++++medicinal Chemistry

Medicinal Chemistry: Combinatorial Chemistry-Parallel Synthesis
Agenda
1. Introduction: The Drug Discovery Process

2. Lead Discovery and Lead Optimization-Drugability
-Drug-like molecules-the rule of 5
-Drug-like vs lead-like: the rule of 3
-Privileged scaffolds
-Unwanted properties: frequent hitters-aggregate forming molecules
3. Combinatorial and Parallel Synthesis in Medicinal Chemistry

-Historical background-objective
-Compound mixtures versus single compounds
-Solid phase synthesis versus synthesis in solution
4. Combinatorial Synthesis of Biopolymers

-Polypeptides
-Peptoids, Oligoureas, Oligocarbamates
-Oligosaccharides
-Oligonucletides, Oligonucleosides
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Daniel Obrecht, Polyphor Ltd

Agenda
4. Combinatorial synthesis of Biopolymers (cont.)

-Combinatorial synthesis-split-mixed synthesis
-Tagging strategies
5.
Strategies for the Synthesis of Small Molecule Libraries

-Library synthesis planning
-Synthesis strategies
-Classical multi-component reactions (MCRs)
-Sequential multi-component reactions (SMCRs)
-Fragment-based lead discovery
-Dynamic Combinatorial Synthesis; Target-guided synthesis (TGS)
-Disulfide thethering
-Click chemistry
-Building blocks
-Parallel and/or combinatorial synthesis
-Parallel work-up
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Agenda
6. Applications of Parallel Synthesis and Combinatorial Chemistry in

Medicinal Chemistry: Case Studies
-Drug targets
7. Appendix (Definitions; Reviews; Literature)
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The value added chain of pharmaceutical R & D
-From 1960 to 1980 the development time of a new NCE (new chemical entity) have quadrupled
-Since 1980 9-13 years have been necessary for the louching of a new drug
-Costs have gone up from 300-450 MioSFr (1987) to 600-800MioSFr in 2000
-Main reasons: higher degree of scientific knowledge of the drug required; regulations for clinical quality
assurance; change in the professional regulations of physicians; increase for administrative work for health
authorities
-It is of prime importance to reduce the development time and costs for the development of a new NCE in
order to reduce the costs of new drugs while keeping the profitability: increase productivity of R & D
J. Kuhlmann, Int. J. Clinical Pharmacol. Ther. 1997, 35, 541-552
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Drug Discovery Process

Assay
development
capabilities
Target
identification
Screening
libraries
Establishing
primary
screening
Screening
capabilities
Parallel
chemistry
Hit
identification
PEM, small
molecules,
fragments
Medicinal
chemistry
Hit
exploration,
hit-to-lead
Molecular
modeling
Lead
optimization
Preclinical
and clinical
development
ADMET
properties
Genomics; Proteomics; Phage display,

Fragment screening; X-ray crystallography
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Lead
Optimisation
5-12 m
Clinical Candidate Selection

(CCS) from alternatives
(Candidate Profiling)
12-36 months
3-6 m
A tt r it io n in D is c o v e r y
>30%
30%
1 2 .0 0
1 0 .0 0
8 .0 0
>50%
30%
6 .0 0
4 .0 0
Of 10
projects
starting in
Lead
Identification
<3.5 will
reach
CCS
(..but
5
possible?)
2 .0 0
0 .0 0
L e a d Id e n t i f i c a t i o n
Selecting Leads that

are drugable
Avoiding problematic
templates
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L e a d O p t im i s a t io n
Removing
the ADMET
concerns
C C S
Selecting the candidate that provides

the best exposure (e.g. unbound
concentration at target ) without
safety concerns

Phase 1
6-9 m
Phase 2
Proof-ofConcept
12m
Phase 3
Efficacy
Long term
Safety
12-24m
Regulatory
Approval
24m
12-24m
A t t r it io n in D e v e lo p m e n t
30%
10%
1 6 .0 0
1 4 .0 0
40%*
20%
1 2 .0 0
1 0 .0 0
E IH E n a b l i n g
62%*
50%
8 .0 0
6 .0 0
4 .0 0
2 .0 0
Phase 1
Phase 2
45%*
20%
Phase 3
13%*
<5%
R e g is t r a t io n
ND A
0 .0 0
E IH E n a b lin g
Ph a s e 1
Ph a s e 2
Ph a s e 3
R e g is t r a t io n
Ensuring PK, metabolism, exposure,

half-life,, safety, in humans are as
expected. Definition of possible human
safety issues and margins.
Reproductive toxicity
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NDA
4% of
marketed cpds withdrawn
Long term pre-clinical & clinical safety,

carcinogenicity studies.
Final assessment of drug-drug interactions &
of bioavailability of the final marketed
formulation
Medicinal Chemistry : Combinatorial Chemistry-Parallel Synthesis

Brain
Dose
Various Tissues
Tissue
distribution
Systemic
Circulation
Metabolism
and exhalation
by lung
Metabolism and
biliary clearance of
unchanged drug
Metabolism
Dissolution
Liver
Gut Wall
Portal
Vein
Gut
Lumen
Permeation
pH ranges
stomach 1.5-6
upper g.i. tract
pH 4.4-7.8
colon 5
effect of food,
bile acids etc
Metabolism
Renal
excretion of drug
and/or metabolites
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Undissolved
dose
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Conc vs Time Curves
Absorption
phase
12
t max
10
Cmax
Distribution &
Elimination
Phase
C mg/L
8
6
4
t = 6h, k =0.693/t1/2 = 0.12h-1
Cmin
0
0
5
C mg/L_Oral
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10
15
C mg/L_IV
20
25
30
Time (h)
12

bioavailability of drugs
Plasma concentration
Drug injected
AUC (oral)
Bioavailability =
AUC
(injected)
AUC (injected)
Drug given orally
AUC
(oral)
Time
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Drug-like molecules-the rule of 5
What is a drug-like molecule?
leading references:
[1] C. Lipinsky et al. Adv. Drug Delivery Rev. 1997, 23, 2; [2] H. Kubinyi et al. J. Med.
Chem. 1998, 41, 3325; [3] M. Murko et al. J. Med. Chem. 1998, 41, 3314; [4] J. R. Proudfood, Bioorg. Med. Chem. Lett. 2002, 12, 1647
Lipinski's rules of 5:
linezolid (antibiotic)
H-acceptor
F
O
O
N
N
H
H-acceptor
H-donor
H-acceptor
-logP < 5
-molecular weight < 500 (600)
-not more than 5 H-bond donors
-not more than 10 H-bond acceptors
(or 10 hetero atoms)
-not more than 5 (10) rotatable bonds
molecules which obey to Lipinski's
rule of 5 have a high propensity for
penetration into cells and for oral
absorption
C 15H 18FN3 O4 (323.33)

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Drug-like molecules-rule of 5
Ac-RGDSNH2 (fibrinogen antagonist)
H2 N
NH2
-logP < 5
-molecular weight < 500 (600)
-not more than 5 H-bond donors
-not more than 5 H-bond acceptors
(or 10 hetero atoms)
-not more than 5 (10) rotatable bonds
x Cl -
HN
OH
O
O
N
H
H
N
O
O
N
H
H
N
O
NH2
O
OH
Lipinski's rules of 5:
molecules which obey to Lipinski's

rule of 5 have a high propensity for
penetration into cells and oral absorption
C17H30N808 x HCl (511.03)

MG: ok; logP < 5 (ok); 13 H-donors (violation); 16 hetero atoms (violation); 17 rotatable bonds (violation)
-peptides, proteins, oligonucleotides and oligosaccharides in general show violations of Lipinski's rules;
-these molecules have many H-bond donors and acceptors, which in physiological environment are surrounded
by water molecules. They show generally low cell penetration, low passage through the blood brain barrier
and low oral absorption. In addition these molecules are generally quickly degraded by various enzymes, which
can also be an advantage.
-these molecules have to be administered by a topical, i.p. or i.v. route; in recent years significant progress has
been made in administering biomolecules: liposomes, inhalation methods, direct injection techniques into the brain..
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privileged fragments
NMR based screening of fragments binding towards a variety of proteins:

Bcl-2 (an antiapoptotic protein), stromeolysin (MMP), VEGF-RBD, p56lck SH2,
FK-506 BP and others.
S. W. Fesik et al. J. Med. Chem. 2000, 43, 3443-47
O
N
OH
N
N
N
H
OH
P OH
O
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Lead-like: the rule of 3
The properties of 40 fragment hits identified against a range of targets using

high throughput X-ray crystallographic screening technology has been examined.
The results indicated that on average fragment hits possessed properties consistent
with a rule of three:
-MW <300
-Number of H-bond donors <3
-Number of H-bond acceptors <3
-clogP =3
In addition it was noted that:
-The number of rotatable bonds was on average <3
-Polar surface area was <60A2
M. Congreve et al. Drug Discov. Today 2003, 8, 876-77; M. Hann, T. I. Oprea, Curr. Opin. Chem.Biol. 2004,
8, 255-263
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unwanted properties: frequent hitters
In order to exclude as early as possible compounds with undesired properties from

compound libraries several selection criteria (filters) have been developed:
-chemically reactive compounds: alkylating agents, Michael acceptors etc.
(G. M. Rishton, Drug Disc. Today, 1997, 2, 382-4)
-toxic chemical groups (toxophores)

-oral bioavailability
-aqueous solubility
-metabolic clearance
-frequent hitters:
(O. Roche et al. J. Med. Chem. 2002, 45, 137-142)
-the activity of the compound is not specific for the target (promiscuous)
-the compound perturbs the assay or detection method (coloured or
fluorescent molecules)
-molecules prone to form polymers (e.g. catechols)
-molecules have a high tendency to form aggregates
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unwanted properties: reactive groups
N R'
O
H
aldehydes
R
acyl halides (X: Cl, Br)
sulfonyl halides (X: Cl, Br)
O O
S
R
X
R'
N
R''
halopyrimidines
O
R'
F 3C
trifluoro-ketones
aliphatic ketones
aliphatic esters
O
S
R'O
R'S
aziridines
epoxides
R'
R'
R'O
-halo-ketones (X: Cl, Br)
anhydrides
O
X
alkyl halides (X: Cl, Br, I)
R''
imines
R'
O
P
R'O
sulfonate esters
aliphatic thioesters
R'
phosphonate esters
OR'
1,2-dicarbonyl compounds
NR'R''
O
O
Michael acceptors
R'
R'
R'
R''
N
R'
R''
N
R'
R''
N
N
H
R'
heteroatom-heteroatom single bond
Reactive compounds and in vitro false positives in HTS ( G. M. Rishton, Drug Disc. Today, 1997, 2, 382-4)
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unwanted properties: frequent hitters
examples of frequent hitters (Matthew correlation coefficient: >0.8)
O. Roche et al. J. Med. Chem. 2002, 45, 137-142
Cl
H
N
NH2
OH
OH
H
N
Cl
OH
N
HO
HO
HO
OH
OH
diethylstilbestrol (1.00)
dopamine(0.88)
clofazimine(1.00)
fenoterol(0.87)
molecules that form aggregates

SO3 H
N
S
N
NH2
NH2
N
SO 3H
O
N
H
Cl
S
N
drug-like
non-drug-like
Cl
CO2 H
G. Mller, Drug Disc. Today, 2003, 8, 681-91

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privileged structures
A single framework or fragments which can bind to different target families in a

specific way
The term privileged structure was first used by Evans et al. (J. Med. Chem. 1988, 31, 223546) on the development of potent, selective, orally active cholecystokinin antagonists
The benzodiazepin scaffold was the first scaffold termed as privileged. It occurs in valium,
librium, in CCK-A antagonists and several more.
F
O
N
Me
N
Me
N
HN
O
NH
N
Cl
N
F
N
H
Valium
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Privileged structures include often favorable conformational arrangements of aromatic/
heteroaromatic groups. Planar arrangements of aromatic groups give raise to stacking
Which results in unfavorable properties such as low solubility and aggragation.
non-planar arrangement of two aromatic rings avoids stacking

F
O O
S
Cl
NH
N
N
O
O
N
N
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N
NH2
COX-II inhibitor (Vioxx)
Me
p38 MAP kinase (SB-218655)
Cl
dopamine transporter inhibitor
22

S
NH 2
amino-thiazole scaffold
OH
OH
NH
O
N
OH
NH
O
S
S
N
H
N
N
H
CP-146662 (discovery)
5-HT1A agonist, dopamine uptake
BMS-268770 (discovery)
CDK-2 inhibitor
CBS-113A (clinical)
COX, 5-lipoxygenase
N
H
N
S
NH2
Cl
O2N
O
S
N
H
OMe
OMe
Cl
CGS-2466(discovery)
Adenosin A3 antagonist,
PDE-4, p38 MAP kinase
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Ro 61-8048 (discovery)
Kynurenin-3-hydroxylase
23

2-aryl-indole scaffold
N
H
OMe
N
N
HN
O
Br
Br
N
H
NK1 antagonist (0.8nM)
Br
N
H
5-HT6 (0.7nM)
5-HT7 (0.3M)
N
H
CCR5 (1.3M)
CCR3 (0.9M)
C. A. Willoghby, Biiorg. Med. Chem. Lett. 2002, 12, 93-6
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Questions
1. What are the Lipinskis rules of five and what do they stand for?
2. Please determine number of rotatable bonds, number of H-bond
donors and acceptors of the following molecules?
O
N
COOH
O
O
A
COOH
HO
O
HO
OH
Cl
H-Lys-Glu-NH 2
HO
B
3. Describe the difference between drg-like and lead-like
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1961: Ivar Ugi publishes his pioneering paper on his four component reaction: If, for example, 40 of each different
components are reacted with one another, the result is 2560000 raction roducts...
R 1COOH
1
R2NH2
R4N=C
H+
R3
N
C N
R4
O
R1
R3 CHO
3
Ugi 4MCR
O
R1
R2 H R3
+
N
H
O
N
O
R4
R1
NHR4
N
R2
irreversible
R3
O
1
N
R2
NHR 4
O
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1963: Seminal paper by R. B. Merryfield describing for the first time the successful synthesis of a short peptide
on a polystyrene resin (J. Am. Chem. Soc. 1963, 85, 2149)
1965: Letsinger and Khorana applied solid supports for the synthesis of oligonucleotides (J. Am. Chem. Soc. 1965,
87, 2149); J. Am. Chem. Soc. 1966, 88, 3181)
1967: J. Frchet described a highly loaded trityl resin (2.0mmol/g)
1967: Wilkinson et al. Described polymer-bound tris-(triphenylphoshine)chlororhodium as a hydrogenation catalyst
(J. Am. Chem. Soc. 1967, 89, 1574)
1969: Solid-phase synthesis of Ribonuclease (J. Am. Chem. Soc. 1969, 91, 501)
1970: H. Rapoport introduced the term hyperentropic efficacy (effect of high dilution) on solid supports
(J. Am. Chem. Soc. 1970, 92, 6363)
1971: Frchet et al. pioneerd solid-phase synthesis in the field of carbohydrate research (J. Am. Chem. Soc. 1971,
93, 492)
1973: Application of intramolecular Dieckmann-condensation for the solid-phase synthesis of lactones by Rapoport
et al. (J. Macromol. Sci. Chem. 1973, 1117)
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27

1973: Leznoff et al. described the use of polymer-supports for the mono-protection of symmetrical dialdehydes,
oximeformation, Wittig reaction, crossed aldol formation, benzoin-condensation and Grignard reaction
(Can. J. Chem. 1973, 51, 3756)
1974: F. Camps describes the first synthesis of benzodiazepines on solid support (Ann. Chim. 1974, 70, 1117)
1976: Leznoff and Files described bromination and lithiation of insoluble polystyrene, thus pioneering the synthesis
of
functionalized resins (Can. J. Chem. 1976, 54, 935)
1976: Rapoport and Crowley published a review entitled: Solid-phase organic snthesis: novelty or fundamental
concept?
which raised three important questions: -degree of separation of resin-bound functional groups; -analytical
methods to follow reactions on solid support; -nature and kinetics of competing side reactions
(Acc. Chem. Res. 1976, 9, 135)
19761978: Leznoff et al. published a series of papers dealind with the synthesis of insect sex attractants (Can. J. Chem..
1977, 55, 1143)
1977: Wulff et al. Synthesized chiral macroporous resins using carbohydrates as templates for the use of column
materials for the separation (Makromol. Chem. 1977, 178, 2799)
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1979: Leznoff employed successfully a chiral linker for the assymetric synthesis of (S)-2-methyl-cyclohexanone in 95%
e.e. (Angew. Chem. 1979, 91, 255)
1974: F. Camps describes the first synthesis of benzodiazepines on solid support (Ann. Chim. 1974, 70, 1117)
1984: Geysen et al. described the the multi-pin technology for the multiple peptide synthesis (Proc. Natl. Acad. Sci.
USA, 1984, 81, 3998)
1985: Houghten et al. described the tea-bag method for multiple peptide synthesis (Proc. Natl. Acad. Sci. USA, 1984,
81, 3998)
1985: G. P. Smith described in seminal paper the use of filamentous phage for the synthesis of peptide libraries
(phage display method, Science 1985, 228, 1315)
1986: Mixtures of activated amino acid monomers were coupled to solid supports for the synthesis of peptide libraries
as mixtures; the product distribution depended on the relative couplind rates (Mol. Immunol. 1986, 23, 709)
1991: Fodor et al. described the VLSIPS method (very large scale immobilised polymer synthesis; photolitographic
parallel synthesis (Science 1991, 251, 767)
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O

