Nucleoside libray e-conference VRX-Harry
Transcript of Nucleoside libray e-conference VRX-Harry
Solid-Phase Combinatorial Technology
to Speed-up Nucleoside Synthesis
Haoyun An
1st e-CombiChem ConferenceCutting Edge Technologies in Combinatorial Chemistry
June 14-25, 2004
Integrated Infrastructure to Success
ChemistryCombinatorial 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 AreasVirology
Oncology
Immunology
Drugs
SARHTS
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)
Combinatorial & Nucleoside Chemistry
▲ Combinatorial Chemistry
►15 years - peptides, oligos, heterocycles
►>100 companies / labs
▲ Nucleoside Chemistry
► 60 years - natural and unnatural
nucleosides
► >80 universities / companies
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
Resins for Nucleoside Libraries
Resin:
Wang
MMT-Cl
Merrifield
Cleavage:
50% TFA/DCM
1~1.5% TFA/DCM or
Hexafluoroisopropanol
HF orstrong 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
Validation
Validate loaded resins
Validate building blocks
Validate the protocols
Validate reaction conditions
Combinatorial Process
Scaffold/
library
design
Scaffold
synthesis/
feasibility
research
Solid phase
chemistry
Large scale
synthesis of
Resin loaded
with scaffolds
Protocol
validationBuilding block
validation
96-Well
plates
synthesis
Enumeration
plate design
Library Design - Afferent
Reagent selection – ACD
Enumeration - Afferent
Combinatorial Process
96-Well
plates
Dry
samples
Sample
preparation
HTP LC-MS
analysis
Results
analysisRegister
in Database
HTP
purification
60-100%
LC-MS
Pooling
Registration Library - Afferent
HTP purification - Paralex
30-60%
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
NO
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
NNHN
NNH2N
NO
OCH3HO
HO
N
O
NNHN
Et3N / DMAP, r.t., o/n
80%
1, TBAF, rt, 3 h
2, TFA80%Confirmed by NMR and LC-MS
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 / TEADMF / imidazole rt, 24 h
1, TBAF, rt, 5 h
2, TFA, 10 min
Py / DMAPrt, 24 h
4.5 g (7 mmol)
(1.2 equiv)
DMAP, Hunig's basepy, DMF, rt, o/n
LC-MS: 93%ES MS: confirmed
7.2 g ResinMAS NMR confirmed
TPS-Cl(2 equiv)
exo-2-aminonorbornane
2’-O-Me-N4-Substituted Cytidine Library
14 plates
1244 compds
NO
OCH3TBDMSO
NH
O
O
O
NO
OCH3TBDMSO
N
O
O
O
NO
OCH3HO
HO
NH
O
O
MMTCl
R1
N
NO
OCH3HO
N
OHO
Pri
ClSO2
Pri
Pri
py / DMAP / TEA rt, 24 h
TBDMSClPy / DMAP
TEA/DMF imidazole rt, 24 h
1, TBAF, rt, 5 h
2, TFA, 10 min
Py / DMAPrt, 24 h
DMAP, Hunig's basepy, DMF, rt, o/n
TPS-Cl(2 equiv)
