Side Chain Cyclized Aromatic Amino Acids: Great Tools as Local

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Side chain cyclised aromatic amino acids: great tools as local constraints in peptide and peptidomimetic design. Olivier Van der Poorten, Astrid Knuhtsen, Daniel Sejer Pedersen, Steven Ballet, and Dirk Tourwé J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01029 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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Journal of Medicinal Chemistry

Side chain cyclized aromatic amino acids: great tools as local constraints in peptide and peptidomimetic design

Olivier Van der Poorten,†,ǁ Astrid Knuhtsen,‡,ǁ Daniel Sejer Pedersen,‡,* Steven Ballet,† and Dirk Tourwé†,*

†

Research Group of Organic Chemistry, Departments of Chemistry and Bio-engineering

Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium ‡

Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162,

2100 Copenhagen, Denmark ǁ

O.V.d.P and A.K contributed equally to this work.

Abstract Constraining the conformation of flexible peptides is a proven strategy to increase potency, selectivity and metabolic stability. The focus has mostly been on constraining the backbone dihedral angles; however, the correct orientation of the amino acid side chains (χ-space) which constitute the peptide pharmacophore is equally important. Control of χ-space utilizes conformationally constrained amino acids which favor, disfavor or exclude the gauche (‒), the gauche (+) or the trans conformation. In this review we focus on cyclic aromatic amino acids in which the side chain is connected to the peptide backbone to provide control of χ1and χ2-space. The manifold applications for cyclized analogues of the aromatic amino acids

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Phe, Tyr, Trp and His within peptide medicinal chemistry is showcased herein with examples of enzyme inhibitors and ligands for G protein-coupled receptors.

Keywords Constrained amino acids, peptide mimetics, constrained peptides, chi space control

Introduction Peptides regulate many important physiological mechanisms in man, and as such they are interesting candidates for drug development.1 Peptides have historically been disfavored as drug leads but in the last two decades this has changed dramatically due to many technological advances in the structural optimization, formulation and production of peptides and peptidomimetics.2 To date more than 60 peptide drugs have been approved and approximately 140 peptides are currently in clinical trials.3 The use of unnatural amino acids has been central to the success of peptide drugs such as the GnRH blockers Degarelix and Abarelix, and the bradykinin B2 antagonist Icatibant.4 Alternatively, cyclic peptides such as the somatostatin analogue Octreotide and the anticoagulant drug Eptifibatide have been introduced in order to improve metabolic stability. The advantages of peptide backbone cyclization for improving potency, selectivity and metabolic stability were elegantly described by Kessler as early as in 1982.5 Peptide homo- or heterodetic cyclization leads to macrocycles with a more precise organization of function-defining elements.6 In such rings, the backbone Φ and Ψ dihedral angles are constrained and controlled. However, the orientation of the side chain pharmacophoric groups, which is determined by the χ dihedral angles, is at least equally important. The control of χ dihedral angles was described by Hruby as “design in χ-space”7 and is generally perceived as the most difficult to achieve in peptide design.6 In case of the aromatic amino acids, Phe, Tyr, Trp and His, a wide variety of side


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chain-constrained analogues have been prepared and were already reviewed in 1999.8 In such cyclic amino acids, the backbone Φ and Ψ and the side chain χ dihedral angles are constrained (Fig. 1). For the aromatic amino acids many side chain-constrained analogues are known8 that allow control of χ2 (e.g. 29) or χ1 dihedral angles (e.g. 3 and 410). Any constraint of χ1 will also influence χ2 and vice versa.11-12

Figure 1. Definition of the backbone and side chain dihedral angles (1) and examples of χ2 (2) and χ1 (3-5) constraints.11 Herein we will focus on tools that provide control of χ1- and χ2-space via introduction of cyclic aromatic amino acids in which the side chain is covalently connected to the peptide backbone (Fig. 2). In Figure 2 (top), Newman projections along the Cα-Cβ bond of L-Phe and the three low energy staggered conformations with the associated χ1 values are depicted. The population of these low energy conformations can be biased by introducing steric constraints such as methyl substitution at Cβ (e.g. 5, Fig. 1) which has been used to great effect in the field of melanotropins and opioids.13



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Figure 2. The lowest energy side chain conformations (top) and examples of families of cyclic aromatic amino acids that target subsets of χ1-space (bottom). The arrows indicate to which main chain atom the side chains are fixed in 6 to 10. A complete list of all constrained amino acids discussed in this review, with their names, abbreviation(s), a reference for the synthesis and application is given in the Supporting Information. By linking the aromatic ring directly to Nα (n = 0) or through a methylene group (n = 1), as exemplified

by

6

(Ioc

=

indoline-2-carboxylic

acid)

and

7

(Tic

=

1,2,3,4-

tetrahydroisoquinoline-3-carboxylic acid), the gauche (‒) and (+) conformation can be accessed, and the trans conformation is excluded. Similarly, only the gauche (‒) and trans conformation can be obtained by linking the aromatic ring to Cα via a methylene bridge to give e.g. 8 (Aic = 2-amino-indane-2-carboxylic acid structure; n= 1), or an ethylene linker, e.g. 9 (Atc = 2-amino-tetralin-2-carboxylic acid; n = 2). Finally, linking the aromatic ring to Nα of the following amino acid provides access to the gauche (+) and trans conformations, e.g. 10 (Aba = 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one) and excludes the gauche (‒) conformation. In these families of cyclic constrained amino acids the aromatic ring is part of the cyclic structure. Consequently, in addition to the χ1 dihedral angle, the χ2 angle is also 4 ACS Paragon Plus Environment

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constrained. The family of backbone constrained aromatic amino acids (e.g. 7, 9 and 10) provides a method to systematically scan the side chain conformations in order to identify the bioactive conformation of a given peptide. Moreover, because different receptors may require slightly different side chain orientations, receptor (sub)type selectivity can be achieved via introduction of such constrained amino acid analogues. The manifold application for these families of side chain cyclized analogues of the aromatic amino acids Phe, Tyr, Trp and His within peptide medicinal chemistry is showcased herein with examples of enzyme inhibitors and ligands for G protein-coupled receptors.

Farnesyl Transferase The GTP-binding proteins of the Ras family are involved in the regulation of cellular growth and differentiation in mammalian cells.14 In order to function, the proteins must be anchored to the cellular membrane, which requires a series of post-translational modifications to the C-terminal CA1A2X sequence (A being an aliphatic residue and X any residue). The first modification involves the catalyzed transfer of farnesyl to the cysteine residue (when X = Met or Ser) by the Zn2+-dependent enzyme farnesyl transferase (FT).15 Because mutated Ras genes are found in 15% of all human cancers, occurring predominantly in pancreas (90%), colon (50%) and lung (30%) carcinomas, the inhibition of FT in these cancers may block oncogenic Ras signaling.14,16 Thus, the identification of FT inhibitors has attracted much attention, yielding both non-peptidic17 and constrained peptidomimetic inhibitors. Systematic replacement of A1 and A2 in the CA1A2X sequence with various natural amino acids revealed the CVFM sequence as the best native tetrapeptide inhibitor of FT (IC50 = 250 nM).18 Later, Marsters et al. demonstrated that replacing Phe3 with Tic3 in the N-terminally acetylated CVFM tetrapeptide 11 improved the inhibitory activity more than 20-fold (12 in 5 ACS Paragon Plus Environment


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Fig. 3).19 The authors proposed that Tic stabilized a turn-like conformation positioning cysteine and methionine to bind to the Zn2+ in a bidentate binding mode.

Figure 3. Potency increase by substituting Phe3 in tetrapeptide 11 with Tic3 to give 12.19 Clerc et al. synthesized the analogue KCVFM 13 with a lysine residue added to the Nterminus to improve aqueous solubility, while retaining biological activity (Fig. 4). Molecular modelling studies on 13, showed a preference for extended conformations.20 In agreement with the results of Marsters et al.,19 the replacement of Val3-L-Phe4 with Val3-L-Tic4, to give 14a, led to a 50-fold improvement in FT inhibition with a preference for an extended structure. Further incorporation of residues that stabilize extended conformations, such as NMeVal3 in 15, resulted in an increased inhibitory potency, whereas introduction of residues which induce turn structures, such as D-Tic4 (14b), or D-Pro3 (16) resulted in a large reduction in potency (Fig. 4).20


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Figure 4. Modifications to the FT inhibitor KCA1A2M and the resulting effect on ligand potency.20 Researchers at Bristol-Myers Squibb (BMS) exchanged Phe3 in the non-N-terminally acetylated CVFM peptide 17 for the constrained phenylalanine derivatives Tic (18), Ioc (19), Aic (20) and Z-α,β-dehydroPhe (21) (Fig. 5).21 Tic-substitution provided the most potent analogue 18. The importance of the ring size was demonstrated by the low potency of the corresponding Ioc analogue 19, whereas the importance of the type of constraint was illustrated by the Aic analogue 20 and by the α,β-unsaturated analogue 21.21 Previous efforts had demonstrated that replacing the amide bonds with the reduced amide isostere led to compounds with improved inhibition effects, enhanced metabolic stability and enhanced whole cell activity for CA1A2X-based FT inhibitors.22 When this strategy was applied at two positions in lead compound 18, peptidomimetics 22 and 23 with improved potencies were obtained (Fig. 5).21



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The importance of the Tic3 residue was highlighted by removal of the aromatic ring to give 24, which resulted in a significant potency reduction. The authors concluded that the role of the heterocyclic ring in Tic serves to orient the phenyl ring in the optimal binding orientation, rather than orienting the tetrapeptide backbone into a bioactive conformation.21 Other derivatives of 23, such as C-terminal esters and replacement of the Cys residue with an Nterminally linked imidazole produced potent, non-toxic inhibitors (not shown).21 However, the researchers at BMS decided to take a non-peptidic benzodiazepine forward into clinical trials.


