Reaction Processing

1. Introduction, basic concepts, and strategies

1.1. The origin of combinatorial chemistry
1.2. Historical landmarks in combinatorial chemistry
1.3. Combinatorial chemistry in the Drug Discovery process
1.3.1. The four key elements of lead finding
1.3.2. The drug discovery process
1.3.3. Sources for novel lead compounds
1.4. Generation of compound libraries
1.5. The Diversity concept (library design)
1.6. Synthetic strategies in combinatorial chemistry
1.6.1. Solution-phase synthesis or solid-phase synthesis
1.6.2. Parallel synthesis or combinatorial synthesis (“split-mixed”)
1.6.2.1. Deconvolution techniques

1
1. Introduction, basic concepts, and strategies
Solid-Phase Polymer-Assisted
Organic Synthesis Solution-Phase Synthesis
(SPOS) (PASPS)
Combinatorial Chemistry
Combinatorial chemistry embraces a diversity of
methodologies, which allow for the easy and fast
synthesis of many compounds avoiding the

Fluorous Synthesis classical methods of purification Tag-Assisted

(chromatography, crystallization, distillation, Synthesis
aqueous work-up) of each compound

“The words Combinatorial Chemistry have different meanings to different people, ranging from split
and mix strategies to parallel synthesis using robots, and embracing the whole range of preparative
chemistry from organic molecules, to catalyst ligands, and even inorganic solids. All of these activities
have in common an attempt to expand the diversity of structure available to the chemist as well as
the access to this diversity, permitting the discovery of new and valuable biological and material
properties.”......... Sir Jack E. Baldwin

MacLean, D. et al. Glossary of Terms Used in Combinatorial Chemistry, J. 2

Comb. Chem., 2000, 2, 562.
1.1. The origin of combinatorial chemistry
Ø In 1963 R. B. Merrifield demonstrated the potential of the solid-phase approach in the synthesis
of polypeptides.

substrate sixties
Linker X Linker substrate
anchoring

elaboration on solid-
phase using an excess
Linker + product 1 of reagents

or Linker product 1
cleavage
purification: filtration and
+ 2
product washing of the resin

Ø Organic and medicinal chemists, however, were more interested in the synthesis of complex natural
products of pharmaceutical relevance and their analogues.

seventies

taxol 3
 Ø With the aim to find more active drugs (drug
discovery), biologists developed high throughput
screening (HTS) methods to evaluate the biological
activity of thousands of compounds per day.
Therefore, organic and medicinal chemists were
forced to elaborate a new synthetic approach for
the synthesis of libraries of compounds by
combinatorial strategies.

Ø A compound library is defined as a collection of a eighties

number (10-100000) of compounds displaying the

same basic structure (scaffold) but a different
functionalization.

Ø In the eighties, combinatorial chemistry mainly

involved the solid-phase synthesis of polypeptides
and oligonucleotides through a linear assembly
strategy.

4
linear assembly strategy
Ø In the nineties, chemists and biologists discovered the potential of small-molecular-
weight-compounds to interact with a variety of unrelated molecular targets, thus
considering them valuable candidate for the identification of new leads.

Ø With the basic idea that “the highest is the number of synthesized compounds, the highest
is the probability to discover a new lead compound”, medicinal chemists were used to
prepare large libraries of compounds (102-104) through a random design. nineties

Ø In the same years, organic chemists developed combinatorial strategies alternative to the
solid-phase organic synthesis (SPOS) and characterized by the use of an organic solvent
for the solubilization of the starting substrates.

Ø The most import methodologies are the following:

• polymer-assisted solution-phase synthesis, PASPS

• fluorous synthesis

• Tag-assisted synthesis
solid-phase solution-phase

substrate (SPOS) reagent (PASPS)

reagent
substrate product
F substrate (fluorous synthesis)

(Tag-assisted 5
Tag substrate
synthesis)
Ø Following the failure of an increase of marketed drugs by the random design approach
(screening of thousands of compounds), medicinal chemists understood the necessity new century
of a rational design for the synthesis of focused libraries. These are meant as and the
collections of valuable and more complex compounds having a higher possibility to present
become a drug. A drug-like molecule should satisfy well defined parameters about its
adsorption, distribution, metabolism, excretion, and toxicity in vivo (ADMET profile)

1.2. Historical landmarks in combinatorial chemistry

1997: Use of fluorinated solvents for liquid-liquid phase extractions by Curran
(Science, 1997, 275, 283)

2003: Automated synthesis of oligosaccharides by Seeberger (Helv. Chim. Acta

2003, 86, 3975).

2003: Glycoproteins library generation by Wong (Curr. Opin. Struc. Biol.

2003, 13, 637.

7
1.3. Combinatorial chemistry in the Drug Discovery process

Ø The main advantage of the combinatorial approach in the drug discovery process is the possibility of
synthesizing many compounds in a short time thanks to the effective purification strategies adopted.

1.3.1. The four key elements of lead finding

8
1.3.2. The drug discovery process

9
1.3.3. Sources for novel lead compounds

10
1.4. Generation of compound libraries

Ø A1-3 (A1, A2, A3), B1-3 (B1, B2, B3),

e C1-3 (C1, C2, C3).

Ø Heterocyclic compounds are valuable

candidates for the identification of
new leads because of the reliability
of the structure-activity-
relationship (SAR) data resulting
from the screening of this class of
molecules.

