design and synthesis of at receptor selective angiotensin ...165358/fulltext01.pdf2 receptor ligands...

Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 316

Design and Synthesis of AT2Receptor Selective Angiotensin IIAnalogues Encompassing - and

-Turn Mimetics

BY

ULRIKA ROSENSTRÖM

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

Till min familj

Papers Included in the ThesisThis thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I. Rosenström Ulrika, Sköld Christian, Lindeberg Gunnar, Botros Milad, Nyberg Fred, Karlén Anders, Hallberg Anders. (2004). A selective AT2 receptor ligand with a -turn-like mimetic replacing the amino acid residues 4–5 of angiotensin II. J. Med. Chem., 47(4), 859-870.

II. Rosenström Ulrika, Sköld Christian, Lindeberg Gunnar, Botros Milad, Nyberg Fred, Hallberg Anders, Karlén Anders. (2004).Synthesis and AT2 receptor binding properties of angiotensin II analogues. J. Peptide. Res., 64(5), 194-201.

III. Rosenström Ulrika, Sköld Christian, Plouffe Bianca, Beaudry Hélène, Lindeberg Gunnar, Botros Milad, Nyberg Fred, Wolf Gunter, Karlén Anders, Gallo-Payet Nicole, Hallberg Anders.New selective AT2 receptor ligands encompassing a -turn mimetic replacing the amino acid residues 4–5 of angiotensin II act as agonists. Submitted.

IV. Rosenström Ulrika, Sköld Christian, Lindeberg Gunnar, Botros Milad, Nyberg Fred, Karlén Anders, Hallberg Anders.Design, synthesis and incorporation of a -turn mimetic in angiotensin II. Novel pseudopeptides with affinity for both AT1 and AT2 receptors. Manuscript.

Reprints are presented with permission from the publishers: the American Chemical Society and Blackwell Munksgaard.

Contents

1 Introduction .........................................................................................91.1 Peptides as Drug Targets ..............................................................91.2 Strategies for the Development of Peptidomimetics ..................101.3 Secondary Structure Mimetics ...................................................13

1.3.1 -Turn Mimetics ....................................................................131.3.2 -Turn Mimetics.....................................................................15

1.4 Benzodiazepines in Peptidomimetics .........................................16

2 Aims of the Present Study.................................................................19

3 Angiotensin II.....................................................................................203.1 The AT1 Receptor.......................................................................21

3.1.1 Structure-Activity Relationship .............................................233.1.2 Bioactive Conformation.........................................................23

3.2 The AT2 Receptor.......................................................................243.2.1 Structure-Activity Relationship .............................................253.2.2 The Ligand Binding Site........................................................26

4 Design and Synthesis of -Turn Mimetics .......................................284.1 Design of Benzodiazepine-based -Turn Mimetics....................284.2 Synthesis of Benzodiazepine-based -Turn Mimetics................31

4.2.1 N-Terminal in Position 7 of the Benzodiazepine...................314.2.2 N-Terminal in Position 9 of the Benzodiazepine...................34

4.3 Incorporation of -Turn Mimetics into Ang II ...........................36

5 Design and Synthesis of -Turn Mimetics .......................................405.1 Design of a Benzodiazepine-based -Turn Mimetic ..................405.2 Synthesis of a Benzodiazepine-based -Turn Mimetic ..............415.3 Incorporation of -Turn Mimetic into Ang II ............................46

6 Peptidic Ang II Analogues ................................................................476.1 Glycine Scan...............................................................................476.2 N-Terminal Modifications..........................................................476.3 Synthesis.....................................................................................48

7 Structure-Activity Relationships ......................................................497.1 Pseudopeptides Encompassing a -Turn Mimetic ......................49

7.1.1 Receptor Binding and Molecular Modeling ..........................497.1.2 Functional Studies..................................................................55

7.2 Pseudopeptides Encompassing a -Turn Mimetic .....................567.3 Peptidic Ang II Analogues .........................................................57

8 Concluding Remarks .........................................................................60

9 Acknowledgements ............................................................................61

10 References...........................................................................................63

11 Appendix.............................................................................................79

Abbreviations

3D three dimensional Ac acetyl ACE angiotensin converting enzyme Ang II angiotensin II Ala alanine Arg arginine Asp aspartic acid Boc tert-butoxycarbonyl BSA bovine serum albumin Cbz benzyloxycarbonyl DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DIEA N,N-diisopropylethylamine DMAP dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide ECL extracellular loop Fmoc 9-fluorenylmethyloxycarbonyl Gln glutamine Glu glutamic acid Gly glycine GPCR G-protein-coupled receptor HATU N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-

methylene]-N-methylmethanaminium hexafluorophosphate N-oxide His histidine Ile isoleucine Lys lysine MS mass spectroscopy NMM N-methylmorpholine NMR nuclear magnetic resonance PDB Protein Data Bank PG protecting group Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Phe phenylalanine Pro proline PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium

hexafluorophosphate

RAS renin-angiotensin system RP-HPLC Reversed-phase high-performance liquid chromatography SAR structure-activity relationship Sar sarcosine, N-methyl glycine SPPS solid-phase peptide synthesis TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl TEMPO 2,2,6,6-tetramethyl piperidinoxy TMD transmembrane domain TFA trifluoroacetic acid THF tetrahydrofuran Trp tryptophan Tyr tyrosine Val valine

9

1 Introduction

1.1 Peptides as Drug Targets Endogenous proteins and peptides affect a majority of the physiological processes in the human body. Most of these exert their action at specific receptors. Among these receptors a large and important group are the seven transmembrane G-protein-coupled receptors (GPCRs).1,2 With the recent elucidation of the human genome, it can be expected that many new biologically active proteins and peptides with relevance in disease processes will be discovered. This means that many new therapeutic targets will be identified, which can be used in the development of future drugs.3,4

Although there are a growing number of therapeutic targets in which peptides act as agonists or antagonists, the native peptides in most cases have a limited applicability as drug candidates. These limitations include: 1) rapid metabolism by proteolysis, 2) poor absorption by the gastrointestinal tract, and poor transport over the blood-brain barrier, 3) rapid excretion by the liver and the kidneys, and 4) lack of receptor specificity due to the conformational flexibility inherent to peptides.5,6 Thus, there is a need for rapid and efficient ways to convert knowledge of the structure and key pharmacophore groups in native peptides into new, nonpeptidic drug candidates.7 These compounds are often referred to as peptidomimetics.

HO O OH

HN

H2N

HN

NH

HN

NH

OH

O

O

O

O

O

OH

Figure 1. The endogenous ligand Leu-enkephalin (left) and morphin (right).

One of the best-known examples of a peptidomimetic is morphine (Figure 1), which was discovered and used long before its endogenous receptor or peptide ligands, enkephalins, had been identified.8 It is still not known how enkephalin rationally can be converted into morphine. Actually, morphine

10

and related opioids were the only known examples of nonpeptide agonists acting on peptide receptors until 1995.9

The development of peptidomimetic protease inhibitors has been feasible in recent years with the availability of three-dimensional (3D) structural information on proteases from X-ray diffraction and NMR spectroscopy.10

However, the more indirect ligand-based design process is today the only option in the development of nonpeptidic agonists/antagonists to GPCRs. The first high-resolution X-ray diffraction image of a GPCR, that of bovine rhodopsin, was published in 2000.11 Hopefully, the 3D structures of other GPCRs will become known in the future opening the way for more rational design in our efforts to find agonists/antagonists to peptide receptors. Until the 3D structures of other GPCRs have been resolved we will have to rely on homology modeling of the receptors based on the structure of bovine rhodopsin and site-specific mutagenesis to provide important clues in our quest for new drugs for these receptors.12

1.2 Strategies for the Development of Peptidomimetics Peptides have a large degree of conformational freedom associated with their many freely rotatable bonds and can adopt a large number of conformations. Unfortunately, in solution and in the absence of the receptor or enzyme the biologically active conformation may be poorly populated. Conformationally constrained analogues can significantly aid in the identification of these conformations. Peptidomimetics are valuable research tools and provide important information on structure-activity relationships (SARs) of both peptides and complex proteins.8,13,14 Constrained structures mimicking the bioactive conformation will be less exposed to proteolytic cleavage, may give more selective ligands, providing an entropy advantage in receptor binding compared to the more flexible linear peptides.8,15,16

In peptidomimetics the peptide backbone has been replaced but the essential side chains and/or functionalities are most offen still present in the correct three-dimensional orientation. These compounds are able to mimic or block the biological effects of the native peptide.17-19 A number of authors have described general strategies for the development of peptidomimetics from biologically active peptides6,12,18-22 similar to the one outlined in Figure 2.

Once the primary structure of the biologically active peptide has been identified, the first design step is to truncate the peptide. The amino acids are removed from the amino and carboxyl termini, one at a time, to obtain the smallest biologically active peptide fragment. Subsequently, side chain requirements (i.e. the pharmacophore groups) can be determined by systematically replacing each residue in the peptide with a specific amino

11

acid and evaluating the biological activity. The amino acid most often used is alanine (Ala scan), but occasionally glycine is used.

Biologically active peptide

Identification of pharmacophore groups and active core - truncation and deletion - amino acid scans

Synthesis of constrained analogues - local constraints - global constraints

Design and synthesis of secondarystructure mimetics - turn mimetics

Bioactive conformation and 3D pharmacophore models

Design and synthesis ofnon-peptide scaffold mimetics

Peptidomimetic

Biophysical studies- NMR

- molecular modeling

Biophysical studies- NMR

- molecular modeling

Figure 2. A general strategy for the development of peptidomimetics.

After the SAR of each amino acid in the peptide has been determined the next step is to try to establish the bioactive conformation(s). The conformational freedom of the highly flexible peptide can be reduced by the introduction of local constrains and/or global constrains. Methods of obtaining local constraints include incorporation of modified amino acids (e.g. D-amino acids and N-methyl amino acids), introduction of modified amide bonds or short-range cyclizations to get dipeptide mimetics. Global constrains are achieved by medium or long range cyclization forming a link between two backbone termini, between one of the termini and one side chain, between two side chains, or between backbone atoms other than the termini. Secondary structure mimetics can also be used as constrains. A secondary structure mimetic is a building block that, when incorporated into a peptide, enforces a particular conformation. Incorporation of such a moiety will provide additional information about the requirements for receptor binding and/or activation. Secondary structure mimetics will be further discussed in Section 1.3. The SAR of the constrained analogues together

12

with information obtained from biophysical studies such as NMR spectroscopy and molecular modeling can, in an iterative process, give a three-dimensional pharmacophore model of the bioactive conformation. The last step is to introduce the pharmacophore groups onto a nonpeptidic scaffold in the correct spatial arrangement, in agreement with the obtained model of the bioactive conformation. The nonpeptidic scaffolds should preferably be small organic molecules with defined stereochemistry and, in order to avoid hydrophobic collapse in an aqueous environment, these core molecules should be rather rigid.23

Although strategies are available today to convert peptides into nonpeptidic drug candidates the process is difficult and laborious and there is no guarantee that new, nonpeptidic drug candidates will be discovered.3Instead, the receptor-based screening of natural or synthetic product collections has proven to be a more useful method to identify peptidomimetics. Furthermore, design, combinatorial chemistry and classical medicinal chemistry have important roles. A number of nonpeptide drug candidates have recently been identified,9,24,25 e.g. growth hormone secretagogues26 and cholecystokinin (CCK) agonists27, see Figure 3. Hirschmann et al. presented an example in which pharmacophore modeling was used to obtain a peptidomimetic agonist of the cyclic peptide hormone somatostatin.28 As outlined in Figure 4, a -D-glucose core structure was decorated with the desired amino acid side chains in an elegant design process via a cyclic hexapeptide. Progress in the field of peptidomimetics has been thoroughly reviewed.3,4,6,8,12,13,16,19,20,22,23,25,28-36

N

N

O

O O

NH

NH

O

N

N

N

SO2CH3

OO

HN

ONH2

A CCK-A agonist A growth hormone secretagogue

Figure 3. Examples of nonpeptide agonists to peptide receptors.

13

Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

NH

HN

O

N O

ONH

HN

O

O

O NH

NH2

NH

O

OO

O O

O

NH

NH2

somatostatin

OH

S S

Figure 4. The endogenous peptide ligand somatostatin (top), an active hexapeptide intermediate (left) and a peptidomimetic agonist (right).28

1.3 Secondary Structure Mimetics The often most essential conformational components of peptides and proteins are the secondary structure elements: -helices, -sheets, reverse turns and loops. These elements are often located on the protein surface and are thought to act as molecular recognition sites in biological processes. Even small peptide fragments can fold into turn conformations in which the amino acid side chains are displayed on the surface of a compact backbone core.37,38 There is overwhelming evidence suggesting that the side chain groups in the peptide are the most important recognition elements in peptide-receptor interaction.32 -Helices and -sheets have not been mimicked to as great an extent as reversed turns, but the number of mimetics is increasing. The reversed turns can be divided into -turns and -turns and will be further discussed in Sections 1.3.1 and 1.3.2.

1.3.1 -Turn Mimetics One of the most common structure motifs in proteins is the -turn.37 A -turn consists of four amino acids, which cause a reversal of direction of the peptide chain. To be considered a -turn the tetrapeptide sequence should not be part of an -helical region, and the distance between C of the first and C of the fourth amino acid residue should be 7 Å (Figure 5).39 The -turns comprise a rather diverse group and can be further classified according to the backbone dihedral angles and (presented in Figure 6) of the i+1and i+2 residues. The classical turn types include I, I´, II, II´, IV, VIa, VIb, and VIII.40,41 Turn type IV includes all the -turns found in proteins that cannot be classified into any of the classical types. In addition, a number of slightly different turn types have been presented in the literature.37,39,42,43 As

14

indicated in Figure 5 a hydrogen bond between the carbonyl in the first residue and the NH group in the fourth residue is often present to stabilize the turn in a pseudo-ten-membered ring structure.