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Figure 5. Modifications to the CVFM sequence and the resulting effect on potency.21 [double column figure]

The Renin-Angiotensin System: ACE, NEP and Renin inhibitors Modulation of the renin-angiotensin system (RAS) constitutes a successful therapy of hypertension and congestive heart failure.23 This modulation is based on the inhibition of the angiotensin-converting enzyme (ACE) and renin. Renin belongs to the class of aspartyl proteases and liberates the decapeptide Angiotensin I (Ang I) by cleavage of the precursor Angiotensinogen.23 The inactive Ang I is converted into the potent vasoconstrictor Ang II by ACE, a Zn2+-containing dipeptidyl protease. The design of the ACE inhibitors 25-27 (Fig. 6), represent classic examples of rational design of peptidase inhibitors.24

Figure 6. The sulfhydryl- 25, and non-sulfhydryl ACE inhibitors 26, 27. A prototype for the sulfhydryl class of ACE inhibitors is 25 which was the first orally active ACE inhibitor to be used in the clinic.25 To reduce side effects of 25, attributed to the presence of the sulfhydryl group, 26 was developed.26 Various aspects of the structure-activity relationships (SAR) of both sulfhydryl (e.g. 25) and non-sulfhydryl (e.g. 26) classes have been reviewed.24,27 Because ACE is structurally related to neutral endopeptidase (NEP), and 9 ACS Paragon Plus Environment


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both enzymes cleave the vasorelaxant peptide bradykinin,28 it was hypothesized that active site similarities would allow the design of dual inhibitors as antihypertensives.29 Structure-activity studies of 25 revealed that the annulation of an aromatic ring on the proline residue produced potent analogues (Fig. 7). A systematic study by Stanton et al.30 indicated that the indoline-2-carboxylic acid analogue 28 was approximately six times more potent than 25 (the IC50 for ACE inhibition reported in this study was 15 nM). Ring expansion to give analogue 29 reduced the potency tenfold, and a similar trend was observed upon moving the aromatic ring to give analogue 30. A further shift of the aromatic ring to provide analogue 31 resulted in a dramatic decrease in ACE inhibitory potency. The importance of the aromatic ring was clearly demonstrated by comparison of 30 with heterocyclic analogues 32 of 33, that both displayed a large decrease in potency. These results were confirmed in in vivo assays in rats, measuring the inhibition of the Ang I-induced pressure after intravenous (iv) administration in spontaneous hypertensive rats, where the indoline analogue 28 was the most potent. The results of inhibiting ACE with 30 (IC50 = 18 nM) were independently confirmed by Klutchko et al.31 and by Hayashi et al.32, who demonstrated that it was more potent in inhibiting ACE (IC50 = 8.6 nM) than 25 (IC50 = 23 nM), whereas it was equipotent to 25 in vivo after oral administration. Klutchko et al. also reported the ACE inhibitory activity of the diasteromeric mixtures 34 and 35, which both showed a similar lack of potency with IC50 values of 58 µM and 14 µM, respectively, versus 13 nM for 25.31


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Figure 7. Benzo-fused analogues of 25. A similar study on the influence of introducing an aromatic ring on the potency has been conducted in the non-sulfhydryl class of ACE inhibitors (Fig. 8).31 Whereas analogue 36 (IC50 = 44 nM) showed a reduced potency when compared to 26 (IC50 = 1.2 nM), analogues 37 and 38 had a similar potency. In contrast to the sulfhydryl compounds 31, 34 and 35, analogue 39 showed good potency (IC50 = 5.8 nM). This striking difference in potency between nonsulfhydryl and sulfhydryl compounds is assumed to be due to alternating binding modes for the two classes of ACE inhibitors.31



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Figure 8. Fused analogues of 26. These studies led to the further development of the per-saturated analogues 40 and 41.24,33 A combination of the conformational constraints in the indoline-2-carboxylic analogues and in 42,34 resulted in the tricyclic [6H]-azepino-indoline analogue 43.35 In vitro screening of compound 43 showed potent ACE inhibition in rabbit lung (IC50 = 5.2 nM). When administered orally to rats, 43 was found to block the Ang I-induced pressor response for more than 6 hours. Surprisingly, good in vivo activity of this bis-carboxylic acid was obtained without further derivatization to improve oral bio-availability.35


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In another approach to design of ACE inhibitors, Flynn and co-workers designed a mimic of the three C-terminal amino acids of Ang I (Cbz-Phe-His-Leu-OH 44; KM 10 nM, Fig. 9).36 The conformationally constrained bicyclic lactam bis-carboxylic acid 45b was found to inhibit rabbit lung ACE in vitro with a Ki of 0.012 nM. In vivo, the prodrug 45a was orally active in conscious spontaneously hypertensive rats by reduction of the Ang I-induced pressor response.36 Further studies resulted in the conformationally constrained Phe-Gly dipeptide mimetic 4637 that displayed ACE inhibition in vitro with a Ki of 10 nM.

Figure 9. Conformationally constrained tripeptide ACE inhibitors 45a/b and 46.36-37 The replacement of His in the tripeptide Phe-His-Leu fragment by a Phe mimetic in 45 and the knowledge that both ACE and neutral endopeptidase (NEP) cleave bradykinin adjacent to Phe8, suggested that scaffold 45 could be used to design dual ACE/NEP inhibitors. Since NEP is the most prominent enzyme in the metabolism and inactivation of atrial natriuretic factor38, a 28-amino acid peptide with natriuretic and diuretic actions, inhibition of both enzymes would be advantageous to reduce blood pressure.29



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The first described synthetic and potent NEP inhibitor was 47 (Fig. 10).39 Compound 47 was shown to be approximately 70-fold more potent in inhibiting NEP than inhibiting ACE. Peptidomimetic 48 on the contrary is a potent ACE inhibitor with low NEP activity. Modification of 48 by introduction of a mercaptoacetyl group at the N-terminus produced the potent dual ACE/NEP inhibitor 49, with potency at NEP similar to that of the prototype inhibitor 47.40 The authors attributed the high inhibitory potency of 49 towards both enzymes to a limited conformational freedom of the mercaptoacetyl amide side chain, which allowed orientation of the thiol function towards the zinc atom in both active sites. Optimization of compound 49 led to (S)- and (R)-benzyl-substituted thiols 50a and 51, which both showed enhanced in vitro inhibitory potencies for rabbit lung ACE and rat kidney NEP.40 Whereas the (R)-diastereomer 51 did not improve ACE inhibition compared to 49, the (S)-isomer 50a did show enhanced overall inhibitory potency towards both metalloproteinases. In vivo analysis of 50b, the thioester prodrug of 50a, resulted in significant blood pressure lowering effects in spontaneously hypertensive and DOCA-salt hypertensive rats over a 5 hour period at a 30 mg.kg-1 oral dose. This rational approach towards 7,6-fused benzazepinone derivative 50a clearly demonstrates the utility of side chain constraints in peptidomimetic design.40


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Figure 10. Model compound 47,39 bicyclic lactam derivatives 48-5340-41 and 5442 as dual ACE/NEP inhibitors. [double column figure] A potent anti-Phe-Leu-based NEP inhibitor 52a (Ki = 0.4 nM) was described by Warshawsky et al.41a Interestingly, the analogue lacking the isobutyl side chain 52b showed much lower potency at NEP with a slightly better ACE inhibitory potency.41b A shift of the phenyl ring to give 53 produced a potent dual inhibitor with an excellent in vivo activity in inhibiting the Ang I pressor response.41c Reference compound 25 was compared with 50a,b and 53 for in vivo inhibition in the Ang I pressor response after oral administration at 5 µmol.kg-1 in rats. It was observed that 53 was comparable to 25 in terms of potency and duration of action, whilst being more effective than 50a,b.41c Subsequently, Flynn et al. probed the effect of a distinct staggered conformation of Phe-containing dipeptides to examine conformational preferences for the inhibition of metalloproteinases via the synthesis of oxazepine 54 as conformationally constrained gauche (‒) dipeptide mimetics.42 Particularly, these gauche (‒) (χ1 = ‒60°) mimetics were complementary to the benzazepinone



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anti (χ1 = 180°) mimetics in terms of χ1 angle. In this case, however, 54 was found to be a poor ACE and NEP inhibitor (Ki ACE = 50 µM; Ki NEP = 330 nM, respectively).42 In addition, various mercaptoacyl dipeptides were designed as dual ACE/NEP inhibitors by BMS (Fig. 11).43 It was found that mercaptoacetyl dipeptide 55 had ACE inhibitory potency compared to 25, but was more than 1000-fold more potent than 25 against NEP. Mercaptopropanoyl dipeptide 56 showed increased potency for both rabbit lung ACE and rat kidney NEP. However, in vivo ACE activity for 56 was reduced, which was attributed to differences in metabolic S-methylation. Replacement of the proline residue with a constrained Tic moiety to give 57 resulted in a slight loss in ACE inhibitory potency in vitro, but equivalent activity in vivo when compared to 56. Finally, introduction of an indoline to give 58 resulted in in vitro dual enzyme affinity comparable to 56, and improved potency in vivo against ACE. Nonetheless, the in vivo potency and duration of action for 58 was inferior in the Ang I pressor response model, when compared to mercaptoacetyl dipeptide 55.43