11
1.5. The Diversity concept (library design)
Ø In a typical combinatorial approach, the reactants displaying the same reactive functionality but
different groups (R1, R2, R3..) are combined together to yield a basic structure, named scaffold,
which is in common with all the elements of the synthesized library.
R3
O R3 O R3
O
H O
R1O NH R1O NH
R1O + NH2 +
R2 N O R2 N O
R2 O H2N O H H

Scaffold: dihydropyrimidinone (DHPM) ring

structural
diversity

DIVERSITY

stereochemical
diversity

Ø Structural diversity: molecular variety of the library given by the different substituents installed
on the reactive substrates. If the scaffold displays further reactive functionalities (e.g. ester
group in the above example), a “library-from-library” approach can be envisaged.

Ø Stereochemical diversity: molecular variety given by the different stereochemistry of substituents

12
on the scaffold.
Ø The molecular variety of a library can be “measured” by evaluating some key parameters,
named descriptors, such as the molecular weight, shape, flexibility, lipophilicity, and
charge distribution of each compound constituting the library.

Ø The design of a compound library can be based on the concept of chemical space. This is the
system with n dimension having as variables the n descriptors which are intended to be
considered in the synthetic plan.

solubility

lipophilicity
molecular
weight

13
Ø A given synthetic strategy may ”fill” the chemical space in three different ways:

chemical space diversity

target-oriented One point low

synthesis (TOS) (target product)

synthetic combinatorial a limited zone medium

strategy synthesis (focused library)

diversity-oriented a wide zone

synthesis (DOS) (large library) high

Schreiberg, S. et al. A planning strategy for diversity-oriented

synthesis, Angew. Chem. Int. Ed. 2004, 43, 46-58. 14
Synthesis of focused librares

Ø In the last years, some important guidelines have been emerged for the design of focused
libraries.

Ø Professor Lipinski from Pfizer collected all data regarding the physico-chemical properties of
more than 50000 molecules tested in industries and universities. From a statistical analysis of
the resulting data-base, it emerged the rule of five which says that:

RULE
Low permeation and absorption of a given molecule are likely occurring when:
OF

1. There are more than 5 hydrogen-bonding donors (sum of NH e OH) FIVE

2. The molecular weight is higher than 500

3. Log P is higher than 5 (P is a parameter related to the lipophilicity of the molecule)
4. There are more than 10 hydrogen-bonding acceptors (sum of N and O atoms)

Lipinski, A. et al. Experimental and computational

approaches to estimate solubility and permeability in drug
discovery and development settings, Advanced Drug
Delivery Reviews, 1997, 23,3.
15
Ø The importance of the “rule of five” in the design of new compound libraries is demonstrated by
the following example.

Rule of five distribution of a 150000 compound library from Sigma Aldrich. 16

1.6. Synthetic strategies in combinatorial chemistry
Ø Different synthetic strategies can be envisaged for the design of a compound library:

1. Solution-phase or solid-phase synthesis

2. Parallel synthesis or combinatorial synthesis (“split-mixed”)

3. Synthesis of libraries containing single molecules or compound mixtures

1.6.1. Solution-phase or solid-phase synthesis

Ø classical solution-phase synthesis

reagent
substrate product
solvent

Ø solid-phase organic synthesis (SPOS)

substrate (SPOS)

17
Ø MODERN solution-phase synthesis

§ Polymer-Assisted Solution-Phase Synthesis (PASP).

reagent (PASPS)

§ Fluorous Synthesis.

F substrate (fluorous synthesis)

§ Tag-Assisted synthesis.

Tag substrate (Tag chemistry)

18
Solid-phase organic synthesis substrate (SPOS)

ADVANTAGES-DISADVANTAGES

++ use of excess reagents to drive reaction to completion

++ purification by simple filtration and washing of the resin (easy automation)

++ spatial separation of the reactive sites on support (e.g. it can be exploited for intermolecular
cyclizations)

+- linker molecules have to be designed to be compatible with the synthetic plan but they can be part
of the final product

- ex novo optimization of the reaction conditions for each step of the synthetic strategy

- lower reactivity of substrates in solid-phase compared to solution-phase

- supports are often expensive

- 19
difficult reaction monitoring by standard techniques (TLC, HPLC, NMR).
Modern solution-phase synthesis

reagent (PASPS) F substrate (fluorous synthesis)

Tag substrate (Tag chemistry)

ADVANTAGES-DISADVANTAGES

++ literature data for reaction optimization

+ excess reagents is not required

+ exploitation of solvent effect

++ limited steric effects compared to the solid-phase approach

++ reaction scope adaptable to different substrates

-+ necessity of by-product analysis

-- need for chromatographic purification for low-yielding steps

20
-- difficult automation
1.6.2. Parallel syntehsis or combinatorail syntheis (“split-mixed”)

Ø Independently from a solid-phase or solution-phase approach, reactions in combinatorial chemistry

can be performed through either the parallel synthesis or combinatorial (=split-mixed) technique.

Ø In some occasion, the term “combinatorial” is used as synonymous of “split-mixed”.

parallel libraries of
synthesis single compounds
combinatorial
synthesis
combinatorial libraries of
synthesis compound mixtures
(“split-mixed”)

21
Parallel synthesis of single compounds Reactive species are
reported in solid-
2×2×2=8 products phase. The same
(case b section 1.4) methodology can be
applied in solution-
phase.

