Molecular Diversity - InnovaSyn

Molecular Diversity - InnovaSyn

Drew Residential School on Medicinal Chemistry Chemical Diversity Generation and Use in Drug Discovery Philip F. Hughes InnovaSyn, LLC Chapel Hill, NC Chemical Diversity Generation and Use in Drug Discovery Overview Reasons, History, Economics, Definitions Combinatorial Chemistry/ Parallel Synthesis Synthesis Methods split/mix, array solid phase, solution phase Equipment Purification Methods Analytical Methods Conclusions Why Chemical Diversity? Reasons

The biggest reason for continued interest in Chemical Diversity is the recent ability of scientists to evaluate very large numbers of molecules in biological systems. i.e. High Throughput Screening High Throughput Screening Biotechnology Genomics Computers Robotics Chemistry synergy Current Screening capacities of 2000-100,000 Samples/Day in multiple assays Where will the Samples come from?

History 1990: A medicinal chemists made 2-6 compounds / month at $2,500-$10,000 / compound Compounds were tested once in a single assay. Leftover compound sent for storage Old Molecular Diversity Company Chemical Storage 20,000-400,000 compounds, many similar classes, some >100 yrs. old Natural Products large number, not clean, test as mixtures Classical Medicinal Chemistry too slow or too expensive New Requirements We need to increase the compound synthesis rate by 20 to 1000 fold

This is less than the increase in screening capacity because were now willing to test each compound in numerous assays Going Faster 4 Ways to go Faster 1. Use Combinations Reuse 2. Do many things at the same time Parallel processing 3. Speed up the process 4. Get someone else to do it Automation Outsourcing The Answer

Combinatorial Chemistry Combinatorial chemistry is a technology through which large numbers of structurally distinct molecules may be synthesized in a time and resource-effective manner, and then be efficiently used for a variety of applications Nick Terrett From the Tetnet page on Two Major Approaches Split & Mix Real Combinatorial Chemistry Array Synthesis Parallel Synthesis Spatially-Addressable Synthesis Matrix Array Synthesis Split & Mix Originated in peptide synthesis Simple efficient chemistry (amides) Long linear sequence of reactions Solid Phase approaches known O H2 N R1-j

O H N N H R1-i O R1-l O H N N H R1-k O OH R1-m # of reagents = 10

# of reactions = steps reagents; 5 10 = 50 # of products = reagentssteps; 105 = 100,000 Split & Mix A A A B B C C Pool B n n/3 C

AA AB AC BA BB BC A Pool CA CB CC n/9 # of reagents = 3 # of reactions =3+ 3 + 3 = 9 # of products = 3 x 3 x 3 = 33 = 27 B C AAA AAB AAC ABA


CCA CCB CCC n/27 A Big Mixture Dealing with Mixtures Options Test as a mixture Encoded Libraries Tags Nucleotide Chemical Labeled reactors Big Mixture Testing Deconvolution generally requires repeated synthesis of smaller and smaller mixtures followed by retesting. 256 128 512 1024

256 16 64 32 64 8 16 This only made sense back capacity 128 when screening 512 32 etc. was limited. - positional scanning Nucleotide Tags Nucleotide Peptide

Beads selected based on binding to target Nucleotide code can be defined for natural or unnatural monomers Nucleotide sequence can be amplified by PCR 1. S. Brenner, R. A. Lerner, Proc. Natl. Acad. Sci. USA, 89, 5381-5383 (1992) 2.. M. C. Needels, d. G. Jones, E. H. Tate, G. L. Jeinkel, L. M. Kochersperger, W. J. Dower, R. W. Barrett, M. A. Gallop. Proc. Natl. Acad. Sci. USA, 90, 10700-10704 (1995) Chemical Tags - Pharmacopeia Example: Arylsulfonamide inhibitors of Carbonic Anhydrase H2 NO2 S O H N CO2 H N O R3 R1