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Figure 11. Conformationally constrained mercaptoacyl dipeptides 55-58 as dual ACE/NEP inhibitors.41b,41c,43 An alternative route to control hypertension was achieved by inhibiting the aspartyl protease renin (Fig. 6).23,44 Renin catalyzes the cleavage of angiotensinogen into the decapeptide Ang I, which is the rate-limiting step in the RAS cascade. In contrast to ACE, renin has high substrate specificity and thus renin inhibitors could potentially be highly selective agents for controlling Ang II formation.45 Human renin hydrolyzes the Leu20-Val21 amide bond of angiotensinogen and potent transition state inhibitors, containing a hydroxyethylene isostere, have been developed.46 Based on the ChaΨ(CHOH-CH2)Valcontaining inhibitors developed by Buhlmeyer,46 de Laszlo constrained the Phe-Leu subsequence of the enzyme inhibitors (for synthetic reasons simplified to Phe-Nle in 59, Fig. 12) into piperidinone 60 and benzazepinone 61.47 In vitro analysis using human plasma renin showed that 60 was 25-fold less potent and 61 250-fold less potent compared to the acyclic control 59 (Fig. 12). The dramatic loss of potency was explained by molecular modeling studies, which revealed that the conformational constraints in 60 and 61 distorted the optimal geometry at C3, hence preventing the putative required extended conformation for binding in the renin active site.47

O n

R=

O n

R Bu

N H

OH

N H

Bu

Ph H N

AcHN

59 IC50 = 0.84 nM

O Ph 60 IC50 = 21 nM N

AcHN O

NHAc O

61 IC50 = 210 nM

N



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Figure 12. Renin inhibitor analogue 59 and conformationally constrained renin inhibitors 60 and 61.47

Bradykinin The nonapeptide bradykinin (BK, Fig. 13) is involved in various physiological processes including vasodilatation and vascular permeability, as well as in pathophysiological pathways causing inflammation and pain.48-49 It exerts its biological effects by activating the constitutively expressed B249 and B1 receptors that are expressed mainly during tissue injury.50

Figure 13. Comparison of the amino acid sequences of 62 and the B2 antagonist 63. The search for stable, selective peptidomimetic antagonists culminated in the discovery of 63, that is the most potent B2 antagonist to date. Marketed as Icatibant for the treatment of angioedema, 63 has been thoroughly characterized both in vitro and in vivo, with IC50 values in the low nanomolar range in several preparations. Moreover, it displays high potency and stability in vivo in several animal models.51 Within the sequence of 63 the Pro7-Phe8 residues have been replaced by the two constrained amino acids D-Tic7-Oic8. These residues prevent cleavage of the antagonist by angiotensin-converting enzyme (ACE),52 whereas the N18 ACS Paragon Plus Environment

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terminal

0

D-Arg

stabilizes the peptide towards degradation by carboxypeptidase N.51 To

further optimize the potency and stability of antagonist 63 Amblard et al. substituted residues 7 and 8 in BK with a variety of constrained amino acids from known ACE inhibitors (Fig. 14).53-54 All peptides were shown to be full agonists with good potencies.53-54 Introduction of the (S)-benzothiazepinone (D-BT) moiety produced the best B2 receptor affinities for both the BK- and the Hoe140-variants 64 and 71, respectively. Moreover, it was shown that the D-BT motif adopts a type II´ β-turn in both solution and solid state.55 These results are consistent with previous research, which indicates that potent binding to the B2 receptor correlates with the propensity for C-terminal type II´ β-turn formation.56



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A

H-Arg-Pro-Pro-Gly-Phe-Ser-Xxx-Arg-OH

O

H N

Xxx = N

62 (Bradykinin)

O

Ph

S

O

O

*

N

N H

N

O 64, Ki 13 nM

N R1

O

rac-67, pKi 5.9 rac-68, pKi 6.6

O

*

N

O

H N H

N O

N

*

O

CH3 69, (S)-CH3, 54% inhib.@10 mM (R)-65 R1= H, pKi 5.6 rac-66 R1= CH3, pKi 6.1 70 (R)-CH3, pKi 6.3

B

H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-Xxx-Arg-OH

Xxx = H H

N

O

63 (Icatibant/Hoe140) O

N

Y O N H

O

N

N H

O

71 Y = S, Ki 0.7 nM 72 Y = O, Ki 2.5 nM

*

N O

(R)-73, pKi 6.5; pA2 5.6 (S)-74, pKi 6.7

O

* N

N O

(R)-75, pKi 7.6; pA2 5.5 (S)-76 pKi 8.5; pA2 5.7

Figure 14. A: Bradykinin analogues where the Pro-Phe residues have been replaced. B: Hoe140 analogues where the D-Tic-Oic residues have been replaced.


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Similarly, Ballet et al. replaced the Pro7-Phe8 residues in 62 and the D-Tic7-Oic8 in 63 with a variety of constrained Aba-Gly and Aba-Ala dipeptide mimics.57 It is known that Abadipeptides (e.g. 65) prefer extended conformations,58 whereas spiro-Aba-dipeptides (e.g. 67) induce turn conformations. However, all these analogues were inferior compared with the parent compounds. The spiro-Aba-mimics 75 and 76 showed superior binding when compared to 73 and 74, which supports the hypothesis that a turn motif is important for binding. Molecular modeling suggested that the positioning of the aromatic ring in the thiazepinone scaffold (e.g. 71), is better than that for the azepinone scaffold (e.g. (R)-73) for efficient receptor binding.57 It is noteworthy that the orientation of the aromatic ring appears to play a major role in determining biological activity (i.e. agonism vs antagonism).53-54,57,59 Thus, introduction of the thiazepinone scaffold gave rise to agonists, whereas the azepinone motif led to weak antagonists. Kyle et al. have reported five mimics of 63 aimed at correlating binding with structure (7781, Fig. 15).56b Using contour plots, they were able to show a trend in which the best antagonists (77 and 79) at the B2 receptor had the highest degree of a type II´ β-turn structure imposed by bulky residues. In contrast cyclized peptides 80-81 that adopt another type of βturn conformation did not display any activity at the B2 receptor.



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Figure 15. Hoe140 analogues 77-81.56b In summary, when the four C-terminal amino acids of 62 are constrained by the D-Tic-Oic dipeptide motif which produces 63, excellent affinity and potency towards the B2 receptors is achieved. This is likely due to an optimal backbone conformation induced by the 6-membered constrained amino acid D-Tic. In contrast, bradykinin mimics constrained by 7-membered amino acids appear to adopt a suboptimal backbone conformation resulting in inferior affinities and potencies.

Angiotensin IV The hexapeptide hormone angiotensin IV (82, Ang IV, Fig. 16) is derived from the bioactive precursor Ang II by cleavage of the two N-terminal amino acids. Ang IV has been shown to improve memory acquisition, protect against ischemic stroke, as well as hyperglycemia, and to have functions in the vascular and renal systems, similar to its 22 ACS Paragon Plus Environment

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precursors.60 Ang IV binds to the putative AT4-receptor, also known as the insulin-regulated aminopeptidase (IRAP), but the exact mechanism of how the biological functions are exerted is not fully understood.61-62 One hypothesis suggests that Ang IV inhibits IRAP and as a consequence the catalytic breakdown of other endogenous peptides, such as vasopressin, oxytocin and bradykinin.60a,63 Native Ang IV also binds to and is degraded by aminopeptidase N (AP-N) and is a weak full agonist at the AT1-receptor, which has Ang II as its primary ligand. Moreover, Ang IV has a short half-life in vivo (t½ < 1min). Thus, to evaluate the potential of Ang IV for drug development, more stable and selective analogues were required in order to fully understand the mechanisms of action of the hormone.64-65

Figure 16. Sequences of angiotensinogen and angiotensin I, II and IV. In order to improve the stability of Ang IV, Lukaszuk et al. performed a β-homo amino acid scan and identified 83 as a potent, selective and stable Ang IV analogue (Fig. 17).66 Using the radiolabeled analogue [3H]-83 in vitro studies with the IRAP receptor were undertaken.67-68 These studies revealed that IRAP undergoes semi-continuous cycling between the surface of the cell and the endosomal compartments.68 Subsequently, the effects of a conformational constraint strategy for the aromatic amino acids Tyr and Phe (as well as Pro) on IRAP inhibition were examined.69 Tyr was exchanged with 6-HO-Atc (84), 6-HO-Tic (85) and 7-HO-Tic (86). These three analogues showed a large drop in IRAP inhibition indicating that they did not result in a favorable orientation of the χ1 and χ2 angles. Thus, the structure and orientation of the Tyr residue appears essential for the biological function of



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Ang IV. This was supported further by the detrimental effect resulting from introduction of a single β-methyl group (87) or two methyl groups at the 2’,6’-position (Dmt).69-70 Homologation of Phe (i.e. 83) had a positive influence on IRAP versus AP-N activity, and abolished AT1 affinity.66 Although IRAP potency was reduced in the Phe-modified analogues 88-91, these compounds showed good selectivity. The Atc- (88) and Aic-analogues (89) favor the gauche (+)/gauche (‒) conformations, whereas the Tic-analogue (90) induces a gauche (+) conformation. Because these three modifications resulted in a similar drop in IRAP inhibition when compared to Ang IV, it was speculated that the bioactive conformation at χ1 for the Phe residue is gauche (‒). This assumption was supported by introducing a β-methyl substituent 91, which is known to favor the gauche (+)/gauche (‒) conformations, and as a result produced the best analogue in this series. H-Val-Tyr-Ile-His-Pro-Phe-OH

