Design, Synthesis, and Evaluation of Small-Molecule Libraries

Combinatorial Chemistry: molecular diversity "Synthesis and Applications of Small Molecule Libraries." Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555-600. "Design, Synthesis, and Evaluation of Small-Molecule Libraries.” Ellman, J. A. Acc. Chem. Res. 1996, 29, 132 -143. Combinatorial Chemistry, Nicholas K. Terrett, Oxford University Press, London, 1998 pharmaceutical industry- drug discovery Lead Cmpd optimization (synthesis) Drug 10 years $ 400 - 800 million $$$ Lead identification: literature (open & patent) nature (natural products) Careful optimization of a lead structure via chemical synthesis “methyl-ethyl-butyl-futile game” Number of marketable drugs per compounds that undergo preliminary biological testing 1 10,000 Rational drug design Combinatorial chemistry 105 Peptides: poor bioavailability, poor transport, easily metabolized: poor drug candidates. However, they are the natural substrates for many enzymes and receptors (drug targets) and have well-defined conformations: excellent lead compounds Enzymes: converts a substrate to a distinct product Receptor: binds a ligand (no reaction), causing a chain of physiochemical events leading to a pharmacological response. Agonist: substance that interacts (binds) with a receptor and elicits an observable response Antagonist: substances inhibits the affect of an agonist, but has no biological activity of its own 106 53 Example of molecular diversity: tetra-peptide: H2N-A-B-C-D-CO2H consider only the 20 natural amino acids (L-series) 204 = 160,000 different tetra-peptides ! now include the 19 D-amino acids (20 L + 19 D = 39) 394 = 2.3 million different tetra-peptides !! now include 20 unnatural amino acids 594 = 12 million different tetra-peptides !!!! Combinatorial chemistry: method by which a family (library) of related compounds (structurally & synthetically) can be prepared and evaluated (screened) For multi-step synthesis, one must use solid-phase synthetic approach in order to expedite purification of intermediates 107 Split synthesis (mixture libraries) A D A D B B D C C D A A A B B combine, mix,split E A E combine, mix,split B B E C C E C C A F A F B B F C C F 108 54 Split synthesis (con’t) A D A F A E A D G A E G A F G F B D G B E G B F G C D G C E G C F G A D H A E H A F H G B D B B C D C E C F A D A E A F E H B D B E B F B D H B E H B F H C D C E C F C D H C E H C F H A D A E A F B E B C E C F A D I A E I A F I B D I B E I B F I C D I C E I C F I I B D C D F 109 Why not? A A D A E A F B B D B B C C D C E G,H, I D,E,F A,B,C E F etc C F Reactivity of the coupling reaction may be different and this could bias the library Split synthesis approach, all compounds are equally represented (each coupling is individually controlled) 110 55 Deconvolution of the library via biological activity and sub-library re-synthesis A D G A E G A F G B D G B E G B C D G C E G C F G A D H A E H A F H B D H B B F H C D H C E H C F H G F G Biological activity of the library H E H G H I select library H A D I A E I A F I B D I B E I B F I C D I C E I C F I I A D A D H 111 Biological