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