82 Ang. IV pKi 7.25 O H2N

Tyr-Ile-His-Pro

H N

COOH Ph

83 pKi 7.56

H-Val-Xxx-Ile-His-Pro-Phe-OH

H-Val-Tyr-Ile-His-Pro-Yyy-OH

OH

Xxx =

Yyy = OH H3C H

N H

O 84 pKi 5.24

N H

N H

O 87 pKi 5.69

N H

O

88 pKi 6.73

O

89 pKi 6.67

R1 R2 H N

85 R1 = OH; R2 = H pKi 4.90 86 R1 = H; R2 = OH pKi 5.06

O

H3C H N O 90 pKi 6.76

N H

O

91 pKi 7.12

Figure 17. β-homo analogue 83, and conformational constraint strategy for Tyr2 and Phe6 with the pKi (IRAP) values of the most potent stereoisomers.69


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Constraining the His residue and the His-Pro fragment proved to be a more fruitful strategy (Fig. 18). His was exchanged by the constrained amino acids Spi (92) and Tic (93) or the HisPro fragment was replaced by Ata-Gly (94),71 Aba-Gly (95) or Aia-Gly (96).72 Compounds 92 and 93, which induce the gauche (+) or gauche (‒) conformation, showed a significant decrease in inhibitory potency for both IRAP and AP-N. Compounds 94-9671-72 on the other hand adopt the gauche (+) or trans orientation. Both 94 and 95 showed inhibitory activities similar to Ang IV, whereas 96 resulted in a poorer inhibition. Together, these findings suggest that the trans conformation at the position of the His residue is preferred, whereas the gauche (+) conformation is not. In addition, all analogues 94-96 showed good IRAP selectivity.72 It had previously been shown that substitution of the N-terminal valine residue with the backbone extended β2hVal amino acid resulted in improved selectivity for IRAP over AP-N and stability against enzymatic degradation (cf. 83, Fig. 17).66 Thus, in an attempt to optimize the overall properties of analogues 95 and 96 this modification was introduced to provide 97 and 98. Both compounds showed good binding and stability in cell preparations, as well as high IRAP versus AP-N selectivity. However, unlike 98, analogue 97 was found to also bind to the AT1-receptor (pKi = 6.85).72



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Figure 18. Constraining the His or His-Pro segment of Ang IV. [double column figure] Moreover, it had been shown that replacement of the Pro residue for a Gly residue in Ang IV results in a 3-fold loss in affinity.73 Thus, to further improve the properties of analogue 98, Tourwé and co-workers substituted the Gly fragment in 98 with various α-side chains (e.g. 99).74 Introducing L-norvaline (99) improved selectivity and resulted in 40-fold higher affinity for IRAP compared to Ang IV.74 Analogue 99 was shown to be stable in human plasma and 26 ACS Paragon Plus Environment

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did not bind to the AT1-receptor. The radiolabeled analogue [3H]-9975 was synthesized to facilitate in vitro and in vivo mechanistic studies. Utilizing [3H]-99 Nikolaou and co-workers showed that pre-treatment of intact CHO-K1 cells with 99 completely abolished the availability of IRAP at the cell surface in vitro.74 Analogue 99 and a side chain disulfide marcrocyclized peptidomimetic 10076 are the best peptidomimetic IRAP inhibitors reported to date. The latter two compounds nicely illustrate the complementarity of locally and globally constrained peptide analogues.



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Somatostatin Somatostatin also known as somatotropin release–inhibiting factor (SRIF) 101 is a cyclic tetradecapeptide (Fig. 19) which was originally isolated from the mammalian hypothalamus as a potent inhibitor of growth hormone secretion.77 It is widely distributed throughout the endocrine and central nervous systems and peripheral tissues, and its effects are mediated by five G protein-coupled receptors, called sst1-5.78 SRIF has multiple functions, such as the modulation of growth hormone, insulin, glucagon, and gastric acid secretion.79 Systematic truncation studies on a large number of SRIF analogues, as well as an alanine scan,80 identified the Phe-Trp-Lys-Thr tetrapeptide unit as the essential pharmacophore for biological recognition.81 The size of the original peptide could be reduced to several cyclic hexa- and octapeptide analogues containing a turn-stabilizing and potency-enhancing D-Trp residue. A prominent SRIF analogue is 102 (Sandostatin®),79a which is an approved drug for the treatment of acromegaly, diarrhea due to VIPomas and carcinoid syndrome. Extensive structure-activity and conformational studies by NMR81a,82 and X-ray diffraction83 indicated the presence of a β-II’ turn conformation with D-Trp and Lys as the central i+1 and i+2 residues of the turn domain. In addition, close proximity of the D-Trp side chain to that of Lys is an important feature for high potency, as indicated by a high field shift of the Lys CγH2 methylene by NMR.82,84 Potent and subtype selective peptide mimetics have been developed by Merck,85 including the sst2 selective ligand 103 (L-054,522), which contains the (2R,3S)-βMeTrp residue.86 Unlike the (2R,3S)-isomer the (2R,3R)-diastereoisomer was much less potent.86 This difference in potency correlated with the requirement for a trans conformation of χ1 of the Trp side chain, which is favored by the (3S)-methyl substituent.7,86-87 Unlike most of the literature on SRIF inhibitors that focus on the β-turn backbone conformation of somatostatin, Tourwé and co-workers investigated the importance of the side 28 ACS Paragon Plus Environment

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chain orientation of the

D-Trp-Lys

residues.88-89 The design of these somatostatin

peptidomimetics was based on the constrained Trp residues D- and L-Aia (104-108, Fig. 19). Initially, D-Aia was introduced in the linear tetrapeptide 104 that contains the four essential side chains for sst receptor binding to provide compound 105. In stark contrast to the parent tetrapeptide 104 compound 105 displayed nanomolar affinity at sst4.90 Replacing D-Aia with L-Aia

(106) resulted in a complete loss of binding affinity, which reflects the importance of

the Aia

D-stereochemistry

for somatostatin receptor binding. The

D-Tcc

containing

tetrapeptide 107 did not show any biological activity, indicating that the preferred conformation of the D-Trp residue is trans and not gauche (‒).90 In another study α-methylbenzazepinones had been demonstrated to adopt a turn conformation in solution phase and in the solid state,58,91 thus racemic α-Me-Aia was introduced, to give tetrapeptide 108.90 Unfortunately, both epimers did not exhibit any significant sst receptor affinity. The authors suggested that this may be due to an unfavorable steric clash between the α-methyl group and sst1-5 upon binding. Based on the results outlined above, the D-Aia-Lys dipeptide core was used to prepare a series of somatostatin dipeptide mimetics.88-89 Interestingly, different receptor subtype selectivity profiles were observed depending on whether the N- and C-termini were capped with various moieties (109-113). It was proposed that the introduction of a substituent at the N-terminus targets the receptor pocket occupied by Phe of the native ligand. Peptidomimetic 109 displayed 3.3 nM affinity for hsst4 and 1.1 nM affinity for rsst5.88 To further explore this scaffold Feytens et al. prepared a series of peptidomimetics 110-113 using various N- and Csubstituents. Dipeptide 113 represents one of the most potent and selective (>10-fold) agonist of the rsst5 receptor to date. Analogue 110 on the other hand can be considered a broad spectrum sst ligand that binds to all subtypes in the nanomolar to subnanomolar range.89 The authors thus concluded that the D-Aia-Lys dipeptide represents a universally recognized sst1-5 29 ACS Paragon Plus Environment


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pharmacophore. Despite the fact that the Aia scaffold prefers extended conformations rather than turn conformations,58 it displays the ability to adopt trans or gauche side chain orientations. This in turn leads to placement of the Trp and Lys side chains in close proximity, which is necessary for receptor recognition and binding. Recently, Lesma and co-workers reported two somatostatin analogues that incorporate a spirocyclic lactam moiety (114 and 115, Fig. 20).92 Computational, spectroscopic and crystallographic studies supported that these peptidomimetics would adopt a stable β-II’ turn conformation. Unfortunately, similar to the

D-Tcc

containing tetrapeptide 107 and the α-

disubstituted analogue 108, 114 and 115 did not have any significant affinity for the sst receptors.92 It is interesting to note that in the urotensin II-related peptide (URP) Ala-c[Cys-Phe-TrpLys-Tyr-Cys]-Val, which has a pharmacophore very similar to the one found in somatostatin, the replacement of Trp by Aia resulted in a drop in potency.93 In contrast, replacement of Trp with Tcc produced a potent agonist. This indicates that despite the fact that SRIF and URP have very similar pharmacophores subtle changes in the orientation of their respective Trp side chains determines receptor recognition.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 19. SRIF derived inhibitors of the somatostatin receptors (sst1-5).86,88-89 [double column figure]

Figure 20. Somatostatin mimetics 114 and 115, containing a tetrahydro-β-carboline-based spirocyclic lactam.92


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Neurokinin-1 Substance P (SP, Fig. 21), neurokinin A (NK-A) and neurokinin B (NK-B) are members of the tachykinin family. They are endogenous neuropeptides that each bind with a different preference to the G protein-coupled receptors NK-1 (SP), NK-2 (NK-A) and NK-3 (NK-B).94-95 The NK-1 receptor is widely expressed in the CNS and peripheral tissues and its preferred endogenous ligand, SP, is involved in pain transmission, noxious stimuli, and inflammation.94-95 This renders NK-1 antagonists potential therapeutics for pathological states such as postoperative pain,96 arthritis,97 asthma95 and emesis98. To investigate the size of the binding pocket in the NK-1 receptor that accommodate the two centrally placed Phe residues in 116, as well as the optimal topology for these residues, Josien et al. performed a study where these amino acids were systematically replaced with constrained amino acids. (Fig. 21)99

