This document describes the catalysis of substitution reactions on heterocycles bound to a solid phase. It discusses immobilizing 2,6-dichloropurine on a Rink resin, performing C6-substitution with various amines in high yields, and C2-substitution via palladium-mediated coupling reactions such as Suzuki and amination reactions. It also investigates modifying the polymeric support by treating it with a brominating agent to install bromine complexes on the resin for further substitution chemistries. The reactivity of the bromine complex is explored, finding it performs selective side reactions in dry benzene that are not possible in NMP solvent.

1. Catalysis of Substitution Reactions
on Heterocycles Bound
to a
Solid Phase
wolfgang.brill@am.pnu.com

2. Catalysis of Substitution Reactions
on Heterocycles Bound
to a
Solid Phase
O
Nuc
N
N N
H
N
X
X
H,X
Nuc
Nuc
Radical, Nuc

3. Levoglucosan based Libraries
epoxide opening
epoxide opening
difficult alkylation or acylation
avoid
glycosidic scissionO
O
XO
O
O
NH2
R1 R2
R3
Pd mediated
coupling
Pd mediated
coupling
Brill, W.-K.-D. et al. Tetrahedron Lett 39 (1998) 787-790
Brill, W. K.-D. et al. Synlett (1998) 1085-1090

4. O
O
OHOH
OH
O
O
OTsTsO
OH
O
O
OTs
O
O
O
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
O
O
O
O
O
O
R1
Li
+
W. K.-D. Brill, June 96
Cerny Anhydride

5. O
O
O
OH
OTsO
O
R1
I
(CH2)10
R1:
The Hydrolysis of Levoglucosan Containing Esters
C(CH3
)2
Li
+
O
O
O
O
O
O
R1
I
(CH2)10
R1:
C(CH3)2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
LiOH, MeOH, H2O

6. Schematic Representation of Resins
crosslink
shrunken resin
(poorly solvated)
compound bound
to 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)

7. O
O
N
H
O
O
O
ORink
R2R2'NH
Li+ Ph4B-
2,6-lutidine
100°C
R1
> 90%
R2OH
P4-t-Bu
dioxan, 60°C
> 90%
R2SH
LiHMDS
dioxane
80°C
> 90%
O
O
N
H
O
O
O OR
OHRink R1
O
O
N
H
O
O
O SR
OHRink R1
O
O
N
H
O
O
O N
OH R
R
Rink R1 2'
2
2
2
O
NH2
O
O
O
O
O
O
O
OR1
Li
+
DIC, HOBT, DMF
levunbas16
Brill 1999Opening of Levoglucosan-2.3-epoxides with various Nucleophiles
N
N P
N
N
P
NMe2
NMe2
Me2N
P NMe2
NMe2
NMe2
PMe2N
NMe2
NMe2
P4-t-Bu:
Rink-linker:

8. O
O
O
O
O
N
H OH
OTs
O
O
O
O
O
N
H O
O
O
O
O
O
N
OH
O
O
O
O
O
N
H
S
OH
O
O
H2N
OH
O
O
NH3
+
OH
S
NH
O
O
O
R
TFA
-TFA
-
S
NH
R
O
OO
T>80°C
+
TFA
DBU
PhSH
T<80°C
TFA
Intera and Intramolecular Substitution on Levoglucosan-2,3-epoxide
levunibas4
Brill 1996

9. O
O
O
O
OH
O
N
H
I
N
ORink
Palladium Mediated Coupling Reactions on the Spacer Residue
Pd(OAc)2, K2CO3
H2O, dioxane,
110°C
84%
O
O
O
O
OH
O
N
H
N
O
OMe
Rink
HO
HO
B OCH3
Bu3Sn
cha11
O
O
O
OO
N
H
I
O
Rink
Pd2dba3, AsPh3
dioxane, 110°C
80-90%
O
O
O
OO
N
H
O
Rink
H
W. K.-D. Brill, June 96
O
O
O
OO
N
H
I
O
Rink
Pd(PPh3)2Cl2, CuI
NEt3, dioxane, rt.
80-90%
O
O
O
OO
N
H
O
Rink

