A combinatorial approach towards pharmaceutically relevant cyclic peptides - Thesis

A combinatorial approach towards pharmaceutically relevant cyclic peptides

Springer, J.

Publication date

2008

Document Version

Final published version

Link to publication

Citation for published version (APA):

Springer, J. (2008). A combinatorial approach towards pharmaceutically relevant cyclic

peptides.

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Pharmaceutically Relevant Cyclic Peptides

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

Prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op donderdag 6 november 2008, te 10:00 uur

door

Jasper Springer

Promotor:

Prof. dr. H. Hiemstra

Co-promotor:

Dr. J.H. van Maarseveen

Overige leden:

Prof. dr. P. Timmerman

Prof. dr. G.J. Koomen

Prof. dr. ir. R.V.A. Orru

Prof. dr. F.P.J.T. Rutjes

Prof. dr. P.H.H. Hermkens

Prof. dr. C.J. Moody

Dr. E.J. Meijer

Faculteit der Natuurwetenschappen, Wiskunde en Informatica

This research was carried out in collaboration with Solvay Pharmaceuticals (Weesp) and

Schering-Plough (Oss), as part of the NWO-Combichem program. The project has been

financially supported (in part) by the Council for Chemical Sciences of the Netherlands

Organisation for Scientific Research (CW-NWO).

Chapter 1

General Introduction

1.1

Cyclic peptides

2

1.2

Difficulties in peptide cyclizations

4

1.3

Combinatorial chemistry

8

1.4

Combinatorial synthesis of cyclic peptides

9

1.5

Outline of the thesis

13

1.6

References and notes

15

Chapter 2

Improved Auxiliary for the Synthesis of Medium-Sized Bis(lactams)

2.1

Introduction

24

2.2

Development and synthesis of the different auxiliaries

28

2.3

Evaluation of the TBS-substituted auxiliary

29

2.4

The isopropyl-substituted auxiliary proved to be optimal

30

2.5

Synthesis of eight-membered bis(lactams)

33

2.6

Towards a solid phase immobilized auxiliary

36

2.7

Conclusions

41

2.8

Acknowledgments

42

2.9

Experimental section

42

2.10

References and notes

56

Chapter 3

Backbone Amide Linker Strategy for the Synthesis of 1,4-Triazole

Containing Cyclic Tetra- and Pentapeptides

3.1

Introduction

60

3.2

Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition reaction

60

3.3

Synthesis of triazole-containing cyclic pseudopeptides

61

3.4

Combinatorial approach towards triazole-containing cyclic peptides

64

3.5

Synthesis of the backbone amide linker

66

3.6

Synthesis of the azido acids

66

3.7

Synthesis of the amino alkynes

67

3.8

Solution phase approach towards the triazole-containing

cyclic pseudopeptides

69

3.9

Solution phase copper-catalyzed cyclization of the linear peptides

71

3.10

Solid phase approach towards the triazole-containing

cyclic pseudopeptides

72

3.11

Solid supported copper-catalyzed cyclization of the linear peptides

73

3.13

Synthesis of a triazole analogue of the cyclic pentapeptide segetalin B

75

3.14

Conformational analysis of the cyclic peptide analogue

77

3.15

Conclusions

82

3.16

Acknowledgments

83

3.17

Experimental section

83

3.18

References and notes

100

Chapter 4

Synthesis of 1,5-Connected Triazole-Containing Cyclic

Pseudotetrapeptides

4.1

Introduction

106

4.2

Towards the synthesis of cyclo-[Pro

−ψ

(triazole)

−

Gly

−

Pro

−

Tyr]

110

4.3

Synthesis of cyclo-[Tyr

−

Pro

−

Gly

−ψ

(triazole)

−

Gly]

111

4.4

Synthesis of cyclo-[Tyr

−

Pro

−

Val

−ψ

(triazole)

−

Gly]

113

4.5

Conclusions

116

4.6

Acknowledgments

116

4.7

Experimental section

116

4.8

References and note

124

Chapter 5

Combined Ugi-4CR and Azide-Alkyne Cycloaddition Reaction for the

Fast Assembly of Small and Diverse Cyclic Peptides

5.1

Introduction

128

5.2

Small cyclic pseudopeptides, first generation isonitrile

131

5.3

Small cyclic pseudopeptides, second generation isonitrile

133

5.4

Towards small cyclic pseudopeptides, third generation isonitrile

135

5.5

Towards cyclic pseudo tetrapeptides

142

5.6

Triazole-containing analogue of chlamydocin

146

5.7

Conclusions

153

5.8

Acknowledgments

154

5.9

Experimental section

154

5.10

References and notes

181

Chapter 6

Retrospection and Outlook

6.1

Auxiliary mediated synthesis of medium-sized bis(lactams)