Figure 21. Substance P 116 and the modified agonists 117-120.99 When Phe7 was replaced with Tic (117) the gauche (+) conformation induced by the Tic residue distorted the α-helical structure that the SP core normally adopts, and the binding affinity to all NK-



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receptors decreased substantially. A similar trend was noticed when Phe8 was replaced with Tic (118). This was confirmed in the guinea pig ileum (GPI) assay, a functional tissue bioassay representative of NK-1 receptor activity, where 117 and 118 were shown to be poor agonists of the NK-1 receptor. In addition, this study also included other side chain constrained Phe analogues, such as indanylglycine (119) and fluorenylglycine (120). Using both of these in place of Phe7 or Phe8 and testing all 4 SP derivatives the authors concluded that the binding pocket for Phe7 is relatively small and only able to accommodate one aromatic ring, since [(2S,3S)Ing7]SP (119), and not the (2S)-Flg7-derivative (not shown) was the most potent analogue.99 These observations also indicated that Phe7 most likely binds in gauche (‒) orientation. In contrast, the high activity of [(2S)Flg8]SP (120) compared to [(2S,3S)Ing8]SP (not shown) indicated that the binding pocket for Phe8 was large enough to fit two aromatic rings, one in the gauche (‒) conformation and one in the trans conformation.99 These conclusions were confirmed in CHO cells expressing the NK-1 receptor, where cAMP and inositol phosphate (IP) production were significantly lowered when stimulating with 117 and 118, whereas compounds with side chains in positions 7 and 8 in the gauche (‒) or trans conformation, respectively (as for 119 and 120), were able to significantly increase cAMP/IP production.100 These strategies, however, did not result in a substantially more potent agonist. Based on the NK-1 antagonist lead 121 (L-732,138, Fig. 22) which showed low nanomolar binding (IC50 = 1.6 nM) and inhibition (Kb = 25 nM),101 Millet et al. prepared the constrained esters 122-125

containing

either

a

tetrahydro-β-carboline

or

tetrahydrocarbazole

skeleton.102

Unfortunately, the constrained derivatives 122-125 all showed weaker NK-1 binding compared to 121. Compounds 123 and 125 were, however, 6- and 4-fold better NK-1 binders than 122 and 124, respectively, indicating that short peptide sequences may produce better NK-1 ligands than small molecule ligands. Superpositioning of 122 and 124 with 121 after energy minimization indicated


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that the rigidity in 122 and 124 due to the constraints was preventing the compounds from attaining the bioactive conformation. Based on the same lead, Ballet et al. also prepared the N-methyl amide Tcc-analogue 126, the Aia-constrained amide 127 as well as the Tic analogue 128.103 Neither 126 nor 127 showed any significant NK-1 antagonism. Remarkably, Tic analogue 128 was more potent than ester 122 and showed decent antagonism (pA2 = 7.5).103

Fig. 22. Constraining the Trp residue in the NK-1 antagonist 121.102-103 [double column figure] The latter result indicated that in these constrained peptidomimetics, an indole ring is not essential for NK-1 affinity, and can be replaced by a benzene ring, as had already been demonstrated in a variety of non-peptide NK-1 antagonists.104 Additionally, the benzodiazepinone antagonist 129104c served as an inspiration to design the 1-phenyl-substituted Aba analogue 130,



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which unfortunately turned out to be inactive (Fig. 23).103 However, removal of the 1-phenyl substituent led to a potent NK-1 antagonist 131 (hNK-1 Ki = 27 nM and pA2 = 8.4).

Fig. 23. Non-peptide NK-1 antagonist 129 as inspiration for more potent peptidomimetic NK-1 antagonists (131). However, 131 was still inferior compared to the parent peptide 116 or to 121. This could indicate that the χ1 and χ2 constraints imposed by the applied amino acid analogs result in a suboptimal relative orientation and distance of the two aromatic groups that are required for high receptor affinity. Thus, the flexible ligands (e.g. 116, 121) are better able to adapt their shape to recognize and bind to the NK1 receptor. Nevertheless, the discovery of the NK1 antagonist 131 was central to the later success in the development of compact opioid agonist/NK1 antagonist hybrids (vide infra, Opioids).

Melanocortin The endogenous melanocortin peptides include α-, β- and γ-melanocyte stimulating hormones (MSHs) and adrenocorticotropic hormone (ACTH). They play a role in a wide range of biological and physiological responses like obesity, anorexia, learning behavior, pain modulation, pigmentation,

sexual

function,

cardiovascular

function,

energy

homeostasis

and

thermoregulation.105 The MSHs act via interactions with the G protein-coupled melanocortin


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receptors (MCRs) in the PNS and the CNS, and activate the adenylate cyclase second messenger signal transduction cascade.105a-h To date five melanocortin receptor subtypes have been identified.106 Both endogenous antagonists such as Agouti protein and Agouti-related protein, as well as agonists, have been identified for the MCRs. The primary native ligand for MC1R, MC3R, MC4R and MC5R is the α-melanocyte stimulating hormone (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-PheArg-Trp-Gly-Lys-Pro-Val-NH2). However, none of the known endogenous ligands have high selectivity for any MCR subtypes. In order to establish structure-activity relationships and relate specific biological functions to the MCR subtypes, potent, biologically stable and selective agonists, antagonists and inverse agonists were needed.105h,107 The side chains of the central tetrapeptide His6-Phe7-Arg8-Trp9 represent the α-MSH pharmacophore.108 Early on, α-MSH was modified through the introduction of the methionine pseudoisosteric amino acid norleucine (Nle) to prevent oxidation.109 In addition, Phe7 was replaced with D-Phe to enhance stability against proteases. The resulting peptide [Nle4,D-Phe7]-α-MSH (132, MT-I) was shown to be more potent than α-MSH at MC1R, MC3R, MC4R and MC5R, and to possess a significantly higher in vivo stability and bio-distribution.109 Later, Al-Obeidi et al. introduced a cyclic lactam analogue MT-II (133, Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2), which proved to be a very potent, but non-selective, agonist at the human MC1R, MC3R, MC4R and MC5R.110 This peptide showed high stability against all proteolytic enzymes and tissue homogenates, and the ability to cross the blood-brain barrier (BBB). Both 132, 133 and their derivatives were used for further in vitro and in vivo pharmacological and biological studies.111 Hruby and co-workers used 133 to develop potent antagonists for the human MCRs, such as the potent antagonist of mammalian MC3R and MC4R, Ac-Nle4-c[Asp5-His6-D-Nal(2’)7-Arg8-Trp9-



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Lys10]-NH2 (134, SHU9119).111a Antagonist 134 has been explored further as a possible lead towards ligands with different pharmacological profiles. When His6 in 134 was replaced with the constrained amino acid Pro6 a full hMC5R selective agonist 135 (EC50 = 0.072 nM), and full antagonism at hMC3R and hMC4R was obtained (Fig. 24). Hence, the His6 position serves as a handle to modulate potency as well as selectivity at the melanocortin receptors.112

O

Trp

H2N

NH

NH O

Lys O

Nle O

135

HN

HN

HN

O

H N O

Arg NH2 HN NH

NH

O HN

Asp O

Pro N

D-Nal(2')

O

O N

O

H N H

HN

O O

136 Oic

137 Aic

N

138 Ioc

139 Tic EC50 hMC3R > 105 nM (0% act.) hMC4R > 105 nM (0% act.) hMC5R 2.05 nM (102% act.)

Figure 24. Analogues of 134/135 modified at position 6.113 In parallel, Grieco and co-workers designed several novel analogues of 134 by inserting various constrained amino acids at position 6 (136-139).113 Analogues 136-138 were inferior to 135 at all MCRs. However, 139 displayed a unique profile with respect to affinity and selectivity for hMC3R, hMC4R and hMC5R. Moreover, 139 was an hMC3R and hMC4R antagonist, and a full agonist at the hMC5R. Thus, a specific conformational restriction at position His6 can influence both binding affinity and selectivity for the MCRs as well as the mode of action (agonism vs. antagonism). The observed MCR selectivity profiles for 135-139 were rationalized by molecular modeling.113


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To understand the physiological role of hMC5R Bednarek and co-workers prepared analogues of [Pro8]-MT-II by introducing constrained amino acids.114 A D-Tic-analogue (Ac-Nle4-c[Asp5-His6-DTic7-Pro8-Trp9-Lys10]-NH2) was not able to interact efficiently with any of the hMCRs. The most promising ligand contained D-4,4’-biphenylalanine (D-4,4’-Bip) at position 7. At the hMC5R, this ligand (Ac-Nle4-c[Asp5-His6-D-4,4’Bip7-Pro8-Trp9-Lys10]-NH2) was a highly potent agonist (IC50 = 4.1 nM; EC50 = 2.5 nM), but only a micromolar agonist at hMC3R and hMC4R, and a partial agonist at hMC1R. Next, His6 and Pro8 were replaced by conformationally constrained amino acids such as Oic and Tic.114 The highest hMC5R receptor subtype selectivity (>5000-fold) was obtained for peptide 140 (Fig. 25), with poor binding affinity to and lack of activation of hMC1R, hMC3R and hMC4R.114 To achieve MCR subtype selectivity the His6-D-Phe7 residues in 133 have also been replaced with the Aba-Xxx motif (141-144).115 Molecular modeling indicated a good backbone overlap of all azepinone-containing analogues with the putative conformation of 133.115 Gratifyingly, the cyclic lactam analogue 141 proved to be a selective hMC3R antagonist (IC50 = 50 nM), whereas the linear analogue 144 was inactive, indicating that Aba containing hMCR ligands require a global conformational constraint to be effective.115



