synthesis of ²-turn and pyridine based peptidomimetics - diva portal
TRANSCRIPT
Synthesis of β-Turn and Pyridine Based
Peptidomimetics
David Blomberg
Department of Chemistry, Organic Chemistry Umeå University
Umeå 2007
Department of Chemistry, Organic Chemistry Umeå University SE-901 87 Umeå
Copyright 2007 by David Blomberg ISBN 978-91-7264-305-5 Printed in Sweden by Intellecta DocuSys, Västra Frölunda, 2007
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Contents
1. List of Papers .................................................................................................. 3
2. List of Abbreviations ..................................................................................... 5
3. Introduction .................................................................................................... 7 3.1 Structure and function of peptides and proteins .................................... 7 3.2 Peptidomimetics ...................................................................................... 9
3.2.1 Development of a peptidomimetic drug - Exanta ....................... 10
4. Objectives of the thesis ................................................................................ 12
5. Peptidomimetics of Leu-enkephalin ........................................................... 13 5.1 Biological action and conformation of Leu-enkephalin ..................... 13 5.2 Design of Leu-enkephalin peptidomimetics........................................ 14 5.3 Synthesis of peptidomimetics incorporated in Leu-enkephalin ......... 15
5.3.1 Synthesis of a ten membered β-turn mimetic .............................. 15 5.3.2 Conformational studies of the ten membered β-turn mimetic using NMR spectroscopy ....................................................................... 20 5.3.3 Synthesis of a seven membered β-turn mimetic on solid phase. 21 5.3.4 Synthesis of linear Leu-enkephalin analogues ............................ 22
5.4 Biological evaluation ............................................................................ 24 5.4.1 Opioid receptor binding assay ...................................................... 24 5.4.2 Binding to µ- and δ- opioid receptors .......................................... 25
5.5 Summary ................................................................................................ 27
6. β-Strand peptidomimetics............................................................................ 29 6.1 β-Strands ................................................................................................ 29 6.2 Design and retrosynthetic analysis of a β-strand mimetic.................. 30 6.3 Attachment of an N-terminal leucine analogue at position 4 of the pyridine ring................................................................................................. 32 6.4 Attachment of a C-terminal glycine analogue at position 2 of the pyridine ring................................................................................................. 34
6.4.1 Nucleophilic aromatic substitution............................................... 34 6.4.2 A reductive amination strategy..................................................... 40 6.4.3 Changing the substitution order and starting with the SNAr reaction .................................................................................................... 41
6.5 Completing the synthesis − A successful Boc strategy ...................... 42
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6.6 Incorporation of a second chiral amino acid analogue and attempts to elongate the β-strand mimetic..................................................................... 45
6.6.1 Introducing a chiral amino acid analogue instead of glycine as C-terminus ................................................................................................... 45 6.6.2 Attempts to elongate the β-strand mimetic.................................. 46 6.6.3 Conclusions.................................................................................... 49
6.7 Summary ................................................................................................ 49
7. Thrombin inhibitors ..................................................................................... 51 7.1 Biological action of thrombin............................................................... 51 7.2 Structure based design .......................................................................... 53 7.3 Retrosynthetic analysis of the thrombin inhibitors ............................. 53 7.4 Synthesis of thrombin inhibitors .......................................................... 54
7.4.1 Attempts to obtain thrombin inhibitors via a Grignard exchange reaction followed by an SNAr reaction using substituted benzylamines.................................................................................................................. 54 7.4.2 A reductive amination approach................................................... 57 7.4.3 Conversion of the cyano group to the desired benzamidines ..... 58
7.5 Biological evaluation ............................................................................ 61 7.6 Crystal structure .................................................................................... 61 7.7 Summary ................................................................................................ 62
8. Thrombin inhibitors with reduced basicity................................................. 64 8.1 Introduction............................................................................................ 64 8.2 Structure based design and retrosynthetic analysis ............................. 64 8.3 Synthesis of thrombin inhibitors .......................................................... 65
8.3.1 Synthesis of Boc-protected alaninal and glycinal ....................... 65 8.3.2 A Grignard reaction and nucleophilic aromatic substitution with cyclic amines........................................................................................... 66 8.3.3 Completing the synthesis .............................................................. 67
8.4 Biological evaluation ............................................................................ 68 8.5 Summary ................................................................................................ 68
9. Concluding remarks ..................................................................................... 70
10. Acknowledgement ..................................................................................... 73
11. References .................................................................................................. 75
Appendix........................................................................................................... 85 Experimental section for chapter 8. ....................................................... 85
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1. List of Papers
I David Blomberg, Mattias Hedenström, Paul Kreye, Ingmar
Sethson, Kay Brickmann and Jan Kihlberg; Synthesis and conformational studies of a β-turn mimetic incorporated in Leu-enkephalin. J. Org. Chem., 2004, 69, 3500-3508.
II David Blomberg, Paul Kreye, Kay Brickmann, Chris Fowler
and Jan Kihlberg; Synthesis and biological evaluation of leucine enkephalin turn mimetics. Org. Biomol. Chem., 2006, 4, 416-423.
III David Blomberg, Kay Brickmann and Jan Kihlberg; Synthesis
of a β-strand mimetic based on a pyridine scaffold. Tetrahedron, 2006, 62, 10937-10944.
IV David Blomberg, Tomas Fex, Yafeng Xue, Kay Brickmann
and Jan Kihlberg; Design, synthesis and biological evaluation of thrombin inhibititors based on a pyridine scaffold. Submitted.
V David Blomberg, Tomas Fex, Kay Brickmann and Jan
Kihlberg; Design, synthesis and biological evaluation of thrombin inhibitors lacking a strong basic functionality in P1. Manuscript.
Reprinted with kind permission from the publishers.
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2. List of Abbreviations
Bn benzyl Boc tert-butoxycarbonyl BSA bovine serum albumin Cbz benzyloxycarbonyl DAMGO [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC dicyclohexyl carbodiimide DCE 1,2-dichloroethane DIBAL diisobutylaluminium hydride DIC N,N’-diisopropyl carbodiimid DIPEA diisopropylethylamine DMAP N,N-dimethylaminopyridine DPDPE [3H] [D-Pen2, D-Pen5]enkephalin DTI direct thrombin inhibitor EWG electron withdrawing group Fmoc 9-fluorenylmethyloxycarbonyl GPCR G protein-coupled receptor HATU O-(7-azabenzotriazole-1-yl)-N, N,N’N’-
tetramethyluronium hexafluorophosphate HMDS hexamethyldisilazane HOAt 1-hydroxy-7-azabenzotriazole HOBt N-hydroxybenzotriazole K-selectride potassium tri-sec-butylborohydride LCMS liquid chromatography mass spectrometry LHRH luthenizing hormone releasing hormone N,O-DMHA N,O-dimethylhydroxylamine NMO N-methyl morpholine N-oxide NMR nuclear magnetic resonance NOE nuclear overhauser enhancement NOESY nuclear overhauser enhancement spectroscopy PAM 4-(Hydroxymethyl)phenylacetamidomethyl PMB p-methoxybenzyl chloride Q tetrabutylammonium rt room temperature
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TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethyl silyl TEA triethylamine TMP 2,4,6-trimethylpyridine TFA trifluoro acetic acid TFAA trifluoro acetic anhydride TFE 2,2,2-trifluoroethanol TMS trimethylsilyl Tris tris hydroxymethylaminoethane UHP urea hydrogen peroxide 3-D three dimensional
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3. Introduction
3.1 Structure and function of peptides and proteins On a molecular level proteins are built up by small residues, amino acids, that are connected via amide bonds to form chains (Figure 3.1). Shorter amino acid sequences, usually containing 2−50 amino acid residues, are defined as peptides, while longer chains are defined as proteins. There are 20 naturally occurring amino acids with a variety of characteristics in the side chains (R1−R4) to provide a wide scope of peptides and proteins with enormous variation in properties and function. Biologically active peptides often act as hormones or transmittor substances and regulate several vital physiological functions, such as secretion of growth hormone (somatostatin), intake of food (neurotensin), sexual function (melanocortin-4) and blood pressure (angiotensin, vasopressin).1-3 Enzymes, transporters over membranes and receptors are examples of important physiological functions filled by proteins.
