and purified fibrinogen clotting assays. The study also synthesised oligonucleotide analogues using standard solid-phase DNA chemistry on a controlled pore glass support. The oligomers were detached from the support and deprotected by treatment with
Chemoselectivity of chlorine versus methylthio (–SCH3), methylsulfinyl (–SOCH3), or methylsulfonyl [–S(O)2CH3] in either the functionalisation or substitution of pyridopyrimidinones and pyrimidopyrimidinones has been studied. Use of this methodology to prepare final targets for rapid SAR studies via two-dimensional array syntheses has been demonstrated.6 A rapid and efficient strategy for the preparation of spiro-pyridones and spiro-quinolones using sequential Pd(0)/C-catalysed allylation and ring closing metathesis reactions has been reported. Application of the present methodology to nucleoside chemistry has also been investigated, and it has also been used in library preparation.7 A method for the formation of libraries of synthetic macrocycles with different ring sizes from diols has been described. Reacting a simple diol precursor with electrophilic reagents leads to a cyclic carbonate, sulphite, or phosphate in a single step in 25–60% yield. Alternatively, converting the cyclisation precursor to a bis-electrophilic iodide or aldehyde enables preparation of a cyclic sulphide and amine respectively, the latter using a double-reductive amination to induce ring closure.8 A global synthetic strategy based on the ‘garlanding’ concept to design and to produce halo-oligopyridines has been reported. The strategy allows the preparation of an infinite number of these new compounds and hence access to a library of halooligopyridines.9 The PEG-immobilisation and the liquid phase synthesis of some coumarins derived from cardol have been presented. Some preliminary results on their tyrosinase inhibitory activity are also included.10
6
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7
2.3. Scaffolds and synthons for combinatorial libraries Efficient reaction conditions have been developed to prepare highly functionalised chiral pyranosido-pyrimidines by regioselective alkylation of the pyranoside ring and palladium-catalysed cross-coupling reaction on the pyrimidine ring under microwave activation. Such scaffolds contain three different functionalities suitable for selective derivatisation and could find applications in the design of peptidomimetics.11 2.4. Solid-phase supported reagents Acetylation of various alcohols and phenols has been performed successfully in high yield using an immobilised cobalt(II) catalyst. The catalyst shows high thermal stability and was also recovered and reused at least ten times without any significant activity loss.12
ionic lipids with short spacer length and short hydrophobic tails bound to DNA and delivered DNA into HEK293 cells more efficiently than those with longer ones. These results suggest that these newly described aminoglycerol-based lipids should be very promising in liposome-mediated gene delivery and illustrate the potential of solid phase synthesis methodology for non-viral vector discovery.19 The human pathogenic bacterium Pseudomonas aeruginosa produces a fucose-specific lectin, LecB, implicated in tissue attachment and the formation of biofilms. To investigate if LecB inhibition disrupts these processes, high-affinity ligands were obtained by screening two 15,536-member combinatorial libraries of multivalent fucosyl-peptide dendrimers. The most potent LecB-ligands identified were dendrimers that led to complete inhibition of P. aeruginosa biofilm formation and induced complete dispersion of established biofilms in the wild-type strain and in several clinical P. aeruginosa isolates.20
2.5. Novel resins, linkers and techniques References Fluorescein isothiocyanate (FITC) is an amine-reactive derivative of fluorescein dye that has wide ranging applications in labelling peptides and proteins. However, its use in solid phase peptide synthesis is restricted by instability in acidic conditions required for linker cleavage. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is used to release targeted peptide from the resin.13 2.6. Library applications The synthesis and identification of sulphonamidoaryl ethers as potent bradykinin B1 receptor antagonists from a 60,000 member encoded combinatorial library have been reported. Two distinct series of compounds exhibiting different structure–activity relationships were identified in a bradykinin B1 whole-cell receptorbinding assay.14 Cdc7 kinase plays a critical role in the regulation of DNA replication in eukaryotic cells and has been proposed as a target for cancer therapy. A class of Cdc7/Dbf4 inhibitors with a pyrido-thieno-pyrimidine core structure have been identified, and the synthesis of a focused pyrido-thieno-pyrimidine library yielded potent and selective Cdc7 inhibitors with antiproliferative activity against cancer cells in vitro.15 The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the av-integrins avb3 and avb5 has been described. High-throughput screening of an extensive series of ECLiPSTM compound libraries led to the identification of a dual inhibitor of the av-integrins avb3 and avb5.16 The discovery and synthesis of a series of 2-amino-5-benzoyl-4(2-furyl)thiazoles as adenosine A2A receptor antagonists from a small-molecule combinatorial library using a high-throughput radioligand-binding assay has been described. Antagonists were further characterised in the A2A binding assay and an A1 selectivity assay, and selected examples exhibited excellent affinity for A2A and good selectivity versus the A1 receptor.17 A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesised and evaluated for their antiplasmodial activities. Cross screening of the library against cultured Leishmania donovani parasites revealed this class of compound as potent inhibitors of in vitro parasite development.18 The solid phase synthesis of a library of aminoglycerol–diamine conjugate-based transfection agents having urea linkage between diverse length of diamines and various lengths of hydrophobic tails has been described. These compounds were characterised and structure–activity relationships determined for DNA binding and transfection ability when formulated as cationic liposomes. Cat-
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Akinc, A. et al. Nat. Biotechnol. 2008, 26 (5), 561–569. Coppola, T. et al. Bioorg. Med. Chem. 2008, 16 (17), 8244–8253. Qin, L.-Y. et al. Tetrahedron Lett. 2009, 50 (4), 419–422. Guilloteau, N. et al. Tetrahedron Lett. 2009, 50 (5), 562–563. Sunami, S.; Ohkubo, M. Tetrahedron 2009, 65 (3), 638–643. Wan, Z. et al. Tetrahedron Lett. 2009, 50 (3), 370–372. Kersten, L. et al. Tetrahedron Lett. 2009, 50 (5), 506–508. Wingstrand, M. J. et al. Tetrahedron Lett. 2009, 50 (6), 693–695. Voisin-Chiret, A. S. et al. Tetrahedron 2009, 65 (3), 607–612. Tocco, G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (1), 36–39. Samb, I. et al. Tetrahedron 2009, 65 (4), 896–902. Rajabi, F. Tetrahedron Lett. 2009, 50 (4), 395–397. Jullian, M. et al. Tetrahedron Lett. 2009, 50 (3), 260–263. Cole, A. G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (1), 119–122. Zhao, C. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 319–323. Letourneau, J. J. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 352–355. Cole, A. G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 378–381. Musonda, C. C. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 401–405. Yingyongnarongkul, B. e. et al. Bioorg. Med. Chem. 2008, 17 (1), 176–188. Johansson, E. M. V. et al. Chem. Biol. 2008, 15 (12), 1249–1257.
Further reading Papers on combinatorial chemistry or solid-phase synthesis from other journals Chen, M.-L.; Adak, A. K.; Yeh, N.-C.; Yang, W.-B.; Chuang, Y.-J.; Wong, C.-H.; Hwang, K.-C.; Hwu, J.-R. R.; Hsieh, S.-L.; Lin, C.-C. Fabrication of an oriented Fc-fused lectin microarray through boronate formation. Angewandte Chemie, International Edition 2008, 47 (45), 8627–8630. Pattabiraman, V. R.; McKinnie, S. M. K.; Vederas, J. C. Solid-supported synthesis and biological evaluation of the lantibiotic peptide bis(desmethyl) lacticin 3147 A2. Angewandte Chemie, International Edition 2008, 47 (49), 9472–9475. Park, J.-J.; Lee, J. H.; Li, Q.; Diaz, K.; Chang, Y.-T.; Chung, S.-K. Divergent syntheses of all stereoisomers of phytosphingosine and their use in the construction of a ceramide library. Bioorganic Chemistry 2008, 36 (5), 220–228. Rivera-Monroy, Z.; Bonn, G. K.; Guttman, A. Fluorescent isotope-coded affinity tag (FCAT) I: Design and synthesis. Bioorganic Chemistry 2008, 36 (6), 299311. Pettersson, S.; Perez-Nueno, V. I.; Ros-Blanco, L.; Puig de La Bellacasa, R.; Rabal, M. O.; Batllori, X.; Clotet, B.; Clotet-Codina, I.; Armand-Ugon, M.; Este, J.; et al. Discovery of novel non-cyclam polynitrogenated CXCR4 coreceptor inhibitors. ChemMedChem 2008, 3 (10), 1549–1557. Tarrago, T.; Masdeu, C.; Gomez, E.; Isambert, N.; Lavilla, R.; Giralt, E. Benzimidazolium salts as small, nonpeptidic and BBB-permeable human prolyl oligopeptidase inhibitors. ChemMedChem 2008, 3 (10), 1558–1565. Heinze, K.; Beckmann, M.; Hemple, K. Solid-phase synthesis of transition-metal complexes. Chemistry – A European Journal 2008, 14 (31), 9468–9480. Heinze, K.; Reinhardt, S. Platinum (II) complexes with non-innocent ligands: solidphase synthesis, redox chemistry and luminescence. Chemistry – A European Journal 2008, 14 (31), 9482–9486. Nielsen, M. C.; Ulven, T. Selective extraction of G-quadruplex ligands from a rationally designed scaold-based dynamic combinatorial library. Chemistry – A European Journal 2008, 14 (31), 9487–9490. Charton, J.; Charruault, L.; Deprez-Poulain, R.; Deprez, B. Alkylsquarates as key intermediates for the rapid preparation of original drug-inspired compounds Combinatorial Chemistry & High Throughput Screening 2008, 11 (4), 294–303. Tse, B. N.; Snyder, T. M.; Shen, Y.; Liu, D. R. Translation of DNA into a library of 13 000 synthetic small-molecule macrocycles suitable for in vitro selection. Journal of the American Chemical Society 2008, 130 (46), 15611–15626.
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7 Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. Solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates using bicyclic oxazaphospholidine derivatives as monomer units. Journal of the American Chemical Society 2008, 130 (47), 16031–16037. Dolle, R. E.; Le Bourdonnec, B.; Goodman, A. J.; Morales, G. A.; Thomas, C. J.; Zhang, W. Comprehensive survey of chemical libraries for drug discovery and chemical biology: 2007. Journal of Combinatorial Chemistry 2008, 10 (6), 753–802. Lazny, R.; Nodzewska, A.; Zabicka, B. Asymmetric solid-phase alkylation of ketones immobilized via SAMP hydrazone analogue linkers. Journal of Combinatorial Chemistry 2008, 10 (6), 986–991.
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Ko, K.-S.; Park, G.; Yu, Y.; Pohl, N. L. Protecting-group-based colorimetric monitoring of fluorous-phase and solid-phase synthesis of oligoglucosamines. Organic Letters 2008, 10 (23), 5381–5384. Brouillette, Y.; Verdie, P.; Martinez, J.; Lisowski, V. Valuable versatile reactivity of thiaisatoic anhydrides: expedient solid-phase synthesis of thieno[1,4]diazepine2,5-diones. Synlett 2008, (15), 2360–2364. Hahn, F.; Muellen, K.; Schepers, U. 2-Iminothiolane as a useful coupling reagent for polyamine solid-phase synthesis. Synlett 2008, (18), 2785– 2790.
Contents lists available at ScienceDirect
Combinatorial Chemistry - An Online Journal journal homepage: www.elsevier.com/locate/comche
Combinatorial Chemistry Online Volume 11, Issue 2, February 2009 N. K. Terrett Ensemble Discovery Corp., Cambridge, MA 02139, USA
O
1. Current literature highlights R1
1.1. Delivery of RNAi therapeutics from a combinatorial library of lipid-like materials
R1
1) R2NH2 2) MeI
O O
N+ R2
O 1 3 R O
1
Gene expression can be modulated through the application of RNA interference (RNAi) technology. Such modulation offers the potential of a new class of therapeutics that addresses previously untreatable diseases. However, in vivo delivery of these putative RNAi therapeutics is challenging. Certain viruses, such as respiratory syncytial virus, can be inhibited locally after administration of naked small interfering RNA (siRNA), and the systemic delivery of siRNA to the liver of non-human primates has been demonstrated using a lipid formulation. Despite these examples, there are relatively few reports demonstrating efficacy of systemically delivered siRNA in primates. Research to date has focused on cationic lipid delivery systems, but the development of safe and effective methods, both in vitro and in vivo, has proven problematic. The delivery of siRNA has been mediated, for example, by direct conjugation of delivery agents to the RNA moiety or peptide-based delivery systems. A barrier to the exploration of new delivery materials is the synthesis of the delivery material itself. Conventional lipid synthesis typically requires individually optimised, multiple-step synthesis, including protection and deprotection stratagems, use and removal of catalysts, solvent exchanges and purification. The optimisation of each (multiple) synthetic step therefore has limited throughput and so limits the ability to generate libraries of substantial size and diversity. Recent work has attempted to develop chemical methods capable of the rapid, parallel generation of lipid-like molecules.1 Library compounds were synthesised through a conjugate addition of primary or secondary amines to alkylacrylates (1) or alkylacrylamides (2) to generate compounds of general structure 3 and 4. The advantage of this type of chemistry is that, unlike many traditional lipid synthesis chemistries, it allows for reaction in the absence of solvent or catalysts and results only in lipidoid product, thereby eliminating the need for protection and deprotection steps and purification.