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Figure 25. Constrained cyclic (140-143) and linear (144) analogues of 133.114-115 Haskell-Luevano and co-workers reported the use of a combinatorial tetrapeptide library to discover potent ligands at hMC4R.116 Serving as Phe surrogates, Tic or D-Tic were incorporated in selected positions of the tetrapeptide template Ac-His-D-Phe-Arg-Trp-NH2. The study identified four tetrapeptide ligands that were able to restore similar full nM agonist potency at hMC4R (EC50’s = 0.21-0.45 nM, Fig. 26 box).116 Inspired by this study Van der Poorten et al. replaced the


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His residue in the minimal α-MSH pharmacophore (Ac-His6-D-Phe7-Arg8-Trp9-NH2) with Aba, Aia, and Ata azepinone dipeptidomimetics (145-149).117

Ac -His6 -D-Phe7 -Arg8-Trp9 -NH2

Ac -Xxx6 -D-Phe7 -Tic 8- D-Phe9 -NH2

R1

R2

NH O

H N

N HH N

O

O

H N

NH2

N H

O

H 2N

N H

O

O

Xxx = Arg R 1 = Cl R 2 = NO2 EC50 hMC4R 0.21 nM

N N N H

N H

N

Xxx = His R 1 = Cl R 2 = NO2 EC50 hMC4R 0.40 nM

NH2

NH

NH O

O O N H

HN

N

N

Ac-Xxx

O

N

N H

Cl

N

O

O

N

H N

O

O

O

O

N H NH N

R 145 R = H 148 IC 50 hMC5R 37 nM EC 50 hMC3R 52 nM hMC4R 0.3 nM

Xxx = His R1 = I R 2 = NO2 EC50 hMC4R 0.24 nM

149 No subty pe selectivity

H N O

I

O N H

O EC50 hMC4R 0.45 nM

HN H 2N

NH 2

NH

146 R = F EC 50 hMC4R 13 nM IC 50 hMC4R 6.5 nM 147 R = Br EC 50 hMC4R 0.3 nM 80% activity

Figure 26. Box: Tetrapeptide agonists at the hMC4R discovered by a combinatorial approach.116 Locally constrained melanocortin tetrapeptides 145-149, containing the Aia (145-147), Aba (148) and Ata (149) azepinone scaffolds.117 The presence of an Aia-D-Phe dipeptidomimetic in 145 resulted in weak micromolar antagonist affinity for the hMC1R, allosteric partial agonism at hMC3R (EC50 = 52 nM) and hMC4R (EC50 = 0.3 nM), and moderate antagonist activity at the hMC5R. Fluorination of the para-position in DPhe7 in 146 led to enhanced activity, which was most pronounced at hMC4R (IC50 = 6.5 nM; EC50



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= 13 nM, full agonist). In contrast to 146, but similar to 145, brominated tetrapeptide 147 gave potent allosteric partial agonist activity at hMC4R (EC50 = 0.3 nM and 80% activity). These affinities reflect the importance of the presence and type of halogen substituent at the level of the DPhe residue. The Aba-D-Phe analogue 148 proved to be a selective antagonist for hMC5R (IC50 = 37 nM), rendering it an ideal template for investigation of hMC5R antagonism. Contrary to the Aia and Aba analogues, the Ata-D-Phe analogue 149 showed no specific subtype receptor selectivity, which might be related to a closer resemblance to the histidine residue. Hence, the insertion of constrained aminobenzo- and indoloazepinone-based residues into the core of melanocortin tetrapeptides resulted in compact and selective human melanocortin receptor ligands with diverse pharmacological profiles.117

Opioid peptides In search of non-addictive analgesics with a prolonged duration of action and reduced side effects common to opioid therapies, a plethora of structural analogues of endo- and exogenous opioid peptides have been explored. Many peptidomimetic techniques have been applied in this field,

including,

peptoids,118

retro-inverso

analogues,119

amide

bond

isosteres,120

and

macrocyclizations.121 All opioid peptides have a common N-terminal message part and a variable C-terminal address segment, which is responsible for receptor subtype selectivity (Fig. 27). Receptor selectivity is dependent on the conformational space available to the peptide and the relative orientation of the Tyr and Phe side chains is a crucial feature of the pharmacophore.122 R. Geiger at Hoechst was the first to use Tic in the enkephalin sequence to probe the Phe4 orientation.123


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Figure 27. Structures of exogenous peptides dermorphin (MOR selective), deltorphin II (DOR selective), and endogenous sequences endomorphin II

(MOR selective) and [Met or

Leu]enkephalin (DOR selective). The exogenous opioid peptides dermorphin 150 and deltorphin II 151 were isolated from the skin of the South American frog Phyllomedusa sauvagei.124 Whereas 150 has a high potency and selectivity for the µ-opioid receptor (MOR), 151 binds selectively to the δ-opioid receptor (DOR). To explore the χ-space in 150 the Phe-Gly segment was substituted by Aba-Gly.122,125 The resulting peptide [Aba3]dermorphin has the Phe side chain constrained in the trans conformation. [Aba3]dermorphin displayed increased affinity and activity at the DOR, with no significant change of behavior at the MOR. By C-terminal truncation of the native sequence 150, the N-terminal tetrapeptide was found to be the minimal segment for opiate-like activity in vivo.126 The tetrapeptide H-Tyr-D-Ala-Phe-Gly-NH2 had an IC50 value of 36.7 nM at MOR and 1247 nM at DOR.127 Introduction of the azepinoneconstrained amino acids into this tetrapeptide at the first position (i.e., Tyr exchanged for Hba) and/or the third position (Phe exchanged for Aba) did not dramatically change MOR affinity. In



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contrast, DOR affinity increased 50 to 100-fold.128 All of the analogues showed agonist activity in functional assays on the guinea pig ileum (GPI, a representative test for MOR agonist activity) and mouse vas deferens (MVD, a measure of DOR agonist activity). Among the investigated compounds 154 (Fig. 28) was the most potent MOR agonist and hence this analogue was selected for in vivo analgesia testing using the rat tail-flick test.128 On intrathecal administration, peptide mimetic 154 was 40-50 times more potent than morphine and possessed a longer duration of action.128 To examine the influence of ring constraints in peptide mimetic 154 on BBB penetration after iv administration, the opioid receptor-induced antinociceptive effect, a CNS effect, was compared to that of the ‘open ring’ di-N-methyl peptide 155. Both 154 and 155 gave a high antinociceptive effect in the tail-flick test, indicating similar BBB permeation, albeit constrained peptide 154 reached a maximum response after 15 min, twice as fast as the more flexible analogue 155.129


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OH

OH

O N H 2N

O N H

O

N

O

N H2N

NH 2

O

154 K i (MOR) 20.8 nM Ki (DOR) 160 nM GPI 3.6 nM MVD 30 nM

H 2N

O N H

N

N H2N

NH 2

O

N H

O

N

O NH 2

O

157 K i (MOR) 14.8 nM Ki (DOR) 5.0 nM GPI 0.002 nM MVD 0.016 nM

OH

O

NH 2

O

O

O

156 K i (MOR) 0.047 nM Ki (DOR) 2.4 nM GPI 0.08 nM MVD 0.17 nM

H 2N

O

OH

O

H N

O

N

155 K i (MOR) 28.1 nM Ki (DOR) 7 nM GPI 22 nM MVD 79 nM

OH

H N

N H

OH

O N H

N O

H N

O H2N

NH 2

O

O

N H

N O

O NH 2

159 K i (MOR) 0.3 nM Ki (DOR) 63 nM

158 K i (MOR) 4.6 nM Ki (DOR) 859 nM

Figure 28. Constrained and/or substituted analogues of the opioid tetrapeptide H-Tyr-D-Ala-PheGly-NH2. Because replacement of Tyr with the unnatural amino acid 2’,6’-dimethyltyrosine (Dmt) resulted in increased potency in many opioid peptides,130 the Hba moiety in 154 was substituted with Dmt to give 156, which had sub-nanomolar affinity for MOR and low nanomolar affinity for DOR.131 The NMe-D-Ala analogue 157 proved to be a potent agonist at both the MOR and the DOR.132 Compared to the lead [Aba-Gly]-peptides, the turn inducing α-methyl- (e.g. 158-159) and spirocyclic Aba-Gly analogues (not shown, cf. 75) exhibited lower affinity for MOR and DOR.58,91a Diastereoisomer 159 was more potent than the counterpart 158, which was rationalized by molecular docking that showed 159 is able to adopt a more favorable conformation for binding.131



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The dermorphin derivative [Dmt1]-DALDA (H-Dmt-D-Arg-Phe-Lys-NH2)133 has potent agonist activity, high metabolic stability and MOR affinity (Ki = 0.143 nM).134 Inspired by [Dmt1]DALDA, the Phe residue was replaced by Aba to give 160 (Fig. 29).135 However, this modification reduced MOR binding 4-fold and increased DOR affinity. In contrast the Aba-Gly analogue 161 exhibited outstanding MOR potency, and was shown to cross the BBB after iv or subcutaneous administration.136 Moreover, changing the C-terminal Gly in 161 to a βAla residue (H-Dmt-D-ArgAba-βAla-NH2) produced an even more in vivo potent peptide.137 The latter peptide can be regarded as a constrained TAPA analogue (H-Tyr-D-Arg-Phe-βAla-NH2), a peptide reported to induce less physical dependence than morphine.138 H -Dmt-D-Arg-Aba-Xxx-NH 2

