1. Solid-Phase Combinatorial Technology
to Speed-up Nucleoside Synthesis
Haoyun An
1st e-CombiChem Conference
Cutting Edge Technologies in Combinatorial Chemistry
June 14-25, 2004

2. Integrated Infrastructure to Success
Chemistry
Combinatorial Chemistry
Organic Chemistry
Nucleoside Chemistry
Medicinal Chemistry
Crystallography
SBDD
Automation
Compound Control
Data Management
Drug Development
ADMET
Chem-Informatics
Discovery Analytical
High Throughput
LC-MS/Purification
Library Acquisition
Therapeutic Areas
Virology
Oncology
Immunology
Drugs
SARHTS

3. Strategies to Enhance Productivity
▲Traditional Approach:
► 75-100 compounds / year / person
▲ Parallel Solution-Phase Combinatorial:
► 500-800 / year / person (Purification issues)
▲ Parallel Solid-Phase Combinatorial:
► 1000-5000 / year / person (after scaffold development)
▲ Complex / Mixture Libraries:
► 1000-5000 / year (require deconvolution)

4. Combinatorial & Nucleoside Chemistry
▲ Combinatorial Chemistry
►15 years - peptides, oligos, heterocycles
►>100 companies / labs
▲ Nucleoside Chemistry
► 60 years - natural and unnatural
nucleosides
► >80 universities / companies

5. Combinatorial Approaches
For Nucleoside Libraries
▲ Challenges -
►Almost nobody made nucleoside libraries by
solid-phase combinatorial approaches
►Difficult to apply combichem approaches to
nucleosides - special structures
 Difficulty & Low yield
 No harsh condition
 Protection and de-protection
►We have pioneered this combination to
accelerate combinatorial nucleoside drug
discovery process

6. Resins for Nucleoside Libraries
Resin:
Wang
MMT-Cl
Merrifield
Cleavage:
50% TFA/DCM
1~1.5% TFA/DCM or
Hexafluoroisopropanol
HF or
strong base
► >100 Resins available for solid-phase reactions
► MMT-Cl Resin is applicable for nucleoside synthesis
N N
NN
N
O
OPGGPO
O
R1
R2
R4R3
Cl
O
X
Cl
OMe

7. Validation
 Validate loaded resins
 Validate building blocks
 Validate the protocols
 Validate reaction conditions

8. Combinatorial Process
Scaffold/
library
design
Scaffold
synthesis/
feasibility
research
Solid phase
chemistry
Large scale
synthesis of
Resin loaded
with scaffolds
Protocol
validation
Building block
validation
96-Well
plates
synthesis
Enumeration
plate design
 Library Design - Afferent
 Reagent selection – ACD
 Enumeration - Afferent

9. Combinatorial Process
96-Well
plates
Dry
samples
Sample
preparation
HTP LC-MS
analysis
Results
analysis
Register
in Database
HTP
purification
60-100%
LC-MS
Pooling
 Registration Library - Afferent
 HTP purification - Paralex
30-60%

10. Development of 2’-O-Me-N4-Substituted
Cytidine Scaffold in Solution
NO
OCH3TBDMSO
MMTO
NH
O
O
NO
OCH3TBDMSO
MMTO
N
O
OTPS
NO
OCH3HO
HO
NH
O
O
NO
OCH3HO
MMTO
NH
O
O
N
O
OHHO
HO
NH
O
O
Pri
ClSO2
Pri
Pri
TPS-Cl
py / DMAP
MMTCl
Py
TBDMSCl
Py / DMAP
85% 89%
76%
NO
OCH3TBDMSO
MMTO
N
O
N
NHN
N
NH2N
NO
OCH3HO
HO
N
O
N
NHN
Et3N / DMAP, r.t., o/n
80%
1, TBAF, rt, 3 h
2, TFA
80%
Confirmed by
NMR and LC-MS

11. 2’-O-Me-N4-Substituted Cytidine Scaffold
on Solid Support
NO
OCH3TBDMSO
NH
O
O
O
NO
OCH3TBDMSO
N
O
OTPS
O
NO
OCH3HO
HO
NH
O
O
NH
NO
OCH3TBDMSO
N
OO
NH2
MMTCl
NH
NO
OCH3HO
N
OHO
Pri
ClSO2
Pri
Pri
py / DMAP / TEA
rt, 24 h
TBDMSCl
Py / DMAP / TEA
DMF / imidazole
rt, 24 h
1, TBAF, rt, 5 h
2, TFA, 10 min
Py / DMAP
rt, 24 h
4.5 g (7 mmol)
(1.2 equiv)
DMAP, Hunig's base
py, DMF, rt, o/n
LC-MS: 93%
ES MS: confirmed
7.2 g Resin
MAS NMR confirmed
TPS-Cl
(2 equiv)
exo-2-aminonorbornane