10. O
O
O
OO
N
H
ORink
A Second Palladium Mediated Coupling Reaction on the Scaffold
HO
HO
B OCH3
O
O
O
OO
N
H
OH
NH
I
Rink
O
O
O
OO
N
H
OH
NH
OCH3
Rink
O
O
OO
NH2 OH
NH
OCH3
20% TFA
72% from
aminoresin
I
H2
N
Li+ Ph4B-
cha12
W. K.-D. Brill, June 96
Pd(OAc)2 K2CO3
H2O, dioxane

11. O
O
N
H
O
O
O OR
OHRink R1
2
O
O
N
H
O
O
O OR
OHRink R1
2
R3
O
NH2
O
O
O OR
OHR1
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 ClCH2CH2Cl
b) toluene before evaporation
R3-X
Schwesinger
base or
KOtBu
Final Derivatizations and Cleavage

12. 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

13. N
H N
N
N
N
H
R
R
R
N
H
N N
N
N
N
R
NH
H H
R
R N
H N
N
N
N
R
N
R
H
H
R
N N
N
N
N
H
R
R
R
N
H
H
N N
N
H
N
N
R
NH
H
R
R
Purines may bind to Proteins in different ways
They address nucleoside binding pockets

14. 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 important2. Nucleosides or nucleoside cofactors are involved in all important
cellularcellular
processes.processes.
DNADNA synthesissynthesis
RNARNA synthesissynthesis
proteinprotein synthesissynthesis
carbohydratecarbohydrate synthesis and oligomerizationsynthesis and oligomerization
lipidlipid synthesis and processingsynthesis and processing
synthesis ofsynthesis of homohomo andand heterocyclicheterocyclic aromaticsaromatics
signalingsignaling via phosphoylation and sulfatationvia phosphoylation and sulfatation
signaling assignaling as second messengersecond messenger oror hormonehormone
alkylationalkylation andand 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!

15. i, ii: Attachment to the polymer
iii: 6-Substitution
iv: 2-Substitution
v: Bromination
vi: Stille coupling
vii: Cleavage
Reaction Scheme
OH
N
N
N
N
Cl
Cl
N
N
N
N N
N
N
N Cl
O CF3
O
N
N
N
N
H
Cl
Cl
N
N
N
N
Br
N
N
N
N N
N
N
N
H
Rink resin
R1
R2
R1
i ii
iii
iv
1 2
3
4
R1-H:
R2-E
6 5
1
2
3
4
5
6
7
8
9
7
R1
R2
v
R1
vi R3-SnBu3
8
R3
R2
R1
R3
R2
vii
9

16. OH
N
N
N
N
Cl
Cl
O CF3
O
N
N
N
N
H
Cl
Cl
Rink resin
1 2
3
4
1
2
3
4
5
6
7
8
9
TFAA, 2,6-lutidine
4 eq.NMP
crystallization of
excess
recycling
Immobilization of 2,6-dichloropurine

17. 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 24.3
3 2,6-lutidine/NMP - 60 24 31.7 68.3
4 “ HNEt3
+
F3CCO2
-
60 24 11.7 88.3
N
H
F
Cl
N
N
N
H
N
Cl
N
F
Cl
N
H N
N
N
Cl
Cl
C6-Substitution

18. N
N N
NCl
Cl
N
N N
H
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 for
very 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

19. N
N N
N
Cl
N
N
Cl
N N
N
N
N
O
N
N
Cl
N
H
O
Pd-catalyst
N
N N
N
Cl
N
N
N N
N
N
N
N
B
OH
OH
Pd-catalyst
Solvent Base
Cat. or
Promotor
Co-ligand
Reaction
T [°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. or
Promotor
Co-ligand
Reaction
T [°C]
Time
[h]
Yield
[%]
NMP K3PO4 Pd2dba3 P(tBu)3 100 48 84.5*
Sustitution on C2
* rest is starting mat.