R2 7 X 31 X 31 library: 6727 members (R1-R2-R3) Each reagent encoded by a unique combination of 3-5 tags based on a binary code: coding 2n-1 members requires n tags Tag incorporated by Rh-catalyzed carbene insertion into polymer C-H Tags released from oxidatively labile linker with (NH4)2Ce(NO3)2, followed by Electron Capture GC (silylated tags) Cl O N2 OCH3 O (CH2 )nOAr Cl Cl Cl Ar = Cl

Cl n = 3-12 Cl Cl n = 4-6 13 tags total Chemical Tags - Pharmacopeia A B n C T1 T2 T1 A

Pool T2 A T 3 AB AC T3 BA B T4 T2 T3 T2 C T3 T4 T1

BB BC T1 T3 AA T2 B T1 T2 C T1 T1 T3 CA CB T2

CC T4 T2 T4 T3 T1 T4 T3 M.H.J. Ohlmeyer, R.N. Swanson, L. W. Dillard, J.C. Reader, G. Aronline, R. Koabyashi, M. Wigler, W. C. Still, Proc. Natl.Acad. Sci. USA, 90, 10922-10926 (1993). J. J. Baldwin, J. J. Burbaum, I. Henderson, M. H. J. Ohlmeyer, J. Am. Chem. Soc., 117 5588-5589 (1995). Pharmacopeias web site ECLiPS encoding technology ICCB at Harvard Chemical Tags - Pharmacopeia AA AA

CB T3 1. AB CC T2 T1 T2 AB T3 1. Clip off compounds for testing T1 T3 CB T4

T1 CC T4 T2 T3 2. T3 T2 T3 2. Clip off tags for analysis (23-1)(25-1)(25-1) = 73131 = 6727 compounds 3 + 5 + 5 = 13 tags 7+31+31=69 reagents, 69 x 2 = 138 reactions T2 T3 T4 T1 T2 T4

T3 Labeled Reactors Radio Encoded Tags - Irori Discovery Partners International Labeled Reactors Radio Encoded Tags - Irori Similar to resin split and mix except that each reactor can is tracked throughout the synthesis. Each product is made once and each can contains only one product. Irori calls this directed sorting, which has been automated A similar package is available from Mimotopes Now owned by Fisher Scientific Split and Mix Synthesis Points

Large diversity requires but can also utilize a longer synthetic sequence Generally makes a smaller amount (pM to nM) of a greater number of compounds Efficiency requires multiple sites (3 or more) of diversity Data handling and analysis can be complex Generally applicable to only solid phase synthetic approaches A A B B C C A Pool B n



CCA CCB CCC n/27 Array Synthesis Use parallel synthesis in a matrix format (8 x 12 array) - 20 reagents with 1 or 2 reactions gives 96 products O H 1 2 3 4 5 6 7

8 9 10 11 12 N HO R1-12 NH A O RA--H B C D E

O RA--H N H R1-12 F G H RA--H N H R1-12 Large Array Synthesis Larger numbers of compounds are available from one scaffold or reaction scheme Lay out a Super Grid 72 X 72 reagents or wells 9 X 6 plates = 54 plates

5184 compounds Chemists make multiple plates at a time Need 72 + 72 reagents Reagents A1 A2 B1 B2 C1 C2 8 X 12 Plates Array Synthesis Points Large diversity requires but can also utilize the large diversity of commercially available reagents More efficient when an array of reactions is treated as a unit parallel processing Efficiency requires at least 2 sites of diversity

Data handling simpler - one site, one compound Applicable to both solid and solution phase synthetic approaches With micro-titer plate format, one can borrow equipment from biologists (a first) Efficiencies gained in matrix format make this a combinatorial technique Make greater quantities (uM to mM) of fewer compounds 1 A B C D E F G H 2 3 4

5 6 7 8 9 10 11 12 Solution and Solid Phase Organic Chemistry Definitions for the sake of discussion: Solution Phase Organic Chemistry is chemistry like it used to be (pre 1990). Solid Phase Organic Chemistry (SPOC) is chemistry where some part of the target molecule is covalently attached to an insoluble support somewhere during the synthetic sequence. Solid Phase Reagents (SPR) are insoluble reagents used in solution phase chemistry (like 10% Pd/C or

polyvinyl pyridine). They (SPRs) may be made using SPOC. They (SPRs) have also made solution phase combinatorial chemistry easier. Solid Phase Organic Chemistry O O NO2 S O O n Core Spacer Br Linker Scaffold Core is usually 1% crosslinked polystyrene Spacer, if present, is usually a polyethylene glycol

