Catalysis of Substitution Reactions on Heterocycles Bound

to a Solid Phase

wolfgang.brill@am.pnu.com

Catalysis of Substitution Reactions on Heterocycles Bound

to a Solid Phase

O

Nuc

N

N NH

N

X

X

H,X

Nuc

Nuc Radical, Nuc

Levoglucosan based Libraries

epoxide opening

epoxide opening

difficult alkylation or acylation

avoidglycosidic scissionO

O

XO

O

O

NH2

R1 R2

R3

Pd mediatedcoupling

Pd mediatedcoupling

Brill, W.-K.-D. et al. Tetrahedron Lett 39 (1998) 787-790

Brill, W. K.-D. et al. Synlett (1998) 1085-1090

OO

OHOH

OH

OO

OTsTsO

OH

OO

OTsO

OO

OTs

OH

O

TsCl NaOMe

OH

BF3 . OEt2

LiOH

Derivatization of the Scaffold

cha2

96% 85%

40-90%

95%

O

O

R1

O

O

R1

OO

OO

O

O

R1

Li+

W. K.-D. Brill, June 96

Cerny Anhydride

OO

O

OH

OTsO

O

R1

I

(CH2)10

R1:

The Hydrolysis of Levoglucosan Containing Esters

C(CH3)2

Li+

OO

OO

O

O

R1

I

(CH2)10

R1:

C(CH3)2

O

O

OO

O

O

O

O

OO

O

O

OO

OHO

LiOH, MeOH, H2O

Schematic Representation of Resins

crosslink

shrunken resin(poorly solvated)

compound boundto the polymer

Reactions can only be performed, if reagents can diffuse through the gelphase of the beads. The polymer has to be solvated with the appropriate solvent to form such a phase.

swollen resin(solvated)

OO

NH

OO

OORink

R2R2'NHLi+ Ph4B-

2,6-lutidine100°C

R1

> 90%

R2OHP4-t-Bu

dioxan, 60°C

> 90%

R2SHLiHMDSdioxane80°C

> 90%

OO

NH

OO

O OROHRink R1

OO

NH

OO

O SROHRink R1

OO

NH

OO

O NOH R

R

Rink R1 2'

2

2

2

ONH2

O

O

O

O

OO

OOR1

Li+

DIC, HOBT, DMF

levunbas16

Brill 1999Opening of Levoglucosan-2.3-epoxides with various Nucleophiles

N

N P

N

N

P

NMe2

NMe2Me2N

P NMe2

NMe2

NMe2

PMe2NNMe2

NMe2

P4-t-Bu:

Rink-linker:

OO

O

O

O

NH OH

OTs

OO

O

O

O

NH O

OO

O

O

O

NOH

OO

O

O

O

NH

SOH

OO

H2NOH

OO

NH3

+

OHS

NH

O

OO

R

TFA-TFA

-

SNH

R

O

OO

T>80°C

+

TFA

DBUPhSH

T<80°C

TFA

Intera and Intramolecular Substitution on Levoglucosan-2,3-epoxide

levunibas4

Brill 1996

O

OO

O

OH

O

NH

I

N

ORink

Palladium Mediated Coupling Reactions on the Spacer Residue

Pd(OAc)2, K2CO3H2O, dioxane,

110°C

84%O

OO

O

OH

O

NH

N

O

OMe

Rink

HO

HOB OCH3

Bu3Sn

cha11

O

OO

OO

NH

I

ORink

Pd2dba3, AsPh3dioxane, 110°C

80-90%O

OO

OO

NH

ORink

H

W. K.-D. Brill, June 96

O

OO

OO

NH

I

ORink

Pd(PPh3)2Cl2, CuINEt3, dioxane, rt.