R2
R1R2NH
Y Y
Y
Y
S
Pri
Pri
Pri
O
O
-OCH3
DCM
OCH3
An 1009
N
NO
O
O S
OSi
OMe
O
O
O
CH3
MAS NMR of 2’-OMeU-4-sulfonate MMT Resin
6,8-Disubstituted Adenosine Library
12 plates
1148 compds
NN
NN
NH2
O
OHHO
HO
BrN
N
NN
NH2
O
OHHO
HO
R1
NN
NN
NHAc
O
OAcAcO
HO
R1
R1X
Heck or
Suzuki
1) TBDMSCl
2) Ac2O3) TBAF
ClN
N
NN
N
O
OAcAcO
R1
O
AcR2
NN
NN
HN
O
OHHO
R1
HO
R2
R2-OH
Ph3P/DEAD
Mitsunobu
1) NH3 deprotection
2) TFA cleavage
8-Amide Adenosine Library
12 plates
1152 compds
N
N
NN
NH2
O
OHHO
HO
MeOCON
N
NN
NHAc
O
OAcAcO
HO
MeOCO
N
N
NN
NHAc
O
OAcAcO
MeOCO
O
1) TBDMSCl
2) Ac2O3) TBAF
Cl
N
N
NN
N
O
OAcAcO
MeOCO
O
AcR1
N
N
NN
HN
O
OHHO
HO
R1
TFA
cleavage
R1-OH
Ph3P/DEAD
Mitsunobu
HNR2R3
N
O
R3 R2
2-Amino Adenosine Library
48 X 48, 24 plates
2304 compounds
N N
NN
Cl
O
OHHO
HO
ClN NH
NN
O
O
OHHO
HONH2
I4 steps
N N
NN
N
O
OHHO
ON
R1 R2
R3
R4
1% TFA cleavage or
hexafluoroisopropane
R1R2NH
THF, Toluene
or NMR
rt - 60 ºC
R3R4NH
THF, Toluene
or NMR
80 - 100 ºC
N N
NN
N
O
OHHO
HON
R1 R2
R3
R4
N N
NN
Cl
O
OHHO
OI
Exo-cyclic Triazine Nucleoside Library
N3O
OHHO
HONH2O
OTBDMSTBDMSO
O
NHO
OTBDMSTBDMSO
O
N
N N
Cl Cl
NHO
OHHO
HO
N
N N
HN N
1) MMT-Cl Resine
2) TBDMS-Cl
N
N
N
Cl Cl
Cl
R1NH2 (primary)
Hunig's base
rt 2-5 h
R2NH2 or R
2NHR
heat 5h
Hunig's base
- 20 °C
1) TBAF
2) TFA
R1
R2
R
3) PMe3
48 X 24, 12 plates
1152 compounds
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 800 600 450.0 1.4
4
6
8
10
12
14
16
18
20
22
24 25.0
cm-1
%T
Cl
OCH3
c. (4) 1 - A m i n o - s u g a r o n r e s i n
4000.0 3000 2000 1500 1000 450.0 1.4
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 800 600 450.0 0.24 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 N3MMTO
OTBDMSTBDMSO
O NH2
MMTO
OTBDMSTBDMSO
FT-IF Spectral Comparison of Different Resins
Clitocine Mimic Exocyclic Amino Nucleoside Library
82 compds
O NH
HO
N
N
N
OHHO
R4
R5
O NHMMTO
N
N
Cl
OTBDMSTBDMSO
HNR4
R5
NO2
NO2
O NH2
MMTO
OTBDMSTBDMSO
Cl
N
N
Cl
NO2
Hunig's base
TBAF TFA
N1- and N2-Methylpyrazolopyrimidine Nucleoside Libraries
96 compds
87.5% successful rate
96 compds
97.9%
successful
rate
N
N N
N
H3CS
OO
HO OH
CH3
O
N
N N
N
NH
OO
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
OO
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
OO
BzO
BzO OBz
CH3
1) P2S5/Pyridine
2) NaOMe/MeOH
N
NN
N
NH
OO
HO OH
HO
R2
CH3
N1
N2
Attached at Other Positions on Base
NO
OCH3AcO
N
O
OTPS
AcO
MMTCl
R3
N
NO
OCH3HO
N
OHO
Pyr. / DMAP / TEA,rt, 24 h
Pyr. / DMAPrt, 24 h
TPS-Cl
(2 equiv)
NO
OCH3AcO
NH
O
O
AcO
R4
HNR3