activity of the library A B D H C D D H D H A D H B D H C D H B D H B D D-H H B E H C E C E H A F A F H B F H C F H F C F H E-H C D H F-H Lead Cmpd B E B H C A E H A E select library D-H Biological activity of the library A-D-H B-D-H C-D-H select library C-D-H 112 56 Problems: • deconvolution of the library can be labor intensive • can be fooled by low concentrations of highly active compounds • activity is dependent upon the compound and its concentration • activity observed is the the combined activity of the entire library Advantage: • can synthesize a very large number of compounds very quickly and relatively easily depending on the chemistry. 113 Encoded Libraries 1 4 1 4 2 4 3 A 2 1) D 2) 4 A D B B D 3 C 1 C D A 1 5 1 B 5 2 C 5 3 A 2 B combine, mix,split 2 1) E 2) 5 A E 3 B 3 combine, mix,split E C E C 1 A 2 1) F 2) 6 6 1 6 2 6 3 A F B 3 C B F C F 114 57 Encoded libaries (con’t) 4 5 1 6 1 4 2 4 3 5 2 5 3 B B D 5 1 2 4 3 5 2 5 3 1 5 2 6 3 5 5 6 3 C D 4= D 5= E 6= F 2 7 4 3 4 5 2 7 5 3 4 2 8 4 3 1) I 2) 9 4 A D 9 4 2 9 4 3 F B D C F C D 7 6 2 7 6 3 5 1 8 5 2 8 5 3 B 8 6 1 8 6 2 8 6 3 E H C E H 5 1 A E I 9 5 2 9 5 3 B E I C E I A F G B G F C F G A E H 9 1 1 C E G C D H 9 6 B E G B D H 3 7= G 8= H 9= I 7 A D H 8 7 A E G 8 1 F 2 C E 1= A 2= B 3= C 1) H 2) 8 A F B 1 C D G 1 B E 3 4 C F 6 2 B D 7 8 B 5 A D G B D G A F 6 7 1 F 1 6 1 1) G 2) 7 4 C F A E 2 CODE: B C E A D 4 3 B E C D 4 6 A E B D 4 2 6 A D 4 6 E 1 7 A F C E C D 4 1 A E A D 9 6 1 9 6 2 9 6 3 I I I A F H B F H C F H A F I B F I C F I Cleave the marker and analyze the code. Each unique 115 marker codes for a unique peptide sequence Synthesis of an encoded library Code (1-10%) library 1) linker- AA1-NH-FMOC HO BOC NH code 1 OH linker O O linker 2) linker-code1-NH-BOC H AA1 N FMOC library region and coding region must be orthogonally protected 1) piperidine 2) add AA 2 BOC BOC NH code 1 H N Code 2 NH code 1 linker linker O O O O linker linker H AA1 N H AA1 N H AA2 N H AA2 N 1) H+ 2)add code 2 FMOC FMOC 116 58 sub-library tag Cy pool tag Cx N O C7H15 CH3 N O C7H15 C2H5 N O C7H15 C3H7 N O CO2H N BOC C8H17 N CO2H BOC C8H17 N CO2H BOC C8H17 CH3 N O C2H5 N O C3H7 N O N CO2H BOC N CO2H BOC C9H19 CO2H N BOC C9H19 CO2H N BOC C9H19 CH3 N O C2H5 N O C3H7 N O CO2H N BOC N CO2H BOC N CO2H BOC Reading the code C9H19 selectively remove the code from the resin N CH3 C7H15 O O HN N O N O N hydrolyze and analyze O N C8H17 C3H7 C9H19 N H CH3 + C7H15 N C3H7 + C8H17 N C2H5 H H CO2H C2H5 117 “On-Bead” Assay for Receptor Binding using a Fluorescently Labeled Receptor 1) Add labeled receptor labelled with fluorescent tag 2) allow to bind to ligands on the beads 3) wash to remove unbound receptor Beads 118 59 Spatially Addressable, Parallel Synthesis E D F A A D A E B B D B E C C D C E A F B F C F 119 Spatially Addressable, Parallel Synthesis (con’t) H G I A A D G A E H B B D G B E H C C D G C E H A F I B F I C F I 120 60 Spatially addressable, parallel libraries Advantage: • synthesizing pure compounds, no need to deconvolute the library Disadvantage • libraries tend to be much smaller 121 Diversomer apparatus Acc. Chem. Res. 1996, 29, 114-122 61 ACE Inhibitors Asp–Arg–Val–Tyr–Ile–His–Pro–Phe–His–Leu–Val–Ile–His–Asn angiotensinogen aspartyl protease ACE inhibitors renin N N O N NH Asp–Arg–Val–Tyr–Ile–His–Pro–Phe–His–Leu angiotensin I (little biological activity) CO2H N Diovan HS angiotensin converting enzyme (ACE) Zn2+ protease O N HO2C Captopril Asp–Arg–Val–Tyr–Ile–His–Pro–Phe angiotensin II (vasoconstriction → high blood pressure) 123 Proteases: catalyzes the hydrolysis of peptide bonds O H3N 1. 