A1B1 A1B2 A2B1 A2B2

A1B1C1 A1B1C2 A1B2C1 A1B2C2 A2B1C1 A2B1C2 A2B2C1 A2B1C2

22
parallel synthesis of single compounds (12 reactions)
 ADVNTAGES-DISADVANTAGES

++ Precise identification of each compound within each flask.

- Need of multiple reaction work-ups for the purification of each compound. Difficult automation.

Combinatorial (“split-mixed”) synthesis

Ø In a typical combinatorial synthesis all A1-An substrates react with the B1-Bn reactants to give
the target products in all the possible combinations.

Parallel synthesis: Combinatorial

A single reaction is synthesis: n × n
run within each flask reactions occur within
a single flask

Ø The split-mixed technique has been optimized to compensate for the different reactivity of the
substrates A and B (different reaction kinetics).
23
 Though applicable to
2×2×2=8 products solution-phase approaches,
(case b section 1.4) this methodology is mainly
used in solid-phase
strategies

A2B1 A1B1 A2B2 A1B2

A2B2C1 A2B1C1 A1B1C1 A1B2C1 A2B2C2 A2B1C2 A1B1C2 A1B2C2

24
combinatorial (split-mixed) synthesis (4 reactions)
 Combinatorial synthesis: n × n
reactions occur within a single
flask

ADVANTAGES-DISADVANTAGES

++ The “split-mixed” technique is ideal for the synthesis of polypeptides and oligonucleotides where is
necessary to economize each synthetic step

+ Each bead is functionalized by a single product because in each step the resin is treated with a
single reagent used in excess.

- A limit of this technique is the low amount of product obtained after the cleavage from the resin
(low loading of the starting support, see chapter 2).

-The final pools contain mixture of products thus making necessary the use of deconvolution
techniques.

25
 number of products
synthetic methodology screening method
generated

traditional slow, from slow to fast,

synthesis slow optimization very accurate

synthetic parallel fast, fast,

plan synthesis very efficient accurate

requirement of
split-mixed
very fast, deconvolution
synthesis
efficient

26
1.6.2.1. Deconvolution technique

Ø The deconvolution technique is based on the evaluation of the biological activity of the final
pools resulting from a split-mixed approach and allows for the identification of the most (or one
of most active) compound.

Iterative deconvolution
1 2 3

A1 A2 A3

mix and divide

A 1
A3

A2

4 5 6

A1A1 A1A2 A1A3

2 1 2 2
A A A A A2A3
3 1 3 2
A A A A A3A3

mix and divide

A1 A3

A2

7 8 9

A1A1A1 A1A2A1 A1A3A1 A1A1A2 A1A2A2 A1A3A2 A1A1A3 A1A2A3 A1A3A3

2 1 1 2 2 1 2 3 1 2 1 2 2 2 2 2 3 2
A AA A A A A A A A AA A A A A A A A2A1A3 A2A2A3 A2A3A3 27
3 1 1 3 2 1 3 3 1 3 1 2 3 2 2 3 3 2
A AA A A A A A A A AA A A A A A A 3 1 3 3 2 3
A AA A A A 3 3 3
A A A
 screening
position 3 7 8 9
(3 pools with 9 compounds)

X-X-A1 X-X-A2 X-X-A3

1. cleavage
biological
2. screening
activity
3. selection of pool 8
4. synthesis
7 8 9

position 2 (4) (5) (6)

(3 pools with 3 compounds)

X-A1-A2 X-A2-A2 X-A3-A2

1. cleavage
2. screening
3. selection of pool 8
4. synthesis

(4) (5) (6)

position 1
(1) (2) (3)
(3 pools with 1 compound)

A1-A1-A2 A2-A1-A2 A3-A1-A2

1. screening
2. selection of the ligand

(1) (2) (3)

A3-A1-A2

•The most active pool cannot contain the most active compound (the overall biological activity
of the pool is given by the activity of each compound and its relative abundance)
28
•The iterative synthesis of a large library of compounds is time-consuming and expensive
 Chapter B
Solid-Phase Organic Synthesis (SPOS)
1. Solid supports
1.1. Crosslinked organic polymers
1.1.1. Polystyrene resins
1.1.1.1. Microporous polymers (gels)
1.1.1.2. Macroporous (macroreticular) polymers
1.1.1.3. Polystyrene resin functionalization
1.1.2. Acrylamide resins
1.1.3. Tentagel
1.2. Linear organic polymers ((MeO)-PEG)
1.3. Inorganic supports
2. Linker molecules, cleavage strategies, and synthetic applications
2.1. Linker molecules releasing one specific functional group
2.2. Linker molecules promoting cyclization-assisted cleavage
2.3. Linker molecules promoting a multidirectional cleavage
2.3.1. Direct cleavage by nucleophilic substitution
2.3.2. Direct cleavage by electrophiles
2.3.3. Safety-catch linkers
29
1. Solid supports
Ø Polymers typically used as support in SPOS:

microporous polymers
(gels)
polystyrene
resins
macroporous polymers
(macoreticular) insoluble
crosslinked in organic solvents
organic polymers TentaGel

acrylamide
resins

linear soluble in organic solvents

PEG (but they precipitate under
organic polymers (polyethylene gycol) particular conditions)

porous glass
SiO2
inorganic insoluble
Al2O3
supports in organic solvents
clays

graphite 30
1.1. Crosslinked organic polymers
1.1.1 Polystyrene resins
Ø Polystyrene beads are of spherical shape with variable dimensions (80-200 µm).

