high-throughput synthesis on solid phase · high-throughput synthesis on solid phase towards...

1E. Felder - Combinatorial Chemistry September 2002

HighHigh--throughput synthesis on solid phase throughput synthesis on solid phase

Towards rigorous quality standards and quantitative characterization without compromising efficiency

Eduard Felder et al.Dept. of Chemistry - Discovery Research - Pharmacia Italia


Network of ActivitiesNetwork of Activities

Assay Technologies

ExternalCollaborationsAnalyticsAnalytics

Structural Chemistry

LogisticsIT Tools

Screening

ExperimentalMethods

ComputationalChemistry

Chemical Kinase Platform




Combinatorial LibrariesCombinatorial Libraries

Medicinal Chemistry

Preparative Organic

Chemistry

Preparative Organic

Chemistry

CombinatorialTechnologies

ChemistryChemistry Lab AutomationLab Automation


Provide high-quality compounds / high-quality data

Drug discovery processDrug discovery process

compound amounts

number of compounds

assay throughput

HumanGenome

Functional GeneCharacterization

TargetValidation

HitFinding

DiseaseGenes

LeadOptimization

PreclinicalCharacterization

HitOptimization

Combinatorial Technologies


UHTS Plate: > 2000 wells / plateAssay Volume: 1-2 µl

Microtiter Plate: 96 wells/ plateAssay Volume: 200µl

Assay Sensitivity and ThroughputAssay Sensitivity and Throughput


LEAD FINDINGLEAD FINDINGLEAD FINDING LEAD OPTIMIZATIONLEAD OPTIMIZATIONLEAD OPTIMIZATION

Hit GenerationHit Generation Hit Optimization Parallel Medicinal Chemistry

Avenues:Avenues:Technical Development Areas:Technical Development Areas:

‘Brute Force’• cmpd # ↑↑ (combinatorial)• screening data ↑↑ (Ultra-HTS)

‘‘Brute Force’Brute Force’•• cmpdcmpd # # ↑↑↑↑ (combinatorial)(combinatorial)•• screening data screening data ↑↑↑↑ (Ultra(Ultra--HTS)HTS)

Screening by NMR(novel scaffolds)

Screening by NMRScreening by NMR(novel scaffolds)(novel scaffolds)

Combinatorial target-guided ligand assembly

Combinatorial targetCombinatorial target--guided guided ligandligand assemblyassembly

Synthesis → AutomationSeparations → High resolution / throughput

MS-directed fraction collectionLogistics

Analytics → Process control analytics in‘new formats ’:- on solid phase: IR / NMR- in microtiterplates: LC-MS; NMR, quant. N-detection

Chemistry Progress: Solid PhaseChemistry Progress: Solid Phase

Process Development AreasProcess Development Areas


Diversity PlatformDiversity Platform

binuc.skc

R'

O

R2

R3

R1 R

2

R3

N N

R4

HN NH2

R4

R1

N N

R2

R5CO

R5

R6

R3

R4

R1 R2

R3

N

OR4 H

R1 R

2

R3

N

R5

R4

R4

O

NH2

varioustransformations

R1

R2

R3

N NR4

R4NHNH2

R4R5

NH2

R4HN NH2

O

R1 R2

R3

N N

OR4 H

A.L. Marzinzik and E.R. Felder, J. Org. Chem. 63, 723 (1998).


NH

R1

N R1

NH

N

N

N

Cl

R2

N R1

N

N

N

N

ClR

2N

N R1

N

N

N

X NH

R3

R4

R3

NO

R3

R3

O

OM e

OMe

O

1 PAL

R1-NH2

a

PAL

b

23

3c

Mitsunobu

4

Pd-chemistry

5

X =

Derivatizing solid supported core scaffolds with common reactionDerivatizing solid supported core scaffolds with common reactionss

S. Ding et al. J. Am. Chem. Soc. 124, 1594 (2002).


Ø Single Bead IR

Ø Magic Angle Spinning NMR

Ø Quantitation by NMR

Ø Direct-Injection NMR for High-Throughput Spectroscopy

Ø LC-MS

Ø HPLC Online Nitrogen Detection

Ø Single Bead IR

Ø Magic Angle Spinning NMR

Ø Quantitation by NMR

Ø Direct-Injection NMR for High-Throughput Spectroscopy

Ø LC-MS

Ø HPLC Online Nitrogen Detection

Solid Phase Synthesis Solid Phase Synthesis -- Process ControlProcess Control


Synthesis Pathway (Example)Synthesis Pathway (Example)