CC 2

O

NHHN

O NH

O

O

NHR1

R2R3

R4

C1 N4

i

i+1 i+2

i+3

Bond 1

Bond 2

Bond 3

Bond 4

Bond 1

Bond 2 Bond 3

Bond 4

i+1 i+2

Figure 5. A -turn (left) with the i, i+1, i+2 and i+3 residues indicated and the angle defined by Ball et al.42

The -turn classification is based on the peptide backbone and does not clearly define the relative positions of the bonds 1, 2, 3, and 4 (see Figure 5). This is often inconvenient in the design of nonpeptide mimetics where the peptide backbone has been replaced. Therefore. an alternative description of the -turn has been introduced, the dihedral angle in the atom sequence C1-C 2-C 3-N4 defined as (Figure 5). The angle describes the topological relationship between the entry and exit peptide bonds of the -turn as well as the relative orientation of the side chains i+1 and i+2.42

C NC N

C

O

H O

HR Hi-1 i i+1

Figure 6. Definitions of the torsional angles and .

An ideal mimic of a reversed turn should position the appropriate functional groups on a relatively rigid framework. This can be a rather challenging synthetic task since turn mimetics often are highly functionalized molecules with defined stereochemistry.16

A large number of compounds suggested to mimic or induce a -turnhave been presented in the literature.8,13,15,19,38,42,44-48 These turn mimetics have been incorporated into peptides to reduce the conformational freedom, or used directly as peptidomimetics. The -turn mimetics can be divided into dipeptide mimetics that replace the i+1 and i+2 residues at the corner of the turn, and tetrapeptide mimetics that replace all four residues in the -turn.38

Among the -turn mimetics reported, those of the dipeptide type are most common. An early example of a -turn mimetic, compound A49 and somemore recent examples50-56 are presented in Figure 7. One of the challenges in the synthesis of -turn mimetics is to introduce all the desired side chains onto the turn scaffold. The first generation of turn mimetics often lacks most

15

of the side chains but in recent years the number of -turns comprising side chains has increased.

During the past years a number of libraries of -turn mimetics with a diverse set of pharmacophore groups arranged in different orientations have been produced. These substances have in most cases not been incorporated into peptides but rather been tested as peptidomimetics.46,52,57,58

H2N

O

OH

O

A

NH O

OMe

O

OtBu

S

NH O

Boc

OOH

H

N

NN

OO

OO

HNNH

O

OO

O

O2N

NH2O

HO

HN NH2

O

N

OH

OOMe

HN

OCbz

NH N

S

OO

H2NNH

E

B C

D

F G

N

O

O

N3

H

OOTMSE

HFigure 7. Examples of -turn mimetics (A49, B50, C51, D52, E53, F54, G55 and H56).

1.3.2 -Turn Mimetics A -turn, which is a more rare reversed turn, consists of three amino acid residues. It is defined by the existence of a hydrogen bond between the carbonyl group in residue (i) and the NH-group of the (i+2) residue forming a pseudo-seven-membered ring (Figure 8). The -turns are divided into two classes, inverse ( i+1 -70 to -85 and i+1 60 to 70 ) and classic ( i+1 70 to 85 and i+1 -60 to -70 ). The i+1 side chain is orientated in an equatorial position in the more common inverse -turn, while the rare classic -turn has an axial i+1 side chain. -Turns are thought to be rare in proteins but are found more frequently in small peptides, especially in small cyclic peptides.37,59-61

16

HN

O HN

O

NHO

R1

R2

R3i

i+1

i+2

Figure 8. A -turn with the i, i+1 and i+2 residues indicated.

The number of -turn mimetics and -turn inducers presented in the literature has increased in recent years,48,61-82 but compared to the -turn mimetics there are still very few. Most of the proposed -turn mimetics consist of a monocyclic structure with a six- or seven-membered heterocyclic or carbocyclic compound, see Figure 9. There are still very few -turn mimetics that have all side chains present and/or have been tested in a biological system.

O

N

HN

O

O

H

AcHNH

O NH

NFmocHN

OOH

OBn

N

O

O OHN3

ONHTrt

O

B CA

Figure 9. Examples of heterocyclic -turn mimetics (A77, B71 and C69).

1.4 Benzodiazepines in Peptidomimetics Benzodiazepine is a molecular framework that has been used to provide high-affinity ligands for many different receptor types and is therefore considered to be one of the most important privileged structures for drug discovery.83,84 Benzodiazepines been extensively studied since the mid 1950s, when researchers at Hoffman LaRoche discovered that chlorodiazepoxide (Figure 10) had an effect on the central nervous system.85

The seven-membered ring in a benzodiazepine can be formed as a cyclic dipeptide and a number of benzodiazepine alkaloids have been isolated as natural products.86

Benzodiazepines have been widely used as drugs with hypnotic, anticonvulsant, anxiolytic, and muscle relaxant properties.87 In addition, several benzodiazepine derivatives have shown antibiotic, antitumor, phytotoxic and antiviral activity.86 One of the most well-known benzodiazepine-based drugs is diazepam, better known as Valium (Figure 10).85 It has been suggested that benzodiazepines such as diazepam may be peptidomimetics of endogenous peptides.19,88,89

17

Cl N

NO

Cl N

N

HN

O

Chlordiazepoxide Diazepam

Figure 10. Chlordiazepoxide, one of the first lead structures with a benzodiazepine core, and diazepam, Valium , one of the most well-known benzodiazepine-based

drugs.

A diverse set of benzodiazepine-based structures has also been used as peptidomimetics and in the discovery process of nonpeptidic drug-like molecules.16,90 The synthesis of and biological data of a number of decorated 1,5-benzodiazepines and 1,4-benzodiazepines have been presented in the literature.67,79,91-98 Many of these derivatives are peptidomimetics that have been used as enzyme inhibitors or ligands to GPCRs.

N

N

NH2O

O OMe

N

N

O

O

H2N

NH

A

C

B

NH

NO

OHO

HN

OHN

NH2

Figure 11. Three examples of benzodiazepines used as turn mimetics. A, a nonpeptide ligand for the somatostatin receptor93, B, a -turn mimetic47 and C, a

peptidomimetic with a -turn moiety.79

The tetrapeptide part of a peptide that comprises a -turn has successfully been substituted by a decorated benzodiazepine scaffold and it has been concluded that 1,4-benzodiazepines can mimic a -turn.16,47,99-102

Furthermore, both 1,5-benzodiazepines and 1,4-benzodiazepines have been incorporated into peptides as constrained dipeptide mimetics.98,103-105 It has

18

also been proposed that the seven-membered diazepine ring in a 1,4-benzodiazepine mimics a -turn.78,79

19

2 Aims of the Present Study

This study is part of an ongoing research project with the overall aim of gaining knowledge on the conversion of peptides in an iterative fashion into nonpeptidic drug-like molecules with maintained biological activity.

The octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), the natural ligand for the AT1 and AT2 receptors, was selected as a model peptide for five major reasons.

An octapeptide has a suitable size for the challenging task of converting a peptide into a nonpeptide agonist. It is a well-studied peptide, and the structure-activity relationship concerning its binding to and activation of the AT1 receptor is well-established.It has been proposed that angiotensin II adopts a turn conformation centered at Tyr4 when interacting with the AT1 receptor. While the physiological role of the AT1 receptor is well-understood, the role of the AT2 receptor was less clear when this study was initiated. AT1 and AT2 receptor in vitro models for receptor binding and functional assays were available.

The specific objectives of this study were: to study the structure-activity relationships of angiotensin II when interacting with its AT2 receptor;to design and synthesize benzodiazepine-based secondary structure mimetics, both - and -turn mimetics, for incorporation into angiotensin II. Selective constrained AT2 receptor agonists should serve as research tools in the search for the bioactive conformation(s); to develop robust and general synthetic strategies that in principle could allow preparation of a variety of side chain decorated scaffolds;

A brief background on the model peptide, angiotensin II, and its receptors is given in Chapter 3.

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3 Angiotensin II

The endogenous hormone angiotensin II (Ang II) is a linear octapeptide with the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe. Ang II is the active component of the renin-angiotensin system (RAS), which plays an important role in the regulation of blood pressure, body fluid and electrolyte homeostasis (Figure 12). Ang II is produced by stepwise cleavage of angiotensinogen by the enzymes renin and angiotensin converting enzyme (ACE) via the inactive decapeptide angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu).106,107 When Ang II is degraded a number of biologically active peptide fragments are formed, for example, the heptapeptides angiotensin (2-8) (Ang III) and angiotensin (1-7), and the hexapeptide angiotensin (3-8) (Ang IV).107,108 The RAS was initially described as a systemic system, but it is now known that many tissues, including the vasculature, brain, kidney, and heart can produce Ang II in local systems. These local systems can synthesize Ang II both by ACE and by other pathways, e.g. via chymase.106,109,110

Renin

ACE

AT1 AT2

Angiotensinogen

Angiotensin I

Angiotensin IIACE

inhibitors

AT1 receptorantagonists

VasoconstrictionSalt/water reabsorptionAldosterone secretionSympathic activationCell growth and proliferation

VasodilationApoptosisCell differentiationAntiproliferation

Figure 12. The renin-angiotensin system, RAS.

The actions of Ang II are mediated by at least two distinct receptor subtypes, designated AT1 and AT2. These receptors were identified using selective receptor ligands including the AT1 receptor antagonist losartan (DuP 753)

21

(Figure 13), the AT2 receptor selective antagonist PD123,177 (Figure 16) and the agonist CGP 42112A (Figure 17).106,111,112

Two more Ang II related receptors have been proposed106,111, the AT3 and the AT4 receptors. The AT4 receptor, for which Ang IV is the natural ligand, has recently been purified and identified as an insulin-regulated aminopeptidase (IRAP).113 The transduction mechanisms for the proposed AT3 receptor are not known and the proposed receptor has not yet been cloned.

3.1 The AT1 Receptor The classical physiological effects of Ang II, such as vasoconstriction, salt/water reabsorption, aldosterone release, stimulation of sympathetic transmission and cellular growth, are mediated by the AT1 receptor. The AT1receptor belongs to the seven transmembrane GPCR family and it has been cloned in humans and several other mammals.106,107,109,114,115 The homology between various species is high. The AT1 receptor is a member of the rhodopsin family of the GPCRs and highly homologous with rhodopsin.116

The receptor gene encodes a 359-amino acid protein and has a molecular weight of 41 kDa, corresponding to the non-glycosylated protein. The human AT1 receptor is mainly distributed in the brain, adrenal gland, heart, vasculature and kidneys.106,107,109,114,115

In contrast to most other mammals, including man where one unique gene is codes for the AT1 receptor, rodents have two distinct isoforms of the AT1receptor, termed AT1A and AT1B. These receptors share 94% sequence homology. The two receptor subtypes have similar properties regarding ligand binding and activation, but whereas the AT1A receptor is predominantly expressed in the kidneys, liver, lungs, and vascular smooth muscle, the AT1B receptor is mainly found in the adrenal and anterior pituitary glands.106,107,114

Many of the amino acids involved in ligand binding, agonist activation, G-protein coupling and internalization of agonist-receptor complexes in the AT1 receptor have been identified using receptor mutagenesis and Ang II SAR. It has been shown that several residues located in the extracellular surface region are essential for ligand binding together with some polar or charged residues located within the transmembrane regions. Two disulfide bonds located in the extracellular part of the receptor are very important for the 3D structure of the AT1 receptor and for Ang II binding.106 In contrast to the binding pocket of peptide analogues, the nonpeptide AT1 antagonists interact mainly with the amino acid residues in the transmembrane part of the receptor. Mutational studies have shown that some parts of the transmembrane region are common binding sites for nonpeptide antagonists and peptidic agonists.117 The development of nonpeptide antagonists of the

22

AT1 receptor has gained a great deal of attention since Ang II is a major regulator of blood pressure and an important factor in cardiovascular diseases. A number of orally active selective AT1 receptor antagonists, the sartans, have been developed from lead compounds discovered at Takeda Chemical Industries.118-122 The first compound to reach the market was DuP 753 or losartan (Cozaar ).123 To date, six AT1 receptor antagonists have been approved by the Swedish Medical Agency for the treatment of hypertension and congestive heart failure,123 see Figure 13. They serve as a complement to the ACE inhibitors, which instead of blocking the AT1 receptor prevent the formation of Ang II (Figure 12).120,122

N

N

Cl

OH

NHNN

N

Losartan, DuP 753

NOH

O

O

Valsartan

N

NO

OO

O

Candesartan

HO

O

N

NOH

O

S

Irbersartan

N

NNH

NNN

O N

N

N

N

Telmisartan

OO

Eprosartan

O OH

NHNN

N

NHNN

N

Figure 13. Selective AT1 receptor antagonists approved for use on the Swedish market.123

An analogue to losartan was one of the first reported, high-affinity nonpeptide agonists for a non-opiate peptide receptor. This compound, the AT1 receptor agonist L-162,313 illustrated in Figure 14, has been reported to have balanced AT1/AT2 receptor affinity.124,125 Recently, it was confirmed by our group that it also acts as an AT2 receptor agonist in vivo.126

N N

N

S

S

HN

OO O

O

Figure 14. The AT1 and AT2 receptor agonist L-162,313.124,126

23

3.1.1 Structure-Activity Relationship The SAR of peptide analogues of Ang II has been reviewed (see references 127-131) and a summary is presented in Figure 15. The contribution from each amino acid residue has been examined and the Tyr4 residue with its hydroxyl group, the side chain of His6, the phenyl ring and the carboxyl group in the Phe8 have been found to be central for agonism, and are considered to be pharmacophore groups. The residues Val3, Ile5, and Pro7 are considered to have mainly conformational stabilizing roles and have therefore been frequently used for cyclization.132-143 In many of the published Ang II analogues Asp1 has been replaced with Sar (sarcosine, N-methyl glycine). [Sar1]Ang II has shown enhanced binding, potency, and stability.144

NH

HN

NH

HN

NH

NH2NO

O

O

O

O

O

HN

OOH

O

HO

O

NH

NHH2N OH

NHN

Residue 1Not required for agonism.Tolerant to extension.