186

6.2

Backbone amide linker strategy for the synthesis of

1,4-triazole-containing cyclic peptides

186

6.4

Combined Ugi-4CR and azide-alkyne cycloaddition reaction for

the synthesis of cyclic pseudopeptides

188

6.5

References and notes

189

Summary

191

Samenvatting

195

4CR 4-component reaction Ac acetyl

All allyl

Alloc allyloxycarbonyl Ar aryl

atm atmospheric pressure aq aqueous

BAL backbone amide linker Bn benzyl Boc tert-butoxycarbonyl B.p. boiling point br broad (spectral) Bu butyl t-Bu tert-butyl ο_{C degrees Celcius} calcd calculated Cbz benzyloxycarbonyl CC combinatorial chemistry COSY correlation spectroscopy d doublet (in NMR) dd double doublet (in NMR) DBU diazabicyclo[5.4.0]undec-7-ene

DCC N,N-dicyclohexylcarbodiimide

DCM dichloromethane

DIBAL-H diisobutylaluminium hydride

DIC N,N-diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP 4-dimethylaminopyridine Dmb 2,4-dimethoxybenzyl DMF N,N-dimethylformamide DMSO dimethylsulfoxide DPPA diphenylphosphoryl azide dr diastereomeric ratio

EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

ee enantiomeric excess

EI electron impact (in mass spectrometry Et ethyl equiv equivalent Fmoc 9-fluorenylmethoxycarbonyl g gram(s) h hour(s) HATU O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

HMBC heteronuclear multiple bond correlation HSQC heteronuclear single quantum coherance HOAt 1-hydroxy-7-azabenzotriazole

HOBt 1-hydroxybenzotriazole HPLC high performance liquid

chromatography

HRMS high resolution mass spectrometry HTS high throughput screening Hz Hertz

IR infrared

J coupling constant (in NMR) L liter(s)

m multiplet (in NMR)

mCPBA meta-chloroperoxybenzoic acid

MeCN acetonitrile

MCR multicomponent reaction Me methyl

min minute(s)

M.p. melting point (range) Ms mesylate

nd not determined

NMR nuclear magnetic resonance NOE nuclear Overhauser effect

NOESY nuclear Overhauser effect spectroscopy OMe methoxy

PE petroleum ether (40-60) PG protective group Ph phenyl

ppm parts per million Pr propyl

i-Pr isopopyl

pybox bis(oxazolidine)pyridine q quartet (in NMR) rt room temperature

s singlet (in NMR) t triplet (in NMR)

TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl

TBTA tribenzyltriazoleamine TEA triethylamine

Tf trifluoromethanesulfonyl TFA trifluoroacetic acid THF tetrahydrofuran TIC total ion count TIPS tri(isopropyl)silyl

TLC thin layer chromatography

tr retention time (in chromatography)

Ts para-toluenesulfonyl

UV ultraviolet v volume xs excess

amino acids

Xaa any amino acid

Ach 1-aminocyclohexanecarboxylic acid Ahe (2S)-aminohept-6-enoic acid Aib 1-aminoisobutyric acid Ala alanine

Aoa (2S)-aminooctanoic acid Apa (2S)-aminopentanoic acid Arg arginine

Asn asparagine Asp aspartic acid Cys cysteine Gln glutamine Glu glutamic acid Gly glycine His histidine Ile isoleucine Leu leucine Lys lysine Met methionine Phe phenylalanine Phg phenylglycine Pip pipecolic acid Pro proline Sar sarcosine Ser serine Thr threonine Trp tryptophan Tyr tyrosine Val valine

7XijhWYj0J^_iY^Wfj[hYedjW_diWXh_[\_djheZkYj_edjeiec[e\ j^[cW_djef_Yie\j^_ij^[i_i$J^[ÄhijfWhW]hWf^m_bbekjb_d[j^[ YedY[fjie\f[fj_Z[iWdZYoYb_Yf[fj_Z[i$J^[i[YedZfWhW]hWf^m_bb _djheZkY[j^[Z_\ÄYkbj_[i[dYekdj[h[Z_dj^[iodj^[i_ie\YoYb_Y f[fj_Z[i$7\j[h[nfbW_d_d]j^[YedY[fje\YecX_dWjeh_WbY^[c_ijho" j^[\ekhj^fWhW]hWf^m_bbZ_ifbWoj^[YecX_dWjeh_Wbiodj^[i_ie\ YoYb_Yf[fj_Z[iWdZj^[Z_\\[h[djWffheWY^[ijemWhZij^[i[ijhkY# jkh[i$J^[ÄdWbfWhW]hWf^m_bb]_l[Wdekjb_d[j^_ij^[i_i$