80-90%O

OO

OO

NH

ORink

O

OO

OONH

ORink

A Second Palladium Mediated Coupling Reaction on the Scaffold

HO

HOB OCH3

O

OO

OONH

OHNH

I

Rink

O

OO

OONH

OHNH

OCH3

Rink

OO

OONH2 OH

NH

OCH3

20% TFA

72% from aminoresin

I

H2N

Li+ Ph4B-

cha12

W. K.-D. Brill, June 96

Pd(OAc)2 K2CO3H2O, dioxane

OO

NH

OO

O OROHRink R1

2

OO

NH

OO

O OROHRink R1

2

R3

ONH2

OO

O OROHR1

2

R3

How to avoid glycolysis:

1) conc. of TFA < 20%

2) solvent of TFA must have higher boiling point than TFA

3) resin must swell in solvent

a) 20% TFA in ClCH2CH2Clb) toluene before evaporation

R3-X

Schwesingerbase orKOtBu

Final Derivatizations and Cleavage

The Synthesis of Purine Derivatives on Polymeric Supports

•Potential Targets

•Development of the Chemistry

•Designing the Synthesis

•Selection of Building Blocks

•The Sort & Combine Method

•Performing the Synthesis with IRORI-MiniKansTM

•Workup, Purification and Archiving

NH N

NN

NH

R

R

R

NH

N N

NN

N

R

NH

H H

R

R NH N

NN

N

R

N

R

H

H

R

N N

NN

NH

R

R

R

NH

H

N N

NH

N

N

R

NH

H

R

R

Purines may bind to Proteins in different ways

They address nucleoside binding pockets

Nucleoside Cofactors Lead us to Drug TargetsNucleoside Cofactors Lead us to Drug Targets

11. Many cofactors contain nucleoside motifs. Many cofactors contain nucleoside motifs

2. Nucleosides or nucleoside cofactors are involved in all important 2. Nucleosides or nucleoside cofactors are involved in all important cellular cellular processes.processes.

DNADNA synthesis synthesisRNARNA synthesis synthesisproteinprotein synthesis synthesiscarbohydratecarbohydrate synthesis and oligomerization synthesis and oligomerizationlipidlipid synthesis and processing synthesis and processingsynthesis of synthesis of homohomo and and heterocyclicheterocyclic aromaticsaromaticssignalingsignaling via phosphoylation and sulfatation via phosphoylation and sulfatationsignaling as signaling as second messengersecond messenger or or hormonehormonealkylationalkylation and and dealkylationdealkylation of DNA and other substrates. of DNA and other substrates.

3. Nucleoside binding sites are not optimized toward very tight binding! 3. Nucleoside binding sites are not optimized toward very tight binding!

i, ii: Attachment to the polymeriii: 6-Substitutioniv: 2-Substitutionv: Brominationvi: Stille couplingvii: Cleavage

Reaction Scheme

OH

N

NN

N

Cl

Cl

N

NN

N N

NN

N Cl

O CF3

O

N

NN

NH Cl

Cl

N

NN

NBr

N

NN

N N

NN

NH

Rink resin

R1

R2

R1

i ii

iii

iv

1 2

3

4

R1-H:

R2-E

6 5

1

23

4

56

7

8

9

7

R1

R2

v

R1

vi R3-SnBu3

8

R3

R2

R1

R3

R2

vii

9

OH

N

NN

N

Cl

Cl

O CF3

O

N

NN

NH Cl

Cl

Rink resin

1 2

3

4

1

23

4

56

7

8

9

TFAA, 2,6-lutidine

4 eq.NMP

crystallization ofexcess

recycling

Immobilization of 2,6-dichloropurine

Yield[%]Entry: Solvent Catalyst T [°C] Reaction

Time [h] Start.mat. Product

1 dioxane DIPEA 80 24 100 -2 2,6-lutidine/dioxane - 60 24 79.3 24.33 2,6-lutidine/NMP - 60 24 31.7 68.34 “ HNEt3

+ F3CCO2- 60 24 11.7 88.3

NH

F

Cl

NN

NH

N

Cl

N

F

Cl

NH N

NN

Cl

Cl

C6-Substitution

N

N N

NCl

Cl

N

N NH

N

Cl

N N

N N

Cl

N

N N

N

Cl

6a

6b

R1

amines (R1-H):

4

R1

7a (95%)

7b (97%)

5a R1=

5b R1=

NMP, cat. H+, 2,6-lutidine

20%TFA

DCE

C6-Substitution

Reaction temperature has to be 53°C to avoid substitution on C2 forvery nucleophilic amines (N-CH2CH2NH2, C-CH2CH2NH2 piperazines, piperidines).