NH

O

O

O

H

ONH

O

OO

NHR

NH

O

OO

NRXR

2

NH

R

O

R2

S NH

R

O

O

R2NH

RO

NH

R2

RNH2

X= SO2 , CO, CONH

4-(4-formyl-3-methoxyphenoxy)butyryl AM resintheoretical loading 0.94mmol/g

R'COClor R'NCOR'SO2Cl

Step a

Step b


O

OR

1

OO

OR2

O

O

OR

3

O

O

O H

Step 1Step 2 Step 3

Solid Phase Synthesis: Monitoring progressSolid Phase Synthesis: Monitoring progress

Solid Phase IR

Solid Phase NMR Solid Phase NMR

Solid Phase IR Solid Phase IR

Solid Phase NMR

NMRHPLC

MSHPLC-MS

OH R1

O

Cleavage

HPLC NMRMS

HPLC-MS

OHR 2

O

Cleavage

HPLCNMRMS

HPLC-MS

OH R3

O

Cleavage


STARTING RESIN IR

Reductive Amination Reductive Amination -- Feasibility StudyFeasibility Study

Different analytical techniques were compared

solid phase IR,

MAS NMR,

HPLC

NH

O

O

O

H

O NH

O

O O

NHR

N

NH2

NH2

NaBH3CN or NaBH(OAc)3

DCM, THFanh, DMA or TMOF(CH3COOH)

or

2759,3cm-1

CH stretching


± 57%

Homogeneousbead population

Heterogeneouscommercial batch

The uniformity of bead loading is measured with microscope IR spectroscopy. The information obtained is unaffected by cleavage efficiency and post-cleavage handling.

The uniformity of bead loading is measured with microscope IR spectroscopy. The information obtained is unaffected by cleavage efficiency and post-cleavage handling.

OnOn--bead IRbead IR

ATR crystalSi or Ge

IR in IR out

0.5 - 2µ m

∼ 100 µ m

AT Reflectance

200 µ m100 µ m

∼15 µ m

∼ 400 µ m

Transmission

IRin

IRout

LCC Polystyrene (new) Commercial Batch


F2 (ppm)12345678

F1(ppm)

708090

100

110120

F2 (ppm)12345678

F1(ppm)

10

11

12

1314

15

ppm2345678

NMR: Our Procedure for SPSNMR: Our Procedure for SPS--boundbound cpdscpds

1D

2D-DQ (or TOCSY)

gHSQCe(or gHMQC)

1-3 mg of resin in 40 µ l of CD2Cl2, Inova-500 equipped with a PFG-ID NanoProbe: 1-2 hours

O

Pol

NH

OO

NH

O

O

O


10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

DCM

H2O

10.27 (9a)7.75 (3)

4.35 (18)

4.08 (12)

3.80 (8)

2.41 (14)

2.16 (13)

Polymeric matrix : Broad signals

Instrument VARIAN INOVA 500nano probe 40µl

1-3 mg of resin swollen in CD2Cl2

scan # 16 t=10ms 3 min exp

1H MAS NMR Spectra of the formyl resin1H MAS NMR Spectra of the formyl resin

NH

O OCH

3

H

O

O

4-(4-formyl-3-methoxyphenoxy)butyryl AM resintheoretical loading 0,94mmol/g


10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

6.65 (14)

6.61 (16, 12)

6.50 (6)

6.41 (4)

4.31 (24)

4.23 (9)

3.98 (18)

3.77 (8)

2.37 (20)

2.11 (19)

28

29

27

30

26

25

24

NH23

21

20

19

18

O17

5

6

4

1

3

2

O7

CH38

9

NH10

11

16

12

15

13

14

O22

Reaction one potaniline 5eq., CH 3COOH 1 eq.,

NaBH(OAc)3 3 eq, in THF o.n.

Aniline derivativeAniline derivative


aniline, CH3COOH, NaBH (OAc)3THF o.n.