O
H N
O
R1
O O R1
N H
NH2
H2N
R1
O
2
R1
H N
N
N H
R1
O
N
4
N H
Using this methodology, several libraries totalling more than 1200 lipidoid members were synthesised. Library members were then screened for their ability to deliver siRNA to a HeLa cell line that stably expresses both firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase. Efficacy of siRNA delivery by lipidoids was determined by treating cells with these complexes, prepared using a firefly luciferase-targeting siRNA, and then measuring the ratio of firefly to Renilla luciferase expression. A number of compounds were found to enhance the delivery of siRNA to HeLa cells. In a further round of experiments, Factor VII, a blood clotting factor, was chosen to assess targeted siRNA delivery to the liver. At least seven lipidoid formulations were identified that mediated significant reduction of serum Factor VII protein levels, with the largest reduction observed for compound 5. The development of a library of lipid-like materials represents an expansion in the diversity and range of intracellular delivery materials, and has clearly demonstrated potential for in vivo utility. (CH2)11CH3 HN
O
O
H N
(CH2)11CH3
O H3C(CH2)11
N
N H O
H3C(CH2)11
N H
N H
N
(CH2)11CH3 NH
N 5
O
1.2. New thrombin binding aptamer analogues containing an acyclic nucleotide
E-mail: [email protected] doi:10.1016/j.comche.2009.01.001
The serine protease thrombin plays a central role in blood clotting, converting soluble fibrinogen to fibrin, activating the factors
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7
V, VIII, and XI, and stimulating platelet aggregation. There are many examples of endogenous molecules that bind to thrombin allowing regulation of the clotting process, and so preventing any loss of blood and inappropriate clotting in healthy individuals. The development of anticoagulant strategies to inhibit thrombogenesis in patients is of great interest and among these the most important are focused on direct or indirect inhibition of thrombin. One such inhibitor that specifically binds to human thrombin (with an EC50 of 20 nM) is a 15-oligomeric aptamer. The solution-state three-dimensional structure of the thrombin binding aptamer has been determined by NMR methods. On the basis of previous NMR results produced by studying the folding of modified aptamers, it can be deduced that the tertiary structure of the thrombin binding aptamer adopts a chair-like conformation. Recent work has attempted to obtain new insights into the recognition event between thrombin and the thrombin binding aptamer.2 This work has undertaken to study the synthesis and the structural and biological characterisation of a new series of modified thrombin binding aptamers in which the thymidines have been replaced, sequentially, by the acyclic nucleoside N1-(3-hydroxy-2hydroxymethyl-2-methylpropyl)-thymine to give 6. Oligonucleotides incorporating acyclic nucleotides are of interest, in part, because they are less susceptible to nuclease action than their natural counterparts. The structural changes between the acyclic 6 and the unmodified T residue 7 result in a one bond shortening between the T base and one of the phosphate groups. Additionally the replacement results in the introduction of a hydrophobic methyl group, and the presence of a prochiral carbon in the linker that develops into a chiral center when the modified nucleotide is inserted into the DNA sequence.