CH3 CH3
H3C N N C CH3 Ph Ph Ph Ph
CN CN
+
Ph
H2O, Δ Ph Ph Ph
suspension stabilzer, Ph
STY mechanical stirring
DVB
Ph
Ph

Ph

crosslinked
bead polystyrene
(80-200 µm).

31
SWELLING CAPACITY

Ø DVB (1-20% in respect with STY) is responsible for the crosslinking between the linear
polymeric chains and thus for the mechanical resistance of the polymer.
Ø The DVB amount has to be modulated to allow the resin to swell in the presence of an organic
solvent.
Ø The swelling capacity reflects the internal flexibility of the polymeric structure and thus the
possibility by the reactants to diffuse within the pores and reach the reactive sites.

volume of wet resin

swelling capacity =
volume of dry resin

Ø Depending on the swelling capacity, polystyrene resins can be classified as:

1) microporous resins or gels (DVB, 1-2%)

2) macroporous or macroreticular resins (DVB, >5%)

32
1.1.1.1. Microporous polymers (gels)

Ø Microporous resins display a low degree of crosslinking (DVB content, 1-2 %) and high swelling
capacity.

Ø The swelling capacity depends on the nature of the attached functional groups.

33
1.1.1.2. Macroporous (macroreticular) polymers

Ø Macroreticular or permanently porous resins are prepared by polymerization in the presence of an

inert solvent named porogen (e.g. toluene).

Ø Whether a macro- or micro-porous resin is obtained depends largely on the ratio of the content of
solubilizing (porogen) and crosslinking agents:

§ a low content of porogen and a low percentage of DVB give microporous resins;

§ high contents of porogen and DVB give macroporous resins.

Microporous resins Macroporous resins

ADVANTAGES-DISADVANTAGES ADVANTAGES-DISADVANTAGES

- Functional groups are homogeneously distributed + Functional groups are distributed on the surface
inside and on the bead (need for a solvent with high of the bead (reactivity is not related to the
swelling capacity). swelling properties of the solvent).
+ Being in the form of gel, microporous beads are
- The macroreticular nature of the resins make
not mechanically degraded (compatibility with
them fragile (need for a shaker for mixing).
magnetic stirring).

- The need for solvents with high swelling capacity + The possibility of using standard quantities of

complicates the optimization/automation of solvent makes the use of macroporous resins

34
multistep syntheses. preferential in automated, multi-step syntheses.
1.1.1.3. Polystyrene resin functionalization

LOADING

Ø The loading of a resin indicates the level of functionalization of the polymer and it is expressed as
the mmol of functional group (FG) per gram of resin.

Ø There are principally two different ways to obtain functionalized polystyrene/DVB-copolymers:

Ø approach A: building first the

polystyrene/DVB-copolymer
followed by the subsequent
chemical introduction of FG.

Ø approach B: FG is already
introduced in a modified
styrene-derived co-monomer.

Ø Approach A offers the advantage that only the accessible aromatic rings and positions on the
aromatic rings are functionalized. The drawback is that the reactions on the polymer are slow and
difficult to be monitored.

Ø Approach B assures the exact positioning of FGs and usually a high degree of loading but it
necessitates the synthesis of the appropriate co-monomer. 35
Chloromethylated polystyrene

Ø Chloromethyl polystyrene was introduced by Merrifield into peptide synthesis.

Ø Approach A: the synthesis of chloromethylated polystyrenes is best achieved by Friedel-Crafts

alkylation of polystyrene with methoxymethylene chloride in the presence of a Lewis acid catalyst.

- SnCl4 is the most widely used catalyst.

- ZnCl2 gives resins with lower loadings.

- BF3.OEt2 is the catalyst that allows for the better control of the loading.
36
approach B: use of co-monomers

B2 Ø B1: co-polymerization of STY, DVB,

and chloromethylstyrene.This
approach, however, leads to a
substantial loss of chlorine content.

B1 Ø B2: co-polymerization of STY, DVB,

and 4-methoxymethylstyrene and
subsequent conversion using BCl3.
Merrifield resins with a chloride
content up to 22% (corresponding to
B3
1.0 chloride per aromatic ring) can be
obtained by this strategy.

Ø B3: co-polymerization of STY, DVB, and 4-methylstyrene followed by chlorination with NaOCl in
the presence of a phase transfer catalyst. This is a valuable method for the preparation of
microporous (DVB, 1%) and macroporous (DVB, 20%) chloromethyl polystyrenes.
37
Various functionalized polystyrene resins

Precursor: chloromethyl polystyrene (nucleophilic displacement)

38
Precursor: polystyrene

Ø electrophilic aromatic
substitution reactions

Ø Lithiated polystyrene resins

can be obtained either via
convenient bromine-lithium
exchange using nBuLi starting
from 4-bromo-substututed
polystyrene or by direct
lithiation of polystyrene in
cyclohexane in the presence
of TMEDA.
39
Precursor: lithiated polystyrene

40
1.1.2. Polyacrylamide resins
Ø In the course of a synthesis on a polymer support the swelling properties of the resin can change
significantly. For instance, in the Merrifield peptide synthesis the starting polystyrene derived
resin is highly hydrophobic while it becomes more and more hydrophilic as the peptide chain grows.
Due to such phenomena certain peptide sequences are not accessible.