1991: Almost simultaneously Furka et al. described the `portioning-mixing method (Int. J. Pept. Prot. Res. 1991,
37, 487); Hruby et al. the `split synthesis (Nature 1991, 354, 82); and Houghten et al. the `divide, couple
and recombine`process (Nature 1991, 354, 84)
1992: Oligonucleotide-encoded chemical synthesis by Lerner and Brenner (Proc. Natl. Acad. Sci. USA, 1992, 89, 5181)
1992: Synthesis od 1,4-benzodiazepines on solid support described independently by S. Hobbs-DeWitt (Diversomer
technology, US-Pat. 5324483, 1993) and J. A. Ellman (J. Am. Chem. Soc. 1992, 114, 10997)
1993: Binary encoded synthesis using gas chromatographically detectable chemically inert tags by W. C. Still et al.
(Proc. Natl. Acad. Sci. USA, 1992, 89, 5181)
1993: Use of multi-cleavable linkers for the synthesis of peptide-like libraries by M. Lebl et al. (Int. J. Protein Res.
1993, 41, 201)
1994: Use of the `safety-catch` linker principle developed by Kenner et al. (J. Chem. Soc. Chem. Commun. 1973, 636)
by J. A. Ellman for multidiretional cleavage from the resin (J. Am. Chem. Soc. 1994, 116, 11171)
1995: Synthesis of a potent ACE inhibitor by combinatorial organic synthesis on solid support using a 1,3-dipolar
cycloadddition reaction by Gallop et al. (WO 95/35278, 1995)
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30

1995: Use of a genetic algorythm for the selection of the products of an Ugi four component reaction (Angew. Chem.
Int. Ed. Engl. 1995, 34, 2280)
1996: Use of the Ugi four component reaction in combination with a 1,3-dipolar cycloaddition reaction of intermediary
formed `Munchnones` with electronpoor acetylenes by R. Armstrong et al. (Tetrahedron Lett. 1996, 37, 1149)
1997: Combination of a cyclo-condensation reaction, multicomponent diversification and multidirectional resin cleavage
using a novel `safety-catch`- and traceless linker yielding highly diverse pyrimidines by D. Obrecht et al. (Chimia
1996, 11, 530; Helv. Chim. Acta 1997, 80, 65) and L. M. Gayo et al. (Tetrahedron Lett. 1997, 38, 211)
1997: Synthesis of a taxoid library using radiofrequency-encoding (J. Org. Chem. 1997, 62, 6092)
2001: Click Chemistry: Diverse Chemical Function from a few good reactions: H. C. Kolb, K. B. Sharpless, Angew.
Chem. Int. Ed. 2001, 40, 2004-21; ibid Drug Discovery Today 2003, 8, 1128-37.
2001: Dynamic Combinatorial Chemistry: J. M. Lehn et al. Science 2001, 291, 2331-32.
2001: Using an enzymes active site to template inhibitors: R. Nguyen, I. Huc, Angew. Chem. Int. Ed. 2001, 40, 1774
2005: Receptor-assisted Combinatorial Chemistry: Thermodynamics and Kinetics in Drug Discovery: J. D. Cheeseman
et al. Chem. Eur. J. 2005, 11, 1708-16
2006: In situ click chemistry: a powerful means for lead discovery: B. K. Sharpless et al. Expert Opin. Drug Discov.
2006, 1(6), 525-38
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2004: Fragment-based drug discovery: D. A. Erlanson, R. S. McDowell, T. OBrien, J. Med. Chem. 2004, 47, 34633482; D. C. Rees, M. Congreve, R. Carr, Nat. Rev. Drug Discov. 2004, 3, 660-672.
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Chemical Biology: Combinatorial Chemistry-Parallel Synthesis

The role of combinatorial chemistry and parallel synthesis in drug discovery
Aim:
Large Screening Libraries
for High Throughput Screening
100'000 to 3'000'000 compounds
-Hit identification
Methods:
-Combinatorial synthesis on solid support
-High throughput parallel synthesis in solution
Focused Libraries for

Hit Confirmation and Validation
100 to 1'000 compounds
Aim:
-Hit confirmation, validation and exploration of SAR
Methods:
-High throughput parallel synthesis in solution
Aim:
Focused Libraries for
Hit-to-Lead Optimization
20 to 100 compounds per cycle
-Hit optimization, SAR, ADMET properties, TPP
Methods:
-Medicinal chemistry approaches; parallel synthesis
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Compound mixtures:
-Mixtures (most often 10-20 compounds) of purified compounds in equimolar amounts
-Mixtures of products synthesized in one reaction in equimolar ratio:
Mol. Immunol. 1986, 23, 709
R1-20
O
H2 N
R
OH
(Boc)FmocHN
O
H
N
O
R1-20: coding amino acids
ratio determined according
to coupling rates
(Boc)FmocHN
O
O
O
R1-20
-Most often products originating from a reaction mixture are not formed in equimolar ratio are
contaminated with impurities
Advantage: compound mixtures can reduce the screening effort in expensive and laborious screens
Drawbacks: compounds in mixtures can interfere with one another; prone to false positive hits
Trend today: screening of single compounds
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Single compounds:
-Synthesis on solid supports without final purification:
requires a lot of development work; allows to make large libraries
-Synthesis in solution using high yielding reactions without further purification:
limits the scope of reactions that can be used; often used in the context of multicomponent reactions; useful for large libraries
-Synthesis in solution followed by high-throughput preparative HPLC-purification:
whole repertoire of organic reactions can be used; is todays standard method for
the synthesis of focused libraries (hit validation; lead optimization)
Trend:
as screening technologies have increased the throughput, screening of single compound

libraries is more and more becoming the standard
as companies are looking for highly diverse general compound libraries of high quality
(purity, stability) library synthesis has shifted from solid phase synthesis (large libraries)
to solution phase synthesis followed by high-throughput purification (normal and
reverse phase)
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Solution phase chemistry:

++
most reactions and reagents have been studied in solution
usually no excess of reagents have to be used
solvent effects can be studied and altered readily
++
steric effects are usually less pronounced in solution and can be overcome more easily
by using more drastic reaction conditions
++
reaction conditions are usually adapted to a large variety of substituents
--
extensive and time consuming, chromatographic purification procedures are often

necessary
-+
side products have to be separated and analysed (can also be an advantage in the first
exploratory stage of a given project
--
parallelisation and automation usually requires more initial effort
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Solid phase chemistry:
++
excess of reagents can be used to drive reactions to completion
++
purification procedures achieved by simple filtrations which can be easily automated
++
assuming complete spatial separation of the reactive sites on a given solid support, the
principle of high dilution (hyperentropic effect, Acc. Chem. Res. 1976, 9, 135) can be
used beneficially; e.g. for intramolecular cyclisation reactions
+-
overall costs for the synthesis of large libraries (assuming no purification of the final
compounds is necessary) can compare favourably with solution synthesis
+-
linker molecules have to be designed which are compatible with the polymeric matrix and
the chemistry used for library synthesis: labour intense development work; ok for large
libraries
--
development of reaction conditions requires more work than in solution reactions on solid
support are more sensitive to steric effects: limitations in the design of highly diverse
libraries
--
reactions are more difficult to monitor; especially a drawback in the development phase
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General
General trends:
Solid-Phase chemistry: large libraries (no purification of individual compounds)
split mixed approach
linear approaches:
polypeptides
peptoids
oligosaccharides
oligocarbamates and ureas
use of solid support as a protective group: for guanidines, amidines, hydroxamic
acids, carboxylic acids, alcohols..)
Solution-phase chemistry:
small focused libraries of high chemical diversity (purified products)

parallel synthesis
convergent approaches
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Questions
1. What are the advantages of using mixtures of compounds in the

biological screening?
2. What are the disadvantages?
3. What are the advantages of using solid phase chemistry?
4.
For which type of molecules is it advantageous to use solid phase

chemistry?
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Examples for libraries synthesized on solid-phase: linear approaches
bond
bond
Monomer A1
bond
Monomer A2
Monomer A3
bond
bond
Monomer A4
monomers
bond formation
polymers
amino acids
amide bond
peptides, proteines
nucleotides
phosphorester bond
oligonucleotides
mono- and disaccharides
glycosidic bond
polysaccharides
N-alkylated glycines
amide bond
peptoids
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40

Examples for libraries synthesized on solid-phase: peptides
-Peptides synthesized as individuals or as mixtures on solid supports (polystyrene, polyacrylamide,

polyacrylamide-polystyrene co-polymers) and cleaved to be assayed in solution
-Peptides synthesized and assayed as individuals or as mixtures on solid supports such as pins (H. M.
Geysen et al. Mol. Immunol. 1986, 23, 709), resin beads (K. S. Lam et al. Nature 1991, 354, 82), cotton
(R. A. Houghton et al. Biochemistry 1993,32, 11035), microchips (S. P. A. Fodor et al. Science 1991, 37, 481),
or cellulose membranes (A. Kramer et al. Pept. Res. 1993, 6, 314)
-Peptides synthesized on the surface of a filamentous phage: Phage display technology
(G. P. Smith et al. Meth. Enzymol. 1993, 217, 228; J. K. Scott et al. Curr. Opin. Biotechnol. 1994, 5, 40)
Mixtures of peptides can be obtained by by using two different strategies:
-As true mixtures where a peptide coupling step involves the coupling of a mixture (typically the 20
coding amino acids) of side-chain protected Boc- or Fmoc- protected amino acids (D or L) in a
predetermined molar ratio which compensates for the different coupling rates.
-as mixtures of resin beads which resulted from synthesis: òne bead-one compound concept`
`portioning-mixing` (A. Furka et al. Int. J. Protein Res. 1991, 37, 487)
`couple and recombine` (R. A. Houghton et al. Nature 1991, 354, 84)
`split synthesis` (V. Hruby et al. Nature 1991, 354, 82)
winter semester 09
41

solid-phase peptide synthesis: overview
1
P1
R
OH
FmocHN
R
OH
R
O
FmocHN
Linker
Boc-strategy
Polymer
O
P1
-cl eavage, wash

Linker
O(NH)
FmocHN
Fmoc-strategy
Polymer
Linker
O
P1
BocHN
O(NH)
FmocHN
O
P2
H2N
Polymer
-coupling, wash
-cleavage, wash
1 cycle
R1
R2
Linker
O(NH)
N
H
Polymer
n cycles
R n+1
H 2N
O
H
N
R
P
N
H
P n
R1
O(NH)
Linker
Polymer
Rn +1
H 2N
O
H2N
O
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N
H
R
P
Rn+1
H
N
OH(NH 2)
O
R1
H
N
R1
N
H
OH(NH2)
O
42

solid-phase peptide synthesis: resins-polymer supports
P1
P1
R
OH
FmocHN
R
O(NH)
FmocHN
Fmoc-strategy
Polymer
Linker
1. Functionalized polystyrene resins:
Ph
Ph
Ph
Ph
FG
Ph
Ph
(1-2%)
cross-linked polystyrene resin
Ph
+
FG
randomly functionalized cross-linked polystyrene resin
Ph
FG
FG: CH2X (X: Cl, OH, NH2 )
Ph
(1-2%)
selectively functionalized cross-linked polystyrene resin
* suspension polymerisation: water, free radical catalyst (dibenzoyl peroxide, AIBN), dispergator: particle size depends
upon stirring speed, the relative amounts of aqueous and monomer phases, amount and nature of dispergator
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43

1. Functionalized polystyrene resins
microporous: 1-2% crosslinking

Ph
macroporous: 20% crosslinking
Ph
Cl
Ph
Cl
(95:5 para/ortho)
chloromethyl-polystyrene resin (Merryfield resin: J. Am. Chem. Soc. 1963, 85, 2149)
OMe
styrene, DVB
OMe
styrene, DVB
styrene, DVB
Cl
ii
(up to 2.5 mmol/g loading)

Cl
i: BCl3, CCl4, 0, 2h; ii: NaOH, CHCl 3 (or ClCH2 CH2 Cl), BnN+ Et3, Cl- , SO2 Cl2 , AIBN, 60; Macromolecules 1986, 19, 2470
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44

Swelling properties of Merryfield type microporous resins:

Solvent
MeOH
EtOH
AcOH
MeCN
pyridine
DMF
THF
dioxane
Et2 O
CH2 Cl2
toluene
crosslinked PS (1% DVB)*

0.95
1.05
crosslinked PS (2% DVB)*
1.0
1.0
2.0
3.5
5.5
4.9
2.6
5.2
5.3
3.0
2.0
2.5
2.8
*swelling capacity: volume of swollen resin/original volume
winter semester 09
45

solid-phase peptide synthesis: linkers
2. TentaGelR resins
Bayer and Rapp; Angew. Chem. Int. Ed. Engl. 1991, 30, 113; contain up to 60-80% of PEG units
i
CH2O(CH2CH 2O)3 CH2CH2O- K+
CH2O(CH2CH2O)4+nH
OH
ii
Me
iii
Me
OH
O(CH2 CH2O)n H
i: ethylene oxide; ii: propylene oxide, SnCl 4, CH2 Cl2; iii: ethylene oxide, KOH, dioxane, 110
Good swelling properties in: water, MeOH, CH2Cl 2, MeCN, THF and DMF; used preferentially in continous flow reactors
3. Polyacrylamide resins
pioneerd by Sheppard: Bioorg. Chem. 1979, 8, 351
O
O
N
basic monomer
N
H
H
N
O
N
H
crosslinking agent
H
N
NHBoc
CO2Me
O
functionalized monomers
Persulphate initiated copolymerisationin 66% aqueous DMF, 1,2-dichloroethane and cellulose acetate/butyrate as emulgator
winter semester 09
46

solid-phase peptide synthesis: linkers
2. Linkers for releasing carboxylic acids
structure
a bbreviation
cleavage conditions
reference
Cl
Merryfi eld resin
HF, CF 3SO3H
OH
hydroxymethyl-PS
HF, CF 3SO3H
Wang resin
95% TFA
J. Am. Chem. Soc. 1973, 95, 1328
1% TFA
Tetrahedron Lett. 1988, 29, 4005
1% TFA
Tetrahedron Lett . 1987, 28, 3787
J. Am. Chem. Soc. 1963, 85, 2149
OH
O
OH
O
SasrinR resin (Bachem)
OMe
OH OMe
Rink resin
O
OMe
Cl
Cl
chloro-trityl resi n
(Barl os)
Ph
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47

Solid-phase peptide synthesis: linkers
2. Linkers for releasing amides
structure
abbreviation
cleava ge conditions
reference
NH 2
BHA (R=H)
MBHA (R=Me)
O
HF, CF 3SO3H
J. O rg. Chem. 1985, 50, 5291

Peptides. 1981, 2, 85
NH 2 OMe
Rink resin
O
95% TFA
OMe
OMe
NH 2
PAL re sin
TFA
Int. J. Prot. Pept. Res. 1987, 30, 206
TFA
O
OMe
OMe
NH2
O
OH
Kaiser oxime resin
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NO2
NH3
J. Org. Chem. 1980, 45, 1295
primary and secondary amines

NH 2NH2 x 1H2O
48

H Me
HOOC
NHFmoc
HOOC
glycine; Gly; G
NHFmoc
HOOC
alanine; Ala; A
H
NHFmoc
valine; Val; V
NHFmoc
HOOC
leucine; Leu; L
HOOC
NHFmoc
phenylalanine; Phe; F
OH (Boc)
SMe
OH (tBu)
OH (tBu)
H*
H
HOOC
NHFmoc
HOOC
serine; Ser; S
H
NHFmoc
NHFmoc HOOC
HOOC
methionine; Met; M
threonine; Thr; T
*
NHFmoc
isoleucine; Ile; I
NHFmoc
HOOC
tyrosine; Tyr; Y
(Boc) H
N
COOH (tBu)
H
HOOC
H
NHFmoc
HOOC
aspartic acid; Asp; D
COOH (tBu)
CONH2 (NHTr)
NHFmoc
HOOC
aparagine; Asn; N
CONH2 (NHTr)
H
NHFmoc
glutamic acid; Glu; E
HOOC
H
NHFmoc
glutamine; Gln; Q
HOOC
NHFmoc
tryptophan; Trp; W
NHPbf
(Tr) H
NH2 (Boc)
HN
NH
N
N
SH (Tr)
H
HOOC
H
NHFmoc
lysine; Lys; K
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HOOC
H
NHFmoc
arginine; Arg; R
HOOC
H
NHFmoc
HOOC
cysteine; Cys; C
NHFmoc
cystine; Cys2;
HOOC
NHFmoc
histidine; His; H
49