TentaGelTM, or ArgoGelTM ( Give more solution-like reactivity with lower resin loading Linker, if present, provides an orthogonal method for releasing the scaffold Scaffold is the part that youre interested in doing chemistry on and releasing at the end of the synthesis Linkers O CL R3 R2 N N Merrifield Si H3 C O CH3

Ellman OH Wang O O (N or O) O R R1 N H O Greenberg photolinker H3 CO Rink amide Rink acid OCH3 NO2

O or R O An Example NO 2 1) n-BuLi, TMEDA HO cyclohexane CO 2H 2) CO 2 NH 2 NO 2 O DIC, pyridine, DMAP O NH2

DMF O Cl O R A--H NO 2 O O pyridine, DMAP CH 2Cl 2 NH O RA--H O DMF O H

N O Cl RA--H NaOCH 3 4:1 THF/MeOH HO N R1-12 NH O R 1-12 RA--H pyridine, DMAP O H R1-12

NH NH O O O NH2 O SnCl2. (H2O) 2 RA--H H. V. Meyers, G. J. Dilley, T. l. Durgin, T. S. Powers. N. A. Winssinger, H. Zhu, M. R. Pavia, Molecular Diversity,1,13020 (1995) CH 2Cl 2 Reaction Path Analysis Expose

Wash 2 Acylation 96-well reactor 1,3 Wash Wash Cleavage 4 Collection & purification Submit Solid Phase Organic Chemistry

Products are insoluble Easier to manipulate physically Easier to clean up, can wash exhaustively Can use excess reagents to drive reactions to completion No bimolecular reactions (infinite dilution) Cant use Solid Phase Reagents (SPR) Modified kinetics (generally slower, greater rate distribution, all sites not equal) Requires new analytical methods Requires linking chemistry (limits reaction conditions, constrains product structure) Solution Phase Organic Chemistry More compounds means less time per compound This requires: Good generalized procedures Short synthetic sequences High yield reactions Stoichiometric addition of reactants Parallel or high throughput purification methods Solution Phase Organic Chemistry

Multiple Component Condensation Reactions R2 O O O O + R1CHO + H2 N NH2 R2 O NH O R3 R3 O

NH Biginelli O O R1 COOH + R2 NH2 + + R5 NC R3 Ugi R3 R1 R4 R3 R4 H N R1 N R2

R5 N R1 O R3 R3 R5 R4 R6 R4 R5 R6 NH2 + R1 CHO + R2 Grieco Kobayashi

R2 R2 N H R1 Armstrong, R.W., Combs, A.P., Tempest, P.A., Brown, S.D., & Keating, T.A. Account. Chem. Res., 29, 123-131 (1996). Solution Phase Organic Chemistry R2 O (CH3O)2 P CO2CH3 H3C-NO2 R2

R2CHO CO2Me KOMe DBU EtOH, reflux CO2Me O2N R2 6 Nitrobutyrates 3072 Compounds O2N R3-NH3 AcOH R1 N

O R3 Single isomer > 95% R1CHO Br Br IC50 = 420 nM FTase Competitive Inhibitor iterate O2N N O2N HO O IC50 = 1.9 nM FTase for enantiomer shown

N HO HO N N Shinji Nara, Rieko Tanaka, Jun Eishima, Mitsunobu Hara, Yuichi Takahashi, Shizuo Otaki, Robert J. Foglesong, Philip F. Hughes, Shelley Turkington, and Yutaka Kanda. J. Med. Chem.; 2003, 46, 2467-2473 Solution Phase Organic Chemistry Products are soluble Byproducts and excess reagents are also soluble and accumulated with each step Direct analysis is much easier (tlc, nmr, ms, hplc, lc/ms) Kinetics are uniform and familiar Use of solid phase reagents (SPRs) is possible No linkers required, less excluded chemistry Requires development of parallel workup and purification methods Called Parallel Synthesis or Rapid Parallel