160 R = (CH2)3NH2 K i (MOR) 0.61 nM HO K i (DOR) 38.6 nM GPI IC50 76 nM MVD IC50 64 nM (IC25) 161 R = H K i (MOR) 0.15 nM K i (DOR) 0.6 nM GPI IC50 0.32 nM MVD IC50 0.42 nM

O

H N

H2N

Ac-Aba-Gly-NMe-3',5'(CF 3)2-Bn

O N H

O

N

N H NH

O

O

+

NH2

R

N

N H

O

O

162 K i (NK1) 27 nM pA2 8.4

N

CF3

NH2 F3C H -Dmt-D-Arg-Aba-Gly-NMe-3',5'(CF 3)2-Bn

HO O

H N

H2N O

N H

NH

H2N

O

N

N

O

CF3

N H

163 K i (MOR) 0.42 nM K i (DOR) 10.4 nM GPI IC50 8.5 nM MVD IC50 43 nM K i (NK1) 0.5 nM pA2 7.8

F3C

HO O

H N

H2N O H2N

O N H

NH

N O

N H


N

164 K i (MOR) 0.08 nM K i (DOR) 0.28 nM GPI IC50 1.86 nM MVD IC50 2.16 nM K i (NK1) 13 nM pA2 6.44

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Figure 29. Opioid agonist and NK-1 antagonist chimeras.103,136-137 [double column figure] Prolonged usage of opioids in pain treatment has been shown to increase the secretion of SP and upregulate the expression of tachykinin receptors, which counteracts the effect of opioid analgesics.139 To address this issue bifunctional ligands that simultaneously act as opioid agonists and NK-1 antagonists have been developed.140 There are several advantages to such chimeras, including a single bio-distribution, simple pharmacokinetic profiles and higher efficacy.121b,139b,141 Hence, the groups of Hruby and Lipkowski designed chimeras containing the enkephalin-based opioid pharmacophore (H-Tyr-D-Ala-Gly-Phe-Met-) at the N-terminus, fused with the C-terminal NK-1 pharmacophore (-Met-Pro-Leu-Trp-NH/O-3’,5’-(CF3)2-Bn) (not shown).139b,140b-e Although, these hybrid peptides maintained the desired low nanomolar dual opioid agonist – NK1R antagonist profile, such ligands remain large and contain a high peptide character. In order to make more compact dual ligands, the NK-1 antagonist 162 was fused with the potent MOR/DOR agonist 161 to give the opioid/NK-1-chimera 163.103 Chimera 163 has improved affinity and slightly reduced antagonistic activity at NK-1 (compared with the parent 162) but retains good agonism at MOR and DOR (compared with 161). An in vivo study in mice using the tail-flick assay, compared 163 (2 mg/kg or 4 mg/kg iv) with morphine (4 mg/kg iv). 163 was found to have an antinociceptive profile with a maximum effect after 2 hours.136 Unfortunately, chimera 163 did not show any effect on opioid-induced tolerance, which the NK-1 antagonism was expected to counteract. A complete loss of antinociception after five (once per day) administrations was seen. Importantly, the constraint of the Aba scaffold in hybrid 163 was demonstrated to be essential, since the linear analog H-Dmt-D-Arg-Phe-Sar-NMe-3’,5’-(CF3)2-Bn showed a considerable loss of NK-1 affinity (Ki = 734 nM), while maintaining affinity for MOR (Ki = 0.62 nM) and DOR (Ki = 1.7 nM).137 More recently, a SAR study was performed in order to improve the affinity and potency of



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163.137 In an attempt to avoid steric clash between the two pharmacophores and reduce the molecular weight Gly in 163 was exchanged for βAla and the trifluoromethyl substituents were removed to give analogue 164.137 Analogue 164 showed improved affinity at both MOR and DOR, as well as improved MOR and DOR activation. However, the extra methylene unit in βAla decreased NK-1 binding and significantly lowered antagonism. Despite a lowered affinity for and antagonism at NK-1, 164 proved to be more active than 163 in in vivo neuropathic pain models for allodynia and hyperalgesia. Moreover, 164 had a markedly improved toxicity profile, compared to 163, and no toxicity was visible at the highest investigated dose.137 This example elegantly illustrates the broad applicability of the azepinone scaffold at different receptor targets. Several small peptidomimetics for e.g. the opioid and somatostatin receptors have been prepared based on this scaffold88-89 as well as enzyme inhibitors.36,40-41 Endogenous opioid peptides such as endomorphin II (Fig. 27) have also been used as starting point in search of improved opioid ligands. It is a potent MOR selective endogenous opioid peptide.142 Substitution of the proline residue in 152 with Tic gave the two potent TIPP-peptides 165 and 166 (Fig. 30).143 In the design of MOR- and DOR-selective peptides, the Tyr-Tic and DmtTic pharmacophores have been extensively investigated, with Dmt-Tic-OH being the smallest fragment having potent opioid properties.144 Replacement of Tic-Gly dipeptide in DOR-selective HDmt-Tic-Gly-NHBn 167 by Aba-Gly gave analogues with reversed MOR/DOR selectivity (e.g. 168).145 Replacement of Tic in 167 for D- and L-Aia (169-170) gave moderate MOR and DOR affinity and a selective MOR agonist, respectively, with activity comparable to 152. In contrast, N,N-dimethylation of the Dmt residue in the

L-Aia

peptide 170 produced potent DOR

antagonism.145 Application of the turn-inducing spiro-Aba-Gly motif (vide supra, bradykinin analogue 75) in the design of endormorphin-2 mimetics (H-Tyr-(R)-spiro-Aba-Gly-Phe-NH2) gave


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high MOR selectivity and potency supporting the hypothesis that the parent peptide was recognized by the MOR in a folded conformation.146 165 TIPP H -Tyr-Tic-Phe-Phe-OH R=H 166 DIPP H-Dmt-Tic-Phe-Phe-OH R = Me

R

HO

Me

HO N

R

N NH

H2N

167 Ki (MOR) 0.16 nM K i (DOR) 0.031 nM GPI IC50 2.69 nM MV D pA2 9.3

O

NH

Me H2N

O O

O

O O

HN

HN OH O NH

O H2N

HO

N H

N O

O

R N H

168 Ki (MOR) 0.46 nM K i (DOR) 11 nM GPI IC50 51 nM MVD IC50 830 nM

R N

O N H

N O

HO

O

169 D-Aia R = H, R' = NHBn K i (MOR) 3.35 nM R' K i (DOR) 160 nM GPI IC 50 14.9 nM 170 L-Aia R = Me, R' = OH K i (MOR) 874 nM K i (DOR) 6.64 nM MVD pA2 8.3

Figure 30. Constrained low molecular weight opioid peptides that mimic TIPP-like ligands. The X-ray co-crystal structure of the DOR in complex with the Tic-containing peptidomimetic 166 was reported in 2015 by Cherezov and co-workers.147 The Dmt residue of peptidomimetic 166 is buried deeply into the core of the receptor, with the methyl substituents complementing the hydrophobic pocket at the bottom of the orthosteric binding site, thus illustrating the importance of this modification.147 The presence of Tic in endomorphin II analogue 166 resulted in a mixed MOR agonist/DOR antagonist profile, which suppresses the propensity for tolerance and physical dependence.148 In 166 the peptide bond between Dmt and Tic adopts a cis configuration with the Tic side chain in the gauche (+) conformation. This specific topology has been proposed to be central for defining DOR agonist versus DOR antagonist properties of opioid peptide ligands.147



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Figure 31. Compound 171 which was co-crystallized with the KOR by Stevens and co-workers.149 In 2012, Stevens and co-workers reported the co-crystal structure of 171 (Fig. 31) in complex with the KOR.149 Peptidomimetic 171 is a selective KOR antagonist with potent activity in models of depression, anxiety, stress-induced cocaine relapse, nicotine withdrawal etc.150 Structurally, the D-Tic

residue in 171 contributes significantly to the extensive hydrophobic contacts at the bottom of

the KOR binding cleft. While the Asp138 residue of the KOR anchors the ligand in a final V-shape by ionic interactions with the ligand’s two amino groups, the phenol is maintained in an optimal orientation for interaction with the receptor, induced by the adjacent D-Tic residue. Other constrained amino acids, such as the amino-tetralin carboxylic acid derivatives Atc and 6HO-Atc and amino-indane derivatives Aic and 5-HO-Aic, have also been integrated into opioid sequences (Fig. 32). Upon substitution of Tyr in sequence 151 (Fig. 27) with (R)- and (S)-6-HOAtc, similar Ki values to the native ligand, high DOR selectivity, and potent agonist responses were observed.151 From NMR analysis of these peptides, the preferred gauche (‒) and gauche (+) conformation for (S)- and (R)-6-HO-Atc, respectively, could be assigned unambiguously (Fig. 32). Similarly, when the Phe residue in the message domains of 151 was replaced by the (S)- or (R)-Atc residues, sub-nanomolar DOR agonists were obtained. These peptides were proposed to adopt the trans conformation at χ1,152 based on complementary studies with the Tic and Aba derivatives, in which gauche (+) / gauche (‒), or gauche (+) /trans conformations are being induced, respectively. Only the latter gauche (+) / trans constraint is well-tolerated for efficient binding and activation of the DOR.125,153