Solid-phase peptide synthesis: coupling reagents
azides
uronium salts
NMe2
PhO
N
PhO P 3
O
NMe2 , X-
R-COOH
X-: PF 6- HBTU; BF4 - TBTU
N
N
N
O
Tetr ahedr on Lett. 1989, 30, 1927
base
X-: PF6- HATU
N
N
N
O
NMe2, X-
X-: PF6 - , BF4 -, (Castro's reagent)
N
N
N
NMe2
O P NMe2
NMe2 , X-
Tetrahedron Lett. 1975, 1219
N
R1 N C N R2 ,
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N
N
OH
Y= CH, N
Y
R1 =R2 = iPr (DIC)
R1 =R2 =cyclohexyl (DCC)
R1 =Et; R2 =CH 2 CH 2N +Me2 , Cl- (EDCI)
acid fluorides
base
R-COOH
F
N
X P N
N
R-CON3
carbodiimides
J. Chem. Soc., Chem. Commun. 1994, 201
NMe2
DPPA (diphenyl-phosphoryl azide)
N
N
, PF6 - (X=Cl, PyCloP)

(X=Br, PyBroP)
R-COF
F
N
F
J. Org. Chem. 1994, 59, 2437
50

Solid-phase peptide synthesis: protective groups
Fmoc strategy:
Cleavage
Main chain (backbone) amino groups: Fmoc
20% piperidine/DMF, rt
Side chain amino groups (Lys, Orn, Dab): Boc
TFA, CH2CH2 , triisoprpoylsilane*
Side chain carboxylic acids (Glu, Asp): t-butyl esters
TFA, CH2CH2 triisopropylsilane*
Side chain primary amides (Gln, Ans): N-trityl
TFA, CH2Cl2 , triisopropylsilane*
Side chain hydroxy(phenol) groups (Ser, Thr, Tyr):

t-butyl ethers
Side chain indole and imidazole groups (Trp, His): N-trityl
Side chain guanidine groups (Arg): Pmc, Pmb

*other scavengers like thioanisol
phenol, H 2O, thiocresol and others
are used
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51

Examples for libraries synthesized on solid-phase: peptoids
Peptoids: ideal scaffold for parallel and combinatorial synthesis
R1
N
H
N
R1
R2
N
O
N
H
R2
R3
H
N
peptide backbone
O
N
H
R1
N
H
N
O
O
N
R2
N
H
R3
N
azapeptide backbone
O
O
N
R3
peptoid backbone
O
-protease stability increased

-number of H-bond donors reduced (can be also disadvantage)
-number of rotatable bonds increased (tertiary amides have lower trans-cis barrier)
-prediction of peptoid backbone conformation quite difficult (flexibility)
-ideally suited for library synthesis: large number of building blocks available
available by solid-phase synthesis
split-mixed synthesis possible
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52

Approach A: sequential coupling of N-substituted glycines
O
N
H
R1
N
O
i, ii
N
H
Fmoc
R1
N
N
R2
Fmoc
H2 N
R1
N
N
R2
R3
N
Approach B: sequential coupling of glycine followed by reductive amination with aldehydes

O
N
H
H
N
R1
N
O
iii, iv
N
H
Fmoc
O
v
H
N
H
R1
N
O
N
H
Fmoc
iii, iv
O
N
H
R1
N
O
N
R2
H
H2 N
R1
N
N
R2
R3
N
i: DBU, DMF; ii: PyBop or PyBrop, R2 NFmocCH2 COOH; iii: DBU, DMF; vi: RCHO, Na(OAc)3 BH or NaCNBH3, MeOH; v: Fmoc-Gly,
PyBop or PyBrop; vi: DIC, DMF, BrCH2COOH; vii: R-NH2, DMSO
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53

Approach C: coupling of bromo-acetic acid followed by nucleophilic displacement with amines
O
vi
NH2
vii
Br
N
H
N
H
R1
N
O
vi, vii
H
N
H
R1
N
O
N
R2
Proc. Nat. Acad. Sci. USA 1992, 89, 9367

O
H2 N
R1
N
N
R2
R3
N
i: DBU, DMF; ii: PyBop or PyBrop, R2 NFmocCH2 COOH; iii: DBU, DMF; vi: RCHO, Na(OAc)3 BH or NaCNBH3 , MeOH; v: Fmoc-Gly,
PyBop or PyBrop; vi: DIC, DMF, BrCH2COOH; vii: R-NH2 , DMSO
O
O
N
HN
CONH2
Screening 18 pools originated from split-mixed synthesis

for [3 H]-DAMGO (-specific) binding to opiate receptor.
Chir 4531: 6nM
Chir 4531
OH
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54

Examples for libraries synthesized on solid-phase: oligosccharides
Ph
O
OBn
Ph
ii
N3
O
O
HO
OPiv
OH
PivO
PivO
S
N3
ii
O
Ph
O
OPiv O
O
N3
O
OBn
BnO
iii
O
O
O
O
BnO
BnO
OBn
N3
O
OPiv
PivO
PivO
Ph
O
S
OPiv Ph
O
O
O
O
O
BnO
BnO
Ph
BnO
O
O
HO
iii
O
O
OPiv O
O
Ph
OH
N3
PivO
PivO
OH
N3
OPiv
i: Cs2CO3 , Merryfield resin; ii: Tf2 O, 2,6-di-tert-butyl-4-methylpyridine, CH2 Cl2, -60 to -20; iii: Hg(OCOCF3) 2, CH2 Cl2, H2 O, r.t.
D. Kahne et al. J. Am. Chem. Soc. 1994, 116, 6953; ibid J. Am. Chem. Soc. 1994, 116, 1766; ibid J. Am. Che m. Soc. 1989, 111, 6881
winter semester 09
55

Questions
1. Name at least three different types of solid supports?

2. Give at least two different ways to synthezise chloro-methyl polystyrene?
winter semester 09
56

Drug-like molecules-the rule of 5
What is a drug-like molecule- how are drugs adminstered
Topical administration:
type of molecules
-intra-, trans- and extradermal: sprays, ointments, powders, plasters

-inhalation: sprays, powder inhalators
small molecules (<600)

peptides, proteins (antibodies,
fusion proteins) and others
Subcutaneous administration:
-by syringes; special devices (slow release)
especially well suited for

proteins (e.g. antibodies)
Intravenous (i.v.):
-injections (syringes) of solutions of the drug into venes (blood-stream)
Intraperitoneous (i.p.):

-injections (syringes) in the peritoneous

Oral (parenteral):
-formulation of the drug into various forms of pills; absorption through the
mucosa into the blood stream
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57

Examples for libraries synthesized on solid-phase: parallel synthesis of single compounds
R 1-20
H
N
H2N
N
H
R1-20
H2N
OH
R2
R3
R10
R1
R20
O
O
N
H
H2N
R1
OH
R2
R 10
R3
R1
R1
R20
H 2N
O
R1
N
H
R20
R1
O
OH
R2
R3
R 10
R20
R2
H
N
H2N
N
H
R1
FmocHN
R20
H
N
H2N
40 times couple and cleave
R1
N
H
R2
O
O
800 reaction vessels
20
R20
R10
R3
R2
40 individual products
FmocHN
H 2N
R2
O
O
N
H
R1
N
H
OH
FmocHN
40 times couple and cleave
R1
H2N
R1
R 20
O
O
FmocHN
H 2N
R2
40 reaction vessels
R1
H2N
H
N
800 products
R1
R1
R 1/2
R1
N
H
R20
O
O
H2N
O
H
N
R2
O
R20
N
H
O
O
800 individual products

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58

Examples for libraries synthesized on solid-phase: parallel synthesis of mixtures
R1-20
H2N
H
N
R1/2
N
H
R1-20
O
O
R2
800 products
R1
O
H2N
2 reaction vessels
R1
OH
FmocHN
R2
H2N
R1
R1
O
O
H2N
R20
H2N
OH
R2
R10
R3
R20
R1
R1
R20
O
O
H2N
H
N
mixture of 400 products

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R20
R1
R1
O
N
H
O
O
OH
FmocHN
R3
O
*cocktail of 20 amino acids
R2
H
N
H 2N
R2
R20
R10
2 times couple* and cleave
N
H
R20
R1
FmocHN
H2N
N
H
H2N
H
N
R2
O
O
N
H
R1
R20
R10
R3
R2
O
O
N
H
R2
R1
OH
FmocHN
2 times couple* and cleave
R1
N
H
R1
R20
R10
R3
H 2N
R1
N
H
R20
O
O
H2N
O
H
N
R2
O
R20
N
H
O
O

59

Examples for libraries synthesized on solid-phase: one bead-one compound
R 1-20
H2 N
O
R1
R1/2
H
N
N
1-20 H
R2
O
H2N
H 2N
mix
800products
divide
R1
R1
R2
O
H2N
H 2N
H 2N
R1
OH
N
H
Pool 20
R20
OH
couple, cleave
OH
FmocHN
R1/2
Pool 3
Pool 2
Fmo cHN
R1
R2
couple, cleave FmocHN
H2 N
H2 N
Pool 1
R2
O
R1 /2
O
H2N
Pool 1 (2 products)
N
H
R1/2
O
O
H 2N
20
Pool 2 (2 products)
N
H
O
O
Pool 2O (2 products)
mix
divide
R1
H2 N
O
H
N
R1/2
R1-20
N
H
R2
O
Pool 1 (40 products)
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H
N
H 2N
O
R1 /2
O
R1-2 0
N
H
R20
O
Pool 2 (40 products)
H 2N
O
R1/2
H
N
1-20
N
H
O
O
Pool 20 (40 compounds)
60

Parallel synthesis of compound mixtures:
++
---
high-throughput with little synthetic manipulations

difficult interpretation of screening results (synergistic and non-synergistic effects)
resynthesis of individual compounds necessary
generally not used anymore
Parallel synthesis of single compounds

++
++
++
--
clear screening results

identification of structure unambiguous
resynthesis generally not necessary; repurification required
many parallel synthetic steps and reaction vessels required; usually expensive robotic equipment
required
method of choice for relatively small compound libraries
Split mixed synthesis of mixtures (one bead- one compound):

++
++
--
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usually clear screening results can be obtained; on bead or in solution

large libraries with few synthetic steps can be obtained in real combinatorial fashion
only small amounts are usually obtained and structure of hits have to be determined by cleavage
and MS or deconvolution or tagging (binary codes or radio-frequency labels) startegies
method of choice for large combinatorial libraries
61

Parallel synthesis of single compounds
-tea bags:
e.g. R. A. Houghton et al. Proc. Natl. Acad. Sci. USA.,

1985, 82, 5131; G. Jung et al. Pept. Res. 1991, 4, 88
label
polypropylene net
resin beads (up to 100mg)
Spatially separated reaction compartments, where peptides can

be synthesized by capitalizing on the fact that all washing,
neutralisation and deprotection steps can be performed simultaneously. For parallel synthesis the bags are separated before
the coupling steps.
-multi pins: H. M. Geysen et al. Proc. Natl. Acad. Sci. USA.,

1984, 81, 3998
96 wel ls
96 pins
polyacrylic acidgrafted polyethylene
Spatially separated parallel synthesis of compounds

in microtiter format
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62

Examples for libraries synthesized on solid-phase: peptides: photolithography
Photolithography: light-directed combinatorial synthesis (S. P. A. Fodor et al. Science 1991, 251, 767)
Spatially separated multiple parallel synthesis using photocleavalbe protective groups such as the N-nitroveratrylcarbonyl group (NVOC), allows the controlled synthesis of (peptide) libraries by the spatially controllable
addition of specific reagents to specific locations.
NO2
H
N
O
R'
O
H2 N Xn
h
h
COOH
NO 2
O
O
O
h
96 well format
R
N
H
H
N Xn
O
O
R'
NO 2
h
R'
R
H
N Xn
H 2N
CHO
O
O
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63

Questions
1. What are the advantages of a split-mixed approach over a parallel

synthesis approach and for which types of molecules will you apply
this technology? Please discuss.
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64

Examples for libraries synthesized on solid-phase: peptides: split-mixed technology (one bead-one compound)
RA-G
H2 N
O
RA-G
H
N
RA-G
N
H
O
O
73 = 343 tripeptides
O
O
HO
step 1
X NHFmoc
O
X1 NH
X2-NHFmoc
couple
O
A NHFmoc
Pool 1A
B NHFmoc
Pool 1B
O
O
O
O
C NHFmoc
O
mix
cleave
split
Pool 1C
Pool 1D
D NHFmoc
O
E NHFmoc
step 2
Pool 1E
O
O
F NHFmoc
Pool 1F
O
O
G NHFmoc
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Pool 1G
A
Pool 2A (7)
Pool 3A (49)
Pool 2B (7)
Pool 3B(49)
B
C
D
E
F
Pool 2C (7)
mix
cleave
split
Pool 3C (49)
Pool 3D (49)
Pool 2D (7)
Pool 2E (7)
O
X1 NH
O
X2-NH
X3-NH2
couple, cleave
tripeptides
dipeptides
O
step 3
Pool 3E (49)
Pool 2F (7)
Pool 3F (49)
Pool 2G (7)
Pool 3G (49
A
Pool 4A(49)
B
Pool 4B (49)
C
D
E
F
Pool 4C (49)
Pool 4D (49)
342 tripeptides
on bead
Pool 4E (49)
Pool 4F (49)
G
Pool 4G (49)
65

Iterative deconvolution (Nature 1991, 354, 84; Science 1994,266, 2019; Proc. Nat. Acad. Sci, USA 1993, 90, 10811)
Sreening reveals in which of the Pools 4A to 4G are the most active compounds; determines most active building block
in the 3rd step (position): assumption it is B; Pools 2A to 2G are resynthesized but not mixed and coupled with building
block B in the third step. The compounds are retested and this determines the favoured building block in the second
step (position): assumption it is G. Now the initial 7 resins are coupled with G (2nd step) and B (3 rd step) and the resulting
Compounds tested again. The most active tripeptide is now identified: assumption it is A-G-B.
Recursive deconvolution (e.g. Nat. Acad. Sci, USA 1994, 91, 11422)
By using this technique samples of the initial resins as well as Pools 2A-2G and Pools 4A-4G are stored away for
resynthesis of sublibraries similarly to the iterative deconvolution procedure.
Positional scanning (e.g. Nat. Acad. Sci, USA 1994, 91, 11422; Life Sci. 1993, 52, 1509)
Indexed or orthogonal libraries (e.g. Chem. Biol. 1995, 2, 621; Tetrahedron Lett. 1997, 38, 491)
Binary encoding (e.g. W. C. Still et al. Proc. Nat. Acad. Sci, USA 1993, 90, 10922)
Radio-frequency tags (Irori system): (J. Am. Chem. Soc. 1995, 117, 10787; J. Org. Chem. 1997, 62, 6092)
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66

6.5. Examples for libraries synthesized on solid-phase: peptides: split-mixed technology : binary encoding
Binary encoding
(e.g. W. C. Still et al. Proc. Nat. Acad. Sci, USA 1993, 90, 10922)
RA -G
H2N
O
H
N
RA-G
7 building blocks
RA-G
O
N
H
3 steps
requires 9 tags
7 3= 343 tripeptides
123 456 789
GC/MS
step 1 step 2 step 3
NO 2
O
O
OAr
n
Cl
Cl
Cl
Cl
step 1: building block A: 1 0 0 tag 1

B: 0 1 0 tag 2
C: 0 0 1 tag 3
D: 1 1 0 tags 1 + 2
E: 1 0 1 tags 1 + 3
F: 0 1 1 tags 2 + 3
G: 1 1 1 tags 1 + 2 + 3
step 2: building block A: 1 0 0
B: 0 1 0
C: 0 0 1
D: 1 1 0
E: 1 0 1
F: 0 1 1
G: 1 1 1
step 3: building block A: 1 0 0

B: 0 1 0
Ar:
C: 0 0 1
D: 1 1 0
Cl
Cl
Cl
E: 1 0 1
F: 0 1 1
G: 1 1 1
n: 0-x variations of Ar and n gives rise to the different tags,
which can be detected in minute amounts by GC/MS
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tag 4
tag 5
tag 6
tags 4 + 5
tags 4 + 6
tags 5 + 6
tags 4 + 5 + 6
for tripeptide E-C-G:

1 3
6 78 9
GC/MS
101 001
111
step 1 step 2 step 3
tag 7
tag 8
tag 9
tags 7 + 8
tags 7 + 9
tags 8 + 9
tags 7 + 8 + 9
67

Examples for libraries synthesized on solid-phase: peptides: radio-frequency tags
Radio-frequency tags (Irori system): (J. Am. Chem. Soc. 1995, 117, 10787; J. Org. Chem. 1997, 62, 6092)
radio frequency tag (code)
fret
code A
resin beads
Irori can (polypropylene)
codeB
A
codeC
mix
couple B
B
codeE
codeF
F
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DB
CB
codeD
codeG
AB
BB
EB
cleave
sort
(radio frequency
reader)
FB
GB
68

Application of the split-mixed method for discovery of Factor Xa inhibitors

Factor Xa is implicated in the blood coagulation cascade: inhibitors of Factor Xa could
be potentially useful as anti-thrombotic agents
(Biochemistry 1998, 37, 1053-1059; Drug Discovery Today 1998, 3, 223))
octa-peptide library (split-mixed technology)