No C2-substitution even at 70°C: anilines, benzylamines, morpholin, primary amines with higher order of substitution on their Ca

N

N N

N

Cl

N

N

Cl

N N

NN

N

O

N

N

Cl

NH

O

Pd-catalyst

N

N N

N

Cl

N

N

N N

NN

N

N

BOH

OH

Pd-catalyst

Solvent Base Cat. orPromotor Co-ligand

ReactionT [°C]

Time[h]

Yield[%]

NMP DIPEA - - 100 48 -NMP Cs2CO3 Pd(PCy3)2Cl2 - 100 “ 48.7

“ K3PO4 “ - “ “ 45.7“ Cs2CO3 Pd2dba3 P(tBu)3 “ “ 65.6“ K3PO4 “ “ “ “ 66.3*

Solvent Base Cat. orPromotor Co-ligand

ReactionT [°C]

Time[h]

Yield[%]

NMP K3PO4 Pd2dba3 P(tBu)3 100 48 84.5*

Sustitution on C2

* rest is starting mat.

X'

L

L

PdAr

X"

L

L

PdAr

X"

B(OH)2

Ar

R

Pd

Ar-X'Ar-RX"-B(OH)2

PdLn

MX"

MX'R-B(OH)2

X'

L

L

PdAr

X"

L

L

PdAr

X"

NRR'

Ar

L

Pd

Ar-X'Ar-NRR'PdLn

MX"

MX'R-NHR'

X'' -

H+

•The mechanism for aminations and Suzuki-couplings is similar.•Amines and boronates couple under the same conditions•A wide selection of building blocks possible•Reaction temperatures compatible with Kans.

Sustitution on C2

Br2

NNN

Br2

N+

Br N+

Br

solv. polar solvent

N N

NN

N

NR

R1 R1'

R2

R2'

N N

NN

N

NR

BrN N

NN

N

NR

R1 R1'

R2

R2'

N N

NN

N

NR

Br

EtOEt

yelloworange

soluble in benzene insoluble in benzene

solv.

Br2solv. Br+solv. + Br-solv.

solv.

2,6-lutidine

rapid equilibrationwith solvent

5 h, rt., DMF

R1 R1'

R2

R2'

R1 R1'

R2

R2'

pentane

Two Types of Bromine Complexes Were Investigated

Synthesis:

Reactivity:

Entry Resin Conditions Time [h] Bromine content1 Merrifield Br-complex, NMP, 2,6 lutidine 3x 241 <0.3 %2 Merrifield Br-complex benzene,

2,6-lutidine24 1.1% 0.14mmol/g

3 product ofentry 2

sat. KOtBu, dioxane 88°C 72 <0.3%

1) Three consecutive treatments.

Modification of the Polymeric Support by the Brominating agent

N

Br2

Bromine-complex

Br-content of resin, treated with the Bromine Complex (EA)

N

Br2

Br

no reaction:

Rink

N N

NN

Cl

Cl

Rink

N N

NN

N

Cl

R1 R1'

Rink

N N

NN

N

NHAc

R1 R1'

Rink

N N

NN

N

N

R1 R1'

R2

R2'

completeconversion:

side reactions: not tolerated groups: activated aromatics: brominationamines: many oxidation products

N

Br2

Reactivity of the Bromine Complex with Purines:

Solvent: NMP

R1: H, alkyl, R1’: alkylR2: H, alkyl, R2’: H, alkyltolerated groups: CONR2, CONRH CONH2,ether functions, aromatics moredeactivated than Bn

5% conversion

The Reactivity of the Bromine Complex in dry Benzene: N

Br2• disproportionation is very slow• little electrophilic substitution• oxidizing power is enhanced relative to reactions in NMP

Selective side reactions:

Rink

N N

NN

N

NH

HR

R

N N

NN

N

NH

HR

RO

Br

Rink'

N

Br2

benzene

Rink

N N

NN

N

N

N

O

N

O

N N

NN

N

N

O

N

O

N

HO

HO

Br

Rink'

N

Br2

benzene

The Reactivity of the Bromine Complex in wet Benzene: N

Br2

Rink

N N

NN

N

NH

HR

R

N N

NN

N

NH2

HR

BrN

Br2Rink'

benzeneH2O

Side chain oxidations are not mediaded by the solid phase.They work are observed also in solution phase.