20. X'
L
L
PdAr
X"
L
L
PdAr
X"
B(OH)2
Ar
R
Pd
Ar-X'Ar-R
X"-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

21. Br2
NN
N
Br2
N
+
Br N
+
Br
solv. polar solvent
N N
N
N
N
N
R
R1 R1'
R2
R2'
N N
N
N
N
N
R
Br
N N
N
N
N
N
R
R1 R1'
R2
R2'
N N
N
N
N
N
R
Br
EtOEt
yelloworange
soluble in benzene insoluble in benzene
solv.
Br2
solv.
Br+solv.
+ Br-solv.
solv.
2,6-lutidine
rapid equilibration
with solvent
5 h, rt., DMF
R1 R1'
R2
R2'
R1 R1'
R2
R2'
pentane
Two Types of Bromine Complexes Were Investigated
Synthesis:
Reactivity:

22. Entry Resin Conditions Time [h] Bromine content
1 Merrifield Br-complex, NMP, 2,6 lutidine 3x 24
1
<0.3 %
2 Merrifield Br-complex benzene,
2,6-lutidine
24 1.1% 0.14mmol/g
3 product of
entry 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

23. no reaction:
Rink
N N
N
N
Cl
Cl
Rink
N N
N
N
N
Cl
R1 R1'
Rink
N N
N
N
N
NHAc
R1 R1'
Rink
N N
N
N
N
N
R1 R1'
R2
R2'
complete
conversion:
side reactions: not tolerated groups:
activated aromatics: bromination
amines: many oxidation products
N
Br2
Reactivity of the Bromine Complex with Purines:
Solvent: NMP
R1
: H, alkyl, R1
’: alkyl
R2
: H, alkyl, R2
’: H, alkyl
tolerated groups: CONR2, CONRH CONH2,
ether functions, aromatics more
deactivated than Bn
5% conversion

24. 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
N
N
N
N
H
H
R
R
N N
N
N
N
N
H
H
R
RO
Br
Rink'
N
Br2
benzene
Rink
N N
N
N
N
N
N
O
N
O
N N
N
N
N
N
O
N
O
N
H
O
HO
Br
Rink'
N
Br2
benzene

25. The Reactivity of the Bromine Complex in wet Benzene:
N
Br2
Rink
N N
N
N
N
N
H
H
R
R
N N
N
N
N
NH2
H
R
BrN
Br2
Rink'
benzene
H2O
Side chain oxidations are not mediaded by the solid phase.
They work are observed also in solution phase.

26. N N
N
N
N
N
Br
Br
N N
N
N
N
N
H
O
Br
N N
N
N
N
NH2
Br
Br
N N
N
N
N
+
N
Br
H
R1"R1'
R2'
N N
N
N
N
N
Br
R1 R1"
R2'
N N
N
N
N
N
H
Br
Br
Br
R1"R1'
¨
R2' N N
N
N
N
N
Br
H
Br
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

27. Attempted Synthesis to Yield 2,6,8-Trisubstituted Purines
N
H N
N
N
NH
Cl
N
H N
N
N
Cl
Cl
N
H N
N
N
NH
N
N
O
N
H N
N
N
NH
N
N
O
Br
N
H N
N
N
NH
N
N
O
Dehalogenation
during Suzuki couling
conditions
ArB(OH)2
Pd-cat

28. Entry Solvent Cu cat. Pd cat. Co-ligand
Product
yield
[%]
dehalog.
[%]
1 dioxane - Pd2dba3 As(Ph)3 - >5
2 NMP CuO Pd2dba3 dpppf
no reaction
3 NMP Cu(OAc)2 Pd2dba3 dppp - 91.6
4 NMP
O
Bu
Et
O
2
Cu
2+
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 reaction
8 NMP Cu2O Pd(OAc)2 dppp >98 -
SnBu3
N
NN
N
Br
NH
NH N
NN
N
NH
NH N
N
N
N
NH
NH
cat.
+
Substitution on C8
P P
dppp:

