Expedite Hit Discovery: Split & Pool in the 21st Century. Dr. Andreas Marzinzik

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1 Expedite it Discovery: Split & Pool in the 21st Century Dr. Andreas Marzinzik

2 Lead Finding in Drug Discovery Lead Target ID Assay Devel. Screen TS / FBS it to Lead Lead ptimization D0 D1 D2a D2b D3 spc it Finding Single, Purified Libraries ICLF Fragment Based Screening Library synthesis for screening: Diversity riented & Target Class ew chemistry development Integrated Combinatorial Lead Finding (ICLF) ne-bead-ne-compound approach

3 Lead Finding in Drug Discovery Target ID Assay Devel. Screen TS / FBS it to Lead Lead Lead ptimization D0 D1 D2a D2b D3 spc compound amounts number of compounds assay throughput Provide hits with modular structures amenable to rapid optimization by previously validated combinatorial chemistry

4 Assay Sensitivity and Throughput UTS Plate: > 2000 wells / plate Assay Volume: 1-2 µl Microtiter Plate: 96 wells/ plate Assay Volume: 200µl

5 The Power of Split-Pool Synthesis Split-and-Mix: 27 products in 9 synthesis steps P1-P9 P10-P18 P19-P27 nebeadonecpd Parallel Synthesis, 27 products in 81 synthesis steps P1 Synthesis step Mix/split P27

beads variable loading and reaction rates 200 µm beads, S.D.

6 Monodispersed Beads equirements: we need a well defined reaction compartment Commercial Batch LCC Polystyrene (new) inhomogeneous beads variable loading and reaction rates 200 µm beads, S.D. ± 2% 1g appr beads Challenge: how do we analyse reactions on beads?

Single Bead Analysis L: 1.20E8 Base Peak F: + c ESI Full ms [ 150.00-1500.00] MS F07 4.38E2 A/D Card Ch. 2 A/D card F07 1.67E2 A/D Card Ch.

7 Single Bead Analysis L: 1.20E8 Base Peak F: + c ESI Full ms [ ] MS F E2 A/D Card Ch. 2 A/D card F E2 A/D Card Ch. 1 A/D card F07 ff-bead LC/MS/ Identity n-bead I Transmission 400 µm AT eflectance I in I out T: elative Abundance MS / MS Time (min) 15 µm I out µm AT crystal Si or Ge Purity I in 100 µm 200 µm 100 µm T: millivolts LC/UV 2.84 L: Time (min) Quantity T: millivolts detection 2.84 L: Time (min)

8 Library Design Medicinal chemistry aspects physicochemical, potency, diversity, ADME/tox ovelty Similarity to active compound classes focus, complementarity Modularity of the scaffold BB availability, library size, feasibility Complexity of Chemistry time of development, purity...

9 Fischer Indole Synthesis 1 reduction 1 TFA, osenbaum C., Katzka C., Marzinzik A., Waldmann. Chem. Comm. 2003, 15,

10 Traceless Fischer Indole Synthesis 1) as 4 *Si 2 Me abs, rt Cs + 2) CsC 3 1) 50 C, KI, DMF 1 Cl 1 Cl. 2 DIC/Bt, 3 eq, Et 3 3 eq, DCM abs, rt, 1 d 3 eq 2) Li, TF/ 2, 1 d, rt B 3 /TF 10 eq 18 h, 60 C ketone 2 1 DCE / TFA 75 % 80 C, 1-3 d 1 3 osenbaum C., Katzka C., Marzinzik A., Waldmann. Chem. Comm. 2003, 15,

11 esults of Fischer Indole Synthesis S 3 Br Br 41% 7% 7% F Br Br F 9% 39% 33% F 3 C Br Br 6% 7% 12% Br 14% 24% osenbaum C., Katzka C., Marzinzik A., Waldmann. Chem. Comm. 2003, 15,

12 Diversity Platform =alkyl 5 4 =alkyl 5 =Calkyl 2 ' Ar C 2 CC 3 Li, DME or PPh =Ar, alkyl 4 =Ar, alkyl 4 =alkyl

13 Annelated eterocycles 4 3-amino-5-hydroxypyrazole ( 5 = ), aet DMS, rt, 16 h 2-aminobenzimidazole aet, DMS, rt, 16 h ' several steps 6-amino-1,3-dimethyluracil, 2, DMA, 16h, rt 5 C