N N

NN

N

N

BrBr

N N

NN

N

NH

OBr

N N

NN

N

NH2

Br

Br

N N

NN

N+

N

Br

H

R1"R1'

R2'

N N

NN

N

N

Br

R1 R1"

R2'

N N

NN

N

NH

Br

BrBr

R1"R1'¨

R2' N N

NN

N

N

Br

HBr

R1' R1"

R2'

R1' R1"

R2'

R1' R1"

R2'

R1 R1"

i

ii

ii

Proposed Mechanism for the Modification at C2

i: Br2-complex; ii: H2O

Attempted Synthesis to Yield 2,6,8-Trisubstituted Purines

NH N

NN

NH

Cl

NH N

NN

Cl

Cl

NH N

NN

NH

N

N

O

NH N

NN

NH

N

N

O

Br

NH N

NN

NH

N

N

O

Dehalogenationduring Suzuki couling

conditions

ArB(OH)2

Pd-cat

Entry Solvent Cu cat. Pd cat. Co-ligandProduct

yield[%]

dehalog.[%]

1 dioxane - Pd2dba3 As(Ph)3 - >52 NMP CuO Pd2dba3 dpppf no reaction3 NMP Cu(OAc)2 Pd2dba3 dppp - 91.6

4 NMPO

Bu

Et

O

2

Cu2+

Pd2dba3 dppp 51.5 48.5

5 NMP CuI Pd2dba3 dppp 20 -

6 NMP S

CuO

O Pd2dba3 dppp 35.7 64.1

7 NMP Cu2O - - no reaction8 NMP Cu2O Pd(OAc)2 dppp >98 -

SnBu3

N

NN

NBr

NH

NH N

NN

N

NH

NH N

NN

N

NH

NHcat.

+

Substitution on C8

P P

dppp:

Selection of Buildingblocks:1) Size and shape constraints: selection of privileged structures based on modeling previous screening results, docking excercises

2) Availability of building blocks: emphasis on proprietary building blocks, commercial building blocks 3) Chemtox considerations:exclusion of: -NO2,-NO,-N2

+, I, -I=O, -N3, heavy metals, alkylating agents, acylating agents, hydrazines, SH, aniline-functions in product

4) Tests of building blocks (several hundred reactions)

5) Generation of a virtual library with the building blocks which work:Agreement to rule of 5 checked, exclusion of dramatic outliers

6) Final selection for Synthesis

The Synthesis is performed on PS-Beads

PS-beads 60 mg per Kan

Rf -transponderswith unique IDRead only !Reading interval: 0.1sRF-frequency: 125 KHzReading distance : 1 cm

reusable many times !

1 cm1 per Kan

Now all Kans can be distinguished

The Sort and Combine Strategy

• 5 Building Blocks : A, B, C, D, E

• 5 Vessels:1 2 3 4 5

How to make 125 single moleculeshaving 3 points of diversity

using :

1st and 2nd Combinatorial Step 125 MiniKansTM with Rf Transponders sorted into 5 reaction flasks