HN
NH2
on-bead screening
H-Tyr-Ile-Arg-Leu-Ala-Ala-Phe-Thr-NH2 (SEL1691)
O
N
H
H
N
O
O
N
H
H
N
O
X-
O
N
NH2
SEL2602
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69

Blood coagulation factor Xa is implicated in hemostasis (bloodcoagulation)
Thrombosis: pathological form of hemostasis:
intrinsic pathways
XII
myocardial infarction (arterial thrombosis)

pulmonary embolism (venary thrombosis
infection by gram-negative organisms
XII
XI
XIa
IX
Factor X
extrinsic pathways
VIIa
TF*/Ca2+
IXa
VIIIa/Ca2+
Factor Xa
/Va/Ca2+
VII
Fibrinogen
Factor Xa inhibitors
Prothrombin
Thrombin
Fibrin
Thrombin inhibitors
cross-linked fibrin clot
*tissue factor
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XIIIa
70

6.5. Examples for libraries synthesized on solid-phase: peptides: split-mixed technology (one bead-one compound)
Current anti-thrombotic therapies include: aspirin
Thrombin inhibitors:
heparin (sulphated poly-saccharide); heparin analogues; hirudin; small molecular

weight thrombin inhibitors (not on the market yet)
high levels of thrombin inhibition necessary; unacceptable bleeding
Factor Xa inhibitors:
trypsin-like serine protease

current molecules in clinical trials
HN
NH2
HN
NH
NH
H
N
S
O
NH2
N
N
H
H2N
S
Cor-Therapies (IC50 factor Xa: 0.65nM)

(IC50 thrombin: 10.0M)
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O
O S
N
NH
COOH
NH
N
Yamanouchi (IC50 factor Xa: 1.3nM)

(IC50 thrombin: >100M)
71

Synthesis of a octa-peptide library by split-mixed synthesis and colorimetric assay on bead:
Streptavidin-AP
biotinylated
Factor Xa
Factor Xa
complex
biotin
inhibitor
AP: alkaline phosphatase
Factor Xa
biotin
inhibitor
inhibitor
Factor Xa
biotin
Streptavidin-AP
biotin
Factor XA
inhibitor
biotin
Factor Xa
AP: alkaline phosphatase de-phosphorylates 5-bromo-4-chloro-3-indolyl phosphate forming a blue precipitate,

which stains the beads
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72

Synthesis of a octa-peptide library by split-mixed synthesis and colorimetric assay on bead:
All active compounds contained Tyr-Ile-Arg at the N-terminus
H-Tyr-Ile-Arg-Leu-Ala-Ala-Phe-Thr-NH2 (SEL1691; IC50: 4-15M))
NH2
HN
N
H
H
N
O
O
N
H
H
N
NH2
HN
O
O
N
NH2
N
H
SEL2316 (IC50: 80nM)
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HN
NH2
HN
OH
Drug Discovery Today 1998, 3, 223
H
N
O
O
N
H
H
N
O
N
NH2
SEL2489 (IC50: 25nM; half-life in

rats and rabbits 8 to 10 minutes)
73

OH
NH2
HN
NH 2
HN
O
H
N
N
H
N
H
NH2
HN
O
H
N
HN
O
N
NH2
H
N
N
H
N
H
H
N
O
N
NH2
SEL2489 (IC 50 : 25nM; half-life in

rats and rabbits 8 to 10 minutes)
SEL2316 (IC50 : 80nM)
HN
NH2
O
N
H
H
N
O
N+
O
N
H
H
N
X-
NH2
O
N
O
NH2
N
H
SEL2602 (IC50: <25nM; improved half-life)

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H
N
O
N+
O
N
H
H
N
O
N
NH2
SEL2602 (IC50: 285nM)

74

Examples for libraries synthesized on solid-phase: peptides: thrombin inhibitors)
Application of solid-phase chemistry for discovery of thrombin inhibitors
multi-directional cleavage
R
R
i, ii
N O
O
N
Fmoc
R1HN
iii, iv
N O
N
O
N
O
NHBoc
O
Ph
O
Ph
Ph
i: 20% piperidine, DMF; ii: amino acid, HBTU, HOBt, DIEA, DMF;
iii: R1NH2, DMF; iv: TFA
NH2 x TFA
Ph
Cl
J. Med. Chem. 1998, 41, 401-406; J. Med. Chem. 1998, 41, 1011-1013
Cl
O
O
IC50: 3nM
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Ph
NH2 x TFA
Ph
75

Examples for libraries synthesized on solid-phase: inhibitors of protein-protein interaction
Characteristic of large surface protein-protein interactions
Fundamental to the functioning of biological systems
many proteins function as part of complexes
cell to cell signalling
cell adhesion
long distance communication (hormones)
Specific inhibition offers important therapeutic potential:
Generally form across a large area of interacting surfaces: 700-1300 A2 average
High binding energy
Difficult to inhibit with small molecules? Small molecule discovery approaches have largely failed
Antibodies and fusion proteins (biopharmaceuticals) have emerged as important drugs:
however, these act only on extracellular targets
Slow to mature : initial binding is thought to occur through hotspots in selected areas
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76

6.5. Examples for libraries synthesized on solid-phase: inhibitors of protein-protein interaction
average contact surface area in protein-protein interactions: 600-900 A 2
Bogan, A. A.; Thorn, K. J. Mol. Biol. 1998, 280, 1-9
Hotspots
topology of the hotspots determine specificity
O-Rings
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77

Extracellular
GH-receptor
growth
hormone
Kd 0.3 nM
Buried surface on each protein
1300 2
Wells, PNAS, 1996, 93, 1-6; Science, 1995, 267, 383

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78

Petidic -helix mimetics as inhibitors of protein-protein interactions
Dr. Sjoerd Wadman
~40% of all HTS campaigns in GSK were targeted to find small PPI inhibitors in 1998
Very low success rate
Many assays suitable for HTS developed
Most were shelved during portefolio review
Addressed one important target with full resource
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79

Oncostatin M
-4-Helical Cytokine
-Pro-inflammatory hormone
-Therapeutic applications:
-Rheumatoid Arthritis
-Asthma
-Interacts with 7TM receptor
-Part of a large family of important proteins
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80

Examples for libraries synthesized on solid-phase: four helix bundle proteins
Family
Long Chain
4-helix bundle
Short Chain
4-helix bundle
Growth Hormone
Prolactin
IL-6
IL-3
IL-7
LIF
OSM
CNTF
CDF
IL-2
IL-4
IL-13
IFN-a
IL-5
GM-CSF
M-CSF
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Dimeric-dimeric
4-helix bundle
IL-10
IFN-G
IFN-B
81

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82

Note side-on interactions of -helices

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83

--helices cluster with hydrophobic residues poiting at

the inside (red) whereas hydrophilic residues (yellow)
are located at the outside
-challenge:
inhibit formation of 4-helix bundle formation by
interacting with the helical momomers
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84

-Helix side-chains are arranged like the steps on a
spiral staircase
-Regular distance
-Regular angle
-Model potential antagonists and pick the ones that
fit the model best
-Aimed to antagonise side-on -helix
interactions through 3 side-chains
-Large - 100k compounds
-Non-peptidic
-Split - mix synthesis on solid phase
-Fully Encoded / Partial Release Technology
-384 screening format
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85

Amino acid-like side-chains

1-14
1-14
5-7 angstrom
FG1
LINK1
CORE 1
FG2
5-7 angstrom
CORE 2
FG3
LINK3
LINK2
1-7
1-7
Spacers hold side chains in correct orientation

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86

Propose Connectivity
and potential monomers
Model compounds
proposed library
Take best connectivity

and best monomers
Measure fit against

Helix Vector Model
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87

R1
R2
H N
O
H
N
Core 1
R3
Core 2
-Amino acids Amino acids
Amines
Amino acids
14
14
14
Tags
required
Amines
Total number of compounds: 134'456

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88

amines
neutral
NH2
NH2
H2 N
NH2
H2N
NH2
NH2
NH2
NH2
TrO
NH2
O
acidic
O
NH2
NH2
O
basic
N
NH2
NH2
N
N
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Boc
NH2
BocHN
NH2
89

6.5. Examples for libraries synthesized on solid-phase: four helix bundle proteins
Core 1 amino acids
HOOC
H
NHFmoc
HOOC
HOOC
NHFmoc
HOOC
NHFmoc
NHFmoc
HOOC
HOOC
NHFmoc
Fmoc
NHFmoc
HOOC
OMe
OMe
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90

-Amino acids
HOOC
NHFmoc HOOC
HOOC
H
Ala
Gly
HOOC
NHFmoc
NHFmoc
H
HOOC
Phe
NHFmoc
HOOC
H
Leu
Val
NHFmoc
H
HOOC
Tyr
NHFmoc
H
NHFmoc
HOOC
NHFmoc
H
OtBu
H
OtBu
Ser
Thr
OtBu
HOOC
NHFmoc
HOOC
H
Met
MeS
Hyp (hydroxyproline)
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NHFmoc
H
COOtBu
Asp
HOOC
NHFmoc
HOOC
H
COOtBu
Glu
NHFmoc
H
Arg
NBoc
HN
NHBoc
91

Core 2: Diacids (Anhydrides)
O
H
O
H
O
O
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O
O
H
O
O
H
O
O
O
O
92

Major conformers closely match -helix in side-chain display vectors
H
Me
O
HN
N
O
O O
N
H
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COOH
93

Concepts
3 building blocks
3 products in pools of 1
Mix
-Split Mix synthesis

-Library encoding
-Differential release
-Single Bead screening

Mix
1 bead = 1 compound
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94

Codes for
each building block
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Building Blocks
95

Affimax encoding strategy
Linker
H
N
O
O N
N
H
O N alloc
N
H
O
HN
Product on acidor photolabile linker
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-Codes are different amines

-Cleaved with cHCl
-Dansylate and analyse by hplc
96

Differential release
PHOTO-LABILE LINKER
O
HN
H
N
NO2
O
OMe
O
CODE
Product
O
HN
O
N
H
ACID-LABILE LINKER
50% on acid labile linker

50% on photolabile linker
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OMe
Product can be released

twice at different times
97

Single bead screening
Acid cleave
Screen pools
Active pool
Plate out individual beads

photocleave
screen single beads
Active bead
Cleave Tag from bead

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Identify active molecule
98

Single bead screening
-Compounds prepared on Tentagel
-Reactions done on an ACT synthesis robot
-All building blocks were rehearsed
-Analysis throughout
-1st stage by magic angle nmr
-later stages by lc/ms and tag reading
-lc/ms aided using analytical constructs
-All done by one chemist in 5 months
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99

Resin differentiation
OH
B ocNH
O
10:1
OH
FmocNH
H2N
NH2
FmocNH
N
H
DIC, HOBT, DMF
Amino Tentagel
O
NHBoc
N
H
10
DIPEA, DMF
O
O
Aloc
H
N
O
Code 1
O
Code 1
O
N
H
NHBoc
N
H
OH
O
H2N
O
NHB oc
N
H
N
H
DIPEA, HATU, DMF
95%TFA
O
Aloc
N
O
Code 1
H
N
Photolinker
Acid Linker
O
N
H
N
H
Aloc
NH2
DIPEA, HATU, DMF
N
O
Code 1
H
N
HN
HN
Photo linker
N
H
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H
N
N
H
AcidLinker
100

Prepared resin
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101

Reductive amination
NO2
O
HN
NO 2
H
N
O
O
OMe
Me4NBH(OAc) 3
AcOH, DMF
O
O
N
H
HN
H 2N
CODE N
H
HN
O
O
OMe
CODE N
H
O
HN
OMe
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N
H
H
N
N
H
H
N
OMe
102

CODE
Linker
1)Split/mix
Library
synthesis 1
N
H
2)
CODE
NHFmo c
Linker
MeO
HO
OM e
MeO
OMe
NHFmoc
HATU,DMF,DIPEA
1) Split/mix
2) Code deprotection
3) Codecoupling
4) Fmoc deprotection
5) HATU,DIPEA,DMF
O
NHFmoc
HO
CODE
Linker
MeO
MeO
1) Split/mix
3) Codecoupling
4) Fmoc deprotection
5) Pyridine
CODE
NH
O
O
O
O
O
O
OH
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Linker
MeO
MeO
O
O
NHFmoc
O
103

Library synthesis 2
CODE
Linker
MeO
MeO
1) Split/mix
3) Codecoupling
4) pyridine, DMF
F
F
F
F
F
O
F
F
O
F
O
NH
H 2N
Linker
MeO
5) DIPEA, DMF
O
O
O
CODE
MeO
OtBu
O
O
O
O
NH
O
O
O
HN
OH
OtBu
O
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104

6.5. Examples for libraries synthesized on solid-phase: four helix bundle proteins
Final construct
O
O
TARGET
H
N
HN
HN
N
H
CO2H
C27H34N4 O8
[542.6]
OMe
OMe
CONSTRUCT
O
NO2
HN
H
N
O
C3C10
MdD o
O'O'
NH2
O
O
O
O
N
O
NH2
OO
PD
DD
HN
O
N
O
NH2
Mo
PP
HH
H
N
N
O
CO 2t-Bu
MeO
OMe
N
H
HN
O
N
H
OMe
O
OM e
N
tag
OMe
acid-labile
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N
H
photo-labile
NH2
EB
MP
C3C4
OMe
O
N
H
N
NH2
O
N
BB
MH'
BP
O
O
HN
O
H
N
CO 2t-Bu
N
H
105

Screening of library
Primary screen :168 x 96 wells / ~30 beads per well
42 plates in 384 format
Half of acid-cleaved material used
Screening concentration ~ 2mM/component
Histogram of 1ry screening data for GL1495 in OSM
Frequency
2000
1500
1000
500
e
or
10
10
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
-5
0
-1
5
-1
-2
% Inhibition
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106

Screening results
-21 sub-micromolar hits re-made as discretes
-5 Compounds potent and selective
-17 also inhibit TNF in same cell line : signalling inhibitors?
Number
Hits
Leads
GSK compound collection
250.000
3134
Natural product extracts
70.000
18
Aptamers
2000.000
78
13
Apha helix library GL1495
134.456
21
Source
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107

Acknowledgements
-Chemistry:
Helen Jenkins
-Biology:
Paul Life, John Spaul
-Screening:
Liz Clarke, Sandra Arpino
-Modelling:
Darren Green
+ many others
And Dr. Sjoerd Wadman
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108

Examples for libraries synthesized on solid-phase: peptides: phage display
Gene3 protein
The native phage contains a DNA genome
surrounded by a protein coat. At one end of the
phage are 5 copies of the Gene3 gene product
expressed from the phage genome.
The phage infects a host bacterial cell (e.g. E.
Coli) and uses the bacterium to replicate itself,
leading to secretion of progeny phage.
E. coli
plasmid
In phage display, the E. Coli host contains a

DNA plasmid encoding Gene3 fused to either a
protein of interest, or a library of random
peptides. As the phage replicates, Gene3 fusion
proteins (expressed from the plasmid) are
incorporated into the phage coat. Libraries of
phages can be produced, with each bacterium
producing phages with a unique peptide
displayed at its surface determined by the
plasmid (the phage also contains the at this
point) of the host cell.
G. P. Smith et al. Meth. Enzymol. 1993, 217, 228; J. K. Scott et al. Curr. Opin. Biotechnol. 1994, 5, 4)
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109

Examples for libraries synthesized on solid-phase: peptides: Phage display panning techniques
A library of phages, each

displaying a unique peptide
sequence, is allowed to bind to
a plate coated with the target
molecule (e.g. protein).
Unbound phages are washed
away.
Specifically bound phages are

eluted.
After 3-4 rounds of panning, individual

phage clones are isolated and sequenced
to determine the sequence of the displayed
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09
peptide.
The eluted phages are

amplified and panning
process is repeated
several times.
110

Examples for libraries synthesized on solid-phase: phage display
Functional mimicry of a protein hormone by a peptide agonist: EPO receptor complex; Science 1996, 273, 464-471
Erythropoetin (EPO) is the primary hormone that regulates the proliferation and differentiation of immature erythroid
cells. Recombinant human EPO is widely used in the treatment of patients with anemia due to renal failure, cancer
chemotherapy, and AZT treatment. The EPO receptor belongs to the cytokine receptor superfamily, which includes
receptors for other hematopoetic growth factors, such as interleukins (IL) and colony-stimulating factors (CSF), as well
as growth hormone (GH), prolactin, and ciliary neurotrophic factor (CNTF).
Screening of a phage libray (Annu. Rev. Microbiol. 1993, 47, 535) against immobilized EPOR gave an active
consens sequence, and a very potent member of the family with agonistic activity in vitro and vivo was identified (see
Figure).
COOH
G
G
T
L
S
S
G
P
C
Y
F
G
H 2N
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111
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112