Synthesis (RPS) Trends over the Last Decade Sld P S&M Sld P Array 10,000+ Solu P Array 2004 # of Compounds 1000+ Solu P Array 1996 Classical Organic Synthesis 0 Solution Phase Array or Parallel Synthesis now dominates

time Dev. times for solid phase Equipment for Solid Phase Organic Chemistry Split & Mix Standard labware with gentle stirring Array Geyson Pin Approach Bottom filtration Top filtration Little stuff Big stuff Geysen Pin Method Resins attached to pins in an 8 x 12 array format Reagents or wash solvents in a 96 deep-well plate Drop it in to run reactions or wash resins Kits available from Mimotopes

Equipment for Solid Phase Organic Chemistry Problem: How do you put 24-96 of these together? Bottom Filter Top Filter Original Sphinx Reactor Solid Phase Chemistry Reactor Plate in a Plate Clamp Strip Caps used to seal reaction after reagent addition Plate removed from clamp for resin washing Plate Bottom acts as a 96-way valve H.V. Meyers, G.J. Dilley, T.L. Durgin, et al Molecular Diversity 1995, 1, 13-20 Commercial Apparatus for Solid Phase

Argonaut Quest 210 Nautilus 2400 Trident Little Stuff FlexChem Big Stuff MiniBlock Bohdan Ram Tecan Combitec Advanced Chemtech 496 Polyfiltronics/Whatman Charybdis Technologies

Myriad Core All Discontinued Big stuff is a bad idea. Parallel Solution Phase Organic Synthesis Equipment An Array of Vessels Heating and cooling Mixing Inert Atmosphere Access for addition and sampling Methods Reactants and reagents added as solutions or slurries Run at equimolar scale Separate the reaction from the workup Equipment for Parallel Solution Phase Organic Synthesis One at a time Synthesis

Parallel Synthesis Equipment for Parallel Solution Phase Organic Synthesis Generic Reactor Block Equipment for Solution Phase Organic Synthesis Reactor Blocks Equipment for Solution Phase Organic Synthesis MicroWave Biotage Solution/Slurry Addition 1 5 Good for Repeated Additions of one Solution

Disposable Polypropylene Syringe Barrels Easily adaptable to Leur fittings (needles) Can deliver from 0.5 uL to 5 mL Inexpensive and Fast (better than robots) Can Deliver Slurries with Modifications 3 2 4 Eppendorf Repeater Pipette eppendorf Solid Addition Solid addition plates/Vacuum systems Solid as a slurry 10% Pd on Carbon in Ethanol NaHB(OAc)3 in Dichlorethane Resins as isopycnic slurries

Purification Methods Solid Phase Wash exhaustively product dependent cleavage Solution Phase - Parallel Purification Extraction liquid-liquid, acid/base SPE, scavenging resins Fluorous Synthesis Chromatography Scavenging Resins O R1 N C 1.5 eq. NH2 CHCl3 , RT, 3h +

R2 R1 O N H N H R2 O NH2 1 eq. N H N H R1

O BuO O N H N O N N H S. W. Kaldor, J. E. Fritz, J. Tang, E. R. McKinney, Biorganic & Med. Chem. Lett.., 6,30413044 (1996). Fluorous Synthesis NO2 O Fluorous (C6F12) Phase S O

Br Replace resin with fluorous handle Aqueous Phase Halocarbon (CH2Cl2) Phase NO2 O CF 3 (CF 2 )n O S Br D. P. Curran, M. Hoshino, J. Org. Chem., 1996, 61, 6480-6481. D. P. Curran and Z. Luo, Fluorous Synthesis with Fewer Fluorines (Light Fluorous Synthesis): Separation of Tagged from Untagged Products by Solid-Phase Extraction with Fluorous Reverse Phase Silica Gel, J. Am. Chem. Soc., 1999, 121, 9069. Liquid Handling Robots