H N
O
O
O
P
CH 3
H 3C O
O-
O O
6
P
O
O P
O-
O
H N
O N
O
O
CH 3
O O
O7
O
P
an aqueous ammonia solution (33%) at 55 °C overnight. The oligonucleotides were evaluated for any effect, following incubation with human plasma, on prothrombin time. The concentration of oligonucleotide required to double the fibrinogen clotting time was also determined. From this library, a number of active oligonucleotides were obtained. One of the most active gave a prothrombin time of 24 s at a concentration of 2 M, and a concentration of 70 nM of the oligonucleotide was found to double time to clot in the fibrogen clotting assay. 2. A summary of the papers in this month’s issue 2.1. Solid-phase synthesis A general solid-phase synthesis of 1,2,5-trisubstituted imidazolidin-4-ones has been described. The key synthetic transformation incorporates a microwave-assisted condensation of an aamino amide on solid support with an aldehyde in solution to give the corresponding resin-bound imidazolidin-4-one in a simple one-pot procedure.3 A new straightforward solid-phase synthesis of perfluoroalkylated dimerisable detergents used as gene delivery systems has been presented. The route used Fmoc-Cys(SASRINTM–Ò)-OH resin as solid support, affording the target products in almost quantitative yields and high purity.4 An efficient method for the solid-phase synthesis of aryl amines, heteroaryl amines, and sterically hindered alkyl amines has been developed. The key step in this process was the formation of resin-bound carbamates by the Curtius rearrangement of aryl carboxylic acids with Wang resin providing the trapping hydroxyl group. The developed methods can be applied to the preparation of libraries containing aryl, heteroaryl, and sterically hindered alkyl amine structures as the pharmacophores.5 2.2. Solution-phase synthesis
N O
5
O-
O
The ability of the synthesised sequences to assume a quadruplex conformation has been evaluated by NMR, UV, and CD spectroscopies, and thrombin inhibitory activity was determined in prothrombin time and purified fibrinogen clotting assays. The study also synthesised oligonucleotide analogues using standard solid-phase DNA chemistry on a controlled pore glass support. The oligomers were detached from the support and deprotected by treatment with
Chemoselectivity of chlorine versus methylthio (–SCH3), methylsulfinyl (–SOCH3), or methylsulfonyl [–S(O)2CH3] in either the functionalisation or substitution of pyridopyrimidinones and pyrimidopyrimidinones has been studied. Use of this methodology to prepare final targets for rapid SAR studies via two-dimensional array syntheses has been demonstrated.6 A rapid and efficient strategy for the preparation of spiro-pyridones and spiro-quinolones using sequential Pd(0)/C-catalysed allylation and ring closing metathesis reactions has been reported. Application of the present methodology to nucleoside chemistry has also been investigated, and it has also been used in library preparation.7 A method for the formation of libraries of synthetic macrocycles with different ring sizes from diols has been described. Reacting a simple diol precursor with electrophilic reagents leads to a cyclic carbonate, sulphite, or phosphate in a single step in 25–60% yield. Alternatively, converting the cyclisation precursor to a bis-electrophilic iodide or aldehyde enables preparation of a cyclic sulphide and amine respectively, the latter using a double-reductive amination to induce ring closure.8 A global synthetic strategy based on the ‘garlanding’ concept to design and to produce halo-oligopyridines has been reported. The strategy allows the preparation of an infinite number of these new compounds and hence access to a library of halooligopyridines.9 The PEG-immobilisation and the liquid phase synthesis of some coumarins derived from cardol have been presented. Some preliminary results on their tyrosinase inhibitory activity are also included.10
6
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7
2.3. Scaffolds and synthons for combinatorial libraries Efficient reaction conditions have been developed to prepare highly functionalised chiral pyranosido-pyrimidines by regioselective alkylation of the pyranoside ring and palladium-catalysed cross-coupling reaction on the pyrimidine ring under microwave activation. Such scaffolds contain three different functionalities suitable for selective derivatisation and could find applications in the design of peptidomimetics.11 2.4. Solid-phase supported reagents Acetylation of various alcohols and phenols has been performed successfully in high yield using an immobilised cobalt(II) catalyst. The catalyst shows high thermal stability and was also recovered and reused at least ten times without any significant activity loss.12