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113
Polyphor2001-1/JPO/DO

5. Strategies for the Synthesis of Small Molecule Libraries
Chemical strategies used for the synthesis of small molecule libraries: overview
Steps required for the design and synthesis of a library
1. Planning (literature search and retrosynthetic analysis of the problem)
2. Synthesis strategy (linear, convergent, multicomponent reactions, tandem reactions...)
3. Building blocks (commercial or self-made)
4. Parallel or combinatorial synthesis (in solution; in solution by aid of solid-supported reagents; on
solid supports)
5. Parallel work-up (two phases: aqueous, organic, fluoruos; solid-phase extraction)
6. Purification: parallel flash chromatography; high-throughput HPLC coupled to MS on normal and
reversed phase
7. Analysis, stability and storage of products
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114

Synthetic strategies: introduction
sub-library A
scaffold
R3
H
N
N
N
R
H
N
O
R2
exit vectors: determine the relative orientation of the high

and low variation substituents and thus the overall shape
of the final molecule
scaffold: MG~290; for the substituents remain MG~210
high variation substituents
low variation substituents

sub-library B
scaffold
R3
H
N
N
N
R2
H
N
O
R1
high variation substituents

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diversity associated with scaffolds: "vertical diversity"; diversity associated

with substituents: "horizontal diversity"
the synthetic strategies generally do not permit simultaneous high variation
of substituents R1-R3; rather sub-libraries (e.g. A and B) are planned; also
SAR data often show that not all substituents are equally importantfor biological
activity
low variation substituents

115

Synthetic strategies: convergent, multi-step
Multi-step synthesis of advanced building blocks (scaffold) by linear or convergent synthetic

strategies and parallel conversion into final products
R1
N
H
R1
O
R2
Cl
building block
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R1
N
R2
NHR2
final products
116

Synthetic strategies: classical multi-component approach
Synthesis of advanced building blocks (scaffold) using multi-component reactions and

parallel conversion into final products
NH
NH2
+
2
H
N
R2
building blocks
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R N=C=S
R3
R3
R3
O
R
O
1
N
R2
final products
Br
117

Classical multi-component reactions
A
A
C
B
intermdiaire
- Classical multi-component reactions (MCRs) are have in common that components (e.g. A, B, C) react in
a reversible way to a reactive intermediate, which reacts in a irreversible way to the product. Thus, the
sequence by which the components are added does nor affect product formation.
- The best known MCRs are the following: Ugi, Passerini, Biginelli, Strecker, Hantzsch, Mannich etc.
- Reactions can be ideally performed in a matrix format
- Classical MCRs generally yield generally the same scaffold
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118

R COOH
1
R3CHO
3
R3
R2NH2
R4N=C
Ugi 4MCR
R1
NHR
N
2
R
H
2
R3
N
C N
R4
O
R1
R2 H R3
N
H+
O
N
O
R4
1
R
irreversible
N
2
R
NHR
O
O-
The classical multi-component reactions are ideally suited for parallel synthesis, however, they
yield generally the same scaffold (limited scaffold diversity)
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119

Passerini 3-MCR
O
R1COOH
R2
R3
+ R4N=C
O R2 R3
H
N 4
1
R
O
R
O
H. Passerini, Gazz. Chim. Ital. 1921, 51, 126
Strecker synthesis
RCHO +
NH3 +
NH2
HCN
R
CN
A. Strecker, Justus Liebigs Ann. C hem. 1854, 91, 345; ibid. 1890, 23, 1474
Bucherer-Bergs variation of the Strecker synthesis

O
O
R1
R2
+ KCN + (NH4)2SO 4
R1
R2
NH
N
H
H . T. Bucherer et al. J. Prakt. Chem. 1934, 140, 69; ibid. 1934, 140, 28
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120

Mechanism of the Passerini 3-MCC reaction
R2 CHO
R1COOH
Passerini 3MCR
R3N=C
R1
R2
NHR3
O
O
H+
R2
O
C N
R3
O
R1
O-
winter semester 09
R2
H+
O
O
N
O
irreversible
R3
R1
R2
O
R1
NHR3
O
O
reactive intermediate
121

Modified Passerini 3-MCR

O
NH4 HCO3
CHO +
R1
R2N=C
+ PhS
COOH
R1
PhS
N
NHR 2
S
O
R. Bossio et al. J. Chem. Res. 1991, 15, 320
O
O
PhS
R1
NHR2
O
O
winter semester 09
H2N
O
NH4+HCO3PhS
R1
R1
NHR2
O
O
PhS
HN
NHR2
HO S
122

Hantzsch MCR's
R1
R2
Br
S
NH2
R3
R2
R1
RN
R2
Br
S
1
NH2
R3
R2
R1N
O
O
R1
R3
COOR2 + NH3 +
R3
COOR2
O
R3
R3
N
H
R1
R3
O
2x
R1
COOR2
+ NH3 +
R3-CHO
R2OOC
R1
COOR2
N
H
R1
A. Hantzsch, Ber. Deutsch. Chem. Ges. 1890, 23, 1474
winter semester 09
123

Classical MCRs
Erlemeyer azlactone synthesis
R2
O
R1
NaOAc
N
H
COOH
R2 CHO
+ Ac2 O +
N
R1
O
3-MCR involving a 1,3-dipolar cycloaddition

MeOOC
O
O
R1
MeOOC
CHO +
HN
N
R1
O
R. Grigg et al. Tetrahed ron 19 93, 49, 867 9
MeOOC N
+
R1
winter semester 09
N
O
124

Mannich 3-MCR
R4
R1
H
N
R2
+ CH2O +
R3
N
R1
R5
R2
H
N
R2
R1
N
R2
+ CH2O +
N
H
N
H
C. Mannich et al. Arch. Pharm. 1 921, 250, 647
Biginelli 3-MCR
R1
O
H2N
R1CHO +
NH2
COOR3
R2
COOR3
HN
O
N
H
R2
P. Biginelli, Ber. Deutsch. Chem. Ges. 1893, 26, 47; ibid. 1891, 24, 2962
Biginelli 3-MCR (Atwal variation)

R2
NH
R1 S
winter semester 09
NH2
R2CHO +
O
R3
COOR
COOR4
R1 S
N
H
R3
125

Classical MCRs
Grieco's 3-MCR
COOH
CHO
COOH
COOH
COOH
HN
H
H
NH2
[4+2]
P. Grieco et al. Tetrahedron Lett. 1988, 29, 5855; R. W. Armstrong et al. Tetrahedron Lett. 1997, 38, 6163
Pauson-Khand MCR
O
R1
R2
R3
R4
+ CO
R1
R3
R4
R2
U. I. Khand et al. J. Chem. Soc. Perkin Trans. 1 1973, 9, 977
winter semester 09
126

Classical multi-component reactions: applications
Applications of MCR's
CHO
O
2x
Me
NO 2
NO2
EtOOC
COOEt
+ NH3 +
COOEt
N
H
COOEt
Me
Nifedipine (Adalat R, Bayer)
COOEt
+ NH 3
H2 N
1
NO2
CHO
O
Me
NO2
COOEt
EtOOC
NO 2
COOEt
EtOOC
+
O
2
- Nifedipine is a widely used anti-hypertensive drug (is off patent now). It belongs to the Ca2+ channel blockers
(other include: Verapamil-type, Dilthiazem-type)
-It can be produced in a Hatzsch-type 3-MCR in a very efficient and cheap way.
winter semester 09
127

Application of the Ugi 4-MCR: genetic algorithm
O
R4
O
OH
+
+
R2
H
R4
+
R3NH 2
R2
R1N=C
N
R3
R2
NHR1
R3
N
H
NHR1
O
L. Weber et al. Angew. Chem. Int. Ed. Engl. 1995, 34, 2280
Genetic algorithms constitute an interesting approach for efficient optimization of multiparameter systems
Parameters: inputs acids, isocyanates, aldehydes, amines; biological activity (inhibition of thrombin)
Genetic operations: replication, mutation and crossover
O O H
S
N
O O H
S
N
N
O
H2 N
winter semester 09
OH
NH2 x HCl
NH
O
H2N
NH 2 x HCl
128

L. Weber et al. Angew. Chem. Int. Ed. Engl. 1995, 34, 2280
O
R4
OH
4 (40)
R4
N
R3
R2
NHR1
NH2
NH2
N
H2N
3
NH
4
0010 011100 0111 010011
H2N
NH
1
NH2
NH2
O
NH
bit pattern
H2N
NH2
H2N
H2N
NHR1
N
H
5 (160'000)
1 (10)
3 (10)
R1N=C
R3NH2
R2
R2
H
2 (40)
H2N
NH
N
NH2
+ 4 amines
HN
H2N
1
NH
2
0010 011100 0110 110011
0011 011100 0111 010111
crossover
mutation
1 st generation: random selection of 20 bit patterns: synthesis

2 nd generation: generated by entering first 20 bit patterns into the genetic algorithm which by means of crossover and
mutations generated the next 20 bit patterns: synthesis and biological testing of all 40 compounds
3 rd generation: the 20 most active compounds (bit patterns) were again entered into the genetic algorithm which generated
the next generation: synthesis and testing
after 16 cycles, the average effective inhibitory concentration (EC50) of the 20 best compounds was submicromolar
winter semester 09
129

Application of the Asinger-Ugi 6-MCR: Penicillin derivatives
OHC * CO2 Me
N
Pht
S
+
Br
NaSH + NH3
1. Asinger
N
2. hydrolysis
CHO
PhtN
S
N
NHPht
O
NHC6 H11
CHO
OHC
Br
* CO2 H
C 6H 11 N=C
HS
CHO
H2 N
S
CO2 Me
NHPht
HN=C * CO2 Me
NPht
O
NPht
PhtN
O-
O
N
C N C6 H11
winter semester 09
S
O
HN
N H
C6 H 11
130

Questions
1. Please name three classical multi-component reactions (MCRs)?

2. Give possible products of the following MCRs
N=C:
CHO
COOH
a)
NO2
50
NH 2
Cl
CF3COOH
b)
MeOH
CHO
CH2Cl2
NH2
winter semester 09
131

Sequential multi-component reactions
B
C
A
A
B
C
C
B
intermediate
- In sequential multi-component reactions (SMRCs) components (e.g. A, B, C) are added in a sequential way to
the reaction mixture. Thus, reaction of A + B form irreversibly intermediate A-B which is subsequently reacted
with C to form the product A-B-C. By changing the sequence of component addition theoretically 6 different
product types (scaffolds) can be obtained.
-The SMCRs offer the same advantages as the classical MCRs, but in addition they have the potential to generate different scaffolds.
winter semester 09
132

R3
O
NH
R -N=C=S
N H2
B
NHR
+ B
Br
A +
N
1
N
N
NHR
+ B
R3
R
N
S
O
R3
R1
O
A + B + CDI + C
O
A
+ 2xC
Sequential multi-component reactions (MCRs) offer the same advantages as the classical MCRs: in
addition several different scaffolds can be obtained employing the same set of building blocks
D. Obrecht, P. Ermert, 5th Ineternational conference on Synthetic Organic Chemistry (ECSOC-5); www.mdpi.org/ecsoc-5/,
September 1-30, 2001, [B0005]
winter semester 09
133

bis-acceptors
bis-donors
+
NHR1
H 2N
NH2 , X-
S
R
NHR1
Cl
R7 N C S
N C O
7
R
Br
H2N
NHNH2
S
5
H 2N
3
R9 X
(X: Cl, Br, I)
nucleophiles
R10 NH2
15
14
13
NHNH2
R3
HO
O
9
NH2 , X
NHN
R8 N C O
electrophiles
OH
12
NH2 , X
acceptor-donors
R4
NHN
6
(X: Cl, Br)
R5
O
10
R6
COOMe
O
11
winter semester 09
134

R
N
'
R HN
'
R HN
R''
N
N
'
R HN
Ph
NH
N
N
HO
O
R''
N
'
R HN
R''
N
N
R
'
R HN
R
R'HN
N
H
N
Me
Olomoucin
R''
N
N
September 1-30, 2001, [B0005]
winter semester 09
135

NH2
S
NHR
R''
SMCR
S
solid-phase
N
R
R'
O
NH2
R
NHR
SMCR
solution
R''
N
R
R''
N
N
H
N
R
N
R
September 1-30, 2001, [B0005]
winter semester 09
136

bis-acceptors
bis-donors
NH2 , X-
S
H2N
NHR 1
O
Cl
NHR1
R7 N C S
N C O
7
R2
Br
S
H2N
NHNH2
O
8
NHNH2
OH
NH2 , X
R8 N C O
R9 X
(X: Cl, Br, I)
nucleophiles
R10 NH2
15
14
13
O
9
+
NHN
12
electrophiles
R3
HO
S
H2N
NH2 , X
acceptor-donors
R4
NHN
6
(X: Cl, Br)
R5
10
R6
COOMe
O
11
winter semester 09
137

NH2 , X-
S
4
ii, iii
+
S
NH2
O
R4
R4
R5
N
R
'
N
H
10
N
N
N
H
i: DIPEA, DMF; ii: m-CPBA, CH2 Cl2; iii: R'NH2 (15), dioxane, 80-100 [8].
September 1-30, 2001, [B0005]
winter semester 09
138

bis-acceptors
bis-donors
NH2 , X-
S
H2N
NHR1
O
Cl
NHR1
R7 N C S
N C O
7
R2
Br
NH2 , X
S
H2N
NHNH2
S
5
acceptor-donors
12
R8 N C O
R9 X
(X: Cl, Br, I)
nucleophiles
R10 NH2
15
14
13
O
8
electrophiles
OH
NHNH2
R3
HO
O
NH2 , X
S
H2N
NHN
3
R4
NHN
6
(X: Cl, Br)
R5
O
10
R6
COOMe
O
11
winter semester 09
139

R2
Br
+
NH2 , XR
S
4
NHR
+ R7 N C S
NH S
12
N
H
NHR7
R2
NH S
R
NHR7
-RCH2 SH
H2N
O
OMe
R2
N
S
N
H
R7
S
N
H2N
i: DBU, DMF, 0; then DBU and 8, r.t.

September 1-30, 2001, [B0005]
winter semester 09
140

Sequential multi-component reactions: application
Ch. Abrecht, P. Ermert, D. Obrecht; Polyphor AG, Gewerbestrasse 14, CH-4123 Allschwil
P. Maienfisch, Th. Pitterna, Syngenta Crop Protection AG, WRO-1060, CH-4002 Basel
Insecticidal lead compound
NC
S
Cl
Library
O
N
H
E
S
R
Cl
Y
N
Me
S
1
R
Cl
Y
N
Cl
O
D. Obrecht et al. Chimia 2003, 57, 262-269
E: RCO; CN
R1 : CF 3, NH2 , 4-Cl-C 6H 4-; thiophen-2-yl-
winter semester 09
141

OMe
NH2
,X-
NH2
N=C=S
NH2 S
R
MeO
OMe
3
N
H
E: R CO ; CN
N
H
OMe
ii, E -CH2-X
E
v, iv
OMe
S
1
iii, iv
N
H
OMe
E
S
1
O
N
H
E
Cl
S
R 4: Me, CH2OE t
Cl
N
H
vi
vi
E
S
1
O
N
4
R
E
Cl
S
1
N
R4
Cl
i: DIEA, DMF; ii: DBU, DMF, ECH 2X; iii: 3-Cl-C 6H4COCl, DCM,pyridine, DMAP; iv: TFA, DCM, H2O; v: 3-Cl-C6H4SO2Cl, DCM,
p yridine, DMAP; vi: K2CO3, DMF, MeI or ClCH2OEt
winter semester 09
142

+
NH2
,X-
NH2
Cl
NH2 S
R1
N=C=S
N
H
E: R3 CO; CN
N
H
Cl
ii, E-CH2-X
E
iii
S
R1
iv
N
H
Cl
S
R
S
N
Me
Cl
R1
Cl
i: DIEA, DMF; ii: DBU, DMF, ECH2X; iii: K2 CO 3, DMF, MeI; iv: K2 CO 3, DMF, ClCH2 OEt
winter semester 09
143

+
NH 2
'
RS
,X-
NH2
NH 2 S
R N=C=S
'
RS
N
H
E: R3 CO; CN
NHR
ii, E-CH2 -X
E
S
H 2N
NHR
iii
H2 N
E
Cl
iv
H 2N
MeO
S
N
Cl
N
H
OMe
H 2N
Cl
N
R4
R 4: Me; CH 2OEt
E: R3 CO; CN
H 2N
N
R4
Cl
i: DIEA, DMF; ii: DBU, DMF; iii: 3-Cl-C6 H4 COCl, CH2 Cl 2, pyridine,
DMAP; iv: TFA, CH 2Cl2 , H2 O; v: K2CO3 ; DMF CH3 I or ClCH 2OEt
winter semester 09
144

Fragment-based screening
enzyme binding pocket
F2'
F2
F2'
F1'
F1'
F1
F3
F3
F3
A. Identification of fragments:
B: Fragment evolution:
C. Fragment linking:
Weak binders mM to 30M are

identified (e.g. F1-3)
-An initial fragment is optimized

(e.g. F1 to F1 and F2 to F2)
-Two ore more fragments,

which bind to proximal
parts of the active site, are
joined together
-very challenging
-D. C. Rees et al. Nature Rev. Drug Disc. 2004, 3, 660-672