2. 3. 4. R N H O H N O R R N H O H N O R protease N H CO2 H2O O H3N R N H O H N O R O R O H N + H N 3 O R N H CO2 Serine protease Cysteine protease Aspartyl protease Zinc (metallo) protease ACE: zinc protease ( no x-ray or NMR structure), compared to carboxypeptidase or thermolysin important catalytic groups: Glu-270, His-196, His-69 (catalytic triad) 124 62 Carboxypepidase: General base mechanism Nucleophilic mechansim (acyl enzyme complex) 125 ACE inhibitor Library O O ArCHO NH2 O N O R1 O O N O R1 Ar O H N O O R2COCl Ar O R2 N O Z Z Z 1,3-dipole R1 = -H, -CH3, -CH2CH(CH3)2, -CH2Ph O CF3CO2H Ar R1 R1 R1 Ar R1 1,3-diploar cycloaddition Ag N Ag O AgNO3, Et3N Ar O R2 N HO CHO CHO ArCHO = Ar R1 Z Z = CO2CH3 CN CHO CHO OCH3 OH O CO2tBu CO2CH2CH3 HS O O HO2C R2COCl = AcS O O Cl AcS Cl AcS Cl N Captopril (ACE Inhibitor) 240 cmpds, each are multiple stereoisomers AcS O HO2C N Ph CO2CH3 Ki ~ 160 pM (3x better than Captopril 126 63 Parallel Synthesis of a Benzodiazepine Library R2 O NH2 R1 N H NH O FMOC O H N R1 HN O 1) F R2 R1' O FMOC R1' 2) O N H R1' H+ O O O R3 N 1) base 2) R3X R2 R3 N cleave from solid support N R1 R2 N R1 O R2 R1 N R1' R1' O Benzodiazepine HO H3C N NHCH3 O N N Cl N Cl O Valium (diazapam) 127 Librium (chlordiazepoxide) Parallel Synthesis of a Benzodiazepine Library 2-am inobenzophenones NH 2 O alkylating agents !-amino acids CO2 H CO 2H NH 2 CO 2H NH 2 CO 2H NH 2 CO 2H NH 2 I I- CH 3 NH 2 OH Cl CO 2H NH 2 O H 2N C O2H N H2 NH 2 CO 2H HO 2C NH 2 CO 2H I-CH 2- CH 3 I-CH 2 -CH 2- CH 3 NH 2 O H+ CO 2H HO NH 2 N H N H2 I H 2N CO 2H CO 2H N H NH 2 Br Br 2 (2-aminobenzophenone) x 12 (amino acids) x 8 (alkyl halides) = 192 compounds Cholecystikinin (CCK) CCK-8: Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2 128 64 Peptide Mimetics or Peptidomimetics Tyr-Gly-Gly-Phe-Met (met-enkephalin) HO SCH3 O O HO N HO2C N H CH3 HO NH2 Morphine O HN O O H N N H Desirable pharmacological properties 1. metabolic stability 2. good bioavailability 3. high affinity (Ki~ 10-9) and specificity for a given receptor or enzyme 4. minimal side effects Natural ligands for receptors or enzymes are often peptides: peptides are good lead structures but poor drug candidates 129 Peptidomimetics Strategies 1. Amino acid modification 2. Dipeptide analogues (isosteres and TS analogues) 3. Peptide backbone modifications 4. Secondary structure mimics Amino Acid Modifications: changes (restricts) conformational degrees of freedom of the peptide CO2H CO2H !-C alkylation H3C NH2 !