NH
HN
NH
HN
R1
O R2
O R3
O R4
Amino acid
residue
Amide
bond
Figure 3.1 A short amino acid sequence with an amino acid residue and an amide bond highlighted. R1−R4 represent amino acid side chains.
The amino acid sequence is referred to as the primary structure of peptides and proteins. Peptides and proteins are usually arranged in more stable secondary structures such as α-helices, β-turns and β-strands.4
8
Figure 3.2 A schematic picture of a GPCR with α-helices as transmembrane elements.
The α-helix is a common structural elements in proteins, for example in the transmembrane regions of GPCR’s (Figure 3.2). The α-helix is stabilized by intramolecular hydrogen bonds between amino acid residues i+4 and i, to form a right handed helical structure. Proline and glycine are rare in α-helices due to the lack of ability to donate a hydrogen bond and high flexibility, respectively. β-Turns occur frequently in proteins and are sequences where the peptide
backbone reverses its overall direction over four amino acids, i to i+3 (Figure 3.3). There are several different types of β-turns defined by the dihedral angels, φ and Ψ, of amino acids i+1 and i+2. A β-turn is generally stabilized by an intramolecular hydrogen bond to form a pseudo ten membered ring and is believed to be of great importance for the recognition and activity of peptides and proteins.5-8
N
HNO
H
HN
O
O
!1
1
"
Figure 3.3 A β-turn with the dihedral angels φ and Ψ shown for residue i+1. The structure only contains Ala for clarity.
A β-strand is an amino acid sequence with the peptide backbone in an extended conformation. It is rarely found as a monomer, but more often as dimers or in tertiary structures called β-sheets, stabilized by hydrogen bonds and hydrophobic interactions (Figure 3.4). The correct overall orientation of the secondary structure elements, i.e. the tertiary structure, of a peptide or a protein is crucial both to get stability and proper biological functions.9,10
9
HN
NH
HN
NH
O
O
O
O
HN
OC
HN
NH
O
O
NH
OHN
O
NH
OCO
HN
HN
HN
O
NH
HN
O
O
Figure 3.4 A β-strand (only containing Ala for clarity) assembled to form a β-sheet. The structure is stabilized by intramolecular hydrogen bonds and hydrophobic interactions.
Despite the versatile and interesting function of peptides in biological systems, metabolic instability and poor bioavailability make them ineffective as orally administered drugs. A few peptides are, however, used as drugs today, such as insulin to control blood sugar levels and oxytocin to induce uterus contractions. Due to the problems with oral administration, insulin and oxytocin have to be given subcutaneously or intravenously. To avoid the problems associated with oral administration, peptides can serve as templates to provide a pharmacophore model in the development of compounds exerting the same action as the native peptide, but with improved pharmacokinetic properties.
3.2 Peptidomimetics The unfavorable pharmacokinetic properties associated with peptides when used as orally administered drugs can, in principle, be avoided by development of peptidomimetics. The general strategy when preparing peptidomimetics is to replace segments related to undesired properties with non-peptidic structures, while attempting to maintain the ability to elicit the same or improved biological response as the native peptide.11,12
Peptidomimetics can be divided into three different classes.3 The first class is characterized by backbone changes, such as incorporation of amide bond isosteres and turn mimetics. The vast literature concerning peptidomimetics of class I including stabilized turn mimetics represented as bicycles,13-19 aromatics20,21 and cyclic compounds.22 The second class of peptidomimetics are referred to as ligands exerting the same biological response as the native peptide ligand without any obvious structural resemblance (functional mimetics). The third class is represented by peptidomimetics with a nonpeptidic core structure, which position key functionalities for interactions with the receptor in a closely related way as the native peptide. Some examples1,3 from the vast literature in the field are peptidomimetics of vasopressin,23 oxytocin,24 LHRH,25 somatostatin and angiotensin II.26
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3.2.1 Development of a peptidomimetic drug - Exanta Today the major drugs used as anticoagulants are warfarin (Waran) and heparin, which inhibit the blood coagulation cascade by two different pathways (Figure 3.5 and Chapter 7.1). Warfarin is a vitamin K antagonist that disturbs formation of the coagulation factors in the coagulation cascade. Heparin induces a conformational change in endogenous antithrombin which increases its activity and thereby inhibits thrombin. Warfarin is the only anticoagulantia that is given orally, but suffers from a narrow therapeutic window and is known to have severe drug-drug and drug-food interactions. To avoid over- or under-treatment, careful dosing and monitoring of the plasma concentration has to be performed regularly. The major drawbacks with heparin is the high molecular weight (∼1500-20000 g/mol) together with highly polar functional groups, which demands subcutaneous administration. Since both current therapies are associated with problems, a new oral anticoagulantia with a wide therapeutic window is highly desirable.
O O
OH O
Warfarin
O
O O
O
O
O3S
O
O3S
O2CO
HO
O
O3S
O3SO O
O2C
O
Heparin
O
AcHN
NH
HOOH
HO
HO
Figure 3.5 The structures of warfarin and heparin; the two most common anticoagulantia.
In the mid 80’s a research team at AstraZeneca R&D in Mölndal initiated a project directed towards an orally administered reversible direct thrombin inhibitor (DTI).27 A DTI exerts its action by blocking the active site of the enzyme thrombin, that is the last factor in the blood coagulation cascade.
The first approach was to mimic a pentapeptide (D-Phe-Val-Arg-Gly-Pro) that had been designed based on a proposed conformation of fibrinogen in the active site of thrombin. The labile amide bonds were replaced by proteolytically more stable ketomethylene isosteres. Acceptable potencies were obtained in this series of compounds, but the pharmacokinetic profile was unsatisfactory. In the early 90’s AstraZeneca gained access to the crystal structure of thrombin and a new approach using computer modeling was started. This new approach eventually revealed melagatran as a potential
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DTI with a molecular weight less than 500 g/mol (Figure 3.6). In vitro and in vivo (iv administration) confirmed melagatran as a reversible DTI with a much wider therapeutic window than warfarin.
NH2
NH
NH
O
N
OHN
HO
O
Melagatran
HN
NH
NH
O
N
OHN
O
O
OH
Ximelagatran
Figure 3.6 The thrombin inhibitor Exanta; structures of the active substance melagatran and the prodrug ximelagatran.
The secondary amine, the carboxylic acid and the benzamidine moiety are all charged to a high extent at physiological pH (7.2-7.4). Not surprisingly the highly polar substance suffered from poor membrane permeability and extremely low bioavailability. Initial attempts to overcome the permeability issue were made by exploring different formulations, but these efforts were unfruitful. Instead an approach to make melagatran more lipophilic by using a dual prodrug, both at the amidine and the carboxylic acid, was investigated. The prodrug strategy resulted in ximelagatran which had good permeability and is converted to melagatran in vivo. Ximelagatran stood up to all project goals as an orally administered DTI and the drug Exanta was released to the market in 2003. The research behind Exanta is an interesting story of the development of a peptidomimetic that finally became a drug and reached the market.
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4. Objectives of the thesis
The overall objective of this study was to develop and apply synthetic organic chemistry in the synthesis of peptidomimetics with the focus set on mimicry of β-turn and β-strand peptide secondary structures. In order to reach this objective the following approaches were taken.
• Stabilized turn mimetics were to be synthesized in order to gain further understanding of the bioactive conformation of the endogenous endorphin Leu-enkephalin (H2N-Tyr-Gly-Gly-Phe-Leu-OH).
• A scaffold based strategy towards β-strand mimetics was to be
developed. The scaffold should promote an extended conformation and allow most of the hydrogen bonding capacity to be maintained.
• Experience acquired during development of the chemistry towards
the turn and strand mimetics, was to be applied to suitable medicinal chemistry projects.
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5. Peptidomimetics of Leu-enkephalin
5.1 Biological action and conformation of Leu-enkephalin Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) is an endogenous peptide which was discovered in the mid 1970s (Figure 5.1).28 The peptide is generated from proenkephalin and released in the brain, where it acts as an agonist at the µ, δ, and κ opiate receptors The opiate receptors belongs to the rhodopsin subclass within the superfamily of G protein coupled receptors (GPCR’s), which are characterized by seven transmembrane α-helices.29,30 The Leu-enkephalin pentapeptide interacts with the receptors and a cascade mechanism that eventually results in relief of pain is initiated.31 Soon after the discovery of Leu-enkephalin a crystal structure of the peptide was reported to contain a 1−4 β-turn conformation in the solid state.32 An intramolecular hydrogen bond between the carbonyl oxygen in Tyr (i) and the amide nitrogen in Phe (i+3) stabilizes the β-turn, thus forming a pseudo ten membered ring.