R4 1) NH3/MeOH
2) TFA
HO
HO
O
Attached at 3’-Position
NO
OCH3
N
O
OTPS
TBDMSO
MMTCl
R3
N
NO
OCH3
N
OHO
Pyr. / DMAP / TEA,rt, 24 h
Pyr. / DMAPrt, 24 h
TPS-Cl(2 equiv)N
O
OCH3
NH
O
O
TBDMSO
R4
HNR3
R4 1) TBAF
2) TFA
OHO
HO
Steps
NH
N
N
O
NH2N
O
OAcAcO
AcO
R1
NH
N
N
O
NH2N
O
OAcOAc
AcO
Br
N
N
N
N
NH2N
O
OHHO
HO
R1
N
N
N
NH
N
O
OHHO
HO
R1 MMT
R2 R3N
R2 R3
N
N
N
Cl
NH
N
O
OAcAcO
AcO
R1 MMT
Suzuki Still Heck
reactions
POCl3
PhNMe2
Et4NCl
Cl-MMT
R2R3NH
NMP
60 oC, 6h
80 o
C
24 h
CH3NH2
CH3OH
50 oC, 24h
HFIP/DCE
50 oC, 24h
Attached at N2-Position on Base
5 plates 480 compds
Protection &
attachment
H2N H2NH2N OHH2N
H2N O H2NO
H2N
H2N H2NS
H2N
N
H2N
O
H2N
H2N
H2N
H2N NH2N
H2N
H2N
NONH2
H2N
H2NCF3
H2N
NNH2N
O
OH2NN
H2N
N
H2N
NH2
H2N
O
NH2NN
O
H2N
H2N
NH2
NH2O NH2
O NH2
NO NH2
NNH2
O
H2N
N NH2
H2N
HO
N NH2
OH2N
SH2N
H2N
CF3H2N
NH2
NNH2
H2N
H2N
H2N
ONH2
O
HO NH2
OH
H2N
NH2
H
H
NH2H
H
H2N OH
H2NO
ONH2
NH2N
NNH2
NH2
HONH2
N NH2
NH2
O
NH2
H2N
H2N
O NH2
H2N
NH2
H2N
H2N
H2NO
H2N
FNH2
H2N
H2NH2N
H2N
H2N
NH2
NH2
F
NH2
F
NH2
FNH2
F
H2NO
H2N
H2NOH
HO
HO
H2N OHH2N
H2N
HO
NH2
NH2
OHNH2
H2N
H2N
H2N N
H2N H2N
H2N OEtH2NOMe
H2N H2N H2N SH2N H2N
NH2N
O
H2N
N
Ph
H2N H2NH2N NH2N
H2NH2N
N O
H2N H2N
H2NPh
H2NN NH2N H2N
H
H
PhNH
H2N
Ph
H2N O
O
H2N H2N
H
H
NO2
N
NHH2N
NH2N
NH2NH2NOH2N
NH2N
N
O
H2N
H2N
OH2N
N OH2N
NH2N H2N
OH
O
H2NPh
HO
H2N
H2N
N
PhOH2N SH2N
H2N
OOH
H2NO
H2N
NH2N
H2N
O
H2N N
NH2
H2N
Ph
H2N H2N
1 2 3
49 50
4 5 6 7 8
9
56
1011 12 13
14 15
16 1718
19
20 21
22 23 24 25
27
26
2928 30
51
52
31
33
3435
36 37
54
38 39
4041
42
55
43 4445
46 47
H2N
32
48
HN H
N HN OHHN
HN NH
OH
O
OHN
PhNHN
NH
OH
N
N
HN NHN
HO
NHN N
O
HN N
N
N
HN
HNHN
HN S
NH
HN
NH
HN HN O NH
PhNHN
1 2 3
5 67
4
8
9
1011
12 13 14
15 16 17
18
1920
21 232422
Some Representative Building Blocks Utilized
Semi-Automated Synthesizer
VanguardVanguard
96-Well
Parallel
Solid-Phase
Synthesizer
Hardware Solutions
TH Parallel Characterization System
Alliance HT LC/MS
MassLynx Control Alliance HT 2790PDA 996
ZQ Mass Detector
Library
C-3
LC-MS Results of A Representative Plate
NO
N
N
OHOH
N
NH
OH
O
NH
O
A1, M.W. 520.55
LC-MS of a Library Member
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
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
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
HT Parallel Purification System
Biotage Parallex Flex HPLC
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
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
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