Ø It can be useful in some occasions using a polymeric support with higher hydrophilicity than
polystyrene such as the polyacrylamide resins.

Ø Synthesis: copolymerization of N,N-dimethylacrylamide 1 (basic monomer), ethylene-bis-

acrylamide 2 (crosslinking agent), and N-acryloyl-N’-(tert-butoxycarbonyl-β-
alanyl)hexamethylene diamine 3 (functionalized monomer). Monomer 3 serves to functionalize the
polyacrylamide polymer after liberation of the primary amino group of the β-alanine moiety.

Ø In order to achieve a homogeneous distribution of the functional groups, monomer 3 was later 41
replaced by acryloylsarcosin methyl ester 4, which is chemically closer to the basic monomer 1.
1.1.3. Tentagel
Ø Tentagel polymers consist of a polystyrene matrix covalently coated with polyethylene glycol (PEG)
chains and have been developed primarily for solid-phase peptide synthesis similarly to
polyacrylamide resins (increased hydrophilicity).

Ø Tentagel resin combines the advantages of PEG such as flexibility and solubility with the mechanical
properties of polystyrene.

Tentagel

Ø Tentagel beads show good swelling properties in protic and polar solvents such as water, MeOH,
CH2Cl2, MeCN, THF, or DMF whereas in apolar solvents such as Et2O they hardly swell at all.
Ø The high flexibility of the PEG chains can be observed in NMR studies.
Ø Loadings of commercially available Tentagel resins range between 0.15-0-30 mmol/g, which is
significantly lower than those of PS-resins.
42
Two main routes are available for the synthesis of Tentagel resins:

1. anionic polymerization of ethylene oxide with hydroxylated crosslinked PS beads;

2. functionalization of β-hydroxy polystyrene followed by anionic polymerization with

ethylene oxide. This strategy avoids the grafting as acid-labile benzyl ether groups to the
polymeric backbone.

1.

2.

43
1.2. Linear organic polymers ((MeO)-PEG)

Ø Linear organic polymers constitute an imports class of supports. These polymers display a
good solubility in organic solvents independently from the nature of attached molecules.
Ø Nevertheless, in particular organic solvents they can be precipitated, thus allowing the
combination of the advantages related to the solution phase (favorable kinetics, easy
monitoring of reaction conversion) with those related to the solid-phase (ease of purification
procedure).
Ø The polyethylene glycol monomethyl ether is the most widely used linear organic polymer used
in combinatorial chemistry. It can be easily precipitated from Et2O, thus allowing an easy and
fast purification of the attached molecules.

MeO-PEG

Ø The hydroxyl functionality is used for anchoring the substrate.

44
1.3. Inorganic supports
Ø The inorganic supports typically used in combinatorial/organic synthesis are:
§ the controlled pore glass (CPG),
§ silica (SiO2),
§ Alumina (Al2O3),
§ clays,
§ graphite.
Ø The advantages that this kind of support offer in comparison with the traditional polystyrene
resins are:
§ compatibility with a wide range of solvents
§ minimum swelling
§ stability at high pressures and temperatures
§ low production costs.

O
SiO2 O Si Cl
OEt
O

45
Ø Comparison between two samples of silica gel and macroreticular polystyrene
under mechanical stirring at different temperatures.

46
2. Linker molecules, cleavage strategies,
and synthetic applications
Ø Linker molecules are bifunctional spacer molecules which contain on one end an anchoring group
for attachment to the solid support and on the other hand a selectively cleavable functional
group used for the subsequent chemical transformations and cleavage procedures.
Ø Linker molecules play a key role in a successful synthetic strategy on solid phase as they
covalently link the polymeric support and the molecules that are synthesized.
Ø Linker molecules can be grouped as follow:

•linker molecules releasing one specific functional group (monofunctional cleavage)

•linker molecules promoting the cleavage through cyclization reactions

•linker molecules promoting a multidirectional cleavage

-by direct nucleophilic substitution

-by direct electrophilic substitution

-by activation of the linker molecules before the cleavage (“safety-catch

principle”).

47
2.1. Linker molecules releasing one specific functional group

Ø Monofunctional resin-cleavage strategies are well suited for the construction of focused
combinatorial libraries, where a given important pharmacophoric group (pharmacophore) that remains
constant is released in the very last step. Therefore, the linkage to the resin serves as a protective
group throughout the synthesis.

Ø The linker molecules can be classified according to the functional group that is released:

• carboxylic acid

• amide

• sulfonamide

• hydroxamic acid

• amines

• alcohols and phenols

Ø A pharmacophore is an abstract description of molecular features which are necessary for molecular
recognition of a ligand by a biological macromolecule. The IUPAC defines a pharmacophore to be "an
ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular
interactions with a specific biological target and to trigger (or block) its biological response”. 48
 carboxylic acid

Ø In the next table are group the most widely used linkers to release the carboxylic acid
functionality.

Ø Scheme for the interpretation of the table:

O O
R
X + substrate Y OC substrate OC product HO2C product + OH
anchoring cleavage conditions
X = OH, halogen solid-phase
conditions (second column)
elaboration
starting resin
(first column)
49
50
Ø Example: synthesis of quinolones (potent antibiotics).