HN
OHN
HN
O
O
H2N
OH
NH
OO
HO
Leu-Enkephalin
Figure 5.1 Leu-enkephalin drawn as a pseudo ten membered ring stabilized by an intramolecular hydrogen bond as observed in the solid state.
Morphine (Figure 5.2) and in particular derivatives thereof are commonly used analgesics to relieve severe pain.31 Like the enkephalines, morphine interacts with the opiate receptors to relieve pain. The fact that the rigid morphine and the highly flexible pentapeptide Leu-enkephalin bind to the same receptor and elicits relief of pain is somewhat surprising. This could, at least partially, be explained by an intramolecular stabilization of Leu-
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enkephalin in the pseudo ten membered ring observed in the crystalline form.33
HO OHO
N
Me
Figure 5.2 The rigid morphine.
5.2 Design of Leu-enkephalin peptidomimetics As mentioned above several reports, propose that Leu-enkephalin could be stabilized by an intramolecular hydrogen bond, either forming a 1−4 or a 2−5 β-turn, in the bioactive conformation.34 In addition a pharmacophore model in which, the charged N-terminal amine, the aromatic residue of Tyr, and the hydrofobic residues of Phe and Leu are key interaction points should be considered when developing peptidomimetics.35-39 In this study, the assumption that the crystal structure of Leu-enkephalin resembles the bioactive conformation was made. In order to stabilize Leu-enkephalin in a 1−4 β-turn conformation, the intramolecular hydrogen bond that forms the pseudo ten membered ring was replaced by an ethylene bridge, affording a stable ten membered ring as in 1 (Figure 5.3).40 The amide bond between Tyr (i) and Gly (i + 1) was replaced with an ether linkage which had been shown to retain biological activity in the linear peptidomimetic 3.41 The methylene ether isostere was employed in order to make the synthesis more feasible while simultaneously striving to ensure biological acceptance for the inserted features. Additionally, to explore the importance of the ring size and the positions of the key features suggested by the pharmacophore model, a Leu-enkephalin analogue 2, containing a seven membered ring, was designed. Linear peptide analogues 3 and 4 were also synthesized as controls to probe the effect of cyclization.
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O
HN
N
O
O
H2N
OH
NH
OO
HO
1
H2N
N
O
OHN
O
O
OH
HO
H2NO
HN
OH
O
H2NO
HN
OH
O
2 4
3
NH
O
NH
O
HN
O
OH
O
OH
O
Figure 5.3 The ten membered β-turn mimetic 1, and the seven membered β-turn mimetic 2, inserted in Leu-enkephalin. Linear analogues 3 and 4. All compounds contain an identical amide isostere.
The designed β-turn mimetic was also aimed to be inserted in other bioactive peptides, and a stabilized β-turn mimetic corresponding to residues of LHRH has been prepared by the same synthetic strategy.25
5.3 Synthesis of peptidomimetics incorporated in Leu-enkephalin
5.3.1 Synthesis of a ten membered β-turn mimetic The strategy behind the synthesis was to introduce all structural features followed by a ring closure via a macrolactamization to afford mimetic 1. A retrosynthetic analysis (Figure 5.4) revealed that 1 could be synthesized from aldehyde I, dipeptide II and glycine III, all suitably protected. Dipeptide II and aldehyde I were to be connected by a reductive amination, followed by an amide bond formation between glycine III and the secondary amine formed in the reductive amination. With all residues in place, ring closure to the desired ten membered ring was planned to be achieved via an amide bond formation. Building blocks II and III were commercially available, but aldehyde I had to be synthesized.
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HN
N
O
H2N
OH
O
NH
O
O
O
HO NH2
H2N
OH
O
NH
O
O
O
HO
OH
OI
II
1
O
IIIHO
H2N
Figure 5.4 Retrosynthetic analysis of the ten membered β-turn mimetic 1.
The synthesis started by preparation of Weinreb’s amide 5 (81%) from Cbz-Tyr(OtBu)-OH using iso-butylchloroformate, N-methyl morpholine and N,O-dimethylhydroxylamine in CH2Cl2 (Scheme 5.1).42 Weinreb’s amide 5 was then treated with allylmagnesium bromide in THF to afford ketone 6 (74%).43
OtBu
NHCbz
O
OH
OtBu
CbzHN
O
N
Me
OMe
OtBu
NHCbz
O
81% 74%
NO-DMHA AllylMgBr
65
Scheme 5.1 Preparation of homoallyl ketone 6.
Different reducing agents were then applied to ketone 6. Chelating reducing agents such as DIBAL or Zn(BH4)2 gave anti aminoalcohol 8 as the major product, while the non-chelating K-selectride favored syn aminoalcohol 7 with a selectivity of ∼ 6:1 (Scheme 5.2).44,45 When using K-selectride as reducing agent, oxazolidinone 9 (14%) was also formed from 7, but the other diastereomeric oxazolidinone was not observed. For the chelating reducing agents no formation of oxazolidinone was observed, probably due to decreased nucleophilicity of the generated alcohol when complexed by Zn or Al.
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OtBu
NHCbz
O
6
K-selectride
OtBu
NHCbz
OH
OtBu
NHCbz
OH ++
HNO
H
O
OtBu
7, 36%
9, 14%
8, 8%
H
Scheme 5.2 The stereoselective reduction of ketone 6.
The configuration of alcohols 7 and 8 was determined after conversion to the corresponding oxazolidinones (Scheme 5.3). Thus, 8 and 7 were treated with aqueous potassium hydroxide in a mixture of methanol and THF, and the NOE-spectra of the generated oxazolidinones were compared.40
OtBu
NHCbz
OH
OtBu
NHCbz
OH
HNO
O
OtBu
7
9
8
HNO
O
OtBu
7.5 M KOH (aq)MeOHTHF
7.5 M KOH (aq)MeOHTHF
10
H-1
H-1
H-2
H-2
Scheme 5.3 Conversion of alcohols 7 and 8 to their corresponding oxazolidinones.
Irradiation of H-2 in 9 gave a 5% NOE (selective) for H-1, whereas a 14% enhancement was obtained for the diastereomeric oxazolidinone 10. This established an anti relationship between H-1 and H-2 in 9 and a syn relationship in 10 (Scheme 5.3). Further into the synthetic sequence it was discovered that only the syn aminoalcohol 7 could be converted to the desired β-turn mimetic. Therefore, K-selectride was chosen as reducing agent to raise the yield of syn aminoalcohol 7 in the transformation of 6. Next, attempts to alkylate alcohol 7 under basic conditions formed oxazolidinone 9 as the only product, therefore an azide was chosen to be explored as protective group. Subsequently, alcohol 7 and oxazolidinone 9 were hydrolyzed to aminoalcohol 11 (80%, Scheme 5.4). Conversion of the free amine in 11 to an azide was achieved using an azidotransfer reaction
18
with triflic anhydride, sodium azide and CuSO4 to afford azidoalcohol 12 (85%).46,47
7 or 9
KOHH2O/EtOH
NH2
OH
OtBu
80%
triflic anhydrideNaN3
CuSO4
N3
OH
OtBu
85%
QHSO4
NaOH (aq)t-Bu bromoacetate
N3
O
OtBu
OtBuO
OsO4
NMO
73%, from 12
11
N3
O
OtBu
OtBuO
OH
OH
N3
O
OtBu
OtBuO
O
12
15
Pb(OAc)4
Na2CO3
85%
13 14
Scheme 5.4 Synthetic procedure to aldehyde 15.
Azidoalcohol 12 was alkylated with α-bromo tert-butylacetate under biphasic conditions, with QHSO4 as phase transfer catalyst, to afford 13.48 Compound 13 was found to be unstable both during purification on silica gel and under storage. Crude 13 was therefore directly oxidized to the stable diol 14 (73%, from 12) using OsO4 and NMO in a solvent mixture containing acetone, THF and H2O. The synthetic sequence was continued by further oxidation of the diastereomeric mixture of diol 14 to aldehyde 15 (85%) using Pb(OAc)4 and Na2CO3 in benzene. Alkylation of the diastereomeric azidoalcohol, generated by same synthetic sequence as 12, but using 8 as starting material, proved unsuccessful when applying the same conditions as above. Most likely this was due to rapid decomposition of the corresponding diastereomer to 13 during synthesis or on purification.