Residue 4The hydroxyl group is essentialfor agonism and the aromaticring for binding.

Residue 2Groups with a positive chargeenhance potency.

Residues 3 and 5Conformational stabilizing roles.

Residue 7Conformational stabilizingrole. A secondary amine is preferred.

Residue 6His is required both forbinding and agonism.

Residue 8The aromatic ring is important for agonism and the carboxylate is crucial for both agonism and binding potency.

12

34

56 7

8

Figure 15. Summary of Ang II SAR at the AT1 receptor. Modified from Hodges et al.127

3.1.2 Bioactive Conformation The bioactive conformation of the highly flexible linear peptide Ang II when interacting with the AT1 receptor has been repeatedly studied and a large number of conformationally restricted Ang II analogues have been synthesized and evaluated for biological activity.77,132-143 Molecular modeling, mutagenesis studies, photoaffinity labeling, and NMR studies of Ang II and its rigidified analogues produced a number of possible 3D models of the receptor-bound conformation.116,132,133,136-139,145-154 It has, for example, been suggested that Ang II adopts a turn conformation around Tyr4

24

when interacting with the AT1 receptor.66,77,136,139,146,149,155,156 There are also models suggesting a strong bend in the Tyr4-Ile5-His6 region with a bioactive conformation characterized by a charge relay system between the hydroxyl in Tyr4, the imidazole in His6 and the carboxylate in Phe8.132,133,137 An extended bioactive conformation of Ang II has also been suggested.145

3.2 The AT2 Receptor The AT2 receptor was discovered in 1989 and interest in it, and knowledge concerning it have increased in recent years.106,107,109,110,114,115,157-164 Today, the biological actions of the AT2 receptor are still elusive but seem to oppose many of the AT1 receptor mediated effects. It has been suggested that the AT2 receptor plays a role in antiproliferation, neuronal cell differentiation, apoptosis, and vasodilation.114,157,158,163,165 In addition, it was recently reported that the AT2 receptor mediates carbonate secretion by the duodenal mucosa.166 The AT2 receptor is the predominant Ang II receptor subtype expressed in fetal tissue, and as its density drops dramatically after birth it is believed to play an important role in fetal development.107,114,158 The AT1receptor is the dominant subtype in adult tissues, but the AT2 receptor is up-regulated in pathological conditions such as heart failure, renal failure, myocardial infarction, brain lesions, vascular injury, and wound healing.107

In the adult the AT2 receptors are mainly found in distinct brain areas, the adrenal gland, vascular endothelium, heart, uterus, and ovary.114,158,164

The AT2 receptor has been cloned from various species, including man, and the amino acid sequence identity between species is above 90%.167,168 It is a seven transmembrane receptor that comprises 363 amino acids. Although the AT1 receptor and the AT2 receptor have similar binding affinities for Ang II they only share 32-34% sequence homology.169,170 The AT2 receptor is a member of the GPCR family but it is one of the most controversial. The signaling mechanisms are diverse and not clearly understood.107,114,157

Some nonpeptide AT2 receptor selective antagonists, e.g. PD123,177 and PD123,319, have been used to identify the AT2 receptor subtype.127,171 Since then a number of AT2 receptor selective compounds and substances with balanced AT1/AT2 receptor affinity have been developed starting from AT1selective antagonists, for example, L-163,579172 (Figure 16). As mentioned above the nonpeptidic AT1 receptor agonist L-162,313 has been reported to also act as an AT2 receptor agonist.126 The first selective nonpeptide AT2receptor agonist was recently described by our group (Figure 16).173

25

N

NN

O

PhPh

R

OH

O

N

N O

HN N

O

S

HN

O

O O

OF

S SHN

O

OO

O

N N

PD123,177: R = NH2PD123,319: R = NMe2

L-163,579

Figure 16. The AT2 receptor antagonists PD123,177 and PD123,319 (left), the balanced AT1/AT2 receptor ligand L-163,579 (midle) and the selective AT2 receptor

agonist (right).

The roles and importance of the AT2 receptor are still not fully understood and the biochemical and physiological functions of the AT2 receptor are still a matter of intense research.106,159 To be able to learn more in this area there is a need for new selective ligands.

3.2.1 Structure-Activity Relationship The peptide SAR for ligands of the AT2 receptor has not been as thoroughly investigated as the SAR of the AT1 receptor ligands. Some investigations have been presented in the literature,131,139,141,144,174-181 but the functional properties of AT2 receptor ligands are only known for a few compounds. The AT2 selective Ang II analogue CGP 42112A (Figure 17) is an agonist and has been regarded as a primary pharmacological tool for AT2 receptor determination and function.181 It has also been shown that a minor modification of Ang II, substitution of His6 for 4-amino-phenylalanine, produces an AT2 receptor agonist that has negligible affinity for the AT1receptor.179

N

NH

O HN

O

HN

O

NH

NO

O

HO

NHN

O

HN OH

O

HN CbzHN NH2

NH

CGP 42112A

NH

HN N

H

HN N

HNH2N

O

O

O

O

O

O

HN

OOH

O

HO

O

NH

NHH2N OH NH2

[4-NH2-Phe6]Ang II

Figure 17. Two AT2 selective agonists, CGP 42112A181 and [4-NH2-Phe6]Ang II179.

Most of the binding studies presented indicate that modifications of the linear peptide Ang II or [Sar1]Ang II are well tolerated by the AT2 receptor.

26

Miura et al. introduced Ala into six of the eight positions in [Sar1]Ang II. Ile5

was not substituted, and the Arg residue was replaced by Gln instead of Ala.182 Only a minor decrease in binding affinity was observed. Arg2, Tyr4,and His6 were most sensitive to changes and may have roles as pharmacophore groups. The N-terminal amino acid Asp1 is not important for binding since Ang III (angiotensin (2-8)) has a similar affinity to that of Ang II119,139,176 and a large number of modifications in position one are allowed.176

The Arg2 residue might be of importance since Ang IV (angiotensin (3-8)) has been reported to have low affinity for the AT2 receptor.176 Furthermore, hydrophobic amino acids in position eight in combination with aromatic side chains in position seven enhance the AT2 receptor selectivity.176,178

Cyclic Ang II analogues have been synthesized and evaluated for AT2receptor affinity.139,141,175 Disulfide monocyclizations in the 3-5 region of [Sar1]Ang II resulted in a drop in AT2 receptor affinity but retained AT1receptor affinity.139 In contrast, compounds bicyclized in the same region had a high affinity for the AT2 receptor but in general low affinity for the AT1 receptor.139 Monocyclic methylenedithioethers have been reported with high affinity for both AT1 and AT2 receptors.175 Conformational characterization of some of these methylenedithioethers suggests that they induce inverse -turn conformations around Tyr4 in Ang II. Furthermore, AT2 receptor selective compounds have been obtained when 4-amino-phenylalanine was introduced into monocyclic methylenedithioethers.175

3.2.2 The Ligand Binding Site The sequence homology between the AT1 and AT2 receptors is low but there is homology in the transmembrane hydrophobic domains and it has been hypothesized that some of the amino acid residues important for Ang II binding to the AT1 receptor are preserved in the AT2 receptor.159,183 The C-terminal carboxylate in Ang II is proposed to interact with the positively charged side chain of the residue Lys199 in the fifth transmembrane domain (TMD) in the AT1 receptor. The corresponding amino acid in the AT2receptor is probably Lys215. It has been shown that positively charged amino acids are required in this position.183,184 Arg167 located at the junction of the second extracellular loop (ECL) and the fourth TMD, and Asp281 in the third ECL of the AT1 receptor, are suggested to interact with the side chain of Tyr4 and Arg2 in Ang II, respectively. Replacing the corresponding amino acid residues in AT2, Arg182 and Asp297, with Ala decreased the binding affinity drastically.174,185,186 When His273 in the sixth TMD of AT2 was replaced by Arg or Glu, the peptidic ligands lost their binding affinity suggesting an important role in ligand receptor interaction.256 The corresponding amino acid residue in the AT1 receptor is probably His. This residue is needed for agonist activation of the AT1 receptor and interacts with the side chain of Phe8 in Ang II.187 Phe308 in the seventh TMD of the

27

AT2 receptor has also been investigated and found to be involved in the formation of a high affinity form of the AT2 receptor.188 However, Phe308 in the AT2 receptor seems not to be of the same importance for receptor activation as the analogous residue in the AT1 receptor, Tyr292. Furthermore, Trp269, Asp279, and Tyr108 in the AT2 receptor are not essential for peptide ligand binding whereas the corresponding residues Trp253, Asp263, and Tyr92

in the AT1 receptor are important for binding.186,189 This indicates that there may be similarities in the ligand-receptor interaction between the AT1 and AT2 receptors but also clear differences.

Moreover, deletion of the N-terminal residues in the AT2 receptor results in reduced affinity for Ang II. In contrast, the AT2 selective compounds CGP 42112A and PD123,319 do not lose affinity suggesting the existence of different binding modes.190 Additionally, radioligand binding experiments have shown that CGP 42112A has unique binding interactions with the seventh TMD and the third ECL suggesting that Ang II, CGP 42112A, and PD123,319 have distinct binding determinants.191

Deraët et al. used photoaffinity labeling in combination with mutagenesis studies and suggest that Ang II binds to the AT2 receptor in an extended conformation.192 In agreement with this finding it was recently suggested that Ang II has an extended backbone conformation in the Tyr4-Ile5 region interacting with the AT2 receptor.141 In addition, it has been proposed that some cyclic Ang II analogues with low nanomolar affinity for the AT2receptor introduce inverse -turn-like conformations around Tyr4 in Ang II.175

28

4 Design and Synthesis of -Turn Mimetics (Papers I and III)

4.1 Design of Benzodiazepine-based -Turn Mimetics

Conformational analysis and the SAR of rigidified Ang II analogues have suggested that Ang II adopts a turn conformation around Tyr4 when interacting with the AT1 receptor.66,77,139,140,143,146,150 There are also indications that there might be a turn region around His6.138 The AT2receptor has so far been less investigated and the bioactive conformation(s) involved in the activation of/binding to the AT2 receptor is not as well established. It has been suggested that Ang II adopts a turn conformation around Tyr4 when interacting with the AT2 receptor.175 We wanted to gain more information about the bioactive conformation of Ang II when interacting with the AT1 receptor and, especially, with the AT2 receptor. Therefore we designed, synthesized and evaluated the two -turn mimetics presented in Figure 18. In both these -turn mimetics the hydrogen bond between CO and NH of the -turn has been replaced by carbon fragments with neither hydrogen accepting nor donating capacities.

N

HN

O

R2

NH

O

NH

I II

NHNH

NO

R2

NH

O

R3

NH

HN

NH

HN

NH

NH2NO

O

O

O

O

O

HN

OOH

O

HO

O

NH

NHH2N OH

NHN

12

34

56 7

8

Figure 18. The octapeptide Ang II (above) and the two investigated -turn mimetics (below).

29

The isoquinoline-based -turn mimetics incorporated into the pseudopeptides 1a and 1b (Figure 19) have previously been presented in the literature and evaluated with regard to AT1 receptor binding.77 In this study, the two diastereoisomers 1a and 1b (a and b denote single diastereoisomers of unassigned absolute stereochemistry) have been evaluated regarding AT2receptor affinity (Table 1, Section 7.1). It was found that one of the diastereoisomers (1a) had binding affinity to the AT2 receptor (Ki = 61 nM). We hypothesized that this diastereoisomer has the stereochemistry that corresponds to the natural L-configuration. In the new benzodiazepine-based -turn mimetic I (Figure 18) an additional NH-group has been introduced

compared to the turn mimetic incorporated in 1a/1b. The benzodiazepine scaffold is more bulky than a peptide -turn but the diazepine ring in a benzodiazepine can be considered a -turn mimetic and has previously been suggested to mimic a -turn.78,79 Since cyclizations between both positions 3 and 5 and between positions 5 and 7 in Ang II have generated bioactive compounds, both a tyrosine side chain and a histidine side chain have been introduced as the R-group in turn mimetic I. The R2-group in turn mimetic Ishould be considered as the i+1 side chain in a -turn. In an ideal turn mimetic the side chains i and i+2 should also be introduced, but since SAR studies for the AT1 receptor have suggested that the Val3, Ile5, and Pro7 side chains mainly have conformational stabilizing roles and induce a turn motif, these side chains were omitted for synthetic reasons.

NH

OHN

OH2N

NH

NH2HN

O

HO HN OH

O

O

N

O

NHN

N

O

NH

O

NH

OHN

OH2N

NH

NH2HN

O

HO HN OH

O

O

N

O

NHN

N

O

NH

O

OH OH

1a 1b

Figure 19. Isoquinoline based -turn mimetics incorporated in Ang II.