MacDonald, A. et al. A solid phase approach to quinolones using the diversomer technology, Tet. 51
Lett. 1996, 37, 4815-4818.
amide

Ø Scheme for the interpretation of the table:

O
R
X + substrate Y XC substrate
anchoring
X = NH2, OH solid-phase
conditions
elaboration
starting resin
(first column)

O O

XC product H2NC product + R'

cleavage
conditions
(second column)
52
Ø Example: synthesis of 1,4-benzodiazepines which are important ‘privileges structures’.
Ø The term "privileged structures" was first coined by Ben Evans (1988, DOI:
10.1021/jm00120a002) who recognized the potential of certain regularly occurring structural
motifs as templates for derivatization to discovery novel ligands for binding to proteins. More
recently Klaus Mueller has tried to define the term privileged structure more specifically: "Small,
non-planar structures with robust conformations that provide interesting 3D exit vectors for
substitution, with drug-like properties and ideally readily accessible synthetically."
Ø In this example the benzodiazepine ring is conjugated to a peptoid chain to generate a hybrid
drug . This is a molecule expected to display the biological functions of both the pharmacophoric
groups (benzodiazepine ring and peptiod chain).

53
Goff, D. A. et al. Solid-phase synthesis of defined 1,4-benzodiazepine-2,5-dione mixtures, J.
Org. Chem. 1995, 60, 5744-5745. 54
sulfonamide

Ø Scheme for the interpretation of the table:

O O
R
X + substrate Y R' substrate R1NS prodotto H2NS prodotto + R"
anchoring cleavage conditions
solid-phase O
conditions O (second column)
elaboration
resin before
cleavage
(first column)

55
Hydroxamic acid

Hydroxamic acids have emerged as an important class of inhibitors of matrix metalloproteinases

(MMPs) such as collagenase, gelatinase, etc. MMs are becoming prime targets in many therapeutic
areas (e.g. anti-inflammatory and anticancer agents).

56
Ø Scheme for the interpretation of the table:

O
R NH2
O + substrate CO2H R NHC substrate
O
anchoring
solid-phase
starting resin conditions
elaboration
(first column)

O O
R'
R NHC product NHC product +
O cleavage HO
conditions
(second column)

57
 amine

Ø Scheme for the interpretation of the table:

R R
X + substrate Y X substrate
anchoring
X = NH2, OH, halogen
conditions
starting resin
(first column)

R R'
X product H2N product +
solid-phase cleavage conditions
elaboration (second column)

58
Ø The dihydropyridine (DHP) ring is a
privileged structure found in many
bioactive compounds including
various vasodilator,
antihypertensive, hepatoprotective,
antitumor, and antibiotic agents.

Ø The strategy herein presented is

based on the classical Hantzsch
three-component reaction (3-CR).

Ø A multicomponent reaction is
defined as a reaction in which three
or more reagents are added
together (or nearly) in a single
vessel to form a new compound that
contains portions of all the starting
components.

Gordeev, M. F. et al. Combinatorial

synthesis and screening of a chemical
library of 1,4-dihydropyridine calcium
channel blockers, Bioorg. Med. Chem. 59
1998, 6, 883-889.
alcohol

Ø Scheme for the interpretation of the table:

R R'
X + substrate Y O substrate
anchoring
solid-phase
conditions
elaboration

R' product
O product HO + R"
clevage conditions
resin before (second column)
the cleavage
(first column)

60
Ø Example: synthesis of a pyrrolidine library.

Hollinshead, S. P. Stereoselective synthesis of highly functionalised pyrrolidines via

1,3-dipolar cycloaddition reactions on a solid support, Tet. Lett. 1996, 37, 9157-9160.
61
2.2. Linker molecules promoting cyclization-assisted cleavage

Ø Cyclization-assisted cleavage offers the following advantages:

- only molecules that have gone through the whole reaction sequence necessary for the
cyclization reaction can be cleaved;

- even if the single reaction steps do not proceed quantitatively, the cyclization will
nevertheless lead to pure products.

Ø Given the importance of heterocyclic products in the drug discovery process and the high
efficiency of cyclization reactions, this cleavage strategy has gained much attention in the last
years.

62
 Reagents and conditions

i) Davies reagent;
ii) trichloroacetic anhydride

!"#"$%&"'(")%)*

iii) DMAP, toluene

+,"$#%--",!"#'),.,%)*

iv) acryloyl chloride, NEt3, CH2Cl2

v) R1-NH2, DMSO
vi) R2-N=C=O
vii) HCl, toluene, 95 °C

63
$""$#%!"#"/"$"),$"%)*
 Reagents and conditions

vii) HCl, toluene, 95 °C

"$')&%")'

viii) TFA

0*)(%$"'(*!")'

iX) BrCN, TFA, CHCl3-H2O

$""$#%12#')%)*

64
Example: generation of a hydantoin library (antidiabetic agents)

Kim, S. W. et al. , Solid-phase synthesis of hydantoin library using a novel

65
cyclization and traceless cleavage step, Tet. Lett. 1997, 38, 4603-4606.
2.3. Linker molecules promoting a multidirectional cleavage
Ø Multidirectional cleavage offers the
advantage that in the final cleavage step
an additional element of diversity is
incorporated. Therefore, the amount of
compounds is multiplied by the number of
elements that can be incorporated.

Ø Most of linkers used for multidirectional

cleavage are also as “traceless linkers” as
no element of the linker remains in the
final molecules.

66
 Ø The main strategies include:
•direct cleavage by nucleophilic substitution reactions
•direct cleavage by electrophilic substitution reactions
•activation of the linker group prior to cleavage ( “safety-catch” principle).