Next, H2N-Phe-Leu-OH was converted to H2N-Phe-Leu-OMe (16) in almost quantitative yield by treatment with TMSCl in MeOH. The following reductive amination was performed in DCE with Na(OAc)3BH as reducing agent under basic conditions to afford 17 (82%) with all motifs of the β-turn mimetic present except for Gly (i+2) (Scheme 5.5).49 The remaining glycine was introduced via an amide bond formation promoted by HATU and DIPEA in DMF to obtain 18 (85%).50,51
19
N3
O
OtBu
OtBuO
15 + 16
NHHN
O
MeO
O
Na(OAc)3BHTEADCE
82%N3
O
OtBu
OtBuO
NHN
O
MeO
O
HATUDIPEADMFFmoc-Gly-OH
85% O
NHFmocformic acid
99%N3
O
OH
OHO
NHN
O
MeO
O O
NHFmoc
17 18 19
N3
O
OH
OPfpO
NHN
O
MeO
O O
NHFmocDCCPfpOHEtOAc
N3
O
OH
NHN
O
MeO
O O
HN
O
20 21
1 R = OH
NH2
O
OH
NHN
O
R
O O
HN
O
DBUdioxanreflux
56%, from 19
SnCl2PhSHTEA
57%, from 21
22 R = OMeLiOH
Scheme 5.5 Completing the synthesis of β-turn mimetic 1.
The phenolic tert-butyl ether and the tert-butyl ester in 18 were simultaneously removed to give 19 (99%) using formic acid, leaving the methyl ester unaffected. Activation of the carboxylic acid to afford pentafluorophenyl ester 20 was accomplished by using pentafluorophenol and DCC in EtOAc.52 Despite purification on silica gel 20 was contaminated with dicyclohexyl urea, but the sequence was continued without further purification. The activated acid 20 was dissolved in dioxane (35 mg/mL) and added to refluxing dioxane (0.15 mL/mg of 20) containing DBU via a syringe pump over a period of 12 h. After the addition was completed, the reaction was refluxed for another 20 minutes to give ringclosed 21 (56%, from 19). Attempts to complete the critical lactamization starting with Fmoc deprotection, followed by using amide bond coupling reagents (DIC, HATU) proved less successful. Finally, reduction of the azide53 and hydrolysis of the methyl ester were achieved in a two step procedure. First reduction of the azide using SnCl2, PhSH and triethylamine in THF, followed by purification by reversed phase HPLC gave amine 22. The methyl ester was then hydrolyzed by treatment with 0.1 M LiOH (aq.) to afford β-turn mimetic 1 (57%, from 21). Thus, the designed β-turn was properly incorporated in Leu-enkephalin over a 15 step synthetic sequence with an overall yield of 3.2%.
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5.3.2 Conformational studies of the ten membered β-turn mimetic using NMR spectroscopy Several attempts were made to obtain high quality spectral data of β-turn mimetic 1 in water, DMSO-d6/water, TFE-d3/water as well as MeOH-d4. However, spectral data with sufficient quality for assignment of 1 could only be achieved in aqueous solution. In water broad peaks and cross peaks with both negative and positive signs in NOESY indicated a slow exchange between several conformations. Therefore compound 21, also containing the ten membered β-turn mimetic, was used as a model for mimetic 1 in the conformational studies. To our delight, for 21 which has both the C- and N-termini protected, spectral data with sufficient quality for conformational determination was obtained in MeOH-d3 at – 70 °C (Figure 5.5).
Figure 5.5 a) A superimposition of the ten conformers with lowest energy generated from the NMR study. b) An overlap between the crystal structure of Leu-enkephalin (dark) and one low energy conformer of 21 (bright). c) An overlap of the β-turn mimetic in 21 (dark) and an idealized type II β-turn (bright).
The extracted conformations were superimposed on different idealized β-turns, which revealed that the highest similarity for an ensemble of low energy conformations was obtained with a type II β-turn (backbone rmsd = 0.43 Å). The ensemble of low energy conformers was also compared with the ten membered ring of crystalline Leu-enkephalin (rmsd = 0.55 Å for the heavy atoms). The only larger deviation of the mimetic from an ideal type II β-turn and Leu-enkephalin in the solid state, was the side chain orientation of the second amino acid (i+1), where a D-amino acid would give an improved fit. The fact that the second amino acid in Leu-enkephalin is glycine, implies that this deviation should not affect binding to the opiate receptors.
21
5.3.3 Synthesis of a seven membered β-turn mimetic on solid phase The seven membered β-turn mimetic 2 constitutes a truncated form of Leu-enkephalin, lacking one of the two glycine residues. The synthetic procedure is mainly based on the strategy developed for 1 (see section 5.3.1) but the assembly of the building blocks was performed on solid phase.
NHBocO
OHN
O
O
ONH2
2. DIC HOBt Fmoc-Phe-OH
1. TFA/CH2Cl2
3. Piperidine DMF
N3
NH
O
HN
O
O
O
tBuO
O
OtBu
15
Na(OAc)3BHTEA
TFA/CH2Cl2
N3
NH
O
HN
O
O
O
OH
O
OH
N3
NH
O
HN
O
O
O
OH
O
OPfp
PfpOHDIC
23
2425 26
Scheme 5.6 Synthesis of activated acid 26, ready for the ring closing reaction.
The main advantage when performing some of the synthetic steps toward 2 on solid phase was that the crucial lactamization was conducted when attached to solid phase, thereby eliminating the risk for oligomerization. First, Boc-protected Leu on solid phase (Tentagel, PAM linker) was elongated with Phe using the standard Fmoc procedure (Scheme 5.6).54 The solid phase bound dipeptide 23 was reacted with aldehyde 15 in a reductive amination reaction using Na(OAc)3BH and triethylamine in DCE to afford secondary amine 24.49 Next, the phenolic tert-butyl ether and the tert-butyl ester was cleaved using TFA in CH2Cl2 to generate carboxylic acid 25, which was activated as a pentafluorophenol ester 26 using DIC and pentafluorophenol.52 Ring closure was accomplished in refluxing dioxane containing triethylamine to afford 27 (Scheme 5.7).
22
N3
N
O
OHN
O
O
O
HO
TEAdioxanereflux
N3
N
O
OHN
O
O
OMe
HO
H2N
N
O
OHN
O
O
R
HO
NaOMeMeOH
SnCl2TEAPhSH
25%, over solidphase synthesis
26
27 28
2 R = OH
56%, from 28
29 R = OMeLiOH
Scheme 5.7 Completing the synthesis of β-turn mimetic 2.
Ringclosed 27 was released from the solid phase using NaOMe in methanol to afford protected β-turn mimetic 28 (25%, over 8 steps on solid phase). β-Turn mimetic 28 was then deprotected in a two step procedure starting with reduction of the azide53 to the corresponding amine 29 using SnCl2, TEA and PhSH in THF. Hydrolysis of the methyl ester and purification by reversed phase HPLC then afforded target β-turn mimetic 2 (56%, from 28).
5.3.4 Synthesis of linear Leu-enkephalin analogues
Fmoc-Tyr(OtBu)-OH was initially reduced to the corresponding alcohol 30 (90%) by activating the acid with iso-butyl chloroformate followed by reduction with NaBH4 (Scheme 5.8).55 The Fmoc-protected amine was then liberated using morpholine to afford aminoalcohol 31 (90%). An azidotransfer reaction using triflic anhydride and NaN3 in a biphasic system then afforded azidoalcohol 32 (72%).46,47
OtBu
NHFmoc
OH
O
OtBu
NHFmoc
OH
iBu-ChloroformateNaBH4
90%
OtBu
NH2
OH
Morpholine
OtBu
N3
OH90% 72%
Tf2ONaN3
30 31 32
Scheme 5.8 The synthesis of azidoalcohol 32 from the corresponding Fmoc-protected amino acid.