ionic lipids with short spacer length and short hydrophobic tails bound to DNA and delivered DNA into HEK293 cells more efficiently than those with longer ones. These results suggest that these newly described aminoglycerol-based lipids should be very promising in liposome-mediated gene delivery and illustrate the potential of solid phase synthesis methodology for non-viral vector discovery.19 The human pathogenic bacterium Pseudomonas aeruginosa produces a fucose-specific lectin, LecB, implicated in tissue attachment and the formation of biofilms. To investigate if LecB inhibition disrupts these processes, high-affinity ligands were obtained by screening two 15,536-member combinatorial libraries of multivalent fucosyl-peptide dendrimers. The most potent LecB-ligands identified were dendrimers that led to complete inhibition of P. aeruginosa biofilm formation and induced complete dispersion of established biofilms in the wild-type strain and in several clinical P. aeruginosa isolates.20
2.5. Novel resins, linkers and techniques References Fluorescein isothiocyanate (FITC) is an amine-reactive derivative of fluorescein dye that has wide ranging applications in labelling peptides and proteins. However, its use in solid phase peptide synthesis is restricted by instability in acidic conditions required for linker cleavage. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is used to release targeted peptide from the resin.13 2.6. Library applications The synthesis and identification of sulphonamidoaryl ethers as potent bradykinin B1 receptor antagonists from a 60,000 member encoded combinatorial library have been reported. Two distinct series of compounds exhibiting different structure–activity relationships were identified in a bradykinin B1 whole-cell receptorbinding assay.14 Cdc7 kinase plays a critical role in the regulation of DNA replication in eukaryotic cells and has been proposed as a target for cancer therapy. A class of Cdc7/Dbf4 inhibitors with a pyrido-thieno-pyrimidine core structure have been identified, and the synthesis of a focused pyrido-thieno-pyrimidine library yielded potent and selective Cdc7 inhibitors with antiproliferative activity against cancer cells in vitro.15 The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the av-integrins avb3 and avb5 has been described. High-throughput screening of an extensive series of ECLiPSTM compound libraries led to the identification of a dual inhibitor of the av-integrins avb3 and avb5.16 The discovery and synthesis of a series of 2-amino-5-benzoyl-4(2-furyl)thiazoles as adenosine A2A receptor antagonists from a small-molecule combinatorial library using a high-throughput radioligand-binding assay has been described. Antagonists were further characterised in the A2A binding assay and an A1 selectivity assay, and selected examples exhibited excellent affinity for A2A and good selectivity versus the A1 receptor.17 A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesised and evaluated for their antiplasmodial activities. Cross screening of the library against cultured Leishmania donovani parasites revealed this class of compound as potent inhibitors of in vitro parasite development.18 The solid phase synthesis of a library of aminoglycerol–diamine conjugate-based transfection agents having urea linkage between diverse length of diamines and various lengths of hydrophobic tails has been described. These compounds were characterised and structure–activity relationships determined for DNA binding and transfection ability when formulated as cationic liposomes. Cat-
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Akinc, A. et al. Nat. Biotechnol. 2008, 26 (5), 561–569. Coppola, T. et al. Bioorg. Med. Chem. 2008, 16 (17), 8244–8253. Qin, L.-Y. et al. Tetrahedron Lett. 2009, 50 (4), 419–422. Guilloteau, N. et al. Tetrahedron Lett. 2009, 50 (5), 562–563. Sunami, S.; Ohkubo, M. Tetrahedron 2009, 65 (3), 638–643. Wan, Z. et al. Tetrahedron Lett. 2009, 50 (3), 370–372. Kersten, L. et al. Tetrahedron Lett. 2009, 50 (5), 506–508. Wingstrand, M. J. et al. Tetrahedron Lett. 2009, 50 (6), 693–695. Voisin-Chiret, A. S. et al. Tetrahedron 2009, 65 (3), 607–612. Tocco, G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (1), 36–39. Samb, I. et al. Tetrahedron 2009, 65 (4), 896–902. Rajabi, F. Tetrahedron Lett. 2009, 50 (4), 395–397. Jullian, M. et al. Tetrahedron Lett. 2009, 50 (3), 260–263. Cole, A. G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (1), 119–122. Zhao, C. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 319–323. Letourneau, J. J. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 352–355. Cole, A. G. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 378–381. Musonda, C. C. et al. Bioorg. Med. Chem. Lett. 2009, 19 (2), 401–405. Yingyongnarongkul, B. e. et al. Bioorg. Med. Chem. 2008, 17 (1), 176–188. Johansson, E. M. V. et al. Chem. Biol. 2008, 15 (12), 1249–1257.