Molecular modeling showed that a scaffold better fulfilling the criteria for an inverse -turn would be obtained if the 9-position rather than the 7-position in the benzodiazepine were used to connect the N-terminal residues. This is illustrated in Figure 20 where the distances and angles between the three C -atoms within the -turn mimetics II and I are compared to a -turn found in a crystallized protein. The peptide backbone of the proposed -turn mimetic IIand the reference -turn were also compared (Figure 21). This comparison shows that turn mimetic II better resembles the native -turn and therefore -turn mimetic II was synthesized.

30

Figure 20. Comparison of the distances and the angles between the C atoms in turn mimetic I and II. The measured angles are given inside and the distances outside

the triangle. The aromatic carbon connected to the N-terminal handle was selected as C i. The triangle labeled Reference represents a minimized ideal inverse -turn.

Figure 21. The -turn mimetic II (light grey) superimposed on an inverse -turn (black).

We decided to introduce a tyrosine side chain as the i+1 group in the -turnmimetic and confirm our previous assumption that the Ile5 side chain, i+2 in a -turn, could be omitted. Therefore -turn mimetic II was synthesized with a tyrosine side chain as the R2-group and an isoleucine side chain or only a hydrogen atom as the R3-group.

31

4.2 Synthesis of Benzodiazepine-based -TurnMimetics

4.2.1 N-Terminal in Position 7 of the Benzodiazepine The synthesis of the bicyclic scaffold in turn mimetic I is based on the strategy described by Keenan et al.78 The idea was to start from a substituted benzaldehyde and utilize natural amino acids to achieve -turn mimetics with the desired stereochemistry, see Figure 22.

N

HN

O

R2

NH

O

OHFmoc

O

F

O2N H2NO

R2H2N

OH

O

+ +OHH

Figure 22. Retrosynthetic analysis of -turn mimetic I.

Scheme 1

F

O2NH

O

H2NO

O

F

O2NNH

O O

N

HN

OO2N

OH

OH

O

N

HN

ONH

OH

OH

O

Fmoc

N

HN

OO2N

OO

Ot-Bu

NaCNBH3HOAc, MeOH

F

O2NN

O OO

HNFmoc

Ot-Bu2

5

43

6

7

72%

Fmoc-Tyr(t-Bu)-OH

HATU, DIEACH2Cl2

90%

1) DBU THF2) Et3N DMSO H2O

64%

1) TFA CH2Cl22) NaClO2 NaH2PO4 (aq) cyclohexane

t-BuOH, THF77%

1) HCO2NH4 Pd/C, MeOH

2) FmocCl Na2CO3 (aq) dioxane

47%

The synthetic route to turn-mimetic I with a tyrosine side chain is outlined in Scheme 1. The first step is a reductive amination of the benzaldehyde 2 with the glycine equivalent 2,2-dimethoxyethylamine. The facile formation of a

32

diketopiperazine in a later step prevents the use of a natural amino acid such as glycine or its methyl ester, see Figure 23. The secondary amine 3 was coupled with Fmoc-L-Tyr(t-Bu)-OH using HATU as activating reagent and DIEA as base. Fmoc groups are usually removed using piperidine, but in this system the piperidine underwent nucleophilic aromatic substitution at the fluorine ipso-carbon. Therefore, DBU was used to remove the Fmoc group. Under the alkaline conditions used in the deprotection step, the amine 4underwent an internal nucleophilic aromatic substitution to give the benzodiazepine 5. It was observed that the benzodiazepine scaffold was obtained in an improved yield if the amine was first purified by column chromatography to remove side products from the deprotection step and thereafter directly treated with triethylamine and water in DMSO.

N O

R2H2NO

O

R1

O2N

F

N O

O NH

R2

O2N

F

Figure 23. Formation of a diketopiperazine.

After formation of the benzodiazepine core structure the acetal protection and the tert-butyl group were removed using TFA/dichloromethane (1:1). The unstable aldehyde was treated with sodium chlorite in a mixture of THF, tert-butyl alcohol and aqueous sodium dihydrogen phosphate at 0 C to give the carboxylic acid193 6 in a yield of 77%. Very mild oxidation conditions were required. If the temperature was raised or other oxidizing agents (e.g. Jones reagent) were used only traces of the desired carboxylic acid could be detected. Jones reagent gave a mixture of products where benzylic oxidation followed by cleavage of the tyrosine side chain was one of the reactions. Ammonium formiate and Pd/C was used to reduce the nitro group and the zwitterion obtained was treated with FmocCl142. The Fmoc-protected turn mimetic 7 was purified through repeated extraction as it decomposed during column chromatography.

To obtain the -turn mimetic I with a histidine side chain the synthetic route had to be slightly changed, see Scheme 2. The acidic deprotection of the acetal employed in the synthesis of the tyrosine analogue was not compatible with the Boc-protecting group at the histidine side chain, and instead an aminoethanol was employed as a glycine equivalent.

In the first step the benzaldehyde 2 was reacted with unprotected 2-aminoethanol in reductive amination. The reaction was performed at reflux for 1.5 h using five equivalents of amine. A higher reaction temperature or a longer reaction time lowered the yield. The alcohol in compound 8 was protected with TBDPSCl to avoid side reactions, and especially to simplify

33

the purification in the following steps. Fmoc-L-His(Boc)-OH was coupled to the secondary amine 9. The subsequent Fmoc deprotection and cyclization into a benzodiazepine were performed as for compound 5. Spontaneous cleavage of the TBDPS group took place during cyclization, probably assisted by the liberation of fluoride ions.

Scheme 2

N

HN

OO2N

HO

N

BocN

F

O2NNH

HO

N

HN

OO2N

OHO

N

BocN

F

O2NNH

TBDPSO

F

O2NN

TBDPSOO

HNFmoc

NNBoc

N

HN

ONH

OHO

Fmoc

N

BocN

2

1312

1110

98

HONH2

NaCNBH3HOAc, MeOH

85%

TBDPSClDBU, CH3CN

98%

Fmoc-His(Boc)-OH

HATU, DIEACH2Cl2

86%

1) DBU, THF

2) Et3N, H2O DMSO

81%

1) ClCOCOCl DMSO, Et3N CH2Cl2

2) NaClO2 NaH2PO4 (aq) cyclohexene t-BuOH, H2O

55%



33%

An attempt to oxidize the alcohol to a carboxylic acid in one step using TEMPO and sodium hypochlorite194 failed. Instead oxidation was performed in two steps. First Swern oxidation195 was performed to obtain the aldehyde in a high yield. Since the aldehyde was found to decompose during storage and during column chromatography on silica, it was purified by extraction and immediately oxidized to the carboxylic acid using similar conditions as for the tyrosine analogue. In contrast to oxidation of the tyrosine derivative, THF had to be excluded from the reaction mixture to obtain compound 12 in a satisfactory yield. Reduction of the nitro group was conducted in similar fashion as for compound 6, but the Fmoc protection was somewhat more complicated since the Boc group could migrate from the imidazole to the aniline nitrogen. This side reaction was eliminated when the reaction time was kept short and the FmocCl was added before the base. The Fmoc-protected -turn mimetic 13 with a histidine side chain was purified by

34

repeated extraction. The -turn mimetics 7 and 13 were incorporated into Ang II by manual solid-phase peptide synthesis (SPPS) (Section 4.3).

4.2.2 N-Terminal in Position 9 of the Benzodiazepine The same general synthetic strategy described above for the -turn mimetics with the N-terminal in position 7 of the benzodiazepine scaffold was also used in the synthesis of benzodiazepine-based mimetics with the N-terminal in position 9, see Figure 24. Unfortunately, the corresponding starting material, 2-fluoro-3-nitro-benzaldehyde, was not commercially available and the synthetic route had to be further developed, see Schemes 3 and 4.

NHN

R2O

OH

OHNFmoc

R3 O

Cl

H

NO2

H2NO

R2OH H2N

OH

O

R3

+ +

Figure 24. Retrosynthetic analysis of -turn mimetic II.

The commercially available 2-chloro-3-nitrobenzoic acid was reduced to the alcohol and reoxidized to the desired aldehyde 15196 in 91% yield over two steps. Due to the risk of diketopiperazine formation in a later step as discussed above (Section 4.2.1), the glycine equivalent 2-aminoethanol or the isoleucine equivalent (S,S)-2-amino-3-methyl-1-pentanol was used as amine in reductive amination with the aldehyde 15. The primary alcohol 16awas protected with a TBDMS group to achieve cleaner reactions and improved yields. The TBDMS-protected amino alcohol could be used as amine in reductive amination but the yield decreased considerably, from 64% (over two steps) to 20%. Protection of the alcohol in the isoleucine derivative 16b gave compound 17b in 83% yield but the subsequent reaction, i.e. amide formation was very slow and provided a mixture of isomers in low yield. Therefore, the TBDMS protected alcohol 17a and the unprotected isoleucine equivalent 16b were used in the amide coupling with HATU as activating reagent and DIEA as base, see Scheme 4. Reacting the sterically hindered amine 16b at room temperature overnight gave a mixture in a very low yield of the desired product. When the reaction temperature was increased to 100 C using controlled microwave irradiation the reaction time could was considerably shortened to only 10 min, and the desired amide was isolated in 56% yield. The less sterically hindered amine 17a was reacted at room temperature overnight and the amide was obtained in excellent yield.

35

Scheme 3

NO2Cl

OH

O

NO2Cl

H

O

NO2Cl

NHHO

R1

NO2Cl

NHTBDPSO

14 15

16a R1 = H16b R1 = CH(CH3)CH2CH3


R1

91%


1) BF3·Et2O NaBH4, THF

HONH2

R1

NaCNBH3HOAc, MeOH

55-69%

TBDPSClDBU, THF

85-93%

The Fmoc-protecting group was removed using DBU and the intermolecular nucleophilic aromatic substitution at the chlorine ipso-carbon in 18aproceeded smoothly in DMSO/Et3N at 100 C. Reacting 18b using the same conditions gave a 7:3 mixture of two isomers. Column chromatography was used to separate the two diastereoisomers and NMR experiments confirmed that 20b was the major product. Deprotection of the TBDMS group in 19awas performed using TBAF in THF and the alcohol was obtained in high yield. The alcohols 20a and 20b were oxidized in two steps utilizing Swern oxidation195 to the aldehydes and thereafter mild oxidation to the carboxylic acids using sodium chlorite at 0 C193. Reduction of the nitro groups in 21aand 21b was performed using ammonium formiate and Pd/C. The zwitterions obtained were isolated by extraction and reacted with FmocCl in dioxane and aqueous Na2CO3. Repeated extraction gave pure compound 22a, while compound 22b was obtained as an 8:2 mixture of diastereoisomers. After purification by RP-HPLC the major component was assigned to be 22b, as deduced from NMR experiments. The turn mimetics22a and 22b were used for incorporation into Ang II by manual SPPS (Section 4.3).

36

Scheme 4

O2NN

HNO

Ot-Bu

O

R1

R2

HN

NHN

O

HO

Ot-Bu

O

Fmoc R1

TBAF THF

2) NaClO2 NaH2PO4 (aq) cyclohexene t-BuOH

NO2Cl

NO

O

NH2

Ot-Bu

R1

R2

O2NN

HNO

HO

Ot-Bu

O

R1

18a R1 = H R2 = TBDPS18b R1 = CH(CH3)CH2CH3 R2 = H

19a R1 = H R2 = TBDPS20a R1 = H R2 = H20b R1 = CH(CH3)CH2CH3 R2 = H



17a or 16b

1) Fmoc-Tyr(t-Bu)-OH HATU, DIEA CH2Cl2

2) DBU, THF

Et3N, DMSO




46-90%41-82%

89% 76-86%

35-68%

4.3 Incorporation of -Turn Mimetics into Ang II A major breakthrough in peptide chemistry was solid-phase peptide synthesis (SPPS) presented by R. B. Merrifield in 1963.197 In this technique, the carboxylic acid in the first amino acid is covalently attached to an insoluble polymer and the peptide is built up stepwise, as outlined in Figure 25. The peptide is finally cleaved from the solid support and isolated. By utilizing SPPS, tedious purification steps can be replaced by simple washing and filtration. In addition, large excesses of amino acids and reagents can be used to force the reaction to completion.197,198

37

1) Fmoc-Tyr(t-Bu)-OH, PyBOP, DIEA, DMF2) 20% piperidine in DMF

1) Fmoc-Val-OH, PyBOP, DIEA, DMF2) 20% piperidine in DMF

1) Fmoc-Arg(Pbf)-OH, PyBOP, DIEA, DMF2) 20% piperidine in DMF

1) Fmoc-Asp(Ot-Bu)-OH, PyBOP, DIEA, DMF2) 20% piperidine in DMF

TriethylsilaneTFA/H2O

OH2N

O

Linker

Linker

Fmoc-Turn Mimetic-OH, PyBOP, DIEA, DMF

20% piperidine in DMF

Linker

ON

HN

O

NH

NHN

ONH

Fmoc

O

ON

HN

O

NH

NHN

OH2N

O

ON

HN

NH

OOH

N

ONH OH

ONH

OH

OHN

OH2N

O

HO

NH

NHH2N

NHN

23

Figure 25. Synthesis of a pseudopeptide encompassing the benzodiazepine-based -turn-like mimetic with a histidine side chain.