Next, alcohol 32 was alkylated with ethyl bromoacetate using KH and QI in THF at 0 °C to afford 33 (89%, Scheme 5.9). Hydrolysis using NaOH in
23
EtOH gave 34 (80%) suitably protected for incorporation on solid phase via formation of an amide bond. Direct substitution of 32 with bromo acetic acid was less effective, hence the synthetic route via ester 33 was preferred.
OtBu
N3
OH
32
OtBu
N3
OOEt
O
OtBu
N3
OOH
O
33 34
Et-bromoacetate
KH
QI
89% 80%
EtOH
NaOH
Scheme 5.9 Synthesis of the dipeptide isostere 34 suitably protected for solid phase synthesis.
Solid phase bound peptides 35 and 36 were synthesized according to the standard Fmoc procedure starting from solid phase bound Fmoc-Leu (Scheme 5.10).54 Incorporation of carboxylic acid 34 to afford solid phase bound peptidomimetics 37 (from 35) or 38 (from 36), was achieved via an amide bond formation using HATU and DIPEA in DMF (Scheme 5.11).50,51
FmocHNO
OHN
O
O
O
H2N
HN
O
O
O
NH
O
H2N
35 36
Scheme 5.10 Preparation of solid phase bound peptides.
Reduction of azides 37 and 38 was performed using SnCl2, TEA and PhSH in THF to afford the corresponding amines 39 and 40.53 A cleavage mixture containing TFA, H2O, ethanedithiol and thioanisol was added to deprotect and release the peptidomimetics from the solid phase to afford 4 (62%, over the solid phase synthesis) and 3 (70%, over the solid phase synthesis) after purification on reversed phase HPLC.
24
HN
O
O
O
NH
HN
O
O
O
NH
OHN
O
OO
O
N3
OtBu
N3
OtBu
HN
OH
O
O
NH
OHN
O
OH2N
OH
HN
OH
O
O
NH
O
O
H2N
OH
35 36
HN
O
O
O
NH
OHN
O
OH2N
OtBu
HN
O
O
O
NH
O
O
H2N
OtBu
37 38
4 3
39 40
34
HATUDIPEACH2Cl2
34
HATUDIPEACH2Cl2
SnCl2TEAPhSHTHF
SnCl2TEAPhSHTHF
H2OTFAscavangers
H2OTFAscavangers
62% over solidphase synthesis
70% over solidphase synthesis
Scheme 5.11 Completing the synthesis of peptidomimetics 4 and 3.
5.4 Biological evaluation 5.4.1 Opioid receptor binding assay Leu-enkephalin mimetics 1, 2, 3 and 4 (Figure 5.6) were evaluated as specific binders at the µ and δ opiate receptors. Membrane bound opiate receptors, prepared as previously described from rat brain without cerebellum, were used in the binding assay.56 All compounds were tested in triplicates at each concentration using three different rat brain homogenates (Figure 5.7 and Figure 5.8).
25
O
HN
N
O
O
H2N
OH
NH
OO
HO
1
H2N
N
O
OHN
O
O
OH
HO
H2NO
HN
OH
O
H2NO
HN
OH
O
2 4
3
NH
O
NH
O
HN
O
OH
O
OH
O
Figure 5.6 The four synthesized peptidomimetics evaluated in an opioid binding assay.
Receptor specific binding was measured using [3H] DAMGO37 and [3H] DPDPE36 as radioligands for the µ- and δ- receptors, respectively. In order to measure the receptor specific binding the membrane bound radioactivity was counted and then corrected for unspecific binding using naloxone. The reference ligands DSLET (D-Ser-Leu-enkephalin-Thr) and Leu-enkephalin were also included in the binding study and treated in the same way as the peptidomimetics for comparison. To prevent proteolysis of the amide bonds in the different Leu-enkephalin analogues, the protease inhibitor bacitracin was used in all experiments.
5.4.2 Binding to µ- and δ- opioid receptors Among the tested peptidomimetics, linear analogue 3 showed the highest affinity for both receptors (IC50 = 14 nM at µ, 1.3 nM at δ) (Figure 5.7, Figure 5.8, Table 5.1 and Table 5.2). Interestingly, 3 bound with higher affinity to both receptors than the two positive controls Leu-enkephalin and DSLET. The affinity of 3 confirms that the ether linkage used as amide bond isostere in our study is well tolerated without reduction of binding affinity as reported previously.41
26
Figure 5.7 Competitive inhibition at the µ-receptor for peptidomimetics 1 (), 2 (), 3 () , 4 () and the two known ligands DSLET, () and Leu-enkephalin (). For clarity only a few error bars are shown in the figure.
Table 5.1 Calculated IC50 values and Hill slopes for the µ-opiate receptor. Compound pIC50 IC50 (nM) nh
1 <6 >1000 -
2 6.13 ± 0.12 740 0.90 ± 0.24
3 7.86 ± 0.09 14 0.80 ± 0.12
4 <5.5 >1000 -
Leu-enkephalin 6.81 ± 0.12 160 0.66 ± 0.12
DSLET 7.41 ± 0.07 39 0.74 ± 0.01
Ten membered ring 1 showed no affinity (IC50 > 1000 nM) at any of the two receptors. The lack of affinity is not due to the amide bond isoster of 1 since this structural fragment also is present in 3. Instead the rigidity imposed by the cyclization may prevent 1 from adopting the required conformation for binding, implying that a 1−4 type II β-turn is not the biologically active conformation for Leu-enkephalin. Other reasons for the lack of affinity could be the steric demand of the ethylene bridge in 1 that might hinder accessibility to the receptors, or the loss of a potential hydrogen bond from the Phe NH. The truncated linear analogue 4 showed no affinity for the µ-receptor (IC50 > 1000 nM) and only modest binding to the δ-receptor (IC50 = 370 nM). This suggests that the decreased length may prevent 4 from reaching critical interaction points in the binding sites of the receptors.
27
Figure 5.8 Competitive inhibition at the δ-receptor for peptidomimetics 1 (), 2 (), 3 () , 4 () and the two known ligands DSLET, () and Leu-enkephalin (). For clarity only a few error bars are shown in the figure.
Table 5.2 Calculated IC50 values and Hill slopes for the δ-opiate receptor.
Compound pIC50 IC50 (nM) nh
1 <6 >1000 -
2 6.79 ± 0.12 160 0.98 ± 0.26
3 8.88 ± 0.08 1.3 0.92 ± 0.14
4 6.07 ± 0.14 370 1.03 ± 0.37
Leu-enkephalin 8.44 ± 0.15 3.6 0.67 ± 0.15
DSLET 8.83 ± 0.16 1.5 0.58 ± 0.14
Seven membered ring 2 showed affinities superior to linear analogue 4 for both the µ-receptor (IC50 = 740 nM) and at the δ-receptor (IC50 = 160 nM). The significantly increased binding affinities displayed by 2 as compared to the flexible linear analogue 4 indicates that Leu-enkephalin probably binds to both receptor subtypes in a rigid turn conformation.
5.5 Summary Several reports claim that the flexible Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) binds to the opiate receptors in a stabilized manner forming either a 1−4 or a 2−5 β-turn. In an attempt to obtain more information concerning the bioactive conformation of Leu-enkephalin, four peptidomimetics were synthesized and evaluated in an opioid receptor binding assay. Two of the peptidomimetics were conformationally restricted by replacing the proposed intramolecular hydrogen bond between the carbonyl oxygen in Tyr (i) and the amide nitrogen in Phe (i+3) with an ethylene bridge. The amide bond between Tyr (i) and Gly (i+1) in the two mimetics was replaced with an ether linkage. Insertion of this amide bond isostere is known to be compatible with binding to the opiate receptors. To probe the effect of
28
cyclization linear analogues of the two cyclized β-turn mimetics, containing the amide isostere between Tyr (i) and Gly (i+1), were also synthesized. All four compounds were tested in an opioide binding assay. This revealed that Leu-enkephalin probably binds to the µ- and δ- receptor subtypes in a more rigid turn conformation, but not in a 1−4 β-turn as we suggested in the original design.