-P. Hayduk et al. Nat. Rev. Drug Disc. 2007, 6, 211-219
winter semester 09
145

Dynamic Combinatorial Synthesis
COOH
COOH
neuraminidase
+
H 2N
R1
COOH
R2
NH 2
NHAc
N
R1
NH 2
NHAc
R2
COOH
NaCNBH 3
H 2O, pH 6
N
R1
NH 2
2 NHAc
HN
R1
NH2
NHAc
2
"amplifications"
COOH
COOH
HN
HN
NH2
NHAc
NH 2
NHAc
HO
amplification= 84
Ki = 700nM
amplification> 30
Ki = 85nM (strongest binder)
M. Hochgrtel et al. J. Med. Chem. 2003, 46, 356-8
winter semester 09
146

Dynamic Combinatorial Synthesis: Disulfide Thethering
SH
+
IL-2
disulfide
exchange
R1
S
S
R2
S S
binding stabilizes
disulfide
S S
R
IL-2
IL-2
C
S
best R series:
A, B, C: H, CO2H, CO2Me or MeO
B
A
improve design of a known inhibitor with tethering "hit"
Me
N
Cl
Cl
O
C
Me
N
R
Cl
Cl
B
improved inhibitors
IC5 0 = 0.2 M
existing inhibitor
IC50 = 3 M
J. A. Wells et al. Proc. Natl. Acad. Sci. USA 2000, 97, 9367-72; A. C. Brainsted et al. J. Am. Chem. Soc. 2003, 125, 3714-15
winter semester 09
147

Click Chemistry
Click Chemistry: Diverse chemical function from a few good reactions

H. C. Kolb, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004
Development of a set of powerful reactions for the rapid synthesis of

useful new compounds and combinatorial libraries through heteroatom
links (C-X-C); an approach called Click Chemistry.
Reactions that have a high thermodynamic driving force, usually greater
than 20 kcal/mol
-Cycloadditions ([1,3]-dipolar additions; Diels-Alder reactions)
-Nucleophilic Substitution reactions on strained heterocyclic
electrophiles
-Carbonyl Chemistry of the non-Aldol-type: synthesis of ureas, thioureas,
aromatic heterocycles, oxime ethers

-Addition reactions to C-C carbon multiple bonds: epoxidations, aziridinations,
dihydroxylations
winter semester 09
148

Click Chemistry
Nature
X
R1 -N=N=N-
:Nuc
Petroleum
O
R1
R2
R2
X: O, NR
N
N
XH
Nuc
R1
R3 XNH2
R1
O
N
N
N
R3
H
R4
R1
XR3
R2
H. C. Kolb, K. B. Sharpless, Drug Discovery Today 2003, 8, 1128-37
winter semester 09
149

Click Chemistry
[1,3]-Dipolar additions of acetylenes and azides
OH
N N N
N N N-
Cu (turnings)
(ca 1g)
HO
(10.0mMol)
+
Ph
H2 O/tBuO(2:1)
(50ml)
RT, 24h
CuSO4 (cat.)
(10Mol%)
Ph
N
N
OH
N N
HO
Ph
3.7g (95%)
white solid
(20.0mMol)
V. V. Rostovtsev et al. Angew. Chem. Int. Ed. 2002, 41, 2596
winter semester 09
150

Click Chemistry in Drug Discovery
O
N
O
O
O
O
O
O
O- O-
N H
N
N
HO
NH 2
OH
Cu (turnings)
(ca 1g)
N N
N
+
O
R
N
H
H2O/tBuO(2:1) H
(50ml)
RT, 24h
CuSO4(cat.)
(10Mol%)
N=N=Nn
O
N
H
N N
N
4
O
O-
O-
N H
N
N
HO
O
O
O-
N H
N
N
O-
NH 2
OH
O
O
O
O
HO
N
O
Ki: 62nM; inhibition of fucosyl transferase

cancer metastasis; lymphocyte trafficking
NH 2
OH
Lee et al. J. Am. Chem. Soc. 2003, 125, 9588-89
Dramatic rate acceleration of the azide-alkyne cycloaddition by sequestering the two components inside
the host structure (enzyme or receptor)
winter semester 09
151

-Emerging resistance in clinical isolates of bacteria render existing antibiotics such

as Neomycin and Ciprofloxaxin inactive
-Enzymes such as aminoglycoside 3-phsphotransferases inactivate 3 position in
aminoglycoside antibiotics by phosphorylation
-Combination of two antibiotics has emerged as a valuable strategy to overcome
rapid resistance mechanisms
winter semester 09
152

NH 2
N
N
N3
HO
HO H N
2
Y
H2N
NH2
HO
OH
O H2N
HOOC
[(CH 3CN)4Cu]PF6; 7% NEt3 in

H 2O; microwave irridiatio n 40s
(yields: 30-80%)
N
N
HOOC
NH 2
N N
X N
HO
HO H N
2
Y
HO
O
H2N
NH2
O
OH
O H2N
N
F
OH
O
OH
H2N
-biological activities (MICs) depended

significantly on the variable spacer
groups X and Y
-best combinations were X= -(CH2)2and Y= -CH 2OCH2-MIC (minimal inhibitory concentration):
E.coli (R477-100): 3g/ml
E.coli (ATCC 25922): 3g/ml
E.coli (AG100A): 0.38g/ml
B. subtilis (ATCC 6633): 0.75g/ml
H2N
O
OH
OH
O
Cipro-NeoB hybrids
winter semester 09
153

Azide 1
Alkynes
OMe
O
O
x
O S
O N
O
N3
N
H
OH
COOMe
N
N
H
OH
OH
O
N
H
COOMe
OMe
N
OMe
HIV-protease (SF-2), buffer, 23, 24h

OMe
O
O S
N
O
OH
N
N N
Ki = 1.7 nM
O
HN
HO
M. Whiting et al. Angew. Chem. Int. Ed. 2006, 45, 1435-39; K. B. Sharpless, R. Manetsch, Exp. Opin.Drug. Disc. 2006, 1(6), 525-38
winter semester 09
154

Summary of fragment-based approaches:

-fragment libraries are smaller: few hundreds to thousands
-screening effort smaller; however, weak binders have to be detectable
-leads derived from fragments are often smaller; allows more extensive
optimization
-fragments can be assembled in a thermodynamically or kinetically controlled
fashion: dynamic combinatorial synthesis
-fragments can be assembled using click chemistry
-finding the appropriate linkers to assemble fragments is a big challenge
winter semester 09
155

Reactions used for the introduction of high variation substituents
Couplage Suzuki:
Formation dàmides et dùres:

O
HV
OH
BB
NH2
BB
NH(R)R
BB
H
N
BB
H
N
Ar
BB
BB
Rduction au diborane:
RHV
O
BB
Amination rductrice:
N
H
RHV
HV
NHR
BB
Alkylation du groupe thiol:
O
HV
BB
BB
NH(R)R
R -SH
R2
Br
O
winter semester 09
base
R2
RS
O
156

2.5. Parallel reactions
Reactions used for the introduction of high variation substituents
Substitution nucleophillique:
Alkylation de NH activs:
R
R1
BB
BB
N
R2
R2
NH(R)RH V
N
H
Raction de Mitsunobu:
N
O
HV
R
Raction de Mannich:
R
R1
OH
R2
O
R2
R CHO
R1 N
OH
R
N
R2
OH
R
R1
N
H
N
H
O
R1
NH2
R
R1
N R1
R3
NH
O
R1
N3
R2
OH
R2
winter semester 09
O
3
R2
157

Questions
1. Please name five efficient reactions that can be used for

final parallel derivatization?
2. Please name potential advantages of fragment-based lead
discovery over screening large combinatorial libaries?
3. What is the rule of 3?
winter semester 09
158

Examples: various parallel extraction procedures
Extractions : principle
Liquid-liquid extractions
Solid-phase extractions
Solid-supported scavengers
Ion-exchange resins
Fluorous phase extractions
winter semester 09
159

Parallel work-up procedures: principle
1. Two phase extractions: manuel extraction
Upper phase: contains product (EtOAc or fluorous phase): separated manually

Lower phase: contains impurities (aqueous phase)
2. Two phase extractions: robotic system (style Tecan)
Upper phase: contains impurities (aqueous phase): separated by robot

Lower phase: contains product (CHCl3 or CH2 Cl2 ): dried and evaporated
winter semester 09
160

Parallel work-up strategies: liquid-liquid extractions
1. Two phase extractions: solubilize impurities in the aqueous phase

O
NHR1
H2 N
Br
R3
1. MeOH, 60
R3
R2
N
2. 4, 60
R HN
1
Me2N(CH)nNH 2
H
N
NH2
S
HOOC
3. aq.NaOH
CH2Cl 2
H
N
excess 2
HOOC
Products of type 5 are soluble in the basic aqueous phase

A. Chuchulowski, T. Masquelin, D. Obrecht, J. Stadlwieser, J.-M. Villalgordo, Chimia 1996, 50, 530
winter semester 09
161

Parallel work-up strategies: solid-phase extractions
Solid phase extractions/filtrations
Solid phase: one or several solid pahses are filled into a polypropylene syringe or
cartridge
Solid phases: SiO 2; Al 2O 3; `ìon exchange resins (basic, acidic and mixed bed)``;
Kieselguhr; MgSO4 ; polymre functionalis: -NH2 , -SH, -PPh2, COOH, CHO,
CH2 OH, isothioure, N3 ...;
The organic phases are passed through these cartridges in order to get rid of
impurities which are adsorbed onto the solid phase. They can be applied
manually or by a robotic system (Tecan)
winter semester 09
162

Parallel work-up strategies: solid-supported scavengers
R2-N=C=O
R1 -NHCONHR2
2
R -COCl
R1-NHCOR3
R -NH2
R4-SO2Cl
3
R1-NHSO2 R4
4
NH2
excess
NHCONHR
NHCOR
NHSO2R
winter semester 09
163

Parallel work-up strategies: solid-supported scavengers
Parallel synthesis in solution using polymer-bound reagents
P1
R
CbzHN
i
O
1)
COOH
P2 GP
N
H
R'
P1'
O
R
cysteine trap
Cl
R
R
CbzHN
H
N+
CbzHN
N2
DMF, -5
Br
O
R
R R NH
CbzHN
or
R 3SH
DCM, 18h
2) CH2 N2/DCM
85-90%
(5-10% methylester)
or
1 2
BrDMF, 1.5h, 25
-10, 1h
NR1 R2
CbzHN
SR3
O
80-85%
HN
HN
N
CbzHN
O
N
H
Ph
CbzHN
N. Y. Yadav-Bhatnagar, N. Desjonqures, J. Mauger, J. Comb. Chem. 2002, 4, 49-55

winter semester 09
164

Parallel work-up strategies: solid-supported scavengers; intermediate catch
Parallel synthesis in solution using polym er-bound reagents
COOH
R
("intermediate catch" or "resin capture"
N3
O
1
R NH2
R3
NHR
CHO
R1
N
O
O
N O
+N
N
R2
DCM, 0
R1
N
NHR5
N O
P
Ph Ph
PPh 3
R2
wash
toluene, 60
+
4
R N=C
R
R
N
O
NHR
R2
A. Chucholowski, D. Heinrich, B. Mathis, C. Mller, Generation of benzodiazepin and benzodiazocin libraries through resin capture
of Ugi-4CC, conference: 214th ACS national meeting, Las Vegas, 1997
winter semester 09
165

Parallel work-up strategies: fluorous phases
FP: fluorous phase; C6F 13 CH2 CH2 - or C10 F 21 CH2 CH2 -
FP
FP
liquid phase reactions
FP
+ excess reagents
Substrate
Products
Substrate
liquid-liquid extraction
1. cleavage
Products
FP
2. extraction
Products
winter semester 09
166

Multicomponent reactions: fluorous phase extraction
R 1NH2
COOH
i, ii
R 2CHO
(Rf)3Si
(Rf)3Si
N
R1
R2
R2
NHR3
iii, ii
N
R1
NHR3
O
R 3N=C
Rf: C10F21CH2CH2 i: TFE, 90, 48h; ii: liquid-liquid extraction; iii: Bu4 NF, THF, rt
A. Studer, S. Hadida, R. Ferritto, S.-Y. Kim, P . Jeger, P. Wipf, D. Curran, Science 1997, 275, 823
winter semester 09
167

Fluorous phase extraction: cleavage by cyclization
X
i
N
N
(Rf)3Si
(Rf)3Si
1
ii
N Me
N
H
(Rf)3Si
2
COOH
OTf-
Rf: C6F13CH2CH2-
iii
X
O
O
O
iv
N
H
(Rf)3Si
O
4
NH
N
N
H
i: MeOTf, CH2Cl2, 1,1,1-(trifluoromethyl)benzene(BTF); ii: anthranilic acid, DMAP, BTF, CH2Cl2; iii: TBTU, furfuryl amine, THF;iv: Et3 N
D. Schwinn, W . Bannwarth, Helv. Chim. Acta 2002, 85, 255
winter semester 09
168

6. Case studies
What are the prime biological targets?
-Kinases:
22%; market: 2 drugs
-GPCR:
15%;
: 30%
-Ion channels:
5%;
: 7%
-Ser proteases:
4%;
: 1 drug
-Phosphatases:
4%;
-Zn proteases:
2%;
: ACE inhibitors
-Nuclear receptors: 2%;
: 4%
-others*
: 44%;
*Many targets involving large surface protein-protein interactions
-despite the fact that kinases, CPCRs and ion channels constitute only about 42% of
all targets of therapeutic interest, the pharmaceutical industry is devoting about 90% of
their resources to those targets; it is believed that these targets can be adressed with
small molecules.
-The number of biologicals (antibodies, fusion proteins, peptides) reaching the market
is increasing. These molecules target mainly large surface protein-protein interactions
winter semester 09
169

6. Case studies
Targets hit by current drugs
Drugs, their targets and the nature and number of drug targets
P. Imming et al. Nature Rev. Drug Disc. 2006, 5, 821-34
1. Number of drug targets :
1997 : Drews et al. Nature Biotechnol. 1997, 15, 1318-19
-Marketed drugs hit 482 targets ; human genome suggests 100'000 proteins
2002: J. Burgess et al.
-after sequencing of human genome:
~8000 targets
~5000 hit by known drugs: 2400 by
antibodies; 800 by proteins
2002: A. Hopkins et al. Nature Rev. Drug Disc. 2002, 1, 727

-on the basis of ligand binding studies:
399 targets, which belong to 130 target families

~3000 targets amenable to small molecules
bottom line: 300-500 targets hit by current drugs; 3000-8000 drugable targets
winter semester 09
170

6. Case studies
Kinase inhibitors
-Recent reviews: A. J. Bridges, Chem. Rev. 2001, 101, 2541-2571; G. Scapin, Drug Disc.Today 2002, 77,
601-611; S. Orchard, Curr. Opin. Drug Disc. & Dev. 2002, 5, 713-717; D. Fabbro, C. Garcia-Echeverria,
Curr. Opin. Drug Disc. & Dev. 2002, 5, 701-712; S. K. Hanks, The FASEB J. 1995, 9, 576-596 (sequences of
kinases); M. E. M. Noble, J. A. Endicott, L. N. Johnson Science 2004, 303, 1800-5; J. Zhang; P. L. Yang; N.
S. Gray, Nat. Rev. Drug Discov. 2009, 9, 28-39 (Targeting cancer with small molecule kinase inhibitors);
-Three families of kinases:
-Serine-threonine kinases (S/TKs)

-Tyrosine kinases (TKs)
-Dual function kinases (DFKs)
-Roughly 2000 kinases known in the human genome

-Kinases phosphorylate serine, threonine
O and tyrosine and are ATP dependent
OH
TKs)
P O
O O-
* OH
ATP
ATP
phospatases
winter semester 09
TKs)
OO P *
O
O
phospatases
171

6. Case studies
Kinase inhibitors: 3D-structure of kinase domain
DDT 2002,77, 601-611
winter semester 09
172

6. Case studies
Nat. Rev. Drug Discov.

2009, 9, 28-39
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173

6. Case studies

2009, 9, 28-39
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174

6. Case studies

2009, 9, 28-39
winter semester 09
175

6. Case studies
2009, 9, 28-39
winter semester 09
176

6. Case studies
Kinase inhibitors on the market
winter semester 09
177

6. Case studies
CPCRs: introduction
50% of all drugs target G-Protein-Coupled Receptors (sales in 2001: ~50billion USD)
G-protein: guanin nucleotide-binding protein
-240 receptors with known ligands from which only ~30 are currently investigated by pharma
companies
-An additional 160 receptors with unknown ligands (orphan receptors) are known
Family 1: rhodopsin-like or adrenergic-like GPCRs
constitute the largest family; contain a short N-terminus and amino acid residues
in the trans-membrane domain are highly conserved
Family 2: glucagon receptor-like or secretin receptor-like GPCRs
Family 3: metabotropic glutamate receptors
Drug design strategies for targeting G-protein-coupled-receptors: Th. Klabunde, G. Hessler,
ChemBioChem 2002,3, 928-44.
3D-structure of bovine rhodopsin: Science, 2000, 289, 739-45; Biochemistry, 2001, 40, 7761-72.
winter semester 09
178