29. 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

30. The Synthesis is performed on PS-Beads
PS-beads 60 mg
per Kan
Rf -transponders
with unique ID
Read only !
Reading interval: 0.1s
RF-frequency: 125 KHz
Reading distance : 1 cm
reusable many times !
1 cm
1 per Kan
Now all Kans can be distinguished

31. 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 molecules
having 3 points of diversity
using :

32. 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
2
1
4
5

33. Flasks 3rd: Redistribution into 5 Reaction
1 x AEB
1 x BEB
1 x CEB
1 x DEB
1 x EEB
1 x AEC
1 x BEC
1 x CEC
1 x DEC
1 x EEC
1 x AED
1 x BED
1 x CED
1 x DED
1 x EED
1 x AEE
1 x BEE
1 x CEE
1 x DEE
1 x EEE
Washing 125 KansTM
in bulk then order into Racks : one Kan in one tube
1 x AEA
1 x BEA
1 x CEA
1 x DEA
1 x EEA
1 x ADA
1 x BDA
1 x CDA
1 x DDA
1 x EDA
1 x ACA
1 x BCA
1 x CCA
1 x DCA
1 x ECA
1 x ABA
1 x BBA
1 x CBA
1 x DBA
1 x EBA
1 x AAA
1 x BAA
1 x CAA
1 x DAA
1 x EAA
1 x ADB
1 x BDB
1 x CDB
1 x DDB
1 x EDB
1 x ACB
1 x BCB
1 x CCB
1 x DCB
1 x ECB
1 x ABB
1 x BBB
1 x CBB
1 x DBB
1 x EBB
1 x AAB
1 x BAB
1 x CAB
1 x DAB
1 x EAB
1 x ADC
1 x BDC
1 x CDC
1 x DDC
1 x EDC
1 x ACC
1 x BCC
1 x CCC
1 x DCC
1 x ECC
1 x ABC
1 x BBC
1 x CBC
1 x DBC
1 x EBC
1 x AAC
1 x BAC
1 x CAC
1 x DAC
1 x EAC
1 x ADD
1 x BDD
1 x CDD
1 x DDD
1 x EDD
1 x ACD
1 x BCD
1 x CCD
1 x DCD
1 x ECD
1 x ABD
1 x BBD
1 x CBD
1 x DBD
1 x EBD
1 x AAD
1 x BAD
1 x CAD
1 x DAD
1 x EAD
1 x ADE
1 x BDE
1 x CDE
1 x DDE
1 x EDE
1 x ACE
1 x BCE
1 x CCE
1 x DCE
1 x ECE
1 x ABE
1 x BBE
1 x CBE
1 x DBE
1 x EBE
1 x AAE
1 x BAE
1 x CAE
1 x DAE
1 x EAE
1
2
3
4
5

34. 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
Workin
g Sheet
Preparerea
gents
Preparerea
gents
Compone
nt
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
Weighin
g empty
Sort to flasks
and racks
LC-MS
(MicroMass
)
11.
prep.
LC

35. Immobilization of 2,6-Dichloropurine

36. Addition of Resin slurry to IRORI MiniKans

37. Sort & Combinetransponder
Sort Kans
redistribute

38. Washing of KansTM
in Bulk
By R. Gallarini, S. Vinzenz,W. Brill

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

40. TFA
Transponder
TFA-Cleavage

41. 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

42. MicroMass LC-MS
HPLC-MS
correlation of
fractions with
expected mass
fraction
location
LC-fractions
crude reaction
mixtures
location of crude
reaction mixtures,
expected mass

43. High-throughput purification
Crude sample
Fraction collection by LC/MS
Result of purification
Confirmation of identity
HPLC/UV purity : 20% HPLC/UV purity : >95%

44. Evaporation

45. Automated weighing system
Throughput: 1 rack / hr

46. Recycling of used Kans
By S. Vinzenz,W.Brill

47. Batch-Registration of Compounds by Synthman

48. 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