29
6. β-Strand peptidomimetics
6.1 β-Strands β-Sheets account for more than 30% of all protein structures and are built up by β-strands which have an extended conformation. The β-strand is rarely found as a monomer, instead β-strands assemble either in a parallel or an anti-parallel fashion to form β-sheets stabilized by a hydrogen bonding network (Figure 6.1).9,10
N
NN
HN
NH
NN
NHN
NH
HN
O
O
N
N
O
O
O
O
O
H H
H
H
H
H
H
H
O
NH
HN
NH
O
O
O
O
O
O
O
O
Figure 6.1 Extended peptide chains, β-strands, dimerizes to form β-sheets. The peptide backbone of the two involved β-strands can run in the same direction (parallel, to the left) or in opposite direction (antiparallel, to the right).
Often β-sheets serve as scaffolds, which position the side chains of proteins in positions required to elicit a biological activity. β-Sheets, or rather the β-strand, is recognized in several protein-protein interactions, for example by proteolytic enzymes, major histocompatibility complex (MHC) molecules, farnesyl transferases and SRC kinases. β-Strands are also involved in protein-DNA interactions in normal gene regulation by the met repressor that requires a dimerization through β-sheet domains. β-Strands are also involved in development of neurological diseases, such as Alzheimer’s and Parkinson’s disease that are associated with aggregation of proteins to form insoluble β-sheet structures. In the field of medicinal chemistry, the β-strand is regarded as a fundamental structural element for development of therapeutics against diseases associated with β-sheet formation or recognition of β-strands. The attractive and versatile biological activities of peptides, together with their pharmacokinetic shortcomings if used as orally administered drugs, make research regarding design, synthesis and evaluation of β-strand mimetics highly interesting. β-Strand mimetics based on polypyrrolinones have been developed with the biological activity
30
retained in applications toward proteases (Figure 6.2).57-60 Also the 1.2 dihydro-3(6H)-pyridinone has successfully been used as a key building block in the synthesis of mimetics (@-tides) of β-strands containing up to of 13 amino acid residues.61-63 A β-strand mimetic must maintain the capability to mimic the activity of the original peptide, while at the same time provide improved pharmacokinetic properties as compared to the native peptide.
N
O
NH
RHN
OO
NH
R
N
O
HN
OHN
O
HN
OR
R R
Figure 6.2 Two examples of β-strand mimetics. @−Tides (left) and polypyrrolinones (right).
The maintenance of the structural features of the original peptide, combined with introduction of surrogates for amide bonds susceptible to proteolysis, is crucial in the design of a β-strand mimetic. Additionally, the structural elements of a β-strand mimetic should promote an extended backbone conformation and also retain the capability to accept and donate hydrogen bonds to facilitate aggregation into β-sheets.
6.2 Design and retrosynthetic analysis of a β-strand mimetic In this study a synthetic procedure to β-strand mimetic Va, based on a pyridine scaffold, was developed (Figure 6.3). The pyridine ring replaces the central amino acid within an amino acid sequence containing three residues. The NH attached at position 2 of the pyridine ring, together with the pyridine nitrogen atom mimics the second amide bond in the peptide chain, while the keto functionality attached at position 4 of the pyridine ring mimics the first amide bond of the peptide sequence.64 The two amide bonds are thereby replaced with proteolytically stable functionalities and the hydrogen bonding potential, crucial for β-sheet formation, is partly maintained. Previously peptidomimetics (Vb), that act as a dopamine modulators, based on the same pyridine scaffold have been synthesized with the amino acid sequence Pro-Leu-Gly-NH2.64 The second amide bond isostere (X = O) in peptidomimetics Vb lack the hydrogen bonding capability as compared to Va (X = NH) and should be less prone to aggregate into β-sheets (Figure 6.4).
31
NH
HN
NH
O
O
O
R1
N
XOH
OO
H2N
R1
IV Va X = NH
R2
R3
R3
R2
Vb X = O
Figure 6.3 A peptidomimetic containing the pyridine scaffold corresponds well to a peptide in an extended conformation, both according to electrostatic calculations and geometric fit.
Electrostatic calculations (Spartan software, AM1 Hamiltonian) on a peptidomimetic closely related to Va (R1 = R2 = R3 = Me, terminally N-acetylated and having a C-terminal NHMe) confirm a similar charge distribution for the amide bond isostere attached at position 2 of the pyridine ring (NH and pyridine N) and the corresponding amide bond in Ac-Ala-Ala-Ala-NHMe.64 The side chain of amino acid 2 (corresponds to R2 attached at position 3 of the pyridine ring) in our mimetic was chosen to be a proton to avoid the risk for a steric clash between two R2 groups or between R2 and a peptide side chain in a β-strand aggregate (Figure 6.4). If desired, substituents at position 3 of the pyridine ring can be introduced by using highly reactive electrophiles in a halogen dance reaction.65
N
NH
OO
NH
R1
R2
R3
N
HN
O OHN
R1
R2
R3
N
NH
OO
NH
R1
R2
R3
NH
O
HN
O OHN
R1
R2
R3
Figure 6.4 A hypothetical antiparallel β-sheet aggregation between two β-strand mimetics Va (left) and between a peptide and β-strand mimetic Va (right).
Usually polar and nonpolar amino acid side chains alternate in native β-sheets. Therefore nonpolar side chains were selected for the R1- and R3-groups in β-strand mimetic Va, intended to be iso-butyl (side chain of leucine) or sec-butyl (side chain of iso-leucine). A retrosynthetic analysis of Va revealed a synthetic pathway using commercially available 2-fluoro-4-iodopyridine (VII) as starting material (Figure 6.5).
32
N
NH
OH
OO
H2N
R1
O
PgHN
R1
N
H2NOH
O
H
FI
Va
VI VII VIII
H2N
HN
NH
CO2H
O
O
R1
IV
R3
R3
R3
Figure 6.5 A retrosynthetic analysis of β-strand mimetic Va, based on substituted pyridine VII as scaffold.
The aromatic substitution at position 4 of the pyridine ring was planned to take place via a Grignard exchange reaction using iso-propylmagnesium chloride to afford a pyridine Grignard reagent, using aldehyde VI as electrophile.66,67 Nucleophilic aromatic substitution (SNAr) at position 2 of the pyridine ring, by displacing the fluorine atom using an amine as nucleophile, was planned as another key transformation. The preferred order to introduce the two substituents was to perform the Grignard exchange reaction first due to expected selectivity problems between the two halogenated positions of the pyridine ring in the SNAr reaction. In addition, interference from an ‘anilinic’ proton in a strongly basic Grignard reaction was anticipated to cause problems.
6.3 Attachment of an N-terminal leucine analogue at position 4 of the pyridine ring It was reasoned that the Grignard reaction to connect protected leucinal at position 4 of the pyridine ring would suffer from interference from the acidic NH proton in the carbamate functionality at the α-amino group. However, an improved yield was expected if di-Boc-protected leucinal was used as electrophile (Scheme 6.1). As the first step in an attempt to prepare this compound, Boc-protected leucine was reduced to the corresponding alcohol 41 (97%) by activating the carboxylic acid with iso-butyl chloroformate followed by treatment with NaBH4.55 The synthesis was continued by
33
protection of the alcohol moiety in 41 as a silyl ether using TBDMSCl and imidazole in CH2Cl2 to afford 42 (90%).
OH
O
NHBoc
OH
NHBoc
i-Bu Chloroformate
NaBH4
97%
41
OTBDMS
NHBoc
42
TBDMSCl
Imidazole
CH2Cl2
90% 66% OTBDMS
NBoc2
Boc2O
DMAP
dioxane
43
TBAF
THF
OH
NBoc2
44
Oxidation
O
NBoc2
45 Scheme 6.1 The strategy to di-Boc-protected leucinal failed, probably during the deprotection of 43.
In order to add the second Boc-group a large excess Boc2O and DMAP was added to 42. The reaction was heated to reflux in dioxane which afforded the di-Boc-protected 43 (66%). Thereafter cleavage of the silyl ether in 43 by using TBAF was attempted. The 1H NMR spectrum confirmed removal of the silyl ether, but the signal from the Boc-group was now split into two, probably due to migration of one of the two Boc-groups to the primary alcohol. Because of some uncertainty regarding the compound identity, oxidation to the corresponding aldehyde 45 was tried using TEMPO, NaOCl and KBr in CH2Cl2.68 No traces of aldehyde 45 was detected, supporting that a Boc-protected alcohol, which is unable to undergo oxidation, had been formed during the treatment with TBAF.