6. Case studies
CPCRs: introduction
G -protein-coupled receptors
Extracellular
-NH2
-S-S-
e1
TM1
TM2
e3
e2
TM3
TM4
TM5
TM6
TM7
i2
i1
Cytoplasmic
i3
COOH-
winter semester 09
179

6. Case studies
GPCRs: some best-selling drugs
H
N
S
Cl
N
H2 N
N
N
O
COOH
OEt
Claritin (Schering-Plough, H 1 antagonist

allergies, 3.1billion USD, 2001)
N N
N
HN
HO
HO
OH
N
H
COOH
Serevent (Glaxo,
1 agonist
asthma, 0.91 billion USD, 2001)
winter semester 09
Neurontin (Pfizer, GABA B-agonist

neurogenic pain, 2.35 billion USD, 2001)
Zyprexa (Ely Lilly, D2 /D1 /5-HT2

allergies, 2.35 billion USD, 2001)
Diovan (Novartis, AT1 antaginist

hypertension, 0.8 billion USD, 2001)
180

6. Case studies
Extracelluular ligand
gated channels
Intracellular ligand
gated channels
Voltage gated
channels
Extracellular ligand
gated channels
winter semester 09
Nicotinoid AChR
GABA
Glycine
5-HT3
CAMP
cGMP
CA ++
G-proteins
Na+
Ca ++
K+
AChR: Neuromuscular Disorder

300000 patients US
Hydroxytryptamin type
Migraine, depression
Not considered here
14 subtypes
>15 subtypes
35 subtypes
> 100
subtypes
50% not yet
charactarized
NMDA
AMPA
KAINATE
Na: Migraine, back pain,

34 mio. patients US
Ca: Hypertension
patients US, prostate cancer
K: MS, spinal cord injury
250000 patients US
NMDA: Brain ischimia, CNStrauma, epilepsy, huntington
disease
2.3 million patients
181

6. Case studies
Case study 1: Inhibitors of influenza endonuclease
Inhibitors of influenza endonuclease: collaboration between Roche and Polyphor Ltd
R1
R1
F
O
BocHN
winter semester 09
NO2
OH
OPiv
BocHN
N
OPiv
O
RHN
N
OH
182

6. Case studies
Disease and target
- Influenza infects an estimated 120 Mio. people in US, Europe and Japan in a typical year
-The influenza endonuclease is an attractive target for several reasons:
i: It is a key component of the viral transcription mechanism, which has no cellular counterparts and
should therefore provide a good potential for discovering selective, non-toxic drugs
ii: In contrast the neuraminidase inhibitors that do not prevent the formation of new virus particles, but interfere
with virus release from host cells and are therefore virustatic, endonuclease inhibitiors, due to the block of viral
transcription, are expected to have a virucidal effect.
winter semester 09
183

6. Case studies
N-Hydroxy-imides
Ketobutanoates
O
OH
N
OH
N
N
Merck: IC50 : 21.3M

O
H
N
N
O
OH
Flutimide: fungal methabolite
OH
OMe
OH
O
Roche: IC50 95
M
Merck: IC50 : 5.5M

F
O
N
Merck: IC50 : >500M

O
OH
OH
O
Roche: IC50 5M
OH
Roche: IC50 >1000M
N
O
OH
Merck: IC50: 0.9

M
OH
O
O
OH
H
N
OH
N
Ph
Roche: IC50 15M

OH
Roche: IC50 >500M
NH2
Cl
Roche: IC50 800M
Roche: IC50 : 0.43

M
winter semester 09
184

6. Case studies
N-Hydroxy-imides
Ketobutanoates
O
OH
OH
O
N
O
Merck: IC 50 : 21.3M
N
O
OH
Flutimide: fungal methabolite

Merck: IC50 : 5.5M
N-Hydroxy-tetramic acids
R1
R1
OH
R2
winter semester 09
N
OH
O
R2
OH
N
OH
185

6. Case studies
N-Hydroxy-2-indolinones
R1
R1
OH
N
OH
O
2
OH
N
OH
OH
O
N
OH
RHN
1
winter semester 09
186

6. Case studies
-N-hydroxy-2-indolinone derivatives 1 were not described in the literature

R1
OH
high variation site
RHN
1
N
OH
-keto amide moiety
O
hydroxamic acid moiety
-Molecules of type 1 bear two potentially reactive and labile functional groups
for which suitable protective groups have to be found
-Molecules of type 1 are acidic and polar and thus problems of isolation and purification
were anticipated; especially for a parallel approach
winter semester 09
187

6. Case studies
R1
OH
O
R3
O
N
OH
N
H
1A
R1
R1
OH
OH
O
O
N
OH
RHN
R1
R HN
OCOR2
O
N
OH
N
H
H 2N
1B
key precursors for parallel synthesis
R1
OH
O
O
5 S
R
N
H
O
N
OPG
PG: protective group
O
N
OH
1C
winter semester 09
188

6. Case studies
R1
COOH
OCOR2
O
H2N
O
N
OPG
N
OPiv
BocHN
2
BocHN
NO 2
4
key precursors for parallel synthesis
winter semester 09
189

6. Case studies
F
H2N
NO2
i
BocHN
NO2
7
CH(COOMe)2
ii
BocHN
NO 2
5
i: Boc2O, THF, 80; ii: CH2(COOMe)2, NaH, DMSO; iii: aq. NaOH, MeOH, reflux
winter semester 09
190

6. Case studies
problemes:
-partial reduction of nitro group
-cyclization
-isolation of hydroxamic acid
COOH
iv, v
BocHN
NO2
O
BocHN
N
OCOtBu
iv: Pt/C(5%),H2, DMSO, EtOH; then AcOH; v: tBuCOCl, DIPEA, CH2Cl2
winter semester 09
191

6. Case studies
O
R
R COX
(2.5 equiv.)
BocHN
O
ii
O
N
OPiv
O
N
OPiv
BocHN
BocHN
O
N
OPiv
9a (R = Me)
10a (R1= Me)
9b (R1= Et)
10b (R 1= Et)
9c (R = Ph)
i: 9a: X=OCOCH3; 9b: X=Cl; 9c: X=Cl, DMAP, DIPEA, CH2Cl 2, THF; 0-r.t.; ii: tBuCOCl,
tetrabutylammonium cyanide or NaCN, pyridine, CH 2Cl2
winter semester 09
192

6. Case studies
O
O
Me
Me
O
BocHN
10a
H2N
N
OPiv
2a
O
N
OPiv
O
i
O
BocHN
10b
N
OPiv
H2N
2b
O
N
OPiv
O
Ph
Ph
O
i
BocHN
9c
O
N
OPiv
H2N
O
N
2c OPiv
i: 4N HCl/dioxane or CF3 COOH/CH2 Cl2 ; then extraction with aq. sat. NaHCO 3 solution and CH2Cl2
winter semester 09
193

6. Case studies
R1
OCOR 2
O
i
R
N
H
11a
O
N
OPiv
R1
OCOR 2
R1
OCOR 2
O
ii
R HN
N
H
N
OPiv
H 2N
11b
2a (R 1= Me; R 2= tBu)
1
R1
OCOR 2
2b (R = Et; R = tBu)
iii
1
2c (R = R = Ph)
O
N
OPiv
O
S
N
H
O
N
11c OPiv
i: R 3COCl, pyridine (DIEA), DMAP, CH 2Cl2 ; ii: R4 N=C=O, CHCl3 (ethanol-free), 70; iii: R5 SO2 OCl, pyridine (DIEA), DMAP, CH 2Cl2
winter semester 09
194

6. Case studies
- out of the library of 131 compounds 26 had an IC50< 50M
Me
Ph
OH
OH
O
O
N
H
N
OH
O
S
N
H
O
N
OH
IC 50 = 48M
IC50= 9M; EC 50 = 21M

Ph
OH
O
HOOC
N
H
O
N
H
N
OH
IC 50 = 3
M; EC50= 6M
compounds are antiviral in cell cultures

winter semester 09
195

6. Case studies
-Based on a pharmacophor hypothesis, novel 1-hydroxy-indolin-2-ones were proposed

as inhibitors of influenza endonuclease
-A parallel synthesis was developed which allowed to synthesize a library 131 compounds
in significant quantities (6-71 mg) and high purities (75 - 99%) within 4 months
-From 131 compounds tested 26 had an IC50< 50M
-From 26 active compounds 10 showed a good antiviral activity in cell cultures
winter semester 09
196

6. Case studies
Case study 2: Structure-based Design
Inhibitors of Metalloproteinases: TACE; MMP1
-Ligand-based design capitalizes on the presence of existing structural similarities between

a set of compounds and the active ligand (also pharmacophore-based drug design)
-Using a solid-phase, parallel synthesis approach optimization could be achieved efficiently
-TNF-converting enzyme (TACE) represents an attractive target for reducing circulating levels
of the proinflammatory protein tumor necrosis factor alpha (TNF-).
-TACE belongs to the Zn-metalloproteinases. The hydroxamic acid moiety chelates to the Znatom located in the active site
winter semester 09
197

6. Case studies
Case study 2: Structure-based Design
Inhibitors of Metalloproteinases: TACE; MMP1
M. Abou-Gharbia, J. Med. Chem. 2009, 52, 2-9

winter semester 09
198

6. Case studies
Case study 3: Parallel synthesis of analogues of antibiotic GE2270 A
Parallel synthesis starting from a natural product-derived building block
O
O
N
S
CONH 2
OH
O
N
S
N
N
N
N
HN
OH
O
HN
O
HN
N
O
NH
NH
N
O
O
HN
MeHN
O
MeO
OH
O
NH
NH
N
O
S
MeHN
MeO
GE2270 A
1
inhibitor of elongation factor EF-TU
active against many gram positive pathogens

MIC 0.06-1.0 g/ml; low solubility in aqueous solvents
J. W. Jacobs et al. (Versicor), 40th annual ICAAC conference, Toronto, Canada, september 17-20th, 2000, Poster 2193 and 2194
winter semester 09
199

6. Case studies
NO2
F
OH
O
S
F
S
N
R
4
NO2
NO2
O
O
N
R
F
O
HN
OH
O
O
HN
O
NH
NH
MeHN
MeO
N
H
N
O
N
R
1
winter semester 09
200

6. Case studies
GE2270 A
F
O
O
S
F
R R NH
R :
COOH
R =H
1
R :
O
O
1
R R NH
R1
N
2
R
R :
COOH
0.5
N
1
0.91
R =H
COOH
0.5
OH
R =Me
NO2
<0.0001
N
R1
1 2
Solubility
(mg/ml)
R:
COOH
0.73
R =Me
winter semester 09
201

6. Case studies
O
O
N
S
CONH2
OH
O
N
S
N
N
N
HN
O
HN
N
O
OH
O
NH
NH
HN
NH
NH
MeHN
HO
OH
O
O
HN
N
O
MeHN
HO
GE2270 D2
winter semester 09
202

6. Case studies
F
OH
O
S
F
OF
N
N
DCC, Pfp
N
HN
O
HN
O
NH
NH
OH
O
N
O
HN
O
HN
OH
O
NH
NH
N
O
HO
MeHN
N
O
MeHN
HO
winter semester 09
203

6. Case studies
F
Solubility
(mg/ml)
NHR1
OF
S
N
N
NH
NH
N
O
S
2
RS
HO
9
R1 :
0.41
R2 = SCH3
COOH
R1 :
>2.0
MeHN
MeHN
OH
O
NH
NH
N
HN
COOH
HN
0.44
R = SCH3
O
N
OH
O
COOH
HN
R1 :
HN
N
N
<0.0001
GE2270 A
R =
COOH
10
i:R1NH 2, DMF, DIEA; ii: H2O; then precipitation + wash; iii: Ts2 O; CH2 Cl2; DIEA; iv: R2SH, DMF/aq. K 2 CO3; then precipitation + wash;
v: TFA/CH2 Cl2 (1:1); Et2O; then precipitation + wash; then dry
winter semester 09
204

6. Case studies
COOH
O
O
N
S
HN
CONH2
O
S
N
N
S
HN
O
N
O
OH
O
NH
NH
MIC(MRSA)
(g/ml)
OH
12
<0.0001
0.125
>2.0
0.5
O
HN
MeHN
N
HN
NH
NH
GE2270 A
O
HN
Solubility
(mg/ml)
N
N
N
O
MeHN
MeO
GE2270 A
S
12
COOH
winter semester 09
205

6. Case studies
Case study 4: Parallel synthesis based on Natural Products
-Rapamycin is a immunosuppressant natural product, which has two binding sites. It binds
to the FKBP domain and to mTOR (kinase) effector domain. Besides its immunosuppressant
activity the synthetic analogue torisel shows potent anti-tumor activity and is used for treatment
of renal carcinoma. Torisel was obtained through a parallel synthesis approach from rapamycin.
winter semester 09
206

6. Case studies
-Using a parallel synthesis approach starting from the natural product rapamycin, the alcohol
group was derivatized with various different substituents.
winter semester 09
207

6. Case studies
-ILS-920 is a semi-synthetic rapamycin derivative which lacks the immunosuppressant activity

but shows neuroprotective properties. It shows good brain penetration.
winter semester 09
208

6. Case studies
Case study 5: kinase inhibitors
-Recent reviews: A. J. Bridges, Chem. Rev. 2001, 101, 2541-2571; G. Scapin, Drug Disc.Today 2002, 77,
601-611; S. Orchard, Curr. Opin. Drug Disc. & Dev. 2002, 5, 713-717; D. Fabbro, C. Garcia-Echeverria,
Curr. Opin. Drug Disc. & Dev. 2002, 5, 701-712; S. K. Hanks, The FASEB J. 1995, 9, 576-596 (sequences of
kinases)
-Three families of kinases:
Serine-threonine kinases (S/TKs)

Tyrosine kinases (TKs)
Dual function kinases (DFKs)
involved in cell signaling pathways
-Roughly 2000 kinases known in the human genome

-Kinases phosphorylate serine, threonine and tyrosine and are ATP dependent
OH
TKs)
O
P O
O OR
* OH
ATP
ATP
phospatases
winter semester 09
TKs)
OO P *
O
O
phospatases
209

6. Case studies
Case study 5: kinase inhibitors
-Some important kinases:
protein kinase C (PKC: 12 isoforms); PKC: involved in cell

proliferation; target in cancer therapy
Growth factor receptor kinases: EGF (epidermal groth factor);
TGF (transforming growth factor); PDGF (platelet-derived
growth factor); VEGF (vascular endothelial growth factor); TNF (tumor
necrosis factor); NGF (nerve growth factor)
MAP-kinase (mitogen activated kinase); CDKs (cyclin-dependent
kinases); JAKs (Janus family of TKs); Abl (Abelson TK); targets in
cancer and inflammation; and many more
some early natural
product leads
H
N
MeOOC
OHO O
Ph
O
OH
NH
N
H
N
Me
HO
OMe
NHMe
Staurosoporine (IC50(PKC): 2.5nM)
(non-specific to other PKC-isoforms)
winter semester 09
N
N
H
OH O
O
O
COOMe
Olomoucine (IC50(CDK): 4.5M)
OH
Bryostatin 1
210

6. Case studies
Case study 5: parallel synthesis of olomoucine analogues
NH 2
Ph
NH
N
building block synthesis

N
Cl
HO
N
N
N
H
Olomoucine (IC50 (CDK): 4.5M)
N
H 2N
Cl
N
P. G. Schultz et al. Tetrahedron Lett. 1997, 38, 1161
NH
N
N
H
ii, iii
N
H
N
F
N
H
iv
H
N
MeO
OHC
HN
O
HN
MeO
NH 2
L: PAL-linker
NH
N
F
NH
N
N
H
parallel synthesis
NH
N
R1
vi, vii
N
R2 HN
N
R1
i: 0.3M NaNO2, HBF4 (48% in H2O), -15; ii: 4-nitro-benzylamine hydrochloride, DIEA, n-BuOH, 50; iii: H2, Pd/C; iv: NaBH(OAc)3,
1% AcO H, DMF; v: R1-OH, PPh3, DEAD, CH 2Cl2/TFA (1:1); vi: R 2-NH2, n-BuOH/DMSO (4:1), 90-100, 48h; vii: TFA/H2O/Me2S (90:5:5)
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6. Case studies
Case study 6: Orexin receptor (OX1 and OX2) antagonists
-Orexin receptor 1 (OX1) and orexin receptor 2 (OX2) belong to the GPCRs and are
believed to modulate appetite, food intake and sleep.
-The two endogenous neuropeptides orexin A and B bind to both OX1 and OX2:
orexin A: IC50=20nM (OX1); IC50=36nM (OX2)
orexin B: IC50=420nM (OX1); IC50=38nM (OX2)
-Actelion initiated a program in developing orexin antagonists as modulators of sleep
-HTS delivered a hit compound 1 which served as starting point for several follow-up
libraries of type 2.
R4
MeO
MeO
MeO
O
N
O
N
R6
N
H
R7
MeO
R8
N
R2
R1
R3
2
R. Koberstein et al. Chimia 2003, 57, 270-275.