=[d[hWb?djheZkYj_ed

1.1

Cyclic peptides

Peptides are among the important structures in life and a numerous physiological and

biochemical functions are influenced by these compounds. Peptides are formally polymers of

amino acids linked by amide bonds (also called peptide bonds) (Figure 1.1). They are

abundant in all natural sources, but the isolation and characterization has been found to be

problematic due to the low concentrations requiring highly sensitive screening assays. Peptide

research has remained limited, until the 1960’s after which screening techniques were

improved and the role of many peptides in important physiological processes was identified.

To date not only a vast number of peptides have been identified from natural sources, but also

their synthesis now has reached the stage that peptides up to 50 residues may be prepared in

an automated fashion using solid phase peptide synthesis (SPPS).

Several peptides are on the

market as drugs for various diseases.

However, the use of peptides as drugs has been limited owing to their poor drug-like

properties.

Besides their metabolic instability the charged termini of peptides prevent proper

passive membrane transport. Secondly, because of the highly conformational flexibility a

proper spatial positioning of the pharmacophoric moiety cannot be maintained, leading to a

lower selectivity and activity.

Figure 1.1 General representation of amino acids, peptides and cyclic peptides.

N H H N CO2 O R3 R4 H N O O H3N R1 R2 HN N H NH H N O O O O R2 R3 R4 R1 tetrapeptide (linear precursor) cyclic tetrapeptide CO2H H2N R1 amino acid

These drawbacks of peptides may be tackled by the introduction of conformational

constraints.

One such solution is ring-closure of peptides leading to cyclic peptides.

These

structures lack the charged termini resulting in a higher bioavailibility

and a lower

biodegradeability. More important, cyclization leads to a lower flexibility, placing the side

chains of the amino acids at well defined positions in space.

This generally leads to

improved pharmacodynamic and -kinetic properties.

Since the discovery of the first cyclic peptide, the antibiotic gramicidin S,

many cyclic

peptides have been identified from natural sources in various ring sizes and structures.

Alternatively, many cyclic peptides have been synthesized based on natural sources

or de

novo incorporating not only the 20 proteinogenic amino acids, but also many

non-proteinogenic analogues.

Cyclic peptides are divided in two main classes: homodetic

cyclic peptides and heterodetic cyclic peptides (Figure 1.2).

Homodetic peptides are

peptides only linked by amide bonds. In heterodetic cyclic peptides at least one link is not an

amide bond, but a different functional group. These functional groups can be introduced in

the final macrocyclization or can be incorporated during the synthesis of the cyclization

precursor.

Figure 1.2 General classification of cyclic peptides.

X Y Homodetic cyclic peptides

N H O

Heterodetic cyclic peptides

O O depsipeptide S thioether alkene X N R oxazole (X = O) thiazole (X = S) imidazole (X = N) R = H, Me

head-to-tail side chain-to-tail (or head) side chain-to-side chain X Y X Y N N N triazole N N N N tetrazole

Cyclic peptides are divided in three main topologies: head-to-tail cyclic peptides, side

chain-to-tail (or head)

and side chain-to-side chain.

Head-to-tail cyclic peptides generally

have the highest conformational strain, caused by the multiple amide bonds. Heterodetic

cyclic peptides allow a greater degree of saturation and consequently could be less

constrained. Examples of such heterodetic linkages include esters (depsipeptides),

thioethers,

oxazoles

and thiazoles,

all of them often encountered in natural

peptides. In addition, synthetic peptides incorporate alkenes

(derived from cross metathesis

reactions

or enyne cycloisomerization

), imidazoles,

triazoles (derived from

azide-alkyne cycloaddition reactions)

and tetrazoles.

Figure 1.3 Cyclic peptides in different sizes.

N NH N HN O O O O Me Me HO

cyclo-[Pro−Val−Pro−Tyr] cyclotetrapeptide 3 H2N N H NH HN N O H N H Me O H N HN NH2 O TAN-1057C homodiketopiperazine 2 NH N O HN O H N O Me Me O O O chlamydocin cyclotetrapeptide 4 N N O O N H NH NH HN HN O Me O HN O Me O Me Me O segetalin B cyclopentapeptide] 5 OH HO Me Me

cyclo-[N-Me−Tyr−N-Me−Tyr] diketopiperazine

In contrast to the easily accessible diketopiperazines (Figure 1.3, e.g. 1)

derived from two

α

-amino acids, their seven-membered homodiketopiperazine homologues are much more

challenging. These seven-membered ring bis(lactams) are made up of

α

- and

β

-amino acids

and display interesting biological activities (e.g. TAN-1057 C 2).