5 x A--5 x A--5 x A--5 x A--5 x A--

5 x B--5 x B--5 x B--5 x B--5 x B--

5 x C--5 x C--5 x C--5 x C--5 x C--

5 x D--5 x D--5 x D--5 x D--5 x D--

5 x E--5 x E--5 x E--5 x E--5 x E--

Washing 125 KansTM in bulk

2nd Redistribution into 5 reaction flasks

5 x AA-5 x BA-5 x CA-5 x DA-5 x EA-

5 x AB-5 x BB-5 x CB-5 x DB-5 x EB-

5 x AC-5 x BC-5 x CC-5 x DC-5 x EC-

5 x AD-5 x BD-5 x CD-5 x DD-5 x ED-

5 x AE-5 x BE-5 x CE-5 x DE-5 x EE-

Washing 125 KansTM in bulk

3

21

45

Flasks 3rd: Redistribution into 5 Reaction

1 x AEB1 x BEB1 x CEB1 x DEB1 x EEB

1 x AEC1 x BEC1 x CEC1 x DEC1 x EEC

1 x AED1 x BED1 x CED1 x DED1 x EED

1 x AEE1 x BEE1 x CEE1 x DEE1 x EEE

Washing 125 KansTM in bulk then order into Racks : one Kan in one tube

1 x AEA1 x BEA1 x CEA1 x DEA1 x EEA

1 x ADA1 x BDA1 x CDA1 x DDA1 x EDA

1 x ACA1 x BCA1 x CCA1 x DCA1 x ECA

1 x ABA1 x BBA1 x CBA1 x DBA1 x EBA

1 x AAA1 x BAA1 x CAA1 x DAA1 x EAA

1 x ADB1 x BDB1 x CDB1 x DDB1 x EDB

1 x ACB1 x BCB1 x CCB1 x DCB1 x ECB

1 x ABB1 x BBB1 x CBB1 x DBB1 x EBB

1 x AAB1 x BAB1 x CAB1 x DAB1 x EAB

1 x ADC1 x BDC1 x CDC1 x DDC1 x EDC

1 x ACC1 x BCC1 x CCC1 x DCC1 x ECC

1 x ABC1 x BBC1 x CBC1 x DBC1 x EBC

1 x AAC1 x BAC1 x CAC1 x DAC1 x EAC

1 x ADD1 x BDD1 x CDD1 x DDD1 x EDD

1 x ACD1 x BCD1 x CCD1 x DCD1 x ECD

1 x ABD1 x BBD1 x CBD1 x DBD1 x EBD

1 x AAD1 x BAD1 x CAD1 x DAD1 x EAD

1 x ADE1 x BDE1 x CDE1 x DDE1 x EDE

1 x ACE1 x BCE1 x CCE1 x DCE1 x ECE

1 x ABE1 x BBE1 x CBE1 x DBE1 x EBE

1 x AAE1 x BAE1 x CAE1 x DAE1 x EAE

1

2

3

4

5

Automatic Sampler

(Hamilton)

SynthManPlan

synthesis

Plan synthesis

Perform synthesis

Perform synthesis

Purify & Analyze

compounds

Purify & Analyze

compounds

Archive data & stock

Archive data & stock

HPLC

Weighing full

Dilution

Import BB

MS

Import Compounds

Family data

Reagent data

Method data

Salt data

Barcodes

Working Sheet

Preparereagents

Preparereagents

Component

Database

Family Database

2.

1.

9

16.

8.

7.15.

6.

5.

3.

4.

14.

13.12.

10.

Archive Databases

Archive Databases

HPLC (Millennium

)

Structural Analysis

Automatic weighing

(MicroWeight)

Automated Sorting

(Kan Sort)

Software for Controlling of the Production, Purification and Analyzing Process

Weighing empty

Sort to flasks and racks

LC-MS (MicroMass

)11.

prep. LC

Immobilization of 2,6-Dichloropurine

Addition of Resin slurry to IRORI MiniKans

Sort & Combinetransponder

Sort Kans

redistribute

Washing of KansTM in Bulk

By R. Gallarini, S. Vinzenz,W. Brill

Sorting of Kans Into Racks prior to Cleavage: Last step of Sort-and-Combine Method

TFA

Transponder

TFA-Cleavage

Preparative HPLC-MS Purification

• LC-MS, MicroMass “platform LC”• Gilson-Autosampler adapted to hold 2 Novartis

Mega Racks = 192 samples• Capacity: 2 units with up to 384 samples• HPLC columns: 5 cm length, 2 cm diameter, 5 µm

C18• Gradient: optimized for each library• Expected MS-Peak is delivered into 10 mL glass

tube

Micromass, UK http://www.micromass.co.uk

MicroMass LC-MSHPLC-MScorrelation of fractions with expected mass

fractionlocation

LC-fractions

crude reactionmixtures

location of crude reaction mixtures, expected mass

High-throughput purification

Crude sample

Fraction collection by LC/MS

Result of purification

Confirmation of identity

HPLC/UV purity : 20% HPLC/UV purity : >95%

Evaporation

Automated weighing system

Throughput: 1 rack / hr

Recycling of used Kans

By S. Vinzenz,W.Brill

Batch-Registration of Compounds by Synthman

Achnowledgement

Modeling: E. Jacoby

Synthman: R. Fäh, H.-P. Moessner

Synthesis: S. Müller, J. Schaub, C. Riva-Toniolo*, D. Tirefort**

Purification & Registration: F. Gombert, G. Lerch, H. Wettstein

Hardware: S. Vinzenz, R. Gallarini

General: A. DeMesmaeker, J. Zimmermann, S. Wendeborn