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6. Case studies
Library 1
O
1)
MeO
Br
Br
MeO
NH
MeO
MeO
2) DIEA; NHR 1R 2
MeO
O
N
MeO
MeO
MeO
MeO
MeO
R2
MeO
N
H
OMe
MeO
>100 analogues
IC 50 =19nM (OX1); 101nM (OX2)
Library 2
O
X
R3
+
MeO
X: Cl, OH
POCl3
MeO
NH2
NaBH 4
MeO
MeO
NH
MeO
MeO
O
N
R
R
R1
N
2

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6. Case studies
Library 2
MeO
MeO
MeO
Library 3
O
N
MeO
X
N
H
N
H
Y
X=H; Y=NMe2 : IC50=45nM (OX1); 1536nM (OX2)
X=Y=F: IC50=1906nM (OX1); 19nM (OX2)
IC50 =18nM (OX1); 1161nM (OX2)
-Substituent R8 can modulate the selectivity towards OX1 and OX2

-OX2 specific compounds can be made

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7. Appendix (Definitions; Reviews; Literature
Some useful definitions in medicinal chemistry
EC50:
effective dose for a 50% of maximal response
Dose:
in mg/kg: mg of compound per kg of body weight; e.g. 1mg in a 25g mouse is the equivalent
of 2g dose in a 50kg (small) adult.
SAR:
structure activity relashionship. Correlation between chemical structure and biological activity.
Phase I:
In phase I clinical trials a compound is dosed to healthy volunteers and three main questions
are asked:
Is the compound safe at the proposed dose?
What are the limiting side effects likely to be?
How long does the compound stay in the system?
1.
2.
3.
Phase II:
Phase II clinical trials aim at showing efficacy of the compound in a sample of patients having a
particular disease. If there are signs that the compound is active enough it can be promoted to
next phase.
Phase III:
Phase III clinical studies are big and comprise many patients. The key issues are the following:
How well does the drug work?
What are its side effects at the proposed efficacy doses?
What kind of a dosing schedule is optimal?
How does it interact , favorably or unfavorably, with other drugs for the same or related conditions?
Success:
At least 25000 compounds have to be made in order to get one drug expenses are around
500 million USD with a lead time of 7-10 years.
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Some useful definitions in medicinal chemistry
Targets:
Up to now only about 200 discrete molecular targets have been explored. Around 50% of these
belong to the GPCRs (e.g. histamine, dopamine or serotonin receptors). With decoding of the
human genome it is believed that 30000 targets will be unveiled.
Protein structure:
-primary sequence: genomics
-sequence alignment with known proteins: conserved residues are characteristic for function
-gene knockout can reveal importance of a target for a certain disease
-expression and purification
-3D structural determination by X-ray or NMR techniques
-mutagensis studies (site directed mutagenesis) can reveal important residues in receptors or ligands
Protein kinases:
transfer the g phosphate of ATP to side chain hydroxyls of substrate proteins.

It is estimated that about 2000 kinases exist in the human genome
Serine/threonin kinases (S/TKs)
Tyrosine kinases (TKs)
Dual function kinases (DFKs)
Protein phosphatases:
cleave phosphate groups from substrate proteins
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ADMET: Adsorption, Distribution, Metabolism, Elimination and Toxicity
ADMET:
Adsorption, Distribution, Metabolism, Excretion(Elimination) and Toxicity
In vitro ADMET experiments:

-Cytotoxicity assay on different cancer cell lines
-Stability in plasma: rodents (mouse, rat), human
-Caco 2 cell passage of compounds: indicator for oral absorption
-Passage of compounds through artificial membranes (PAMPA)
-Metabolism studies in liver microsomes: first pass metabolism
-Protein binding (binding to serum albumin): indicates availability of compound in plasma
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2. Target classes: -2.1. Enzymes

-2.2. Substrates, metabolites and proteins
-2.3. Receptors
-2.4. Ion channels
-2.5. Transporter proteins
-2.6. DNA/RNA and the ribosome
-2.7. Targets of monoclonal antibodies
-2.8. various
-2.9. unknown
2.1. Enzymes:
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-Oxidoreductases (e.g. MAO-B, aromatases etc.)

-Transferases (kinases, phosphatases, DNA polymerases etc.)
-Hydrolases (serine proteases, metalloproteases etc.)
-Lyases (DOPA decarboxylase, carbonic anhydrase etc.)
-Isomerases ((DNA gyrases, topoisomerases etc.)
-Ligases (dehydropteroate synthase, mTOR etc.)
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2.3. Receptors:
2.4. Ion channels:
-Direct ligand-gated ion channels (GABAA, acetylcholine R,

glutamate R)
-GPCRs (class 1, class 2 (secretin-like), others)
-Cytokine receptors
-Integrin receptors
-Receptors associated with TK
-Nuclear receptors
-Voltage-gated Ca2+ channels (L- and K-type)
-K + channels (epithelial, voltage-gated)
-Na+ channels (epithelial voltage-gated)
-RIR-CaC
-TRP-CC
-Cl- channels
2.5. Transporter proteins:
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-Cation-chloride cotransporter (CCC)

-Na+ /H+ antiporters
-Proton pumps
-Eukariotic sterol transporters
-Neurotransmitter/ Na+ symporter
-Noradrenalin/Na+ symporter
-Dopamine/Na+ symporter
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2.6. DNA/RNA and the ribosome:

-Nucleic acids
-RNA (16S-rRNA; 23S-rRNA)
-Spindle (tubulin, kinesins)
-Ribosome (30S subunit; 50S subunit)
2.7. Targets of monoclonal antibodies:
-Vascular endothelial factor (VEGF; e.g.
bevazizumab; Avastin)
-Lymphocyte function-associated receptor
(LFA-1; efalizumab)
-Epidermal growth factor receptor (EGFR)
(e.g. cetuximab)
-h-EGFR-2 (e.g. trastzumab; Herceptin)
-Immunoglobulin E (IgE; e.g. omalizumab;
Xolair)
-CD-3
-CD-20 (Rituximab; Mabthera)
-CD-33 (Gemtuzumab))
-CD-52 (Alemtuzumab)
-TNF(Adalimumab; infliximab; Enbrel)
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G-Protein Coupled Receptors (GPCRs):

-Acetylcholin receptors (muscarinic receptors;
MCR 1-4)
-Adenosin receptors
-Adrenoreceptors (1, 2, 1)
-Angiotensin receptors
-Calcium-sensing receptors
-Cannabinoid receptors (CB1, CB2)
-Cysteinyl-leukotriene receptors
-Dopamine receptors
-Endothelin receptors
-GABAB recptors
-Glucagon receptors
-Glucagon-like peptide-1 receptor (GLP-1R)
-Histamin receptors (H1, H2)
-Opioid receptors (, ,
)
-Neurokinin receptors (NK1, NK2, NK3)
-Prostanoid receptors
-Prostamide receptors
-Purinergic receptors
-Serotonin receptors (5-HT1A, 5-HT1B/1C,
5-HT2a, 5-HT3, 5-HT4)
-Vasopressin receptors (V1, V2, OT)
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Cytokine receptors:
Integrin receptors:
-Growth hormone receptor

-Erythropoetin receptor (EPO)
-Granulocyte colony stimulating factor receptor (G
-Interleukin-1 receptor (IL-1R)
-Interleukin-2 receptor (IL-2R)
-Tumour necrosis factor (TNF)
-Glycoprotein IIb/IIIa receptor (GPIIb/IIIa)
Receptors associated with TK:

Nuclear receptors:
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-Insulin receptor
-Mineralcorticoid receptor
-Glucocorticoid receptor
-Progesteron receptor
-Oestrogen receptor
-Androgen receptor
-Vitamin D receptor
-ACTH receptor
-Retinoic acid receptor (RXR)
-Peroxisome-proliferator-activated receptors (PPAR;
-Thyroid hormone receptor
222

7. Appendix (Definitions; Reviews; Literature)
4. Appendix-Reviews
-Applications of Combinatorial Technologies to Drug DIscovery. 1. Background and Peptide Combinatorial Libraries.
M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon, J. Med Chem. 1994, 37, 1233
-Applications of Combinatorial Technologies to Drug DIscovery. 2. Combinatorial Organic Synthesis, Library Screening
Strategies, and Future Directions. M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon,
J. Med Chem. 1994, 37, 1385
-Combinatorial Libraries. Synthesis, Screening and Application Potential. R. Cortese (Ed.), W. De Gruyter, Berlin (1995)
-Combinatorial Peptide and Nonpeptide Libraries. G. Jung (Ed.), VCH, Weinheim (1996)
-Kombinatorische Synthese. K. Frobel, T. Krmer, Chemie in unserer Zeit 1996, 30, 270
-Organic synthesis on solid phase. J. S. Frchtel, G. Jung, Angew. Chem. Int. Ed. Engl. 1996, 35, 17
-Combinatorial synthesis of small-molecular-weight organic compounds. F. Balkenhohl, C. Bussche-Hnnefeld,
A. Lansky, C. Zechel, Angew. Chem. 1996, 108, 2436
-Solid-phase organic reactions: a review of recent literature: P. H. H. Hermkens, H. C. J. Ottenhejm, D. Rees,
Tetrahedron 1996, 52, 4527
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4. Appendix-Reviews
-Synthesis and application of small molecule libraries: L. A. Thompson, J. A: Ellman, Chem. Rev. 1996, 29, 132
-Strategy and tactics in combinatorial organic synthesis. Applications to drug discovery. E. M. Goron, M. A. Gallop,
D. V. Patel, Acc. Chem. Rev. 1996, 29, 144
-Design, synthesis and evaluation of small-molecule libraries. J. A. Ellman, Acc. Chem. Res. 1996, 29, 132
-Multiple-component condensation startegies for combinatorial library synthesis: R. W. Armstrong, A. P. Combs,

S. D. Brown, T. A. Keating, Acc. Chem. Res. 1996, 29, 123
-Combinatorial organic synthesis using Parke-Daviess DIVERSOMER method: S. Hobbs-DeWitt, A. W. Czarnik,
Acc. Chem. Res. 1996, 29, 114
-Combinatorial Chemistry, Synthesis and Application. S. Wilson, A. W. Czarnik, Wiley 1997
-The current status of heterocyclic combinatorial libraries: A. Nefzi, J. M. Ostresh, R. A. Houghthen, Chem. Rev.
1997, 97, 449
-Organic synthesis on soluble polymer supports: Liquid phase methodologies: D. J. Gravert, K. D. Janda, Chem. Rev.
1997, 53, 5643
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4. Appendix-Reviews
-Synthesis and application of small molecule libraries: L. A. Thompson, J. A: Ellman, Chem. Rev. 1996, 29, 132
-Recent developments in soliud-phase organic synthesis: R. Brown, Contemporary Organic Synthesis 1997, 4, 216
-Solid-PhaseOrganic Reactions II. A Review of the Recent Literature. P. H. H. Hermkens, H. C. J. Ottenheijm,
D. C. Rees, Tetrahedron 1997, 53, 5643
-Functionalized polymers: Recent developments and new applications in synthetic organic chemistry: S. J. Shuttleworth,
S. M. Allin, P. K. Sharma, Synthesis 1997, 1217.
-Functionalized resins and linkers for solid-phase synthesis of small molecules: C. Blackburn, F. Albericio, S. A. Kates,
Drugs of the Future 1997, 22, 1007
-Solid supported combinatorial and parallel synthesis of small-molecular-weight compound libraries: D. Obrecht, J. -M.
Villalgordo, Tetrahedron Organic Chemistry Series, Vol 17, Pergamon, 1998.
Very recent reviews:
-Combinatorial carbohydrate chemistry: L. A. Marcaurelle, P. H. Seeburger, Curr. Opinion Chem. Biol. 2002, 6, 289-296
-Combinatorial synthesis of natural products: J. Nielsen, Curr. Opinion Chem. Biol. 2002, 6, 297-305
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4. Appendix-Reviews
-Combinatorial synthesis of natural products: J. Nielsen, Curr. Opinion Chem. Biol. 2002, 6, 297-305
-Recent advances in isocyanide-based multicomponent chemsitry: A. Dmling, Curr. Opinion Chem. Biol. 2002, 6,
306-313
-High speed combinatorial synthesis utilizing microwave irridiation: C. O. Kappe, Curr. Opinion Chem. Biol. 2002, 6,
314-320
-Applications of parallel synthesis to lead optimization: M. Altorfer, Ph. Ermert, J. Fssler, S. Farooq, E. Hillesheim,
A. Jeanguenat, K. Klumpp, P. Maienfisch, J. A. Martin, J. H. Merrett, K. E. B. Parkes, J. P. Obrecht, Th. Pitterna,
D. Obrecht, Chimia 2003, 57, 262-269
-Versatile monitoring tools in parallel solid-phase synthesis: E. R. Felder, K. Martina, S. Scarpella, M. Tato, Chimia
2003, 57, 229-236.
-The analytical challenge: Keeping pace with combinatorial chemistry: D. B. Kassel, P. L. Myers, Pharmaceutical News
2002, 9, 171-177.
-Synthetic aspects of combinatorial chemistry: P. Wipf, Pharmaceutical News 2002, 9, 157-169.
-Multicomponent reactions: emerging chemistry in drug discovery from xylocain to crixivan: Ch. Hulme, V. Gore,
Curr. Med. Chem. 2003, 10, 51-80
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4. Appendix-Reviews
-Drug design strategies for targeting G-protein-coupled-receptors: Th. Klabunde, G. Hessler, ChemBioChem 2002,
3, 928-44.
-Protein kinase inhibitors from the urea class: J. Dumas, Curr. Opin. Drug Disc. 2002, 5, 718-27.
-Inhibitors of the JNK signaling pathway: S. J. Harper, P. LoGrasso, Drugs of the Future 2001, 26, 957-73.
-Inhibitors of growth factor receptor kinase-dependent signaling pathways in anticancer therapy-clinical progress:
P. A. Renhowe, Curr. Opin. Drug Disc. 2002, 5, 214-224.
-Kinases as targets: prospects for chronic therapies: S. Orchard, Curr. Opin. Drug Disc. 2002, 5, 713-727.
-Current progress on farnesyl protein transferase inhibitors: S. B. Singh, R. B. Lingham, Curr. Opin. Drug Disc. 2002,
5, 225-44.
-Medicinal chemistry of target family-directed masterkeys: G. Mller, Drug Disc. Today 2003, 8, 681-91.
-Topics in drug design and discovery: Chapter 26. Privileged structures-an update: A. A. Patchett, R. P. Nargund,
Ann. Rep. Med. Chem. 2000, by Academic Press.
-Drugs, leads and drug-likeness: an analysis of some recently launched drugs: J. R. Proudfoot, Bioorg. Med. Chem.
Lett. 2002, 12, 1647-50.
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4. Appendix-Reviews
-Protein kinase inhibitors: insights into drug design from structure: M. M. Noble, J. A. Endicott, L. N. Johnson,
Science, 2004, 303, 1800-5.
-Small-molecule inhibitors of protein-protein interactions: progressing towards the dream: M. R. Arkin, J. A. Wells,
Nature Reviews Drug Discovery 2004, 3, 301-17.
-NMR in drug discovery: M. Pellecchia, D. S. Sem, K. Wthrich, Nature Reviews Drug Discovery 2002, 1, 211-19.
-Chemical inhibitors of Protein Kinases: A. J. Bridges, Chem. Rev. 2001, 101, 2541-2571.
-Fragment-based lead discovery: D. C. Rees, M. Congreeve, W. Murray, R. Carr, Nature Rev. Drug Disc. 2004, 3,
660-72
-Persuing the leadlikeness concept in pharmaceutical research: M. M. Hann, T. I. Oprea, Curr. Opin. Chem. Biol.
2004, 8, 255-63.
-Design and synthesis of of protein superfamily-targeted chemical lbraries for lead identification and optimization,
S. J. Shuttleworth, R. V. Connors, J. Fu, J. Liu, M. E. Lizarzaburu, W. Qiu, R. Sharma, M. Wanska, A. J. Zhang,
Curr. Med. Chem. 2005, 12, 1239-81.
-Receptor-assisted Combinatorial Chemistry: Thermodynamics and Kinetics in Drug Discovery,
J. D. Cheeseman, A. D. Corbett, J. L. Gleeson, and R. J. Katlauskas, Chem. Eur. J. 2005, 11, 1708-16.
-A decade of fragment-based drug design: Strategic advances and lessons learned,
P. Hayduk, J. Greer, Nature Rev. Drug Disc. 2007, 6, 211-19.
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4. Appendix-Reviews
-High throughput Lead Optimization in Drug Discovery; Ed. T.Kshirsagar,CRC Press, Taylo&Francis Group,
2008.
-Discovery of innovative small molecule therapies; M. Abou-Garbia, J. Med. Chem. 2009, 52, 2-9.
-Transforming fragments into candidates; D. E. De Kloe et al. Drug Discov. Today 2009, 14, 630-646.
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Medicinal Chemistry: Combinatorial Chemistry-Parallel Synthesis

1. Introduction: The Drug Discovery Process

3. Combinatorial and Parallel Synthesis in Medicinal Chemistry

4. Combinatorial Synthesis of Biopolymers

Daniel Obrecht, Polyphor Ltd