-N alkylation HN CO2H NH2 NH2 CO2H CO2H NH CH3 L-Phenylalanine CO2H unnatural AA's NH2 CO2H NH2 CO2H NH2 H2N CO2H n 130 65 Dipeptide analogues O H N O H N R N H R O H N O N N H R Bridge two neighboring AA residues O n O R O R O H N n NH N H R O ACE Inhibitor H Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu HO2C ACE angiotensin I angiotensin II N Ph O CO2H 131 Transition state analogues for a protease: O H N R R N H O H N O protease O H N R R N H R O O H N O H N Nu OH R R H2N Nu N H R R O Nu= H2O, -OH, -SH, -CO2H H2O O H N R OH + Nu: N H R O R OH H2N B: O HN R N H OH OH O R R H N N H O HN active site water Transition State H N O H H Statine OH H H N N H H O HO O O OH N H H N N H H N OH O N H + P O O H N _ O Statine 132 66 TS analoges via combinatorial synthesis O Br NHBoc R O AA1 AA2 AAn NH2 H O AA1 AA2 AAn N (iPr)2NH O NHBOC R NaBH4 O H O AA1 AA2 AAn N NH AAn-1 AAz R OH H HO AA1 AA2 AAn N NH AAn-1 AAz NH2 R 1:1 mixture of stereoisomers 133 Peptide Backbone Modifications: Increase biological half-life Retro-Inverso Isomers: Change from L- to D-amino acids and inverse N to C sequence CONH2 Natural O H2N O O H N O H N N H H N N H O O H2NOC O H N N H N H N O OH O NH SCH3 N H CONH2 Retro-Inverso isomer O H2N H N O H N O O O H N N H O H2NOC O H N N H N H O N H NH2 O N HN SCH3 N H Interesting idea, but the retro-inverso isomers usually showed much weaker binding. However, they were significantly more resistant to enzymatic digestion. 134 67 Amide Bond Isosteres: _ O + N H N H O OH O O N H F F F O H2O R H N R O O N H O HO OH N H H F F F F H N N H OH O H N R R Secondary Structure Mimics: main 2° structures: α-helix, β-sheet, β-turns, γ-turns α-helix mimic groups responsible for biological activity Replace with a non-peptide scaffold 135 Vasoactive Intestinal Peptide (VIP): 28 AA helical peptide. Bronchodilatorasthma treatment “α-helical Peptidomimetic for Vasoactive Intestinal Peptide (VIP)” G. L. Olson et al. J. Med. Chem. 1993, 36 (21), 3039-3049. CONH2 Leu Tyr Scaffold Replace with scaffold 12.32 Å N H N Asp His O S O Tyr Phe 12.99 Å NH2 “Ala-scan” determine which residues are needed for binding 136 68 Ter-phenyl as an α-helix mimic g (i+6) c (i+2) f (i+5) g c d (i+3) d a’ (i+7) f a a (i) b (i+1) e (i+4) b e 137 “stapled” α-helices H3C NHFMOC n CO2H 138 69 β-amino acids O H2N O OH N N R N H !-amino acid H H O O O O H2N OH NH R H2N H H O "-amino acid oligomeric "-amino acid will fold into a helical structure with 3 AA's per turn N N O O N H OH N H β-Sheet mimics Ph O H N N N N O O H O HN O H H N N O Scaffold O N N H O H H O NH O O N N N N H O Ph 139 β-Turn Mimics: R O R Scaffold N H O (i+3) (i+1) N H NH O N H N R R H NH Linchpin N R (i) N NH O O O R R R O R (i+2) H O O H R O R O N O NH3 CO2 CO2 !-Turn + + CO2 NH3 + NH3 O O S R HN O R (CH2)n n(H2C) HN O O O H H O N R R N O HN O H N O HN R R O H O O NH O H N R HN O N R N H O O NH 140 70 Combinatorial synthesis of linchpin β-turn mimic O O O Br R1 H N OH NH2 N H N H DCC, HOBT R1 S S-tBu H2N n Br O O R2 O N H H N R1 O S S-tBu N H 1) n OH O N H DCC, HOBT 2) piperidine O H N NHFMOC R1 NH2 N O R2 n S S-tBu O R2 O Br O OH R3 DCC, HOBT N H O H N R1 O R2 n S Br H N N R3 1) Ph3P 2) cyclization 3) CF3CO2H R3 N H O S N n O O HN R1 S-tBu O NH2 Evaluated for Somatostatin receptor binding n= 1,2 x R2= 34 AA x R 3 = 10 = 1292 β-turn mimics 141 71