Cyclic tripeptides are

generally very difficult to addresss because of the instability of these compounds due to a

transannular collapse into diketopiperazines.

For this reason, alkylated amide bonds are

usually

present,

preventing

this

rearrangement

of

the

cyclic

tripeptides.

Cyclotetrapeptides represent an abundant class of cyclic peptides with many examples from

nature.

Most of these peptides are isolated from fungi, marine sources, micro organisms or

higher plants and their biosynthesis occurs via non-ribosomal pathways.

A broad class of the

cyclic tetrapeptides comprise the histone deacetylase inhibitors (e.g. apicidin, HC-toxin and

chlamydocin 4),

which contain substituted 2-amino-8-oxodecanoic acids (Aodas) as one

of their constituents.

A second class consists of cyclic tetrapeptides containing two opposite

proline units (e.g. cyclo-[Pro

−

Leu

−

Pro

−

Leu]),

including tyrosinase inhibitors

(cyclo-[Pro

−

Val

−

Pro

−

Tyr] 3).

Different cyclic pentapeptides have been isolated from plants and

showed interesting biological activities (e.g. segetalin B 5).

A vast number of cyclic peptides are known containing a higher number of amino acids in the

ring, ranging from hexapeptides up to a 31 amino acids containing natural cyclic peptide,

cyclopsychotride A, isolated from a tropical plant.

However, by increasing the number of

amino acids in the ring, these cyclic peptides also increase the conformational flexibility,

reducing their use as effective potential drugs or scaffolds.

1.2

Difficulties in peptide cyclizations

Due to the limited amount of cyclic peptides available from natural sources, the

synthesis of cyclic peptides has been an increasingly important task.

Generally, the

formation of the macrocycle is the limiting step in the synthesis of cyclic peptides and is

accompanied by competing intermolecular reactions leading to oligomerization and

polymerization. The main reason behind this problem is the rigidity of the intermediate amide

bonds.

Scheme 1.1 Torsion angles

ϕ, χ, ω

and

ψ

and resonance stabilization and cis/trans

isomerization of peptide bonds.

N O N O H H N O H N O H transoid ω = 180ο cisoid ω = 0ο N H N O H R χ ω ψ ϕ

The configuration of the amide bond can be described in torsion angles

ϕ, χ, ω

and

ψ

(Scheme 1.1)

.

Rotation about the C-N amide bond in the peptide (

ω

) is

drastically hindered by the strong double bond character (the rotational barrier is ~ 105

KJ.mol

) of which the transoid (

ω

= 180

) conformation is energetically favoured over the

cisoid (

ω

= 0

) conformation by 8 KJ.mol

.

A second problem in the synthesis of cyclic peptides is the site of ring closure. This

phenomenon is also referred to as sequence dependency. This was illustrated by Schmidt et

al.,

who synthesized the cyclic pentapeptide cyclo-[Ala

−

Phe

−

Leu

−

Pro

−

Ala] 6 starting from

all the five possible linear precursors (Figure 1.4). It was shown that depending on the ring

closure site, yields of the final cyclic peptides ranged from 0% up to 21%, accompanied in

some cases by extensive dimerization.

Figure 1.4 Synthesis of cyclo-[Ala

−

Phe

−

Leu

−

Pro

−

Ala] 6 at different ring closure sites.

N H NH N HN HN O O O O O Me Me Me Me

cyclo-[Ala−Phe−Leu−Pro−Ala]

6 27% (dimer) 10% (dimer) 0% 21% (monomer/dimer) 21% (monomer)

However, although the choice of the correct linear precursor that will provide the cyclic

product is important, this is very difficult to predict. Several factors can influence

ring-closure, such as steric hindrance and kinetic competition with the dimerization reaction or the

transition-state energy. Molecular modelling experiments were performed on chlamydocin to

investigate all these factors

and comparison with experimental data revealed that the

transition-state energy proved to be the limiting factor in peptide cyclizations. The best linear

precursor for the cyclization was found to be the one providing the least strain in the

transition-state. This work was extended to the prediction of the best linear precursor of

several other cyclic peptides.