12. 2’-O-Me-N4-Substituted Cytidine Library
14 plates
1244 compds
N
O
OCH3TBDMSO
NH
O
O
O
N
O
OCH3TBDMSO
N
O
O
O
NO
OCH3HO
HO
NH
O
O
MMTCl
R1
N
N
O
OCH3HO
N
O
HO
Pri
ClSO2
Pri
Pri
py / DMAP / TEA
rt, 24 h
TBDMSCl
Py / DMAP
TEA/DMF
imidazole
rt, 24 h
1, TBAF, rt, 5 h
2, TFA, 10 min
Py / DMAP
rt, 24 h
DMAP, Hunig's base
py, DMF, rt, o/n
TPS-Cl
(2 equiv)
R2
R1R2NH
Y Y
Y
Y
S
Pri
Pri
Pri
O
O

13. -OCH3
DCM
OCH3
An 1009
N
N
O
O
O S
O
Si
OMe
O
O
O
CH3
MAS NMR of 2’-OMeU-4-sulfonate MMT Resin

14. 6,8-Disubstituted Adenosine Library
12 plates
1148 compds
N
N
N
N
NH2
O
OHHO
HO
Br
N
N
N
N
NH2
O
OHHO
HO
R1
N
N
N
N
NHAc
O
OAcAcO
HO
R1
R1
X
Heck or
Suzuki
1) TBDMSCl
2) Ac2O
3) TBAF
Cl
N
N
N
N
N
O
OAcAcO
R1
O
Ac
R2
N
N
N
N
HN
O
OHHO
R1
HO
R2
R2
-OH
Ph3P/DEAD
Mitsunobu
1) NH3 deprotection
2) TFA cleavage

15. 8-Amide Adenosine Library
12 plates
1152 compds
N
N
N
N
NH2
O
OHHO
HO
MeOCO
N
N
N
N
NHAc
O
OAcAcO
HO
MeOCO
N
N
N
N
NHAc
O
OAcAcO
MeOCO
O
1) TBDMSCl
2) Ac2O
3) TBAF
Cl
N
N
N
N
N
O
OAcAcO
MeOCO
O
Ac
R1
N
N
N
N
HN
O
OHHO
HO
R1
TFA
cleavage
R1
-OH
Ph3P/DEAD
Mitsunobu
HNR2
R3
N
O
R3 R2

16. 2-Amino Adenosine Library
48 X 48, 24 plates
2304 compounds
N N
NN
Cl
O
OHHO
HO
Cl
N NH
NN
O
O
OHHO
HO
NH2
I
4 steps
N N
NN
N
O
OHHO
O
N
R1
R2
R3
R4
1% TFA cleavage or
hexafluoroisopropane
R1
R2
NH
THF, Toluene
or NMR
rt - 60 ºC
R3
R4
NH
THF, Toluene
or NMR
80 - 100 ºC
N N
NN
N
O
OHHO
HO
N
R1
R2
R3
R4
N N
NN
Cl
O
OHHO
O
I

17. Exo-cyclic Triazine Nucleoside Library
N3O
OHHO
HO
NH2O
OTBDMSTBDMSO
O
NHO
OTBDMSTBDMSO
O
N
N N
Cl Cl
NHO
OHHO
HO
N
N N
H
N N
1) MMT-Cl Resine
2) TBDMS-Cl
N
N
N
Cl Cl
Cl
R1
NH2 (primary)
Hunig's base
rt 2-5 h
R2
NH2 or R2
NHR
heat 5h
Hunig's base
- 20 °C
1) TBAF
2) TFA
R1
R2
R
3) PMe3
48 X 24, 12 plates
1152 compounds

18. a. 4 - M e t h o x y-t r i t y l c h l o r i d e-P S-r e s i n
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.01.4
4
6
8
10
12
14
16
18
20
22
24
25.0
cm-1
%T
Cl
OCH3
c.(4) 1-A m ino - sugar on resin
4000.0 3000 2000 1500 1000 450.01.4
4
6
8
10
12
13.5
cm-1
%T
b . (3) 1-A z i d o-s u g a r o n r e s i n
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.24
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
5.83
cm-1
%T
O N3
MMTO
OTBDMSTBDMSO
O NH2
MMTO
OTBDMSTBDMSO
FT-IF Spectral Comparison of Different Resins