2.3.1. Direct cleavage by nucleophilic substitution

Ø Typical nucleophile reagents are amines, alcoholates, thiolates and C-nucleophiles such as Grignard
reagents.

67
68
 Ø Example: synthesis of a library
of bicyclo[2.2.2]octane
derivatives.

Ø An important strategy in
combinatorial chemistry is the
synthesis of rigid structures
such as polycyclic compounds
because they offer highly
reliable SAR data.

Ley, S. V. et al. Solid phase synthesis of biclyclo[2.2.2]octane derivatives via tandem Michael
addition reactions and subsequent reductive amination, Synlett 1995, 1017-1020. 69
2.3.2. Direct cleavage by electrophiles

Ø Most of the linkers that allow

multidirectional electrophilic
cleavage from the resin are
based on the chemistry of silicon
(aromatic electrophilic ipso-
substitution)

70
2.3.3. Safety-catch linkers
Ø “Safety-catch linkers” are linker molecules that are activated in the very last step before cleavage.

Ø The major advantages associated to this class of molecules are the following:

• during the synthesis of the library the linker moiety is completely stable to a wide range of
reaction conditions.

• the linkage between the resin and the substrate can be specifically designed and planned in view of
the structure and chemical stability of the final products

• the linker group can often be reduced to a single atom such as sulfur, tin, and silicon.

• the “safety-catch” principle generally leads to multidirectional cleavage.

Ø Some examples:

Reagents and conditions

i) CH2N2 or ICH2CN, DBU

ii) Nu- (amines or alcoholates)

71
 Reagents and conditions

iii) m-CPBA, CH2Cl2

ii) Nu- (amines or alcoholates)

vi) R3X, DMF

v) DIEA, DMF

REM-linker 6

72
Reagents and conditions

vi) base
vii) R2CHO

viii) TFA
ii) Nu- (amines or alcoholates)

73
Kahne et al. Glycosylation on the Merrifield resin using anomeric sulfoxide, J. Am. Chem.
Soc. 1994, 116, 6953-6954.

74
 Chapter C- Polymer-assisted solution-phase
synthesis (PASPS)
1. Polymer-assisted solution-phase synthesis (PASPS): introduction and basic concepts
1.1. PASPS: strategies
1.2. Classification of supported reagents
2. PASPS for compound library generation
2.1. “Orchestrated” use of polymer support reagents and sequestrants
2.2. Use of sequestering enabling reagents (SERs)
2.3. The “catch and release” technique
3. PASPS for the synthesis of natural products and biologically relevant molecules
4. PASPS: literature

75
3.Polymer-assisted solution-phase synthesis
(PASPS): introduction and basic concepts

Ø Polymer-assisted solution-phase synthesis (PASPS) is that methodology which utilizes polymer

support reagents to promote and/or purify reactions performed in solution phase.

Ø The main difference between PASPS and SPOS is that in the former methodology the substrate is
elaborated in solution phase and not in solid phase.

reagent
substrate product

solid phase solution phase

substrate (SPOS) reagent (PASPS)

76
Ø Supported reagents are reactive species linked to a heterogeneous support (typically polystyrene
or silica). These promote the conversion of one ore more substrates into a new product. The
excess of the polymer supported reagent is removed by filtration.

Ø Supported catalysts are reactive species linked to a heterogeneous support . They are used in
sub-stoichiometric amount to catalyze the conversion of one or more substrates into a new
product. Importantly, they can be recycled by filtration.

Ø Supported scavengers are reactive species linked to a heterogeneous support . These are capable
to selectively sequester a by-product of the reaction thanks to complementary functionality.
Similarly, they can be be used to remove the excess of a reactant. They are removed by filtration.

reagents

reagents scavenger

catalyst

77
Ø Polymer supported reagents can be classified into two groups:

1) ionic resins (also named ion exchange resins). These resins are often used to support anionic
nucleophiles, oxidants, or reducing agents.

2) covalent resins. In these resins the functional group is covalently attached to the polymeric
matrix.

ionic resins covalent resins

+ -
NMe3OH N basic reagents

SO3H CO2H acid reagents

Ø sequestering enabling reagents (SERs) are not supported reagents and are capable to selectively
intercept a reaction by-product with low reactivity and convert it into a more reactive species
easily removable by means of a polymer supported scavenger.

78
1.1. PASPS: strategies

Ø case A: use of supported reagents only

The substrate is converted into the product using one or more supported reagents without formation
of by-products.

reagent 1 reagent 2
substrate intermediate product

Ø case B: use of supported reagents and sequestrants

Purification of the product requires the use of one or more polymer-supported scavengers to
selectively remove reaction by-products or excess reactants.

reagent 1 product scavenger

reactant A
+ + product
reactant B reactant B (excess)

scavenger reactant B

79
 Ø case C: use of sequestering enabling reagents

If it is not possible to directly remove a by-product with a polymers-supported scavenger because of

its low reactivity it is necessary to use a SER for converting it into a more reactive species, which is
then removed by the action of a suitable polymer-supported scavenger.

reagent 1 product product scavenger

reactant A reagent 2 (SER)
+ + prodotto
+
reactant B reactant B (excess) activated reactant B

scavenger activated reactant B

Ø case D: catch and release technique

The catch and release strategy is complementary to those previously described because it is the
reaction product that is “fished” selectively from a complex mixture containing many by-products.
In this approach a polymer-supported scavenger (reagent 2) sequesters the product, which is then
released in the solution phase purified by the action of a standard reagent (reactant 3)

reagent 1 product reagent 2 reagent 2 product

reagent 3
recatant + product
+
conversion
by-products by-products in solid-phase

by-products
(after filtration)
80
Ø Advantages of PASPS compared to traditional solution phase synthesis

1) easy separation of polymer-supported reagents by filtration

2) use of excess of reagents to drive reaction to completion without purification issues

3) usein the same reaction medium supported reagents with complementary functionalities (acid and
base; nucleophile and electrophile). This important feature of PASPS, name the “principle of wolf
and lamb”, originates from the steric hindrance of functionalities on support, which avoids their
mutual interaction.