Further reading Papers on combinatorial chemistry or solid-phase synthesis from other journals Chen, M.-L.; Adak, A. K.; Yeh, N.-C.; Yang, W.-B.; Chuang, Y.-J.; Wong, C.-H.; Hwang, K.-C.; Hwu, J.-R. R.; Hsieh, S.-L.; Lin, C.-C. Fabrication of an oriented Fc-fused lectin microarray through boronate formation. Angewandte Chemie, International Edition 2008, 47 (45), 8627–8630. Pattabiraman, V. R.; McKinnie, S. M. K.; Vederas, J. C. Solid-supported synthesis and biological evaluation of the lantibiotic peptide bis(desmethyl) lacticin 3147 A2. Angewandte Chemie, International Edition 2008, 47 (49), 9472–9475. Park, J.-J.; Lee, J. H.; Li, Q.; Diaz, K.; Chang, Y.-T.; Chung, S.-K. Divergent syntheses of all stereoisomers of phytosphingosine and their use in the construction of a ceramide library. Bioorganic Chemistry 2008, 36 (5), 220–228. Rivera-Monroy, Z.; Bonn, G. K.; Guttman, A. Fluorescent isotope-coded affinity tag (FCAT) I: Design and synthesis. Bioorganic Chemistry 2008, 36 (6), 299311. Pettersson, S.; Perez-Nueno, V. I.; Ros-Blanco, L.; Puig de La Bellacasa, R.; Rabal, M. O.; Batllori, X.; Clotet, B.; Clotet-Codina, I.; Armand-Ugon, M.; Este, J.; et al. Discovery of novel non-cyclam polynitrogenated CXCR4 coreceptor inhibitors. ChemMedChem 2008, 3 (10), 1549–1557. Tarrago, T.; Masdeu, C.; Gomez, E.; Isambert, N.; Lavilla, R.; Giralt, E. Benzimidazolium salts as small, nonpeptidic and BBB-permeable human prolyl oligopeptidase inhibitors. ChemMedChem 2008, 3 (10), 1558–1565. Heinze, K.; Beckmann, M.; Hemple, K. Solid-phase synthesis of transition-metal complexes. Chemistry – A European Journal 2008, 14 (31), 9468–9480. Heinze, K.; Reinhardt, S. Platinum (II) complexes with non-innocent ligands: solidphase synthesis, redox chemistry and luminescence. Chemistry – A European Journal 2008, 14 (31), 9482–9486. Nielsen, M. C.; Ulven, T. Selective extraction of G-quadruplex ligands from a rationally designed scaold-based dynamic combinatorial library. Chemistry – A European Journal 2008, 14 (31), 9487–9490. Charton, J.; Charruault, L.; Deprez-Poulain, R.; Deprez, B. Alkylsquarates as key intermediates for the rapid preparation of original drug-inspired compounds Combinatorial Chemistry & High Throughput Screening 2008, 11 (4), 294–303. Tse, B. N.; Snyder, T. M.; Shen, Y.; Liu, D. R. Translation of DNA into a library of 13 000 synthetic small-molecule macrocycles suitable for in vitro selection. Journal of the American Chemical Society 2008, 130 (46), 15611–15626.
N. K. Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 4–7 Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. Solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates using bicyclic oxazaphospholidine derivatives as monomer units. Journal of the American Chemical Society 2008, 130 (47), 16031–16037. Dolle, R. E.; Le Bourdonnec, B.; Goodman, A. J.; Morales, G. A.; Thomas, C. J.; Zhang, W. Comprehensive survey of chemical libraries for drug discovery and chemical biology: 2007. Journal of Combinatorial Chemistry 2008, 10 (6), 753–802. Lazny, R.; Nodzewska, A.; Zabicka, B. Asymmetric solid-phase alkylation of ketones immobilized via SAMP hydrazone analogue linkers. Journal of Combinatorial Chemistry 2008, 10 (6), 986–991.
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Ko, K.-S.; Park, G.; Yu, Y.; Pohl, N. L. Protecting-group-based colorimetric monitoring of fluorous-phase and solid-phase synthesis of oligoglucosamines. Organic Letters 2008, 10 (23), 5381–5384. Brouillette, Y.; Verdie, P.; Martinez, J.; Lisowski, V. Valuable versatile reactivity of thiaisatoic anhydrides: expedient solid-phase synthesis of thieno[1,4]diazepine2,5-diones. Synlett 2008, (15), 2360–2364. Hahn, F.; Muellen, K.; Schepers, U. 2-Iminothiolane as a useful coupling reagent for polyamine solid-phase synthesis. Synlett 2008, (18), 2785– 2790.