19. Clitocine Mimic Exocyclic Amino Nucleoside Library
82 compds
O NH
HO
N
N
N
OHHO
R4
R5
O NH
MMTO
N
N
Cl
OTBDMSTBDMSO
HN
R4
R5
NO2
NO2
O NH2
MMTO
OTBDMSTBDMSO
Cl
N
N
Cl
NO2
Hunig's base
TBAF TFA

20. N1- and N2-Methylpyrazolopyrimidine Nucleoside Libraries
96 compds
87.5% successful rate
96 compds
97.9%
successful
rate
N
N N
N
H3CS
O
O
HO OH
CH3
O
N
N N
N
NH
O
O
HO OH
CH3
O
1.0 M Amines
in NMP
80 ºC 24 hrs
R
N
N N
N
NH
O
O
HO OH
CH3
HO
R
30% HFIP in DCE
50 ºC 24 hrs
N
N N
N
H3CS
O
O
HO
HO OH
CH3
MMT resin- Cl/
2,6-Lutidine/DMF
N
N N
N
H S
O
O
HO
HO OH
CH3
MeI/DIPEA
N
N N
N
H O
O
O
BzO
BzO OBz
CH3
1) P2S5/Pyridine
2) NaOMe/MeOH
N
N
N
N
NH
O
O
HO OH
HO
R2
CH3
N1
N2

21. Attached at Other Positions on Base
N
O
OCH3AcO
N
O
OTPS
AcO
MMTCl
R3
N
NO
OCH3HO
N
O
HO
Pyr. / DMAP /
TEA,rt, 24 h
Pyr. / DMAP
rt, 24 h
TPS-Cl
(2 equiv)
N
O
OCH3AcO
NH
O
O
AcO
R4
HN
R3
R4 1) NH3/MeOH
2) TFA
HO
HO
O

22. Attached at 3’-Position
N
O
OCH3
N
O
OTPS
TBDMSO
MMTCl
R3
N
NO
OCH3
N
O
HO
Pyr. / DMAP /
TEA,rt, 24 h
Pyr. / DMAP
rt, 24 h
TPS-Cl
(2 equiv)N
O
OCH3
NH
O
O
TBDMSO
R4
HN
R3
R4 1) TBAF
2) TFA
O
HO
HO
Steps

23. NH
N
N
O
NH2
N
O
OAcAcO
AcO
R1
NH
N
N
O
NH2
N
O
OAcOAc
AcO
Br
N
N
N
N
NH2
N
O
OHHO
HO
R1
N
N
N
N
H
N
O
OHHO
HO
R1
MMT
R2 R3
N
R2 R3
N
N
N
Cl
N
H
N
O
OAcAcO
AcO
R1
MMT
Suzuki
Still
Heck
reactions
POCl3
PhNMe2
Et4NCl
Cl-MMT
R2R3NH
NMP
60 o
C, 6h
80 o
C
24 h
CH3NH2
CH3OH
50 o
C, 24h
HFIP/DCE
50 o
C, 24h
Attached at N2-Position on Base
5 plates 480 compds
Protection &
attachment

24. H2N H2NH2N OH
H2NH2N O H2N
O
H2N
H2N H2N
S
H2N
N
H2N
O
H2N
H2N
H2N
H2N N
H2N
H2N
H2N
NO
NH2
H2N
H2N
CF3
H2N
N
NH2N
O
OH2N
N
H2N
N
H2N
NH2
H2N
O
N
H2NN
O
H2N
H2N
NH2
NH2O NH2
O NH2
NO NH2
N
NH2
O
H2N
N NH2
H2N
HO
N NH2
OH2N
S
H2N
H2N
CF3H2N
NH2
N
NH2
H2N
H2N
H2N
O
NH2
O
HO NH2
OH
H2N
NH2
H
H
NH2
H
H
H2N OH
H2N
O
O
NH2
N
H2N
N
NH2
NH2
HO
NH2
N NH2
NH2
O
NH2
H2N
H2N
O NH2
H2N
NH2
H2N
H2N
H2N
O
H2N
F
NH2
H2N
H2NH2N
H2N
H2N
NH2
NH2
F
NH2
F
NH2
FNH2
F
H2N
O
H2N
H2N
OH
HO
HO
H2N OH
H2N
H2N
HO
NH2
NH2
OHNH2
H2N
H2N
H2N N
H2N H2N
H2N OEtH2N
OMe
H2N H2N H2N S
H2N H2N
N
H2N
O
H2N
N
Ph
H2N H2N
H2N NH2N
H2N
H2N
N O
H2N H2N
H2N
Ph
H2N N NH2N H2N H
H
Ph
NH
H2N
Ph
H2N O
O
H2N
H2N
H
H
NO2
N
NH
H2N
N
H2N
NH2N
H2N
OH2N
NH2N
N
O
H2N
H2N
OH2N
N OH2N
NH2N H2N
OH
O
H2N
Ph
HO
H2N
H2N
N
PhOH2N SH2N
H2N
O
OH
H2N O
H2N
NH2N
H2N
O
H2N N
NH2
H2N
Ph
H2N H2N
1 2 3
49 50
4 5 6 7 8
9
56
10 11 12 13
14 15
16 17
18
19
20 21
22 23 24 25
27
26
2928 30
51
52
31
33
34
35
36 37
54
38 39
40
41
42
55
43 44
45
46 47
H2N
32
48
H
N H
N HN OHHN
HN N
H
OH
O
O
HN
Ph
NHN
NH
OH
N
N
HN N
HN
HO
NHN N
O
HN N
N
N
HN
HN HN
HN S
N
H
HN
NH
HN HN O N
H
PhNHN
1 2 3
5 6
7
4
8
9
10
11
12 13 14
15 16 17
18
19
20
21 23 2422
Some Representative Building Blocks Utilized