Standard Fmoc/t-Bu SPPS methodology was used to synthesize pseudopeptide analogues of Ang II encompassing the four -turn mimetics 7,13, 22a, and 22b. The C-terminal amino acids were coupled to a H-Phe-Wang resin in DMF using HBTU as activating reagent and DIEA as base. The incorporation of the bicyclic -turn mimetics was expected to be slow and require a long reaction time. Therefore, to avoid the risk of formation of tetramethylguanidino derivatives, HBTU was replaced by PyBOP in the incorporation of the turn mimetics and in the subsequent couplings. In -turnmimetic 7 the phenolic hydroxyl group lacked protection and according to MS analysis two equivalents of Fmoc-amino acid were incorporated into

38

each of the following coupling steps. However, after each coupling the presumed phenyl esters were smoothly cleaved in the following Fmoc-deprotection step. After completion of the synthesis, the target peptides were liberated from the solid support by treatment with TFA in the presence of water and triethylsilane. At the same time, the acid labile side chain protecting groups were removed. The crude peptides were isolated by precipitation with ether. The -turn-like mimetic 13 with a histidine side chain was incorporated into Ang II replacing the peptide segment Ile5-His6-Pro7 (23), Figure 25. The -turn-like mimetic 7 with a tyrosine side chain gave the final products 24-26 (Figure 26). In these pseudopeptides the benzodiazepine-based turn mimetic was used to replace Val3-Tyr4-Ile5 (24)or Tyr4-Ile5 (25 and 26) segments of Ang II. Furthermore, -turn mimetic 22a produced the pseudopeptides 27-30, where the benzodiazepine system was used to replace Val3-Tyr4-Ile5 (27 and 29) or Tyr4-Ile5 (28 and 30)segments of Ang II (Figure 27). The final products (24-30) were purified by preparative RP-HPLC and analyzed by LC-MS. A combination of ion-exchange chromatography and RP-HPLC purification was required to purify pseudopeptide 23.

N

HN

NH

OOH

N

ONH

O

NH

NHH2N

H2N

O

HO

O HN

OH

O

O

N

ONH

NHN

HO

25

N

HN

NH

OOH

N

ONH

OH2N

O

HO

O HN

OH

O

O

N

ONH

NHN

HO

26

N

HN

NH

OOH

N

OH2N O H

NOH

O

O

N

ONH

NHN

HO

NH

NH2HN

O

HO

24

Figure 26. Pseudopeptides encompassing the benzodiazepine-based -turn-like mimetic with a tyrosine side chain.

39

NHN

OHN

NH

NH O

ON

HN

H2N NH

OH2N

HO

OO

NHN

OH

HN

OOH

O

27

NHN

OHN

NH

NH O

ON

OH2N

HO

OO

NHN

OH

HN

OOH

O

29

NHN

OHN

NH

NH O

ON

OHN

O

NHN

OH

HN

OOH

OH2N

O

O

HO

NH

NH2HN28

NHN

OHN

NH

NH O

ON

OHN

O

NHN

OH

HN

OOH

OH2N

O

O

HO

30

Figure 27. Pseudopeptides encompassing the benzodiazepine-based -turn mimetic 22a with a tyrosine side chain.

-Turn mimetic 22b was used to replace the Val3-Tyr4-Ile5 or the Tyr4-Ile5

segment of Ang II. Partial epimerization took place during peptide synthesis and the pseudopeptides were obtained as mixtures of two diastereoisomers. Pure pseudopeptides 31a, 31b, 32a, and 32b (a and b denote single diastereoisomers of unassigned absolute stereochemistry) were isolated after RP-HPLC purification (Figure 28). We hypothesize that the stereochemistry of the major products (31a and 32a) corresponds to the natural L-configuration.

NHN

OHN

NH

NH O

ON

HN

H2N NH

OH2N

HO

OO

NHN

OH

HN

OOH

O

31a

NHN

OHN

NH

NH O

ON

HN

H2N NH

OH2N

HO

OO

NHN

OH

HN

OOH

O

31b

NHN

OHN

NH

NH O

ON

OHN

O

NHN

OH

HN

OOH

OH2N

O

O

HO

NH

NH2HN 32a

NHN

OHN

NH

NH O

ON

OHN

O

NHN

OH

HN

OOH

OH2N

O

O

HO

NH

NH2HN 32b

Figure 28. Pseudopeptides encompassing the benzodiazepine-based -turn mimetic 22b with a tyrosine and an isoleucine side chain.

40

5 Design and Synthesis of -Turn Mimetics (Papper IV)

5.1 Design of a Benzodiazepine-based -Turn Mimetic A turn region around Tyr4 has been suggested in Ang II when interacting with the AT1 receptor since 3-5 cyclized Ang II analogues have produced bioactive compounds. Whereas a number of -turn mimetics have been introduced in this region66,77,82 a -turn mimetic has to the best of our knowledge, never been investigated, although it has been suggested that Ang II might adopt a -turn conformation.143,152 Therefore, we designed and synthesized turn mimetic III, presented in Figure 29, with side chains in position i+1 and i+2 of the -turn. Two locations of a -turn have been suggested in Ang II, in the 3-6 region or in the 2-5 region. We decided to introduce our turn mimetic in the 3-6 region. Therefore, a tyrosine side chain was introduced in the i+1 position (R1) and an isoleucine side chain in the i+2 position (R2).

N

HN

NH

R1

O

R2

HN

OIIIFigure 29. The investigated -turn mimetic.

Conformational analysis was performed to classify the proposed -turn mimetic. The low energy conformations were compared with -turnsextracted from the X-ray structures of proteins in the Protein Data Bank (PDB). -Turns belonging to type II and type IV were mainly identified in this comparison but also turns corresponding to type II´. Type IV is a diverse collection of non-defined turns and was therefore not considered further since -turn mimetics are primarily designed to mimic the classical turn types. Thus, turn mimetic III seems to preferentially mimic a type II or a type II´ -turn (Figure 30).

41

Figure 30. A minimized conformation of the -turn mimetic III (light grey) superimposed on a II turn (left) and a II´ turn (right).

5.2 Synthesis of a Benzodiazepine-based -TurnMimetic

Retrosynthetic analysis of the proposed -turn mimetic (Figure 31) shows that a synthetic route very similar to the one utilized in the synthesis of -turn mimetic I (Section 4.2.1) could be suitable for the key intermediate A.Different amino acids could be used as starting materials to introduce the i+2 and i+3 side chains, while different i+1 side chains would be accessible through palladium catalyzed cross couplings. The starting material, 2-fluoro-5-nitrobenzaldehyde, used in the synthesis of compounds 7 and 13, should be further functionalized with a bromo atom to get a handle for the palladium catalyzed coupling reactions.

N

HN

NH

O

R

Fmoc

PGO

OH

O

N

HN

O2N

Br

O

R

O

O

O2N

BrF

H

O

N

HN

O2NO

R

PGO

O

O

H2N

H2N

ROH

O

++O

O

A

Figure 31. Retrosynthetic analysis of a -turn mimetic.

The synthesis of the key intermediate 36 is presented in this thesis see Scheme 5. The experimental part is described in the Appendix (Section 11). The synthesis starts with bromination of 2-fluoro-5-nitrobenzaldehyde199 2.

42

Compound 2 is strongly deactivated for electrophilic aromatic substitution and the powerful brominating reagent dibromoisocyanuric acid (DBI)200,201

had to be used to obtain the functionalized benzaldehyde 33 in 50% yield. Compound 33 was reacted with the glycine equivalent 2,2-dimethoxyethylamine in reductive amination and the secondary amine 34,was coupled with Fmoc-L-Ile-OH utilizing HATU as activating reagent. DBU was used to remove the Fmoc group and the obtained amine was purified by column chromatography and thereafter immediately dissolved in DMSO and treated with triethylamine and water. Intramolecular nucleophilic aromatic substitution delivered the key benzodiazepine derivative 36.

Scheme 5

HOHN

Fmoc

O

N

NH

N

OO

OBrBr

H2SO4

O2N

FBr

N

OOO

HNFmoc

H

OO2N

FBr

O2N N

HN

O

OO

MeOMeO

ZnCl

Pd(OAc)2Ph3P

H2N O

O

HOAcMeOHNaCNBH3

O2N

Br

N

HN

O

OO

HN

O2N

FBr

O

O

HATUDIEACH2Cl2

1) DBU THF2) Et3N DMSO H2O

50%

2

33 34

35 36

37

66%

55% 77%

In an attempt to introduce the i+1 tyrosine side chain a commercially available THF solution of 4-methoxybenzylzinc chloride was reacted with 36 in a Negishi coupling.202 Two palladium catalysts, tetrakis-(triphenylphosphine)palladium or [1,1´-bis(diphenylphosphino)ferrocene]-dichloropalladium(II)·CH2Cl2 [(DPPF)PdCl2·CH2Cl2], different temperatures, reaction times, and additives were tried, but the desired product was not observed.

Compound 39, synthesized through reductive amination of acetone with 2-bromo-6-methyl-4-nitroaniline, was chosen as a model substance (Scheme

43

6). It was reacted with 4-fluorphenylzinc bromide and a catalytic amount of [(DPPF)PdCl2·CH2Cl2] at 160 C for 5 min utilizing microwave irradiation, and the desired product (40) was achieved in 50% yield. However, as with compound 36, no product was formed when compound 39 was reacted with 4-methoxybenzylzinc chloride. The 4-methoxybenzylzinc reagent seemed to be very unstable and therefore the synthetic route outlined in Scheme 5 was abandoned.

Scheme 6

NH2

Br

O2N

OHN

Br

O2NNaBH4THFH2SO4

HN

R

O2N

83%38 39 40

RZnX

[Pd]

Instead, the i+1 tyrosine side chain of the -turn mimetic was introduced early in the synthesis through Friedel-Crafts acylation of anisole with the acid chloride of 2-fluoro-5-nitroisophthalic acid monomethyl ester 45.Benzylic oxidation produced 2-fluoroisophthalic acid (42), and the monoester (44) was synthesized in one step or via the diester (43), Scheme 7. An acid catalyzed esterification produced a mixture of the diester, monoester and diacid (3:10:10), which could be separated through repeated extraction. In the other approach (scheme 7) the diacid (42) was treated with thionyl chloride and subsequently with methanol to give the diester (43), which was hydrolyzed with one equivalent lithium hydroxide to allow isolation of the monoester in 47% overall yield.

Scheme 7

MeOHH2SO4

LiOH (aq)THF/MeOH

47%40%

F F

O OH

OH

O

F

O OMe

OMe

O

F

O OH

OMe

O

KMnO4KOH (aq)

58%

1) SOCl2

2) MeOH

99%41 42 43

44

44

The monoester 44 was nitrated in excellent yield using standard conditions (Scheme 8). Reversal of the reaction steps, i.e. first nitration and then esterification, was not successful. The fluorine was replaced with a methoxy or hydroxy group through nucleophilic aromatic substitution under the alkaline conditions used in the ester hydrolysis, but also in the extraction of the product mixture. Reflux of 45 in thionylchloride gave the acid chloride, which was reacted in a Friedel-Crafts acylation with five equivalents anisole in presence of two equivalents of the Lewis acid AlCl3. A mixture of the ortho/para-substituted products 46a and 46b (2:5) was obtained in 52% yield, and 40% of the starting material 45 could be recovered. A larger excess of anisole or Lewis acid, a longer reaction time or higher temperature did not improve the conversion significantly. The two isomers could be separated by column chromatography and the desired isomer, 46a, was obtained in 37% yield.

Scheme 8

HNO3H2SO4

F

O OH

OMe

OO2N

F

O

OMe

OO2N

OMe

F

O

OMe

OO2N

MeO

+4497%

1) SOCl2

2) AlCl3 anisole CH2Cl2

46a37%

46b15%

45

Selective reduction of the substituted benzophenone 46a to form substituted diphenylmethane 47 was performed in excellent yield using a catalytic amount of trifluoromethanesulfonic acid in TFA with triethylsilane as the hydride donor203 (Scheme 9). The subsequent synthetic steps in Scheme 9 were first tried without reducing the carbonyl group. Unfortunately, a number of side products were formed since the fluorine ipso-carbon was very activated for nucleophilic aromatic substitution. Reduction of the methyl ester in 47 to the alcohol without reduction of the nitro group was carried out in water and dioxane with a large excess of NaBH4.204 The alcohol obtained was reoxidized to the aldehyde by Swern oxidation195.Thereafter, the aldehyde 48 was reacted with 2-aminoethanol in reductive amination and the isolated alcohol was protected with a TBDMS using TBDMSCl and DBU in THF. The subsequent amide coupling of 49 with Fmoc-L-Ile-OH was performed with HATU as activating agent and the obtained Fmoc-protected amine was deprotected with DBU. An internal nucleophilic aromatic substitution produced the benzodiazepine as a 1:3 mixture of the TBDMS-protected alcohol and the free alcohol. Therefore, the mixture was treated with TBAF in THF and the benzodiazepine with a free alcohol (51) was isolated in a yield of 90%. Oxidation of the alcohol

45

group in 51 to a carboxylic acid was performed in two steps under mild conditions. Swern oxidation195 gave an aldehyde that was immediately treated with sodium chlorite in a mixture of tert-butyl alcohol and aqueous sodium dihydrogen phosphate at 0 C193 to give the carboxylic acid 52 in 70% yield.

Scheme 9

TFACH2Cl2

Et3SiHCF3SO2H

F

OMe

OO2N

OMeH2N

OH

F

O2N N O

H2N

OTBDMS

OMe

N

HN

O2NO

O

OHO

Boc

N

HN

O2NO

OMe

OHO

F

O2N NH

OTBDMS

OMe

N

HN

NH

O

O

OHO

Boc

Fmoc

F

HO2N

OMe

N

HN

O2NO

OH

OMe

46a

98%

1) NaBH4, H2O dioxane

65%


NaCNBH3HOAc, MeOH

2) TBDMSCl DBU, THF

71%

1)

1) Fmoc-Ile-OH HATU, DIEA CH2Cl2

2) DBU, THF92%

2) TBAF, THF90%


2) NaClO2 NaH2PO4 (aq) cyclohexene t-BuOH

70%

1) BF3·Me2S, CH2Cl2

58%

1) Et3N, DMSO

2) Boc2O, DMAP Et3N, THF, H2O

1) H2, Pd/C MeOH


32%

O

5049

4847

5453

5251

The protecting group on the phenolic hydroxy group in 52 was changed from a methyl to a Boc group to facilitate SPPS. BF3·SMe2 was used to cleave the methyl group, and the Boc group was introduced by treatment with Boc2O in THF and water utilizing DMAP as a catalyst and Et3N as base. Finally, the nitro group was reduced by hydrogenation and the aniline group was

46

protected with FmocCl. The Fmoc-protected -turn mimetic 54 was purified by column chromatography and incorporated into Ang II by SPPS (Section 5.3).