OH
NHBoc
OH
NH2
OH
N3
Oxidation
O
N3
80%
Formic acid
Tf2O
NaN3
64%
41 46 47 48 Scheme 6.2 The synthetic strategy toward azidoaldehyde 48 failed in the oxidation of azidoalcohol 47.
A second strategy aiming to avoid an acidic carbamate proton in the leucinal to be used in the Grignard reaction, involved the use of an azide as protection for the α-amino group. The synthetic route to azidoleucinal 48 started by removal of the Boc-protection group from 41 using formic acid to afford aminoalcohol 46 (80%). The amine was thereafter transformed to the corresponding azide 47 (64%) with retention of configuration by using NaN3, Tf2O and CuSO4 in CH2Cl2.47,69 The azidoalcohol was then treated with different oxidizing agents. Despite trying TEMPO and NaOCl,68 Swern oxidation70 and Dess-Martin71 periodinane, the desired product was not
34
observed. The neighboring azide might be responsible for the failure of these oxidation reactions. These disappointing results lead us to examine if the Grignard reaction could be performed in a reasonable yield in presence of the carbamate NH (Scheme 6.3). Dess-Martin periodinane oxidation of alcohol 41 generated the desired aldehyde 49 (88%). Due to the reported sensitivity of the chiral center in protected α-amino aldehydes, aldehyde 49 was used without further purification in the next step.72,73 The following Grignard reaction was performed by adding iso-propylmagnesium chloride to 2-fluoro-4-iodopyridine thereby preforming the pyridine Grignard reagent.64,67 Then aldehyde 49, dissolved in THF, was added to afford alcohol 50. Considering the acidic carbamate NH a two fold excess of both 2-fluoro-4-iodopyridine and iso-propylmagnesium chloride was used.
O
NHBoc
49
iPrMgClTHF2F,4I pyridine
41%, from 49
N
FBocHN
OH
50 51
N
FBocHN
OBn
BnBrQHSO4
50% NaOHaqOH
NHBoc
41
88%
Dess-Martinperiodinane
Scheme 6.3 A successful strategy to connect leucinal at position 4 in 2-fluoro-4-iodopyridine ring via a Grignard exchange reaction.
To our delight the secondary alcohol 50 was formed in a reasonable yield from this reaction as indicated by NMR spectroscopy of the crude material. Crude 50 was then protected as a benzyl ether under phase transfer conditions to afford 51.74 Fortunately, purification of benzyl ether 51 was straightforward (in comparison to alcohol 50) and the desired product was obtained in 41% yield (based on 49) over the two steps. The N-terminal leucine derivative was now properly connected to the pyridine scaffold, and the conjugate was protected in the desired manner.
6.4 Attachment of a C-terminal glycine analogue at position 2 of the pyridine ring
6.4.1 Nucleophilic aromatic substitution A model study to displace the fluorine in 2-fluoropyridine with an amine was performed using benzylamine (1.2 equiv.) as nucleophile. A variety of conditions were applied to establish a robust and high yielding SNAr reaction, exploring solvents, bases and reaction temperatures (Table 6.1).
35
Table 6.1 Different reactions performed with benzylamine (1.2 equiv.) as nucleophile in an SNAr reaction using 2-fluoropyridine as electrophile.
N
FH2N
+
N
NH
52 Entry Solvent Base/additive Temp.
°C Time Heating method Yield %
1 NMP TEA 150 48 h Sealed cylinder 30 2 NMP TEA 250 30 min Microwave 42 3 NMP DIPEA 250 60 min Microwave 52 4 Pyridine 250 60 min Microwave 79 5 CH3CN Ag2CO3 120 10 min Microwave 6 CH3CN CuCO3 120 10 min Microwave 7 Toluene DIPEA/NaCl 200 20 min Microwave 8 THF n-BuLi 78→rt 3 h - - 9 THF n-BuLi/Ag Zeolite 78→rt 3 h
As revealed by entries 8 and 9 in table 6.1 strongly basic conditions based on n-BuLi failed to generate product. In contrast, use of amine bases and elevated temperatures, entries 1−4, afforded the target compound 52 in modest to excellent yields. The idea behind entry 5 was to get a coordination between Ag+ and the fluorine attached to the pyridine ring, thereby attempting to enhance the reactivity of the electrophile. In entry 6 the possibility to have a coordination between Cu2+ and the pyridine nitrogen was tested to get an increased reactivity. Toluene is not a good solvent in absorbing microwaves but adding solid NaCl generate hotspots around the salt where the reaction is thought to take place. In entry 7 this method was applied but no traces of product was observed.
As the next step of the exploration of the SNAr reaction the amine nucleophile was chosen to be the sterically demanding Ile. We reasoned that if a method to substitute 2-fluoropyridine using a derivative of Ile could be developed, the method should also be applicable to several less sterically hindered amino acids.
BocHNOH
O
BocHNOH
BocHNOBn
H2NOBn
iBu chloroformateNaBH4
5453
85%, over 2 steps
55
91%
Formic acid
BnBrQHSO4
NaOHaqPhCH3
Scheme 6.4 Conversion of Boc-Ile-OH to benzyl protected aminoalcohol 55.
36
To prepare a suitable Ile derivative Boc-Ile-OH was reduced to the corresponding alcohol by first activating the acid with iso-butyl chloroformate followed by addition of NaBH4 to afford 53 (Scheme 6.4).55 Treating 53 with BnBr under phase transfer conditions, using QHSO4 as phase transfer catalyst, afforded ether 54 (85%, from Boc-Ile-OH).74 Finally, the Boc-group was removed using formic acid to afford 55 (91%), which was used as nucleophile in the aromatic substitution of 2-fluoropyridine (Table 6.2).
Table 6.2 Different reactions performed with 55 (1.5 equivalents) as nucleophile in the SNAr reaction using 2-fluoropyridine as electrophile.
N
NH
OBnN
H2NOBn
F+
55 56 Entry Solvent Base/additive Temp. °C Time Heating
method Yield %
1 HMDS TEA/TMSCl 175 1 h Microwave 2 Pyridine 190 30 min Microwave 3 Pyridine 230 1 h Microwave 7 4 NMP pyridine 250 30 min Microwave 5 THF BuLi 78→ 0 3 h 40 6 THF MeMgCl Reflux 7 h Oil bath 38 7 THF NaHMDS/TMSCl 78→rt 5 h 8 THF NaHMDS/15-
crown-5 78→reflux 7 h Oil bath *
* Compound 56 was not formed, instead a nucleophilic attack from 15-crown-5 on 2-fluoropyridine occurred and gave compound 57
(Scheme 6.5).
Using 55 as nucleophile in this reaction generally afforded no product or low yields. In contrast to the results with benzylamine only strongly basic methods, entries 5 and 6, generated the target compound 56, but only in approximately 40% yields. Interestingly, treating 2-fluoropyridine with NaHMDS, 55 and 15-crown-5 ether in refluxing THF afforded a substitution product where the crown ether had displaced the fluorine atom to afford 57 (68%, Scheme 6.5).
N
F
15-crown-5
NaHMDS
THF
reflux
68%
O
O
OO
O
N
57
+
55
H2NOBn
Scheme 6.5 Unexpected byproduct formed in an attempt to react 2-fluoropyridine with 55.
37
The conditions reported in entry 6, which gave a more stable reaction outcome than those of entry 5, were now applied in an SNAr reaction of fluoropyridine 51 (Scheme 6.6).
H2NOBn
55 51
N
FBocHN
OBn
58
N
NH
BocHN
OBn
OBn
MeMgClTHF
+
Scheme 6.6 A synthetic attempt to afford substituted pyridine 58 using the preferred method obtained in the model study.
Unfortunately, the reaction towards 58 resulted in multiple undesired products and an alternative synthetic route was needed. First an investigation of if less sterically hindered amine nucleophiles could accomplish this transformation on substituted pyridine 51 was initiated. Different glycine analogues were used as nucleophiles and 51 as electrophile (Scheme 6.7). First, an excess of H2N-Gly-OtBu (5 equiv.) was used as nucleophile and reacted with 51 at 150 °C in pyridine by using microwave irradiation. The reaction mixture turned brown but the desired product was only observed in trace amounts (LCMS). It was also found that the Boc-protection group was labile and partially cleaved under these conditions. Boc-protected 51 was therefore converted to the more stable acetamide 60 (86% from 51, Scheme 6.7).