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Insertion of Aic in the [D-Ala2]deltorphin I sequence, resulted in an analogue with strong binding and activity at the DOR (Ki = 4.96 nM; MVD IC50 = 0.08 nM), while cyclic analogues incorporating Aic or Atc in the non-selective dermorphin analogue 172 resulted in selective MOR agonists, such as 173.153 The ‘open ring’ analogues of 173 bearing an α-methyl-Phe or o-methylPhe residue did not induce a similar shift in selectivity. This example again illustrates how constrained aromatic amino acids can be used in conjunction with macrocyclization to impose receptor subtype selectivity. X H3N

Atc X=H

Aic X=H

CO-NHHβ'

6-HO-Atc X = OH

N H

Hδ'

HO H

O

H-Tyr-c[Orn-Phe-Glu]-NH2

HO

X

Hβ'

H N

H2N O

172 K i (MOR) 0.98 nM K i (DOR) 3.21 nM GPI 1.17 nM MVD 1.11 nM

N H

δ'

H-Tyr-c[Orn-Aic-Glu]-NH2

HO

O O

N H O

173 K i (MOR) 4.21 nM K i (DOR) 209 nM O GPI 7.2 nM MVD 36.5 nM O

O

H N

H2N O

HN

O

5-HO-Aic X = OH

N H

O

O

NH2

N H

HN

NH2

N H

HN

O

HN

O

ο −Me-Phe

α −Me-Phe

Figure 32. Integration of conformationally restricted amino-indane and amino-tetralin residues as rigid templates into opioid sequences. The arrows in the Newman projection show the observed nOe effects that allowed the unambiguous assignment of the gauche (‒) conformation.151-153



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Conclusions and perspectives In the past many linear peptides have been rigidified to favor their bio-active conformation, mainly via macrocyclization of the backbone and/or the side chains. This continues to be a reliable and efficient strategy for optimizing the overall properties of potential therapeutic peptides. However, these macrocyclization strategies only provide the medicinal chemist with control of the Φ and Ψ dihedral angles of the peptide backbone and not so called χ-space. χ-space defines the orientation of the amino acid side chains and is of the essence for favorable biological recognition events. Introduction of cyclic constrained aromatic amino acids provides a valuable tool for controlling χ dihedral angles and thus presenting specific topologies. Herein we have focused on unnatural amino acids that are constrained via cyclization of the amino acid side chain to the peptide backbone. The presented studies clearly showcase the advantages of such constrained residues for improving potency, efficacy, selectivity, stability and absorption. For instance, in the opioid peptides, the Tic residue was essential to provide the selective DOR antagonist 165 (TIPP) and the Aba constraint in peptides 154-159 gave potent balanced MOR/DOR agonists which are able to cross the BBB. The Aia residue allowed the preparation of potent somatostatin mimetics 109-113 and stable and selective Ang IV analogs 98-99. The design of peptidomimetics with constrained amino acids as outlined is herein is generally an empirical method and requires the use of a variety of differently constrained amino acids to find the best orientation of a crucial side chain in a peptide for interaction with its biological target, but there is no real alternative to optimize flexible ligands. Although some structural studies to determine the (solution) conformation of the peptides containing the constrained amino acid analogs have been performed (e.g. for the opioids), the design of ligands has generally been based on the known conformational preferences of the different scaffolds (cf. Fig. 2). In some cases peptidomimetic


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design has been aided by molecular modeling (e.g. for the melanocortin, opioids, bradykinin, substance P). In conclusion, the use of constrained amino acids with aim to discover potent bioactive peptidomimetic ligands with greatly improved PK profiles comparted to their native peptide counterparts represents a powerful tool at the hands of peptide medicinal chemists. However, a variety of differently constrained amino acids are required to probe χ-space and optimize the peptidomimetics interaction with its biological target. Many constrained amino acids for peptide synthesis are commercially available, in particular analogs of Phe. However, many other amino acid building blocks showcased herein cannot be acquired from commercial sources and thus require custom synthesis. Gratifyingly, this is easily accomplished by standard synthetic organic methods utilizing optimized and robust synthetic routes reported in the literature (see Supporting Information for a comprehensive referenced list of constrained amino acids).

Acknowledgements OVdP, SB and DT are grateful to the Research Foundation Flanders (FWO-Vlaanderen) and to the Flanders Innovation & Entrepreneurship (VLAIO) for the financial support. DSP thanks the Lundbeck Foundation and the Carlsberg Foundation for financial support. AK was supported by a grant from the Department of Drug Design and Pharmacology, University of Copenhagen.



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Abbreviations Aba: 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one; ACE: angiotensin-converting enzyme; ACTH: adrenocorticotropic hormone; Aia: 4-amino-indolo[2,3-c]azepin-3-one; Aic: 2-aminoindane-2-carboxylic acid; Ang: angiotensin; AP-N: aminopeptidase N; Ata: 4-aminotriazolodiazepinone; Atc: 2-amino-tetralin-2-carboxylic acid; B1: bradykinin receptor subtype 1; B2: bradykinin receptor subtype 2; BBB: blood-brain barrier; Bip: biphenylalanine; BK: bradykinin; cAMP: cyclic adenosine monophosphate; Cha: 3-cyclohexylalanine; CHO: Chinese hamster ovary; CNS: central nervous system; DALDA: H-Tyr-D-Arg-Phe-Lys-NH2; D-BT: (S)-benzothiazepinone; DIPP: H-Dmt-Tic-Phe-Phe-NH2; Dmt: 2’,6’-dimethyltyrosine; DOCA-salt: deoxycorticosterone acetate-salt; DOR: δ-opioid receptor; EC50: concentration at which 50% of the maximal response is achieved; Flg: 9-fluorenylglycine; FT: farnesyl transferase; GnRH: gonadotropin-releasing hormone; GPCR: G protein-coupled receptor; GPI: guinea pig ileum; GTP: guanosine-5’triphosphate; Hba: 4-amino-1,3,4,5-tetrahydro-8-hydroxy-2-benzazepin-3-one; IC50: concentration needed to replace 50% of a receptor-bound ligand; Ing: 1-indanylglycine; Ioc: indoline-2-carboxylic acid; IP: inositol phosphate; IR: infrared spectroscopy; IRAP: insulin-regulated aminopeptidase; iv: intravenous; Ki: inhibition constant; KOR: κ-opioid receptor; MCR: melanocortin receptor; MOR: µ-opioid receptor; MSH: melanocyte stimulating hormone; MVD: mouse vas deferens; NEP: neutral endopeptidase; NK: neurokinin; Nle: norleucine; Oic: octahydroindole-2-carboxylic acid; pA2: negative logarithm of the molar concentration of antagonist that produces a dose ratio of 2; PNS: peripheral nervous system; RAS: renin-angiotensin system; SAR: structure-activity relationship; SP: substance P; Spi: 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid or spinacine; SRIF: somatotropin release-inhibiting factor; sst1-5: somatostatin receptor subtype 1-5; TAPA: H-Tyr-D-Arg-Phe-βAla-NH2; Tcc: 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; Thi:


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thienylalanine; Tic: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TIPP: H-Tyr-Tic-Phe-PheNH2; URP: urotensin II-related peptide

Associated Content Supporting Information A complete list of all constrained amino acids discussed in this review, with their names, abbreviation(s), a reference for the synthesis and application is given in Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author Dirk Tourwé: phone +32 2 6293295; fax +32 2 6293304; e-mail [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Biographies Olivier Van der Poorten was born in Belgium, in 1989. He received his B.Sc. (2011) and his M.Sc. (2013) in Chemistry from the Vrije Universiteit Brussel, Belgium. Since 2013 OVDP is a Ph.D. student in the Organic Chemistry Research Group at the Faculty of Science and Bioengineering Sciences, Vrije Universiteit Brussel, under supervision of Prof. Dr. Steven Ballet with



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work focusing on conformationally constrained amino acids for peptide turn mimicry and drug transport. Astrid Knuhtsen obtained her B.Sc. (2009) and her M.Sc. in Medicinal Chemistry (2012) both from Aarhus University, Denmark. Her Ph.D. was performed as a collaboration between University of Copenhagen, Denmark, and the University of British Columbia, Canada, under the supervision of Assoc. Professor Jesper L. Kristensen and Assoc. Professor Daniel Sejer Pedersen. She received her Ph.D. degree in 2016 and is currently a Postdoctoral Fellow in the Chemical Biology & Medicinal Chemistry department at University of Glasgow, UK. Daniel Sejer Pedersen, received his M.Sc. degree under the supervision of Assoc. Prof. Otto Dahl in 1999 (University of Copenhagen), and the Ph.D. degree under the supervision of Prof. Stuart Warren in 2009 (University of Cambridge). DSP worked for a number of years in the biotech industry and as a Postdoctoral research fellow both in Australia and Denmark. In 2010 DSP was appointed Associate Professor in medicinal chemistry at the Dept. of Drug Design and Pharmacology, University of Copenhagen where he runs an independent research group. Steven Ballet was born in Belgium, in 1979. He received his Ph.D. in Chemistry from the Vrije Universiteit Brussel, Belgium in 2007, after which he performed a postdoctoral stay at the University of Adelaide (Australia) and the Clinical Research Institute of Montreal (Canada). In 2010 he returned to the Vrije Universiteit Brussel as an associate professor in Bioorganic Chemistry. His current research interests are medicinal, peptide and peptidomimetic chemistry. Dirk Tourwé obtained the Ph.D. degree at the Vrije Universiteit Brussel in 1974 under the direction of Prof. Georges Van Binst on the topic “Synthesis and conformational analysis of benzoand indoloquinolizidines”. He obtained the habilitation in 1979 and then switched his research


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interests to the development of peptide mimetics and of constrained amino acids. He became the director of the group on Organic Synthesis in 1995. Since 2012 he is professor emeritus.



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