Aniline derivative: Solid phase IR and HPLC after cleavageAniline derivative: Solid phase IR and HPLC after cleavage

Solid phase IR

O

OCH

3

NH

HPLC @254 A=12495214 after reaction with RSO2Cl and cleavage

O

OCH3

NS O

O

NHO

NHS

O

O

NH

O

TFA 30% r.t. 2h.

RSO2Cl,DIEA, DCM


NH

O

O

O H

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

Pol-Ar-CHO

Pol-Ar-CH2-ohdmf

dmf

H2o

8.03 (6)7.34 (4)

6.50 (5)

6.38 (3)

4.39 (8)

3.98 (18)

3.75 (16)

2.38 (20)

2.11 (19)

NHO

O

O

CH3

NH

N

Reaction one pot2-aminopyridine 5 eq, CH3COOH 1eq.,

NaBH(OAc)3 3 eq, in THF o.n.

Pyridylamine: MAS NMR of a loaded resinPyridylamine: MAS NMR of a loaded resin

* *


Pol31

28

2927

3026

25

24

NH23

21

20

19

18

O1712

11

13

10

14

9

O22

O15 CH316

8NH72

3

N1

4

6

5

H6

H5

H5

H4H3

H13H14

H4

H18 H19

H19H20

1H-MAS-2DDQ:• signals only from the (flexible) coupled 1Hs• the more rigid resin becomes “NMR-invisible”• 20 min experiment• ~2 mg of resin in 40 µ l of CD2Cl2

δx

δx+ δy


Pyridylamine: MAS NMR of a loaded resin Solid phase IR and HPLC of the cleaved product

Pyridylamine: MAS NMR of a loaded resin Solid phase IR and HPLC of the cleaved product

O

ON NS

OO

NH

O

NH

N

S OO

NHO

RSO2Cl, DIPEA,DCM

TFA 30% 2h r.t.

O

OCH3

NH N

2-amino pyridine,CH3COOH, NaBH(OAc)3in THF o.n.

absent


10.5 10.0 9.5 9 . 0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

10.5 10.0 9.5 9 . 0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

pyridine TMOF in DCM o.n.CH3COOH, NaBH(OAc)3 in DMA

aniline TMOF in DCM o.n.CH3COOH, NaBH(OAc)3 in DMA

Some non obvious examplesSome non obvious examples


O

CH3

OO

L-HG elT e nta

H

O

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0ppm

0.00

0.01

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0ppm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

NHO O

CH3

H

O

O

Poli mer

High mobility solid supportHigh mobility solid support

Formyl Tentagel resinFormyl Tentagel resin

Standard Polystyrene resinStandard Polystyrene resin


Yield calculationYield calculation

O

O

H

ONH

O

N

NH2

S

N

NH 2

O

NH

O

R

NH

O

N

NH2

S

N

NH2

NaBH(OAc)3CH3COOH,DCM, anhTHF, TFA 90% in DCM

We compared:

1. Weight

2. HPLC against standard,

3. Deconvoluted 1H MAS spectraHPLC: calibration curve

2-amino pyridine 2-amino thiazole


Pol25

22

23

21

24

20

1918

NH17

1514

1312

O11 5

6

4

1

3

2

O7

CH38

9X (NH-2Py or OH)10

O16

H-9 (X=OH)

H-9 (X=NH-2Py)

H-24

H-12

H-8

1H-HRMAS-NMR: Deconvolution of Overlapping Signals


Yield evaluation by weight, HPLC against standard curve, deconvoluted NMR Yield evaluation by weight, HPLC against standard curve, deconvoluted NMR

entry amineresin mg (01.94mmol/g)

TMOF10eq

CH 3COOH1 eq

NaBH(OAc)3

3eqNaCNBH 3

3eqsolvent NMR yield WEIGHT

Yield(mg)

HPLCYield against Standard (%)

1 Thiaz 50 X X DCM 43%5,9

(59%) 23

2 Thiaz 50 X X DCM 34%10,6

(100%) 27

3 Thiaz 50 X X THF n.d.6,1

(60%) 2

4 Thiaz 50 X X DCM 40%13,2

(131%)32

5 Pyr 50 X X DCM 48%11,8

(121%)40

6 Pyr 50 X X DCM 53%12,7

(123%)43

7 Pyr 50 X X THF 35%12,4

(121%)27

8 Pyr 50 X X DCM n.d.6,2

(63%)10


First implementations of rapid NMR quality assessments from First implementations of rapid NMR quality assessments from titerplatestiterplates