5.3 Incorporation of -Turn Mimetic into Ang II The -turn mimetic 54 was incorporated into Ang II using the classical Fmoc/t-Bu solid-phase methodology as described for the -turn mimetics in Section 4.3. H-Pro-Phe-2-Chlorotrityl or H-His(t-Bu)-Pro-Phe-2-chlorotrityl resin were used in the synthesis of the three pseudopeptides (55-57) in Figure 32. The anilinic nitrogen in the -turn mimetic was assumed to react slowly. Therefore, double couplings of the following amino acid were used. The benzodiazepine-based -turn mimetic was used as a turn template for substituting the Val3-Tyr4-Ile5-His6 (55), Val3-Tyr4-Ile5 (56), or Tyr4-Ile5 (57)segments of Ang II, see Figure 32.

N

HN

NH

O

HO

HN

O

OH2N

O

HO

NH

NH2HN55

N

HN

NH

O

HO

HN

HN

O

OH2N

O

HO

NH

NH2HNHN N

O

N NH

OOH

O

56

NHN

OOH

O

O O

N

HN

NH

O

HO

HN

HN

O

ONH

HN N

O

N NH

OOH

O

57

O

OH2N

HN

H2N NH

HO

O

Figure 32. Pseudopeptides encompassing the -turn mimetic III.

47

6 Peptidic Ang II Analogues (Paper II)

6.1 Glycine Scan In most of the binding studies presented to date, it seems that modifications in Ang II or [Sar1]Ang II are well-tolerated by the AT2 receptor. In an Ala/Gln scan of [Sar1]Ang II recently presented by Miura et al.182 only minor decreases in affinity for the AT2 receptor were observed. We decided to perform a Gly scan of the native peptide Ang II to confirm and further explore these findings. Substitutions with Gly rather than Ala were preferred since we wanted to avoid any possible influence of the methyl group on receptor recognition. It should though be noted that the conformation of the peptide backbone resembles the native peptides better in an Ala scan than in a Gly scan. Thus, an Ala scan is usually preferred when attempting to identify the side chains important for binding. However, in this study we wanted to examine whether the extra steric bulk of the methyl group contributed favorably to binding and the amino acids in Ang II were substituted one by one with glycine. The synthesis of these peptides is presented in Section 6.3.

6.2 N-Terminal Modifications When Miura et al.182 replaced Arg2 in Ang II with Gln only a 10-fold decrease in affinity was observed. In contrast, we observed a decrease in affinity from 3 nM to more than 10 M when substituting Arg with Ala in one of our pseudopeptides encompassing the benzodiazepine-based turn mimetic I (compare compounds 25 and 26 in Table 1, Section 7.1). Thus, we decided to study the importance of the Arg side chain in Ang II with regard to AT2 receptor affinity in further detail. We prepared some N-terminally end modified Ang II analogues and also included some peptides where the distance between the Arg2 side chain and Tyr4 side chain was varied (see Section 7.3).

48

6.3 Synthesis The peptides 58-71 (Table 3, Section 7.3) were synthesized on a Symphony instrument using standard Fmoc/t-Bu based SPPS methodology starting from H-Phe-Wang resin or H-Gly-2-chlorotrityl resin (Figure 33). Double couplings in DMF, using HBTU as activating reagent and NMM as base, were used to introduce all amino acids. Unreacted amine was capped by addition of acetic anhydride at the end of each coupling cycle. The Fmoc-protecting group was removed before the next coupling step using 20% piperidine in DMF. The N-terminal acetylation of peptide 68 was achieved by treatment of the resin with 20% acetic anhydride immediately after Fmoc deprotection. After completion of the synthesis the final Fmoc group was removed and a mixture of triethylsilane, water and TFA was used to cleave and deprotect the target peptides. The crude compounds were isolated by precipitation using diethyl ether and the pure peptides (58-71) were obtained after preparative RP-HPLC purification.

Linker

Linker

Linker

Linker

X Linker

NH

R1

O

O

Fmoc

H2N

R1

O

O

NH

R1

O

O

HN

Fmoc

O

R2

NH

R1

O

O

HN

O

RNH

Fmoc

O

Rn+1

NH

R1

OH

O

HN

O

RH2N

O

Rn+1

1. Attachment of the first Fmoc-protected amino acid

2. Removal of Fmoc-protecting group

3. Coupling of the next protected amino acid

4. Repetition of steps 2 and 3

5. Deprotection and cleavage from the resin

n

n

Figure 33. General strategy for SPPS of the Ang II analogues 58-71. The amino acid side chains were, if necessary, protected with acid labile protecting groups

cleaved at the same time as the final peptide was liberated from the resin.

49

7 Structure-Activity Relationships

All the synthesized pseudopeptides and peptides were evaluated in radioligand-binding assays by displacement of [125I]Ang II from AT2receptors in pig uterus membranes205 and from AT1 receptors in rat liver membranes119. The natural substrate Ang II and the AT2 selective receptor agonist [4-NH2-Phe6]Ang II179 were used as reference substances.

7.1 Pseudopeptides Encompassing a -Turn Mimetic

7.1.1 Receptor Binding and Molecular Modeling(Papers I and III)

The pseudopeptides 23-32 were evaluated for AT1 and AT2 receptor affinity (Table 1). Compound 25, in which the benzodiazepine-based -turn mimetic has replaced the Tyr4-Ile5 segment of Ang II (Section 4.3), displayed considerable binding affinity for the AT2 receptor (3.0 nM) and exhibited high AT2/AT1 receptor selectivity; see Table 1. In contrast, when the Val3-Tyr4-Ile5 segment of Ang II was replaced with the same -turn mimetic producing pseudopeptide 24 no binding affinity for the AT1 or AT2 receptor was detected (Ki > 10 000 nM). The pseudopeptides 1a and 1b,encompassing an isoquinoline-based, -turn mimetic in the 3-5 position of Ang II (Figure 19, Section 4.1), have previously been shown to lack AT1receptor affinity.77 These results were confirmed in this study and it was also shown that compound 1a binds fairly well to the AT2 receptor (Ki = 61 nM), while the diastereoisomer (1b) lacked binding affinity to the AT2 receptor (Table 1).

50

Table 1. In vitro binding affinities of pseudopeptides encompassing a -turn mimetic

Compound

AT1

(Rat liver membranes) Ki (nM) ± SEM

AT2

(Pig uterus myometrium) Ki (nM) ± SEM

Ang II 0.24 ± 0.07 0.23 ± 0.01 [4-NH2-Phe6]Ang II >10 000 2.1 ± 0.5 1a >10 00077 61 ± 2 1b >10 00077 >10 000 23 >10 000 >10 000 24 >10 000 >10 000 25 >10 000 3.0 ± 1.1 26 >10 000 >10 000 27 >10 000 2.8 ± 0.2 28 >10 000 0.8 ± 0.1 29 >10 000 37.4 ± 2.1 30 >10 000 9.1 ± 1.4 31a >10 000 2.6 ± 0.2 31b >10 000 117.4 ± 7.7 32a >10 000 0.3 ± 0.01 32b >10 000 0.08 ± 0.003

NH

O HN N

HO

O

NH

HN

ON

NHNNH

NHH2N

O

OH

O

H2NHN

OOH

O

O

HO

S S

72

Figure 34. A 3,5-cyclized Ang II analogue with Ki of 0.62nM and 44 nM for the AT2and AT1 receptors, respectively. Compound 72 has been shown to preferentially

adopt low energy inverse -turn conformations around Tyr4.175

The pseudopeptides 24, 25, and 1a were compared with compound 72(Figure 34) in molecular modeling studies. The methylenedithioether 72,which has been shown to adopt low energy inverse -turn conformations around Tyr4, exhibits high affinity for the AT2 receptor (0.62 nM).175 When the backbone atoms in compounds 24, 1a, and 72 are compared atom by atom, an exact match can be obtained for the i+1 residue and onwards to the C-terminus. However, the i residue of the -turn-like mimetic region and thus the N-terminal amino acids in pseudopeptide 24 cannot be mapped to

51

compound 72. The i+1 and i+2 residues in the -turn/ -turn mimetic region of 24, 1a,and 72 were superimposed after preliminary conformational analysis (Figure 35). The results indicate that the N-terminal amino acids entering the -turn mimetic are oriented quite differently in compound 24,which lacks AT2 receptor affinity, and compound 72, which has high receptor binding affinity. An Ala scan of Ang II indicated that Arg2, Tyr4,and His6 are sensitive to modifications.182 In addition, Ang IV (Ang II(3-8)) has been reported to have low affinity176 for the AT2 receptor, suggesting a role for Arg2 in receptor binding. Furthermore, mutagenesis studies of the AT2 receptor have indicated that the charge and length of the Arg2 side chain in Ang II are important.174,186

Figure 35. Stereopresentation of the best fit of the -turn mimetic moieties of compound 24 (blue) and compound 1a (orange) to the -turn part of 72 (green).

Our hypothesis was that compound 25, which has an extra Val residue and exhibits high affinity to the AT2 receptor, could compensate for the geometrical differences and position the guanidino group of the Arg2 side chain and/or the N-terminal end in a more favorable position for the receptor. Thus, the Val residue could function as a spacer in compound 25and the two active compounds 25 and 72 could interact with the receptor in a similar manner. Molecular modeling of the model compounds 24m, 25m,and 72m, presented in Figure 36, was performed to test this hypothesis. The cyclic moieties were superimposed and the position of the carbon in the guanidino group and the N-terminal CH3-CO bond vector were plotted. These plots showed that the two compounds with AT2 receptor-binding affinity, 25m and 72m, could access common areas not accessible to the inactive compound (24m). One of these sets of conformations where 25mand 72m position the guanidino group in same region of space is shown in Figure 37 together with a conformation of 24m with the guanidino carbon close to this set. It was also observed that 25m and 72m have a more extended conformation in accordance with a previous hypothesis.192

Compound 26, in which Ala has replaced the Arg2 residue, was synthesized (Section 4.3) and tested to challenge the assumption that the Arg2 side chain in pseudopeptide 25 is important for binding. This

52

compound was found to have a Ki value over 10 000 nM for the AT2receptor, further supporting the importance of the Arg2 side chain.

N

HN

NH

OOH

N

ONH

O

NH

NHH2N

O

NH

N

HN

NH

OOH

N

O

O

NH

NH

NH2HN

24m 25m

72m

HN

O HN

O

NH

HN

O

NH O

O

S S

HN

HN NH2

NHN

OHN

O

O

HN

H2N NH

27m

NHN

OHN

NH

NH O

OO

NH

NH2HN

28m

Figure 36. Model compounds used in conformational analysis and molecular modeling.

A turn mimetic that better fulfills the criteria for a -turn was obtained when the handle for attachment of the N-terminal residues was moved from the position 7 to position 9 of the benzodiazepine scaffold (see discussion in Section 4.1). In addition, molecular modeling of compounds 25m and 24msuggested that with a shift of the N-terminal from position 7 to position 9 the extra valine introduced in pseudopeptide 25 would not be needed (Figure 37). The guanidino group in the Arg2 side chain should be able to reach common areas without the valine spacer group.

53

Figure 37. Similar positionings of the arginine side chain, the N-terminal group and the -turn/ -turn mimetic moiety are shown for the superimposed model compounds 25m (yellow carbons) and 77m (green carbons). When the -turn mimetic moiety of the model compound 24m (violet carbons) is used as a basis for superimposition, the arginine side chain and the N-terminal acetyl group cannot reach the same binding site.

Figure 38. An alignment model with the model compounds 25m (white carbons), 27m (yellow carbons), and 28m (green carbons). The cyan sphere indicates the acceptor site.

54

The new -turn mimetic produced the pseudopeptides 27-30, without the isoleucine side chain, and 31a, 31b, 32a, and 32b with the isoleucine side chain in the i+2 position of the -turn mimetic (Section 4.3). Both pseudopeptide 27 and 28 exhibited good binding affinity for the AT2receptor and had high AT2/AT1 receptor selectivity (Table 1). Compound 28,in which the -turn mimetic replaced the Tyr4-Ile5 peptide segment, displayed a slightly better binding affinity (Ki = 0.8 nM) than compound 27(Ki = 2.8 nM) in which the -turn mimetic replaces the Val3-Tyr4-Ile5 peptide segment.

We were encouraged to see that compound 27 had high affinity for the AT2 receptor and that the valine residue was therefore not needed when -turn mimetic II was used. Molecular modeling similar to that used for 24mand 25m was performed for the model compounds 27m and 28m (Figure 36). The key elements in 25m, 27m, and 28m were superimposed and, as shown in Figure 38, the model obtained could position the critical groups in the same region of space. None of the models found for the inactive compound, 24m, was consistent with this model. Therefore, it seems that the inactive compound, 24, cannot adopt the same binding mode/interaction pattern as the three active compounds 25, 27, and 28. Furthermore, an interesting observation was that the valine side chains of compounds 25 and 28 were located in the same region of the model. This could imply that the valine residue not only allows for flexibility and better receptor alignment, but also might have some importance for direct receptor interaction. This may explain the slight increase in affinity when the valine residue is inserted into 27 to give 28.