25. Semi-Automated Synthesizer
VanguardVanguard
96-Well
Parallel
Solid-Phase
Synthesizer
Hardware Solutions

26. TH Parallel Characterization System
Alliance HT LC/MS
MassLynx Control Alliance HT 2790
PDA 996
ZQ Mass Detector

27. Library
C-3
LC-MS Results of A Representative Plate

28. N
O
N
N
OHOH
N
N
H
OH
O
N
H
O
A1, M.W. 520.55
LC-MS of a Library Member

29. NO
OCH3HO
HO
N
O
HN
N
C18H30N4O5
Mol. Wt.: 382.45
383
(M + 1)+
(1)
382
0.42
100%
LC-MS of a Library Member

30. NO
OCH3HO
HO
N
O
HN
N
C19H34N4O5
Mol. Wt.: 398.50
399
(M + 1)+
398.25
0.44
100%
LC-MS of a Library Member

31. Library Quality
Plate #
Successful
Rate*
(%)
Compds (%)
with 80-100%
Purity
Plate #
Successful
Rate*
(%)
Compds (%)
With 80-100%
Purity
1 33.3 16.6 11 91.7 81.2
2 41.2 16.0 12 93.8 82.3
3 66.3 56.3 13 94.8 69.8
4 75.0 45.8 14 94.8 89.6
5 72.0 57.0 15 95.8 62.5
6 76.0 60.0 16 95.8 84.3
7 87.5 71.9 17 96.9 74.0
8 85.0 85.0 18 97.9 92.7
9 87.6 72.0 19 97.9 93.7
10 92.7 71.0 20 100 91.7
* percentage of compds with >60% purity is defined as successful rate

32. HT Parallel Purification System
Biotage Parallex Flex HPLC

33. Software Solutions
 Afferent TeamWorks
– Enumerate library, generate compd and impurity profiles
 MassLynx Control Software
– Generate sample list as Excel file (ID, MW)
 OpenLynx Software
– Automatic spectral (MS, UV) and chromatographic data
processing
– ID results in color-coded fashion & 96-well format
 Activity Base/Customized Program
– Cherry-Pick compds: registration vs. repurification
– Enter analytical data into database

34. Conclusions
 Developed versatile parallel solid-phase
combinatorial technology for the synthesis of
diverse nucleoside libraries
 Various positions on nucleoside scaffolds can
be attached onto solid supports
 Diverse purine-, pyrimidine, pyrazolopyrimidine
and triazine-based nucleoside libraries were
synthesized in high quality and speed
 All library members were characterized by HT
LC-MS. Some were characterized by NMR
 Applicable to other nucleoside derivatives

35. Acknowledgements
Chemistry Team:
Frank Rong
Qazi Habib
Roberto R. Amador
Kanda Ramasamy
Varaprasad Chamakura
Esmir Gunic
Yung-hy Koh
Stephanie Shaw
Yili Ding
Jean-Luc Girardet
Huanming Chen
Jeffrey W. Abt
Weijian Zhang
Zhi Hong
Stanley Lang
Inventory & HTS:
Maja Stojiljkovic
Vesna Stoisavljevic
David Hyndman
Paul Diaz
Analytical:
Jingfan Huang
David Y. Li
Micheal Landsman
Xiaogang Han
Teresa Cain
CAAD
Biochemistry
Biology
Virology
Oncology