B. Hamper et al.B. Hamper et al.


CHO

HMDS

Flow-through NMR Spectrain 96-well plate (regular DMSO)

B. Hamper et al.B. Hamper et al.


Malonamides

Plate View

Reference intermediates

Residual malonamide intermediates (%)

Complementary HPLC Analysis


Development of accurate NMR Development of accurate NMR quantitation quantitation with BTMSB standardwith BTMSB standard

V. Pinciroli et al.V. Pinciroli et al.V. Pinciroli et al. J.Comb.Chem. 3, 434 (2001)

Si

Si


O

H

OO

OO

NRX

R2

X NH

RR2

NH

OO

R

8 x RNH2

X= SO2, CO, CONH

X= SO2, CO, CONH

2 x R'COCl2 x R'NCO2 x R'SO2Cl

48 x

Application example (small compound array)Application example (small compound array)

After cleavage the products were analysed by:

• HPLC-MS (identity, purity)• 1H NMR against standard (identity, purity, amount)• 1H NMR Vast System against standard (identity, purity, amount)


8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

6.011.86 1.351.24 1.000.97 0.910.230.11 0.09 0.04

CH3-SO2-CH3

BTMSB

CH3-ch2-so-ch3

H4eq

H3,5eq

H2,6eq H2-6ax

6.80 (16)

6.79 (16)

6.76 (13)

6.68 (17)

6.67 (17)

4.03 (11)

O

CH3

21

OCH319

NH

O

NH

Si

Si

Direct-Injection NMRfor High-Throughput Spectroscopy

VAST= Versatile Automatic Sample Transfer

Experimental4mM DMSO 400ml (H2O presence does not matter)60 scans plus scout scan each sample20 min each sample (including temperaturestabilization, gradient shimming on the first sample &2 rinsing cycles per sample)Virtually no contamination between samplesReliable automation2D exps can be run separately or in the run


NMR Routines on NMR Routines on CombiChemCombiChem LibrariesLibraries

M. Tatò et al.M. Tatò et al.

p 96 wells plate run in nearly 16 hours

• 0.5-2 mg / 200 µl DMSO-H6 (H2O does not matter)

• 32-128 scans plus scout scan each sample

• 8 -12 min each sample (including temperature stabilization,

gradient shimming on the first sample and 1 rinsing cycle per sample)

• virtually no contamination between samples

• reliable automation

• 2D exps can be run separately or in the run






(Varian)

Sample in DMSOH6


8 . 5 8 . 0 7 . 5 7 . 0 6 . 5 6 . 0 5 . 5 5 . 0 4 . 5 4 . 0 3 . 5 3 . 0 2 . 5 2 . 0 1 . 5 1 . 0 0 . 5 0 . 0

9 1 . 8 3 2 4 . 6 1 3 . 5 81 . 8 51 . 5 31 . 3 9 1 . 0 00 . 5 3 - 0 . 0 1

Si

Si

1

2

3

4

5

6

NH7

8

O9

10

11

12

13

14

15

16

17

N 18

1

1,39

HPLC A%@ 220 = 72%

NMR sample strength 70%

Flow NMR strength: 70%

Ideal situation

V. Pinciroli et al. J.Comb.Chem. 3, 434 (2001)


Si

Si

1

NH

O O

NH

0,57

2H

0,21

1H

If the starting material is not UV visible

HPLC A% @220 = 72%HPLC A% @220 = 72%

NMR strength 21% NMR strength 21%

Flow NMR 18%


8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5

90.05 22.766.76 1.401.31 1.000.90 0.480.180.160.12 0.04 0.020.01 0.010.01 0.00-0.00 -0.00-0.00 -0.02

NH

O

N

HPLC A% @220= 46%HPLC A% @220= 46%

NMR strength not easily measurable

NMR strength not easily measurable

Flow NMR not measurable

Flow NMR not measurable

If there are no diagnostic NMR signals


8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

6.011.86 1.351.24 1.000.97 0.910.230.11 0.09 0.04

CH3-SO2-CH3

BTMSB

CH3-ch2-so-ch3

H4eq

H3,5eq

H2,6eq H2-6ax

6.80 (16)