Many methodologies have been developed to improve the cyclization of peptides, with the

ideal synthesis being sequence independent, free of racemization and high yielding. The

traditional lactamization between the C-terminus and the N-terminus in solution is still the

most applied method for the synthesis of cyclic peptides, although as outlined before the

result of the final lactamization reaction is difficult to predict. The reactions have to

performed at high dilution (10

to 10

M) to avoid competing oligomerization and

polymerization. Many peptide coupling reagents have been developed for the activation of the

C-terminal carbonyl group,

including carbodiimides,

phosphonium,

uronium,

immonium,

imidazolium,

triazine,

organophosphorus

and acid halogenating

reagents.

Some coupling reagents result in better cyclization yields than others, although

the outcome differs in individual cases. No predictions can be made on the best results and

optimization of the lactamization is just a matter of trial and error. However, some additives

and coupling reagents derived from HOAt have been shown to promote cyclization by

preorganization of the linear precursor (Scheme 1.2) and suppress side reactions such as

racemization.

Scheme 1.2 Preorganization by HOAt-derived coupling reagents.

N N N N O O NH2 NH O N N N N OH

Primarily, prolonged existence of the activated carbonyl group of the amino acids or peptides

A (Scheme 1.3)

gives O-attack of the amide on the activated ester resulting in the formation

of an oxazolone B, which under mildly basic conditions undergoes racemization via the

formation of tautomer C. The resulting oxazolone stereoisomer mixture of A and D directly

reacts with a nucleophile, explaining the loss of stereochemical homogeneity of the coupled

material E and F. Consequently, peptide coupling should always proceed at the N-terminus

and mildly activating coupling conditions are needed in combination with protective groups

preventing oxazolone formation such as carbamates.

Scheme 1.3 Mechanism of the racemization during peptide coupling via oxazolones.

N P O H O X R X P O N O R H base P O N OH R acid P O N O H R Nu N P O H O Nu R H A B C D E + P N O H O Nu H R + F

To overcome the limitations of the classical lactamization strategies, different alternative

approaches have been developed. Preorganization of the linear precursor and strain relief of

the linear precursor are main pivots on which these methods hinge. Different auxiliary-based

methods have been developed for the synthesis of several cyclic peptides, based on the

original strategy by Meutermans et al. (Scheme 1.4). Salicylaldehyde derived linkers are

incorporated into linear peptides, inducing the cyclization by positioning of the C- and

N-termini in close proximity, after which a transannular O→N acyl transfer reaction delivers the

final lactams.

Scheme 1.4 Auxiliary-mediated synthesis of cyclic peptides.

R HN O O N O HO R HN O O N acyl transfer auxiliary cleavage R OH O H

This approach has been successfully applied in the synthesis of all-L cyclic pentapeptides and

tetrapeptides. The auxiliary was attached at the N-terminus of the linear peptide. However,

extensive C-terminal racemization occurred during the formation of the macrolactone.

To

overcome these problems, incorporation of the auxiliary in between the linear peptide resulted

in the formation of bis(lactams) without extensive racemization of the C-terminus in a

sequence independent manner.

The synthesis of cyclic peptides has also been described by means of an intramolecular

Staudinger ligation (Scheme 1.5).

Coupling of a borane-protected auxiliary to azido acid

dipeptides resulted in the formation of the linear cyclization precursors 7 and 8, after which

the intramolecular Staudinger ligation could be effected after liberation of the phosphane. The

1,4-diazepine-2,5-dione 9 was obtained from both linear precursors in reasonable yield.

Scheme 1.5 Synthesis of 1,4-diazepine-2,5-dione by intramolecular Staudinger ligation.

N₃ Ph N H O S O PPh2 BH3 N Ph NH O S O Ph2P HN Ph NH O O dabco H2O, 35% N₃ HN S O PPh₂ BH3 O Ph N NH S O Ph2P O Ph dabco H₂O, 29% 7 8 9

Similar to the synthesis of cyclic peptides in nature by non-ribosomal peptide synthetase

(NRPS), an isolated terminal thioesterase domain (TE) of this multienzymatic complex

catalyzed the cyclization of a decapeptide to form gramicidin S.

Other groups have used a

similar strategy for the synthesis of medium-sized cyclic peptides (9-14 residues).

Core-functionalized dendrimers have been used for the synthesis of cyclic peptides (Scheme

1.6).

Several generations of carbosilane dendrimers were functionalized at the core by

carbodiimides and used in the synthesis of small bis(lactams). Induced by the site-isolation

effect exerted within the dendrimeric core, intramolecular reactions were favoured over

intermolecular reactions and resulted in formation of the bis(lactams) in good yields

compared to usual carbodiimides.