All AT2 receptor affinity was lost when the arginine residue in pseudopeptide 25 was substituted by an alanine (26). When the arginine residue in 27 and 28 was replaced with alanine (29 and 30) only a 10-fold drop in affinity was detected, see Table 1. A similar affinity drop is observed when Arg2 is replaced by Gly (Table 3, Section 7.3) or Gln in Ang II182. It therefore seems that compounds 27 and 28 interact in a similar way with the AT2 receptor as Ang II. The large decrease in affinity for compound 26might be due the non-optimal interaction with the receptor in combination with removal of the Arg side chain.

The pseudopeptides 31a and 32a were synthesized and tested to investigate whether the introduction of the lipophilic side chain Ile5 could improve binding further. Pseudopeptide 32a, with a Val residue, exhibits a Ki of 0.3 nM, and 31a without the Val residue had a Ki of 2.6 nM. Both compound 31a and compound 32a had approximately the same binding affinity for the AT2 receptor as the corresponding analogues without the Ile side chain, 27 and 28. These results indicate that the Ile side chain in the -turn template does not affect the binding affinities.

Partial epimerization took place during the incorporation of the protected -turn mimetic 22b into Ang II, producing two additional compounds, 31b

55

and 32b. Interestingly, pseudopeptide 32b exhibits an even better binding affinity for the AT2 receptor (Ki = 0.08 nM) than its diastereoisomer 32a,while 31b shows moderate binding affinity (Ki = 117.4 nM). None of the pseudopeptides displayed any affinity for the AT1 receptor.

Cyclization in the 5-7 region of Ang II has also been reported to give compounds with binding affinity for the AT1 receptor.138 These results indicate that there might also be a turn region around His6 in Ang II when interacting with the AT1 receptor. We therefore synthesized and incorporated turn mimetic I with a histidine side chain into Ang II as a substitute for the Ile5-His6-Pro7 peptide segment. Notably, the obtained pseudopeptide 23lacked affinity for both the AT1 receptor and the AT2 receptor.

7.1.2 Functional Studies (Paper III)

The effect of compounds 25, 27, and 28 on cell differentiation was studied in neuroblastoma × glioma hybrid NG108-15 cells. It has previously been shown that these cells only express the AT2 receptor and that treatment with Ang II or the selective AT2 receptor agonist CGP 42112 induces neurite outgrowth, one of the first steps of neuronal differentiation.206,207 Treatment of the cells with compound 25, 27, or 28 at a concentration of 100 nM induced the same effect as Ang II (100 nM). Pictures of the cells are shown in Figure 39B-E and a quantification of the neurite outgrowth is presented in Figure 39F. The effect was attributed to the AT2 receptor since co-incubation of the pseudopeptide (25, 27, or 28) with the AT2 receptor antagonist PD123,319 (1.0 M) halted neurite elongation. It has previously been shown that PD123,319 do not effect the cell morphology.207,208 These results indicate that compounds 25, 27, and 28 act as agonists at the AT2 receptor. It was also investigated whether compound 25 could suppress proliferation in PC12 cells. These cells have been shown to express only the AT2 receptor. Compound 25 was shown to inhibit proliferation of cells to the same extent as Ang II at 1.0 M, this finding further supports that compound 25 acts as an agonist at the AT2 receptor.

56

25

28 27

Ctr Ang II 25 28 27

Figure 39. Comparative effects of the three compounds 25, 28, 27, and Ang II on neurite outgrowth in NG108-15 cells. The cells were cultured for 3 days in the

absence (Control, A), or in the presence of 100 nM of compound 25 (B), 100 nM of compound 28 (C), 100 nM of compound 27 (D) or 100 nM of Ang II (E). Neurite outgrowth, quantified are shown in F (neurites longer then one cell body were

counted). All panels are seen at the same magnification.

7.2 Pseudopeptides Encompassing a -Turn Mimetic (Paper IV)

Pseudopeptides 55-57 (Section 5.3) were evaluated with regard to AT1 and AT2 receptor binding affinity (Table 2). Compound 55, in which the -turn mimetic replaces the Val3-Tyr4-Ile5-His6 peptide segment, exhibited moderate binding affinity for the AT2 receptor (Ki = 53.1 nM). This compound lacks the histidine side chain and it has been shown that the binding affinity for the AT2 receptor drops about 10 times when the histidine residue in Ang II is substituted by alanine182 or glycine (Table 3). Accordingly, when the histidine residue was incorporated to give compound 56 (Figure 32) the binding affinity for the AT2 receptor was almost

57

recovered (Ki = 4.7 nM). Compound 57, in which the -turn mimetic replaces the Tyr4-Ile5 peptide segment, has slightly higher binding affinity (Ki = 1.8 nM).

Table 2. Binding affinities of pseudopeptides encompassing a -turn mimetic

Compound

AT1

(Rat liver membranes) Ki (nM) ± SEM

AT2

(Pig uterus myometrium) Ki (nM) ± SEM

Ang II 0.24 ± 0.07 0.23 ± 0.01 [4-NH2-Phe6]Ang II >10 000 2.1 ± 0.5 55 2108 ± 33 53.1 ± 1.7 56 1668 ± 20 4.7 ± 0.3 57 14.9 ± 0.4 1.8 ± 0.04

Compound 55 displays very weak binding affinity for the AT1 receptor (Ki = 2108 nM). This compound lacks the histidine side chain, which for the AT1 receptor is considered one of the pharmacophore groups127.Interestingly, when the Tyr4-Ile5 segment of Ang II was substituted for the -turn mimetic the resulting pseudopeptide 57 displayed high binding affinity for the AT1 receptor (Ki = 14.9 nM). In compound 56 the histidine side chain is also present but not the valine residue and this compound has very weak binding affinity for the AT1 receptor. These results could imply that not only the histidine side chain but also the distance between the pharmacophore groups Arg2 and Tyr4 are important for the binding affinity.

7.3 Peptidic Ang II Analogues (Paper II)

The eight peptides obtained in the glycine scan of Ang II (58-65) (Section 6.3) all showed relatively high affinity for the AT2 receptor (Table 3). Only the substitution of the Arg residue affected the binding affinity substantially, leading to an almost 100-fold decrease in affinity for compound 59. For the remaining Ang II analogues the affinity was reduced by factors of 2 to 13. Thus, the Arg2 side chain seems to be most important for AT2 receptor binding. Miura et al.,182 in an Ala scan of Ang II, observed similar results and the importance of the Arg2 side chain has also been indicated in mutagenesis studies. It has been hypothesized that Arg2 interacts with Asp297

in the third ECL of the AT2 receptor.174,186

58

In addition, the N-terminal modified peptides 66-71 (Section 6.3) were evaluated regarding AT2 receptor affinity (Table 3) to further study the importance of the Arg2 side chain. Removal of the Asp residue gave compound 66 [Ang III, Ang II (2-8)], a metabolite of Ang II, which exhibits a Ki of 2.2 nM. This Ki value is similar to previously reported data.119,139,176

Substitution of the Arg residue in 66 with Gly gave 67. The reduction in affinity of this compound for the AT2 receptor was only a factor of nine to that of Ang II and only 2 compared to Ang III. We speculated that although the distance to the positive charge differs, the N-terminal primary amine in compound 67 might interact in the same area as occupied by the Arg side chain. Therefore, the primary amine in 67 was acetylated to give 68. This compound also exhibited high binding affinity for the AT2 receptor (Ki = 2.8 nM). Thus, it can be concluded that a charged group at the N-terminal end of Ang III is in fact not necessary for binding. In comparison, a 100-fold decrease in affinity was seen when Arg2 was replaced by Gly (59), which suggests that Ang II and Ang III may bind differently to the AT2 receptor.

Table 3. Binding affinities of peptidic Ang II analogues

Comp. Sequence AT1 Ki (nM) ± SEM

AT2 Ki (nM) ± SEM

Ang II 1.0 0.6 [4-NH2-Phe6]Ang II - 0.8 Glycine substituted peptides

58 Gly-Arg-Val-Tyr-Ile-His-Pro-Phe 6.1 ± 0.2 4.0 ± 0.6 59 Asp-Gly-Val-Tyr-Ile-His-Pro-Phe > 10 000 55 ± 5 60 Asp-Arg-Gly-Tyr-Ile-His-Pro-Phe 1.6 ± 0.1 1.2 ± 0.2 61 Asp-Arg-Val-Gly-Ile-His-Pro-Phe 48 ± 2.0 5.2 ± 0.8 62 Asp-Arg-Val-Tyr-Gly-His-Pro-Phe 1.6 ± 0.1 2.3 ± 0.1 63 Asp-Arg-Val-Tyr-Ile-Gly-Pro-Phe 14.3 ± 0.06 7.6 ± 1.0 64 Asp-Arg-Val-Tyr-Ile-His-Gly-Phe 146 ± 7.0 4.3 ± 0.6 65 Asp-Arg-Val-Tyr-Ile-His-Pro-Gly >10 000 1.1 ± 0.1

N-terminally modified peptides 66 Arg-Val-Tyr-Ile-His-Pro-Phe 10.5 ± 0.3 2.2 ± 0.2 67 Gly-Val-Tyr-Ile-His-Pro-Phe > 10 000 5.4 ± 0.4 68 Ac-Gly-Val-Tyr-Ile-His-Pro-Phe 17.3 ± 0.2 2.8 ± 0.3 69 Asp-Arg-Val-Gly-Tyr-Ile-His-Pro-Phe >10 000 255 ± 12 70 Asp-Arg-Gly-Val-Tyr-Ile-His-Pro-Phe >10 000 2.9 ± 0.3 71 Asp-Arg-Tyr-Ile-His-Pro-Phe 204 ± 10 238 ± 7

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The importance of the distance between the Arg2 and Tyr4 side chains for binding was addressed by synthesizing the internally elongated nonapeptides 69 and 70. Insertion of a Gly residue between Val and Tyr gave compound 69, which had a low AT2 receptor affinity (Ki = 255 nM). In contrast, when the Gly residue was introduced between Arg and Val (70) relatively high affinity was maintained (Ki = 2.9 nM). The 100-fold difference in binding affinity between compounds 69 and 70 might be explained by the different conformational preferences induced by the Gly-Val or Val-Gly segments. Removal of the Val residue in Ang II gave 71 with a Ki of 238 nM. Thus it seems that the distance between Arg2 and Tyr4 is of importance and influences the binding affinities for the AT2 receptor.

For comparison, compounds 58-65 were also evaluated with regard to AT1 receptor binding. In contrast to the results for AT2 receptor binding, the eight Ang II analogues in the Gly scan showed a wider range of binding affinities for the AT1 receptor (Table 3). Both [Gly2]Ang II (59) and [Gly8]Ang II (65) completely lacked affinity for the AT1 receptor (Ki > 10

M). Replacement of Tyr (61) or His (63) gave less than a 50-fold affinity reduction, 48 nM and 14.3 nM, respectively. The substitution of Pro with Gly resulted in compound 64 with a Ki of 146 nM. Furthermore, replacement of the Val or Ile residue with Gly (60 and 62) did not affect the binding affinity significantly. These results are similar to those obtained in the Ala scan performed by Miura et al.182 The main difference is the lack of affinity of [Gly8]Ang II (Ki > 10 M) compared to [Ala8]Ang II (Ki = 1 nM) and the wider range of affinities in the present study. It is well known that substitution of the aromatic ring in the phenylalanine residue in Ang II by an aliphatic side chain turns the agonistic response into an antagonistic response.128,131 The lack of the aliphatic side chain in [Gly8]Ang II might be the reason for the difference in affinity.

Removal of the N-terminal Asp residue gave 66 (Ang III) with a Ki of 10.5 nM for the AT1 receptor, which is similar to previously reported data.119,139 Compound 67, in which the Arg residue in 66 has been substituted by a Gly, lacked affinity for the AT1 receptor (Ki > 10 M). When this compound was acetylated (68), the affinity was recovered (Ki = 17.3 nM). The backbone-elongated peptides 69 and 70 lacked affinity for the AT1receptor, indicating that the position of the Arg side chain relative to Tyr, His, and Phe is important. Also, the deletion analogue 71, which showed a 200-fold decrease in affinity compared to Ang II, illustrates the importance of the relative positions of the pharmacophore groups.

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8 Concluding Remarks

In this study each amino acid in the octapeptide Ang II has been evaluated for its contribution to AT2 and AT1 receptor binding. Furthermore, a number of pseudopeptides encompassing a - or -turn mimetic have been designed, synthesized and evaluated for AT1 and AT2 receptor binding. Some of these Ang II analogues have also been evaluated for AT2 receptor agonist activity. The results are summarized below.

A glycine scan of Ang II showed that only removal of the arginine side chain affected the AT2 receptor binding affinity substantially. In contrast to the findings in the glycine scan it was observed that a positively charged group was not needed for AT2 receptor affinity in N-terminal truncated Ang III peptide analogues, suggesting different binding modes for Ang II analogues and smaller N-truncated ligands. Synthetic routes to two benzodiazepine-based -turn mimetics (I and II)and one -turn mimetic (III) have been developed. The turn mimetics have primarily been incorporated into Ang II replacing the area around Tyr4.

N

HN

O

R

NH

O

NH

NHNH

NO

R1

NH

O

R2

I II III

N

HN

NH

R1

O

R2

HN

O

The synthesized pseudopeptides have been evaluated for AT1 and AT2receptor binding affinity. Most of the compounds exhibited high AT2/AT1receptor selectivity and nanomolar affinity for the AT2 receptor. One pseudopeptide encompassing the -turn mimetic (III) exhibited a high AT1 receptor affinity. Molecular modeling of the Ang II analogues encompassing the -turnmimetics (I and II) has suggested that the relative positioning of the arginine side chain and the scaffold with the tyrosine side chain is important for AT2 receptor binding. It has also been suggested that these pseudopeptides bind to the AT2 receptor in an extended conformation. Three of the pseudopeptides have been evaluated in AT2 receptor functional assays and have been shown to exert agonistic activity.