4) recycling of supported catalysts

5) easy of automation and compatibility with reaction in flow mode

6) reduced toxicity and stink of supported reagents compared to their unsupported counterparts.

Ø Advantages of PASPS compared to SPOS

1) easy monitoring of reaction mixtures (TLC, HPLC, NMR e MS)

2) faster optimization of the reaction conditions by simple translation form traditional solution phase
synthesis

3) no need of the two additional steps of SPOS, anchoring and cleavage.

81
Ø Drawbacks of PASPS

1) Elevated cost of supported reagents

2) Lower reactivity of supported reagents compared to their homogeneous counterparts

3) Difficult analysis of supported reagents for establishing their purity and integrity.

1.2. Classification of supported reagents

Ø Supported reagents can be group as follows:

- nucleophiles

- electrophiles

- oxidants

- reducing agents

- bases

- condensation agents

- miscellanea
82
nucleophiles
(reagents)

83
nucleophiles
(scavengers)

84
electrophiles
(scavengers)

85
oxidants

86
reducing
agents

87
bases

88
 bases

condensation
agents

89
miscellanea

90
2. PASPS for compound library generation
2.1. “Orchestrated” use of polymer support reagents and
sequestrants

parallel synthesis of hydroxamic acids

S. V. Ley et al. Synthesis of an array of potential matrix metalloproteinase inhibitors using 91

a sequence of polymer-supported reagents Bioorg. Med. Chem. Lett. 1999, 9, 2049-2052.
parallele synthesis of bicyclo[2.2.2]ocatanes S. V. Ley et al. Parallel solution-phase
syntheses of functionalised
bicyclo[2.2.2]octanes: generation of a
library using orchestrated multi-step
sequences of polymer-supported
reagents and sequestrants J. Chem.
Soc., Perkin Trans. 1 2000, 3465-
3654.

92
parallel synthesis of dihydropyrimidinones

A. Dondoni et al. Parallel synthesis

of dihydropyrimidinones using
Yb(III)-resin and polymer-
supported scavengers under
solvent-free conditions. A green
chemistry approach to the Biginelli
reaction Tetrahedron Lett. 2001,
42, 7975-7978.
93
2.2. Use of sequestering enabling reagents (SERs)

removal of aromatic amines

J. J. Parlow et al. In situ chemical tagging: tetraflurophtalic anhydride as a “sequestration enabling

reagent” (SER) in the purification of solution-phase combinatorial libraries Tetrahedron Lett. 1997,
38, 7959-7962.
sequestering of hindered alcohols
Application to the synthesis of oligosaccharides

A. Massi et al. Hybrid Solution/Solid-Phase

Synthesis of Oligosaccharides Using
Trichloroacetyl Isocyanate as Sequestering
Enabling Reagent of Sugar Alcohols Angew.
Chem. Int. Ed. 2005, 44, 1672-1676.

96
2.3. The “catch and release” technique

Romo, D. et al. Simultaneous deprotection and purification of Boc-amines based on ionic resin
capture J. Org. Chem. 1998, 63, 3471-3473.

A. Kirschning et al. The “resin-capture-release” hybrid technique: a merger between solid- and
solution-phase synthesis Chem. Eur. J. 2000, 24, 4445-4450.
3. PASPS for the synthesis of natural
products and biologically relevant
molecules
Ø The PASP technique has also been utilized successfully in the synthesis of complex natural
products and molecules of pharmaceutical relevance. Indeed, the possibility of avoiding expensive
and tedious separation procedures (especially chromatographic purification) make the entire
synthesis more adaptable to an industrial process. This has been demonstrated by several
examples reported in the literature.

S. V. Ley et al. Multi-step application of immobilized reagents and scavengers: a total synthesis of
Epothilone C Chem. Eur. J. 2004, 10, 2529-2547.
total synthesis of Viagra

S. V. Ley et al. Polymer-supported

reagents for multi-step organic
synthesis: application to the synthesis
of Sildenafil Bioorg. Med. Chem. Lett.
2000, 10, 1983-1986.

99
4. PASPS: literature

S. V. Ley et al. Multi-step organic synthesis using solid-supported reagents and scavengers: a new
paradigm in chemical library generation J. Chem. Soc., Perkin Trans. 1 2000, 3815-4195.

A. Kirschning et al. Functionalized polymers-Emerging versatile tools for solution-phase chemistry

and automated parallel synthesis Angew. Chem. Int. Ed. 2001, 40, 650-679.

S. V. Ley et al. Solid-supported reagents for multi-step organic synthesis: preparation and
application Il Farmaco 2002, 57, 321-330.

100