Scheme 1.6 Encapsulation of the linear peptide prevents intermolecular reactions.

Solid phase chemistry has also been applied for the synthesis of cyclic peptides. In this case,

the pseudo-dilution effect exerted within the resins favours the intramolecular reaction over

intermolecular resins, especially with low resin loadings. Examples of different strategies for

the solid phase synthesis of cyclic peptides will be outlined in the next two sections.

1.3

Combinatorial chemistry

Traditionally, new chemical lead structures for pharmaceutical research relied on the

isolation and modification of natural products or on the mechanism-based or structure-based

design of potential drugs. With the emerging field of high-throughput screening (HTS)

devices in 1980s, these traditional methods no longer fulfilled the need for large amounts of

molecules. Thus, large numbers of molecules were needed for screening purposes. As an

answer, combinatorial chemistry was developed in the early nineties addressing the need by

the construction of chemical libraries from a collection of building blocks, and systematically

combining these to create a vast amount of molecules.

The potential of even a small set of building blocks is substantial and can lead to a large set of

molecules. Traditionally molecule A and molecule B react in a single vessel to form the

product AB. Applying the building block concept of combinatorial chemistry a set of

molecules A

-A

react with a set of molecules B

-B

resulting in the formation of any product

combination A

B

to A

B

. With the basic set of twenty encoded amino acids a tripeptide

would have 20

= 8,000 library entities, a tetrapeptide 20

= 160,000 library entities and a

pentapeptide even 20

= 3,2 million library entities. The advantage of combinatorial chemistry

is its efficiency, being faster and cheaper than the orthodox chemistry.

The construction of a large amount of molecules can be performed as mixtures, or can be

performed in parallel as single compounds, either in solution phase or on solid supports.

Although a diverse set of solid supports has been developed

with different linker units

and swelling properties in particular solvents,

reactions on solid supports afford clean

products after cleavage and reactions can be driven to completion by the use of excess of

reagents, the supports are not compatible with heterogeneous reagents or solid side products.

Solution phase techniques

have become increasingly popular and can employ existing

reactions and more harsh reaction conditions, but the purification of the products becomes

more important. Solid phase extractions

or chromatographic techniques

have been used

to deal with this issue, but more important are the use of immobilized reagents

or

scavengers

to selectively trap side products.

The early experiments with combinatorial chemistry were mainly in peptide chemistry. These

sets of building blocks were easily available and the chemistry of peptide coupling and

protection was well documented. A pioneer in the field of peptide synthesis was Merrifield,

introducing synthesis on solid supports. Most of the methods nowadays still rely on this

original synthesis.

Initial experiments with supports with the so-called split-and-mix

method

resulted in large quantities of mixtures of compounds, from which individual

activities were difficult

to deconvolute and false positives and negatives were commonly

encountered.

To circumvent the synthesis and screening of mixtures, peptides can be

synthesized on pins arranged on microtitre plates.

Another alternative is the so-called

‘teabag’ method where resins in sealed bags are placed in activated monomer solutions and

result in single peptide sequences in each bag.

Simultaneous coupling of mixtures of

activated monomers results in the formation many peptides, but the extent of coupling of each

monomer is difficult to control.

A very elegant method for the identification of single

library members is based on the optical encoding by laser etched grids in polypropene blocks

grafted with polystyrene resins.

1.4

Combinatorial synthesis of cyclic peptides

The main goal of the pharmaceutical industry nowadays has shifted from the careful

investigation of a few well documented targets towards the screening of a large amount of

molecules on a diverse and diffuse set of target structures using advanced methods, such as

bioinformatics, proteomics, genomics, combinatorial chemistry/HTS. However, especially the

latter two techniques have not fulfilled the initial expectations. Although the techniques have

matured to a state in which a large number of molecules can be synthesized and screened, it is

becoming more and more important which molecules are selected as input.

It has been observed in HTS, that a number of molecules have a significantly higher hit-rate

compared to other molecules. The term ‘privileged structures’ has been coined for these kind

of molecules by Evans in reference to the benzodiazepine scaffold.

These molecules have

the ability to bind to a large set of receptors.

Cyclic peptides are also considered as being part of these privileged structures.

Although

these molecules have not been identified as ‘drug-like’, some cyclic peptides have been

exploited as drugs and are currently on the market. However, a library of these molecules

would serve as a perfect molecular toolkit to study pharmacophoric properties. This library

would be optimally diverse, both in terms of conformational space and R-group diversity.