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9 Acknowledgements

I would like to express my sincere gratitude to all present and former colleagues at the Division of Organic Pharmaceutical Chemistry, where I have spent the past years working on this thesis, for a friendly and pleasant working atmosphere.

I wish to express my sincere gratitude to:

Professor Anders Hallberg, for being an excellent supervisor and for his inspiring guidance and enthusiastic support throughout this study. Thanks also for spreading such a positive atmosphere throughout the department and for the much-appreciated “chemistry-skiing” trips.

My co-advisors and co-authors: Anders Karlén for always being helpful and sharing his vast knowledge in molecular modeling and the field of Ang II with me. Gunnar Lindeberg for introducing me to the field of peptide chemistry and for valuable help with peptide synthesis and comments on my manuscripts.

My co-authors: Christian Sköld, for his modeling contributions and for all help, support and good ideas. Milad Botros for working long hours testing and retesting my compounds. Professor Nicole Gallo-Payet, Bianca Plouffe, Hélène Beaudry, Gunter Wolf and Professor Fred Nyberg for rewarding collaboration.

The past and present members of the angiotensin group, for fruitful discussions concerning chemistry and Ang II.

Rikard Larsson and Marcus Rydberg, for skilful assistance during their Master’s and summer projects.

Gunilla Eriksson, for always being helpful and happy. Sorin Srbu, for his help with computer problems. Marianne Åström and Arne Andersson for help with secretarial and technical issues.

Uno Svensson, for being such an expert in nomenclature and medicinal chemistry, and for always being willing to help. Mats Larhed for your advice

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concerning palladium chemistry and for providing a good scientific atmosphere.

Helen Sheppard, for excellent linguistic revision of my thesis. All linguistic mistakes are without doubt my own last minute changes. Gunnar Lindeberg Christian Sköld, Jennie Georgsson, Jonas Nilsson, Charlotta Wallinder, Anja Johansson, and Anders Karlén, for constructive criticism and comments on my thesis. Kristofer Olofsson, for linguistic revision of my manuscripts.

My co-workers and room mates: Karl Vallin, Susanna Lindman, Kristofer Olofsson, Jennie Georgsson, Anna Arefalk, and Robert Rönn, for pleasant times in the lab and interesting discussions concerning everything.

Anja Johansson, Petra Johannesson, Máté Erdélyi, Anna Ax, Pär Holmberg, Charlotta Wallinder, and Karolina Ersmark, for fruitful discussions about chemistry and life.

The Swedish Academy of Pharmaceutical Chemistry is gratefully acknowledged for making my attendance at scientific meetings and national courses possible. The Swedish Foundation for Strategic Research (SFF) and the Swedish Research Council (VR) are acknowledged for financial support of this project.

Jag vill också tacka alla vänner och släktingar för att ni finns och får mig att koppla bort från kemin. Speciellt,

Mamma och pappa som alltid funnits till hands. Tack för kärlek, stöd och för att ni alltid har uppmuntrat mig att anta nya utmaningar.

Sofia, Calle och Fredrik, de bästa syskon man kan tänka sig.

Sandra och Ullis för alla pratstunder och allt roligt vi har tillsammans.

Jonas, för all din kärlek, stöd och tro på mig. För att du finns vid min sida och är min allra bästa vän.

Ulrika 20/10-2004

63

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11 Appendix

Experimental data for substances not included in papers I-IV.

General Methods. 1H and 13C NMR spectra were recorded on a JEOL JNM-EX400 at 400 MHz and 100.5 MHz, respectively. Spectra were recorded at ambient temperature. Chemical shifts are reported as values (ppm) referenced to 7.26 ppm CHCl3, 77.0 ppm CDCl3, and 2.50 ppm, 39.5 (DMSO-d6). Optical rotations were measured on a Perkin-Elmer model 241 polarimeter. Mass spectra were recorded by LC-MS utilizing electrospray ionization (ESI) or GC-MS, using capillary column and electron impact ionization (EI) at an energy of 70 eV.

Thin layer chromatography (TLC) was performed using aluminium sheets precoated with silica gel 60 F254 (0.2 mm, E. Merck). The spots were identified by UV-detection and/or by spraying with 3% methanolic solution of ninhydrin followed by heating. Column chromatography was performed using Merck silica gel 60 (40-63 m). The starting material 2-fluoro-5-nitrobenzaldehyde199 2 and the reagent DBI201 were synthesized according to previously published procedures.

3-Bromo-2-fluoro-5-nitrobenzaldehyde (33). To 2-fluoro-5-nitrobenz-aldehyde 2 (13.00 g, 76.9 mmol) dissolved in 85 mL concentrated H2SO4 asuspension of DBI (12.00 g, 41.8 mmol) in 225 mL H2SO4 was added over 45 min. The red reaction mixture was stirred for 3 days. According to GC-MS some starting material remained according to GC-MS and more DBI (1.12 g, 3.9 mmol) was added. The reaction was stirred for one more day before it was poured onto ice and extracted with EtOAc. The organic layers were combined and washed with water and brine, dried over Na2SO4 and evaporated. The residue was purified by column chromatography (gradient pentane to 5% EtOAc in pentane) and the desired product 33 was obtained as a white solid (9.6 g, 50%). 1H NMR (CDCl3) : 10.37 (s, 1H, C(O)H), 8.71 (m, 2H, 2 × CH), 13C NMR (CDCl3) : 183.9 (d, 5.5 Hz, C(O)H), 164.0 (d, J= 267.9 Hz, CF), 144.6 (C), 133.8 (d, J = 2.4 Hz, CH), 124.9 (d, J = 11.6 Hz, C), 123.3 (d, J = 2.4 Hz, CH), 111.7 (d, J = 22.6 Hz). GC-MS (M+): 247. Anal. Calcd for C7H3FBrNO3: C, 33.90; H, 1.22; N, 5.65. Found: C, 33.97; H, 1.25; N, 5.83.

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2,2-Dimethoxyethyl-(3-bromo-2-fluoro-5-nitrobenzyl)amine (34). HOAc (0.50 mL, 8.8 mmol) and 2,2-dimethoxyethylamine (1.00 ml, 13.5 mmol) were added to 3-bromo-2-fluoro-5-nitrobenzaldehyde 33 (1.51 g, 6.1 mmol) dissolved in 20 mL MeOH. The reaction mixture was stirred overnight before NaCNBH3 (0.77 g, 12.3 mmol) was added in portions. After another night, water was added and the MeOH was evaporated. The remaining aqueous solution was extracted with EtOAc, dried over Na2SO4and evaporated. Purification by column chromatography (gradient system diethyl ether/petroleum ether 1:3 to 1:1) gave 34 as a yellow oil (1.35 g, 66%). 1H NMR (CDCl3) : 8.40-8.34 (m, 2H, 2 × CH), 4.52 (t, J = 5.3 Hz, 1H, CH), 3.99 (m, 2H, CH2), 3.40 (s, 6H, 2 × CH3), 2.78 (d, J = 5.3 Hz, CH2), 2.49 (br s, 1H, NH). 13C NMR (CDCl3) : 160.9 (d, J = 256.8 Hz, CF), 144.2 (d, J = 3.8 Hz, C), 129.8 (d, J = 18.5 Hz, C), 127.9 (d, J = 1.5 Hz, CH), 124.6 (d, J = 6.1 Hz, CH), 109.81 (d, J = 24.6 Hz, C), 103.5 (CH), 54.3 (CH3), 50.3 (CH2), 46.4 (d, J = 2.3 Hz, CH2).

(2S)-2-[(9H-fluoren-9-ylmethoxylcarbonyl)amino]-3-methylpentanoic acid (3-bromo-2-fluoro-5-nitrobenzyl)-(2,2-dimethoxyethyl)amide (35).Fmoc-Ile-OH (1.52 g, 4.3 mmol), HATU (1.62 g, 4.3 mmol) and DIEA (2.00 mL, 11.5 mmol) were added to a solution of the amine 34 (1.31 g, 3.9 mmol) in 20 mL CH2Cl2. The reaction mixture was stirred for 5 h and water was added. The layers were separated and the aqueous layer was extracted with EtOAc. The organic layers were combined and washed with 1 M KHSO4,water, saturated NaHCO3 (aq) and brine, dried (Na2SO4) and evaporated. The residue was purified by column chromatography (gradient diethyl ether/petroleum ether 3:1 to 2:1) to give 35 as white foam (1.43 g, 55%). [ ]22

D = -36 (c = 1.04, CH2Cl2). The product was observed as rotamers at 20 ºC by NMR. 1H NMR (CDCl3) : 8.41 (minor) and 8.36 (m, 1H, CH), 8.09 and 8.04 (minor) (m, 1H, CH), 7.76 (m, 2H, 2 × CH), 7.63-7.56 (m, 2H, 2 × CH), 7.40 (m, 2H, 2 × CH), 7.30 (m, 2H, 2 × CH), 5.44 (minor) and 5.40 (m, 1H, CH), 4.94 and 4.90 (minor) (m, 1H, CH2), 4.65-4.48 (m, 2H, CH + CH2)4.46-4.16 (m, 3H, CH + CH2), 3.75 (m, 1H, CH2), 3.51 and 3.15 (minor) (m, 1H, CH2), 3.46 and 3.42 (minor) (s, 3H, CH3), 3.39 and 3.37 (minor) (s, 3H, CH3), 1.81 (m, 1H, CH), 1.52 (m, 1H, CH2), 1.14 (m, 1H, CH2), 0.98-0.82 (m, 6H, 2 × CH3).

(2S)-9-Bromo-4-(2,2-dimethoxyethyl)-2-(isobutyl)-7-nitro-1,2,4,5-tetrahydrobenzo[1,4]diazepin-3-one (36). DBU (330 L, 2.2 mmol) was added to 35 (1.30 g, 1.9 mmol) in dry THF (4 mL). After 1.5 h the reaction was complete according to TLC (10% MeOH in CH2Cl2). The reaction mixture was evaporated and the residue was purified by column chromatography (CH2Cl2 to elute Fmoc-related side products, then 2% MeOH in CH2Cl2) to yield the free amine. The product (788 mg, 1.7 mmol) was dissolved in 35 mL DMSO and 470 L water and 935 L Et3N were

81

added. The reaction mixture was stirred at room temperature over night, EtOAc and saturated NaHCO3 (aq.) were added. The layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The crude residue was purified by column chromatography (1% MeOH in CH2Cl2) to give 36 as a yellow foam (665 mg, 77%). [ ]22

D = -246 (c = 1.01, MeOH).1H NMR (CDCl3) : 8.25 (d, J = 2.6 Hz, 1H, CH), 7.85 (d, J = 2.6 Hz, 1H, CH), 5.27 (d, J = 16.9 Hz, 1H, CH2), 4.45 (dd, J = 8.2, 4.7 Hz, 1H, CH), 4.31 (dd, J = 5.4, 4.9 Hz, 1H, CH), 4.17 (d, J = 16.9 Hz, 1H, CH2),3.64 (dd, J = 14.0, 4.9 Hz, 1H, CH2), 3.52 (dd, J = 14.0, 5.4 Hz, 1H, CH2),3.35 (s, 3H, CH3), 3.20 (s, 3H, CH3), 2.14 (m, 1H, CH), 1.82 (m, 1H, CH2),1.32 (m, 1H, CH2), 1.08 (d, J = 6.6 Hz, 3H, CH3), 1.00 (d, J = 7.4 Hz, 3H, CH3), 13C NMR (CDCl3) : 168.6 (C), 147.7 (C), 136.9 (C), 128.3 (CH), 125.4 (CH), 119.0 (C), 109.2 (C), 103.2 (CH), 58.8 (CH), 54.9 (CH3), 54.8 (CH3), 52.2 (CH2), 49.3 (CH2), 35.0 (CH), 24.7 (CH2), 16.3 (CH3), 10.7 (CH3).

(2-Bromo-6-methyl-4-nitrophenyl)isopropylamine, 39. Compound 39 was prepared according to a literature procedure209. An ice-cooled suspension of 2-bromo-6-methyl-4-nitroaniline 38 (1.00 g, 4.3 mmol) and NaBH4 (1.70 g, 44.7 mmol) in 17 mL THF was added to an ice-cooled solution of acetone (3.20 mL, 43.6 mmol) in 17 mL THF. Sulphuric acid (2.3 mL, 43.2 mmol) was added and the reaction mixture was stirred at room temperature for 8 h. Ice water was added followed by NaOH (2M) to make it alkaline and the mixture was extracted with diethyl ether. The combined organic layers were washed with water and dried over MgSO4 and evaporated to give 39 (0.99 g, 83%) as a yellow solid. 1H NMR (CDCl3) : 8.28 (m, 1H, CH), 7.95 (m, 1H, CH), 3.87 (septet, J = 6.4 Hz, 1H, CH), 2.41 (s, 3H, CH3), 1.22 (d, J = 6.4 Hz, 6H, 2 × CH3). 13C NMR (CDCl3) : 150.3 (C), 140.2 (C), 127.9 (C), 126.6 (CH), 126.4 (CH), 114.4 (C), 48.3 (CH), 23.9 (CH3), 21.7 (CH3).

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of PharmacyEditor: The Dean of the Faculty of Pharmacy

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A doctoral dissertation from the Faculty of Pharmacy, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Pharmacy.(Prior to July, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Pharmacy”.)

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