A Primer 10 mL Loop Tees Robot Arm 6-Way Valve X Tip Y Z Syringes System Solvent Purification Methods Filtration Use Parallel Filtration and a Liquid Handling Robot Salt Removal Covalent and Ionic Scavenging Resin Removal Extractions Liquid-Liquid

SPE - Solid Phase Extraction Chromatography 20 M Polyethylene frit Small hole drilled into the bottom of each well Polypropylene Silica C18 Fluorous Silica Filtration Salt Removal Covalent and Ionic Scavenging Resin Removal Robot Tip Filter plate Source plate Destination plate

Extractions Liquid-Liquid 1. Positional Heavy Solvent Extraction 2. Positional Light Solvent Extraction 3. Liquid Detection Light Solvent Extraction 1 2 1 3-1 3-2 3-3 2 3 Chromatography and SPE Silica Gel Fluorous Silica Gel C18 Ion Exchange

1. Dissolve Samples in a suitable solvent 2. Transfer to little chromatography columns 3. Elute clean products and/or collect fractions Chromatography Example Cyclic Urea Plate, wells 148, Before and After Filtration through Silica gel Commercial 24 & 96-well Filter Plates Varian Oros technologies Robbins Scientific Polyfiltronics Whatman Spike International Commercial Robotics Robots TECAN Hamilton Gilson Custom solutions Chemspeed Complete reaction stations AutoChem weighing, extraction, transfers InnovaSyn

extraction, transfers, TLC spotting J-KEM High Through-Put Prep HPLC Systems based on UV and/or ELSD Biotage Gilson Argonaut Isco Systems based on Mass Spect MicroMass, PE Sciex, Shimadzu, Agilent Analytical methods Solid Phase - few high throughput methods NMR - gel phase, MAS IR - works well

MS - laser assisted removal and ionization elemental analysis - must analyze starting materials Solution Phase - some high throughput methods TLC - ideal for parallel analysis MS - ion spray, 45 sec./sample, reports at 2 sec./sample NMR - high throughput with flow probes 2 min./sample HPLC, LC-MS 5 min./sample The challenge is not so much to collect the data as to analyze it. Robotic TLC Plate Spotting Example TLC Plate Some Pertinent Points Analyze an entire plate (96 compounds) at once Trends are easy to spot Note similar impact of substituent change Common impurities

Common by-products Can Spot Across or Down to See Trends Non linearity of detection No structural information A B C D Mass Spectroscopy Mass Spectrometers used in Combinatorial Labs Use an Ion Spray technique (ES or APCI) to allow flow injection analysis (FIA) Auto Samplers sample from multiple 96 well plates Use quadrapoles for mass filters Have data analysis and reduction packages for matrix analysis Can run samples at < 1 min. each LC/MS becoming much more routine (5 min. each) Analytical Data Analysis

LC/MS MicroMass Diversity Browser Lilly RTP Analytical Viewer Analytical Data Analysis NMR ACDs SpectView SLAVA SLAVA Trends 1. With higher screening throughput there is a trend away from making or testing mixtures. 2. With better purification methods, SPOC no longer dominates combinatorial chemistry. 3. Everyone is demanding purer products and more material with better characterization. 4. Equipment complexity is dropping as we learn to be clever rather than over-engineer. There are more commercial options though big machines are going away. 5. The methods of Parallel Synthesis are slowly finding their way into all aspects of synthetic chemistry. 6. Handling data (registration, analysis, results) remains a major challenge.

7. Combinatorial Chemistry/ Parallel Synthesis is here to stay. Conclusions By application of robotics, computers, clever engineering and chemistry, the methodology now exists to synthesize, with reasonable purity and yield, medicinally relevant organic molecules at 100 to 10,000 times the rate possible just 10 years ago. The field of Combinatorial Chemistry/ Parallel synthesis is evolving and melding with classical Medicinal Chemistry. Further Information Archiving TLC Plates UV Images Captured using a UV Light Box with a Visible

Camera Visible Images Captured using a Scanner All Images Stored on Disk and Printed for Notebook storage Camera Computer Scanner UV light box

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