This is due to the possibility of introducing not only different side chains of the amino acids,

but also introducing both enantiomers. The exploration of this library could consequently lead

to valuable information on the structural properties of these pharmacophores and could

subsequently lead to lead optimization programs.

The solid phase synthesis of libraries of cyclic peptides has been performed by three main

strategies. The peptides may be synthesized by the anchoring of the different side chains of

the amino acids to a solid support. The peptide can also be linked to the solid support via the

nitrogen of a backbone amide in the so-called backbone amide linkage strategy. Finally, the

peptide can be synthesized immobilised via the C-terminus either with an activated linker or

with a ‘safety catch’ linker.

Attachment via the side chain of the amino acids of the peptide is the most common method

for the synthesis of cyclic peptide libraries (Figure 1.5). The synthesis requires an orthogonal

protection of the C- and N-termini and is usually performed by N-Fmoc and CO

All

protection. The final macrolactamization is performed on the resin and cleavage of the cyclic

peptide and removal of the side chain protective groups is accomplished in one step after

cyclization. A disadvantage of this method is the requirement of specific residues in the

design of the cyclic peptide.

Figure 1.5 Side chain attachment via different amino acids.

HN O R CO2All NHFmoc X O HN O = Lys X−R: X O n = Asp (n = 1), Glu (n = 2) X = O, N Si Me Me = Phe O = Tyr N N = Hys X−R:

Obviously the amine functionality of lysine

together with the acid functionality in the

glutamic

and aspartic acid

side chains serve as perfect resin attachment sites. The

tyrosine phenolic hydroxyl group has been anchored to a solid support via a Mitsunobu

reaction

and histidine has been attached via its imidazole ring to trityl-resins and both have

been used for the synthesis of cyclic peptides.

Besides this, an elegant traceless method

based on arylsilanes has been developed for the immobilization of phenylalanine and the

subsequent synthesis of sansalvamide A and scytalidamide A.

Using the side chain

attachment method, a self-deconvolution cyclic pentapeptides library based on the natural

peptide BQ-123 designed to produce 82,944 cyclic peptides, was prepared.

At four variable

positions a dosen residues were scanned and although no analogues more active than the

natural peptide were found, this example nicely shows the potency of cyclic peptide libraries.

A second method which has been employed for the synthesis of cyclic peptides is by means of

the so called backbone amide linkage strategy (Figure 1.6). This has the advantage over the

earlier described method of side chain attachment that no specific residues are required. The

backbone amide linker can be placed at any residue except for proline. Besides this, N-amide

alkylation has been speculated to prevent aggregation

of the growing peptide chain and can

aid the final macrolactamization by favouring the cisoid character of the amide bond.

Different types of backbone amide linkers have been described depending on the cleavage

conditions. Poly(alkoxy)benzylamine linkers (PAL) can be efficiently removed from the final

product by treatment with TFA.

Monoalkoxybenzylamine type linkers require harsh

condition for their removal, like treatment with HF, but have been successfully applied in the

synthesis of library of cyclic pentapeptides based on somatostatin.

Different to the

benzylamine derived linkers was the use of a hydroxylamine type linker, from which the

peptides could be removed by treatment with SmI

.

Figure 1.6 Different backbone amide linkers.

L N CO2All O NHFmoc L O N O OMe MeO : _{cleaved by:} TFA O N O HF ON O SmI2

Although attachment via the side chains and backbone amide linkers attached via the amide

provide robust procedures, further purification of the peptides after cleavage is usually

required to remove the remainings of the side chain protection and cleavage cocktails. A more

ideal type of linker would eliminate final purification and would render pure cyclic peptides.

Initially these types of linkers were activated linkers, attached via the C-terminus to the

peptide (Scheme 1.7). During the usually N-Boc based peptide synthesis, this bond was

designed to be stable to acidic conditions and nucleophilic attack during the coupling

procedures, but at the end be susceptible for cleavage through head-to-tail lactamization of the

final peptides.

Scheme 1.7 Activated linker method.

Nitro-substituted phenols have been used for the synthesis of cyclotetrapeptides, activated by

the neutralization of the end amine groups with triethylamine.

Ösapay et al.

applied

Kaiser’s oxime resin for the synthesis of several peptides.

A series of cyclic penta-,

hexa- and heptapeptides was synthesized using the thioester linkage in a type of

intramolecular native chemical ligation strategy.

Finally polymer-bound HOBt was used

for the synthesis of a series of small lactams.