14 Diversity riented Synthesis 2 1 isatine, L-azetidine- 2-carboxylic acid, DMA, 4h, 80 C isatine, cis-4-hydroxy- D-proline, DMA, 4h, 80 C 2 1 ' 2 isatine, L-thiazolidin- 4-carboxylic acid DMA, 4h, 80 C isatine, pipecolinic acid DMA, 4h, 80 C 2 1 S 1 isatine, D-tetrahydroisoquinoline-3-carboxylic acid DMA, 4h, 80 C 2 1 2

15 1,3 Dipolar Cycladdition - Mechanism C _ - C 2 rac

16 atural Product-like Scaffold MeC Me MeC Me MeC C 3 Me MeC C 3 Me etero-diels-alder Messer., Pelle X., Marzinzik A. L., Lehmann., Zimmermann J., äner. Synlett 2005, in press.

17 Synthesis of the Scaffold 2 Me 2 2 Me Ph 2 ClC 2 attachment to solid support X Me Messer., Pelle X., Marzinzik A. L., Lehmann., Zimmermann J., äner. Synlett 2005, in press.

18 Libraries from atural Products P have played a considerable part in the exploration and development of new drugs The hit-rate of pure natural compounds is higher than that of synthetic compounds atural products provide unique chemical diversity, distinct from that found in the majority of synthetic libraries Biologically active natural products were evolutionarily selected and validated for binding to particular protein domains. Therefore such natural products are interesting starting points for library development Zearalenone derivative

19 Libraries from atural Products S L [ ] n Y X Z Epothilone B total synthesis, semi-synthesis LF semi-synthesis Zearalenone derivative Cachoux F., Isarno T., Wartmann M., Altmann K.-. Angew. Chem., Int. Ed. 2005, 44, 7469

20 Template Generation 1) 2, Pd/C, TF 1) 2 Cl 2) 2)aB 4, icl 2 Et, -30 C Amberlite I120 AC, dioxane 65 C 2 teoc-p DIEA DMF, 80 C Si Amberlite I-120 2, AC 60 C Si Grosche, P; Akyel, K. G.; Marzinzik, A. L. Synthesis 2005, 12, 2015

21 Zearalenone-derived Library teoc Tos. py, DCM rt Br P1-tBu, DMF teoc rt teoc 1. TBAF TF, 50 C TFA, DCM Me, 2 2. 'CCl DIEA, DCM, rt ' ' Grosche, P; Akyel, K. G.; Marzinzik, A. L. Synthesis 2005, 12, 2015 Challenge: how do we identify active cpds from split-pool synthesis?

22 Split & Pool Synthesis Split & pool synthesis: Sizeable amounts / well ne bead / well Mixture Amount: as much as you want Screening of mixtures + save of screening cost - no SA, mixtures problems Single Compound Amount 1-30 nmol Deconvolution Screening with T formats + no false positives, SA possible - redundancy, amount

23 Success in TS Assay Activity profile MS analysis Circle diameters correspond to inhibition >80% inhibition umbers correspond to M

24 FLIP-Screen: Fractionation Data % Inhib 100% 50% 100% Ion count Mass fraction %Inhibition Mass439 Mass 380 Mass 423 Mass 465 Mass 446 Mass 398 Mass 384 Mass 451 Mass 488 Mass 340 Mass 366 Mass 416 Mass 460 Mass 448

25 Activity of its from Single Beads % Activity activity of of validated ICLF and hits SLIA from compounds split-pool and individual compound libraries > 5 Split-pool IC50 or EC50 CombiChem ICLF SLIA Analysis by F. Stoll, CADD

26 The esult - Proprietary compounds - Complex chemical structures - igh diversity - igh quality - Better success-rate than single compounds from CC - Fast follow-up - Which compound will give the next success story?

27 Acknowledgements Chemistry/Technology Fred Berst Werner Breitenstein Philipp Grosche Markus Vögtle Jürg Zimmermann Analytics occo Falchetto Erwin ermes Serge Moss Technical Support Christoph Freslon aphael Gattlen Faouria assur Xavier Pelle Stephanie Pickett Erich Spieser

Current Literature. Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8

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