6.79 (16)

6.76 (13)

6.68 (17)

6.67 (17)

4.03 (11)

O

CH3

21

OCH319

NH

O

NH

Si

Si



Experimental4mM DMSO 400ml (H2O presence does not matter)60 scans plus scout scan each sample20 min each sample (including temperaturestabilization, gradient shimming on the first sample &2 rinsing cycles per sample)Virtually no contamination between samplesReliable automation2D exps can be run separately or in the run


Identity-Quantity-Purity

HPLC/N/UV/MSn detection platform

Minimal Sample Consumption: Including Nitrogen DetectionMinimal Sample Consumption: Including Nitrogen Detection


Integrated Analytics

Nitrogen detection

0

1000

2000

3000

4000

5000

0 0.2 0 . 4 0.6 0.8 1 1.2

25_4000

p m o l

y = 99.872 + 3824.7x R= 0.99919

pmol

N area

0

50

100

150

200

250

0 0.005 0.01 0.015 0 .02 0.025 0.03 0.035 0.04

25_200

pmol

y = 16.249 + 4753.7x R= 0.99936

pmol

N area

Nitrogen ContentCalibration


Minutes0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

N-r

espo

nse

0.00

0.02

0.04

0.06

0.08

0.10

N-r

espo

nse

0.00

0.02

0.04

0.06

0.08

0.10

NitrogenDPHA, 200 pmol N std

Novartis

Online N-Detection: Standard Sample (200 pmol) R. Falchetto et al.R. Falchetto et al.


Nitrogen detection

Chemiluminescent reaction between ozone generated from oxygen and nitric oxide via high temperature pyrolysis of nitrogen-containing compounds

Linear standard calibration curves over a >104 range of analyte concentrations. HPLC-CLND is compatible with reversed phase, size exclusion, and ion chromatography. Sensitivity is 0.5 ppm Gly-Gln peptide (0.1 ppm N) in water, 4x > absorbance at 215 nm.


0

50

100

150

200

250

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04

25_200

pmol

y = 16.249 + 4753.7x R= 0.99936

pmol

N area

Nitrogen Content R. Falchetto et al.R. Falchetto et al.


Concluding RemarksConcluding Remarks

Ø Derivatizing novel scaffolds on solid phase with standardreactions is an efficient, compound prolific approach

Ø Ample choice of techniques for in-process control of solidphase syntheses (on and off beads)

Ø No significant bottlenecks Ø Beware quantitation by weighing of non-crystalline microsamples

(< 5mg) Ø NMR quantitation against standard is the most accurate system

however ... it is relatively slow. Impractical for samples < 1mg Ø The MAS NMR on solid phase gives good qualitative information,

the quantitative evaluations may be less precise and moredemanding in terms of interpretation (spectra deconvolution)

Ø Nitrogen detection (HPLC online) has excellent throughput andis the method of choice for the quantitation of microsamplesof large libraries

Ø Initial concerns on analytical limitations of solid phase chemistryhave been overcome

Ø Derivatizing novel scaffolds on solid phase with standardreactions is an efficient, compound prolific approach

Ø Ample choice of techniques for in-process control of solidphase syntheses (on and off beads)

Ø No significant bottlenecks Ø Beware quantitation by weighing of non-crystalline microsamples

(< 5mg) Ø NMR quantitation against standard is the most accurate system

however ... it is relatively slow. Impractical for samples < 1mg Ø The MAS NMR on solid phase gives good qualitative information,

the quantitative evaluations may be less precise and moredemanding in terms of interpretation (spectra deconvolution)

Ø Nitrogen detection (HPLC online) has excellent throughput andis the method of choice for the quantitation of microsamplesof large libraries

Ø Initial concerns on analytical limitations of solid phase chemistryhave been overcome


Acknowledgements

Katia Martina - Combinatorial Chemistry

Marco Tatò - Structural Chemistry

Roberto Biancardi - Structural & Predevelopment Analysis

Bruce Hamper - Combinatorial Chemistry, St. Louis USA

The Chemistry Department – Pharmacia Italy, Nerviano (Milano)

high-throughput synthesis on solid phase · high-throughput synthesis on solid phase towards...

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