However, the initial strategies suffered from substantial loss of the peptides during the

synthesis due to the labile activated bond. Moreover, complete side chain protection was still

necessary during the cyclization. A second generation of these types of linkers were based on

the so-called ‘safety catch’ principle (Scheme 1.8). These linkers were attached to the peptide

by a stable bond. After completion of the synthesis, this bond is transformed to a labile bond

inducing the peptide cyclization.

Scheme 1.8 Safety catch linkers pre-activated (X) and activated (X*).

NHBoc O activation NH O H N HN X NHBoc O X* N-deprotection cyclization X X* N N S N H O O S N O O CN O OBn O OH S O S O O O X X*

The first types of these linkers were developed by Marshall and Flannigan and relied on the

oxidation from sulphide to sulphone, but major drawbacks were caused by cysteine residues

and methionines.

A hydrazide-activated linker was used for the synthesis of stylostatin

1, activated by oxidation.

The best known safety catch linker was developed by Kenner

and was used in the synthesis of many cyclic peptide libraries.

For example a

192-membered library of cyclic decapeptides was constructed on the basis of the natural products

tyrocidine, streptocidin and loloatin.

Screening identified nine analogues with potencies

increased up to nine-fold compared to the natural products. Alkylation of side chains could

again be a potential problem, especially employing cysteine and tryptophan. Finally, a

catechol type linker was described that is deactivated by protection of one of the hydroxyl

groups with a benzyl group. Activation is employed by treatment with TFMSA.

In the synthesis of cyclic peptides problems can be encountered within the growing peptide

chain.

The incorporation of difficult sequences,

generally consisting of sterically

hindered building blocks or

α

,

α

-disubstituted amino acids, has initiated the development of

different coupling reagents and coupling strategies.

A second problem is the

back-folding of the growing peptide chain, inhibiting proper couplings of the next amino acids. The

choice of solvent and polymer support is crucial in these cases,

but also repetitive

alkylation of the amides of the growing peptide chain inhibits unwanted back-folding.

Diketopiperazine formation during coupling of the third amino acid on the peptide depends

strongly on the coupling conditions and the linkage to the solid support, but is still

encountered as a persisting side reaction

(Scheme 1.9).

Scheme 1.9 Diketopiperazine formation during peptide synthesis.

X O H N O NH₂ R1 R2 HN NH O O R1 R2 + XH 10

Although different methods exist for the combinatorial synthesis of cyclic peptides, the

ultimate strategy, independent of the sequence design of the linear precursor, should be high

yielding and free from competing side reactions. Therefore, several new strategies will be

presented for the combinatorial synthesis of cyclic peptides, each with their own advantages.

1.5

Outline of the thesis

The main goal of the research described in this thesis was to develop combinatorial

approaches towards the synthesis of small homodetic and heterodetic cyclic peptides. This

work would build on the already existing methods developed in our group for the synthesis of

small homodetic cyclic peptides and triazole-containing cyclic peptides.

In Chapter 2 the existing auxiliary developed for the synthesis of bis(lactams) was refined to

make it more sequence independent and robust. The improved auxiliary was further modified

to make it suitable for solid supported reactions.

A backbone amide linker strategy for the solid phase synthesis of triazole-containing cyclic

pseudotetrapeptides and pentapeptides has been described in Chapter 3. The method was

optimized for the synthesis of a triazole analogue of the cyclic tetrapeptide

cyclo-[Pro

−

Val

−

Pro

−

Tyr] and used for the synthesis of a library of tetrapeptides. An analogue of

the cyclic pentapeptide segetalin B was made and the effects of the introduction of the triazole

linkage on the conformation of the peptide were elucidated.

Chapter 4 describes a new strategy for the introduction of 1,5-connected triazoles in cyclic

peptides. These proposed cisoid amide bond surrogates were introduced into the cyclic

tetrapeptide cyclo-[Pro

−

Val

−

Pro

−

Tyr] at different sites. The effects of the introduction on the

final macrolactamization was investigated.

The usefulness of a combination of the Ugi multicomponent reaction and the

copper-catalyzed azide-alkyne cycloaddition reaction for the synthesis of small cyclic peptides is

described in Chapter 5. The method was optimized for small cyclic pseudopeptides and cyclic

pseudotetrapeptides and used for the synthesis of libraries of triazole-containing cyclic

pseudopeptides. Finally, this methodology was applied to the synthesis of an analogue of the

biologically relevant cyclic tetrapeptide chlamydocin.

In the final Chapter 6 a retrospection on the different topics of this thesis is presented together

with a discussion on the future of the topics outlined in this thesis.

1.6

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