38
N
F
+
+
+
H2NOH
H2NOtBu
O
H2NOH
O
10 equiv.
pyridine190 !C1 h
N
NH
OH
N
NH
Allylamine17 bar2.5 h
5 equiv.
5 equiv.
pyridine
AcHN
OBn
60
N
F
OBn
AcHN
N
F
OBn
H2N
N
F
OBn
BocHN
Formic acidAc2OCH2Cl2
5951 60 (86%, from 51)
N
NH
OBn
AcHNOtBu
O
61, trace amounts
NaHCO3N
FAcHN
OBn
60
N
NH
OBn
AcHNOH
O
+NH
O
OBn
AcHN
62, < 10% 63, < 10%
N
FAcHN
OBn
60
N
FAcHN
OBn
60
AcHN
OBn
AcHN
OBn
64, 54%
65, > 90% crude
A.
B.
C.
D.
E.
Scheme 6.7 The Boc-protection group was partially cleaved during attempted SNAr reaction of 51 and was therefore converted to acetamide 60 (A). Thereafter, different glycine analogues were used as nucleophiles in the SNAr reaction with 60 (B→E).
An excess of H2N-Gly-OtBu (5 equiv.) and 60 were heated to 150 °C in pyridine by using microwave irradiation. However, also in this case the reaction turned brown and 61 was detected only in trace amounts (LCMS). Raising the temperature to 180 °C did not increase the yield of 61, but resulted in formation of a black solid. The side reaction was believed to be due to polymerization of tert-butyl protected glycine. Actually, when the reaction was run without fluoropyridine 60 a similar black solid was formed as well. As the problem thus seemed to originate from the tert-butyl ester, glycine with the carboxylic acid moiety unprotected was instead tested as nucleophile. Solubility problems of glycine in organic solvents required the use of aqueous media in the reaction and saturated aqueous NaHCO3 was chosen. Subsequently, excess glycine (5 equiv.) was heated with 60 to 160 °C for 1 h using microwave irradiation. Indeed the desired product 62 was formed but in small amounts (< 10%) as revealed by LCMS analysis. Unfortunately, a byproduct resulting from an attack of water to afford
39
pyridone 63 (LCMS) was also formed in equimolar amounts. Our interest therefore turned to other glycine analogues with better solubility in organic solvents in order to eliminate water as competing nucleophile. Ethanolamine and allylamine were chosen since both contain functionalities that could serve as masked carboxylic acids. First excess ethanolamine (10 equiv.) was heated together with 60 in pyridine to 190 °C for 1 h. This gave the desired substitution product 64 in 54% yield. When evaluating different experimental conditions it was noticed that the yields of the nucleophilic aromatic substitutions was increased by three factors. High concentrations of the amine nucleophile and long reaction times at high temperatures. Therefore, allylamine was used as solvent in the SNAr reaction with 60, and the reaction was run at 17 bar (~150 °C) for 2.5 h using microwave irradiation. To our satisfaction, all of 60 was cleanly converted to the desired product 65, which was obtained in high yields (> 90%).
With the substitution using ethanolamine and allylamine as nucleophiles accomplished in good to excellent yields the next task was to oxidize the alcohol moiety in 64 or the olefin in 65 to the desired carboxylic acid, or preferably to the corresponding ester to avoid zwitterionic compounds. The development of oxidation conditions was not performed on substituted pyridines 64 and 65, but instead on the more simple compounds 66 and 67 generated from 2-fluoropyridine in a related manner as described above (Scheme 6.8, for details see paper III).
N
N
NH
NH
OH
N
NH
OH
O
N
NH
RuCl3
NaIO4
OH
OH
K-osmate
NMO
51%
66
67
N
NH
N
NH
67
O3
NaOH
MeOH
OMe
O
N
NH
OMe
O
NaIO4Br2
MeOH
70
70
69
68
Scheme 6.8 Attempted oxidation to give the desired carboxylic acid 68, or preferably methyl ester 70.
Oxidation of 66 was attempted by using RuCl3 with NaIO4 as cooxidant in a solvent mixture containing CH3CN, H2O and CH2Cl2.75 Immediately after the addition of RuCl3 a colorless solid was formed that proved insoluble in most organic solvents and problematic to analyze. Oxidation strategies to
40
acid 68, assumed to be a zwitterion, were not explored further due to anticipated problems in handling the charged target compound. Methods to directly generate ester 70, thereby avoiding the carboxylic acid, seemed more appealing. First, oxidation of the olefin in 67 to the corresponding diol 69 (51%) was accomplished by using a catalytic amount of potassium osmate with NMO as cooxidant. The diol was further treated with Br2 and NaIO4 in methanol, but the desired product was not observed.76 A smooth procedure for oxidation of olefins directly to the corresponding methyl ester has been reported. This method was applied to olefin 67 by purging a stream of ozone through a solution of 2 M methanolic NaOH and CH2Cl2 containing 67.77 Unfortunately, in our hands all 67 was consumed but no product was observed by TLC or NMR analysis of the crude product.
6.4.2 A reductive amination strategy Treating 2-aminopyridine with a strong base (e.g. NaH) followed by alkylation is known to alkylate the nitrogen in the pyridine ring instead of the amine in position 2.78 On the other hand applying reductive amination conditions is known to result in alkylation of the amino group.79 Therefore a model study was performed using 2-aminopyridine and anisaldehyde under various reductive amination conditions (Scheme 6.9). Acidic conditions using AcOH and Na(OAc)3BH in CH2Cl2 or MeOH, neutral methods in methanol employing molecular sieves as drying agent and a basic method using Na(OAc)3BH and TEA in CH2Cl2 were investigated. The basic conditions49 turned out to be superior but still afforded 71 only in a modest 36% yield. To afford the desired glycine substituted pyridine 68 the conditions from the model study were applied to 2-aminopyridine and glyoxylic acid, but only starting material was recovered from the reaction. The more soluble tert-butyl glyoxylic acid, generated from tert-butyl acrylate via ozonolysis, was also employed in the reductive amination. 1H NMR spectroscopy of the crude product revealed that, most likely, the tert-butyl ester of α-hydroxy acetic acid was formed as the major product.
N
NH2
N
NH
OMe
N
NH2
N
NH
OH
O
or
N
NH
OtBu
O
Na(OAc)3BHTEADCE
Reductiveamination
68
7136%
72
Scheme 6.9 Attempts to afford glycine substituted pyridine via reductive amination of 2-aminopyridine.
41
Another approach to afford the glycine substituted pyridine 68 by refluxing 2-aminopyridine with concentrated perchloric acid and glyoxal in methanol has been described in the literature.78 In an attempt to use this approach aminopyridine 73 was prepared in 60% yield by heating 60 (~150 °C, sealed steel cylinder) in water containing 25% ammonia (Scheme 6.10).80 Then 73, perchloric acid and glyoxal were heated to reflux in methanol, but unfortunately these rather harsh conditions resulted in decomposition of the starting material without any detectable formation of 74.
N
NH2
OBn
AcHN
N
NH
OBn
AcHNOMe
O
perchloric acidglyoxal25% NH3 in H2O
60%
73 74
N
F
OBn
AcHN
60
Scheme 6.10 An SNAr reaction displacing the fluorine with NH3 followed by an attempt to afford glycine substituted pyridine 74.
6.4.3 Changing the substitution order and starting with the SNAr reaction
As previously mentioned the preferred order of substitution of the pyridine scaffold was to perform the Grignard reaction prior to the SNAr reaction. This was due both to a possible selectivity issue between the two halogenated positions in 2-fluoro-4-iodopyridine and an anticipated interference from the ‘anilinic’ proton when running the Grignard reaction as the second step. If both selectivity and protection of the ‘anilinic’ proton prior to the Grignard reaction could be accomplished, reversal of the reaction order could be successful. This approach was investigated as outlined in Scheme 6.11.
42
N
I F
N
I NH
heatallylamine
TsCl +H2N N
H
Ts
2.5 equiv.
CH2Cl2
99%
N
I N
Ts
63%
DMSOKHheat2-F,4-I-pyridine
iPrMgCl49
N
N
TsOH
BocHN
7776
78
75
Scheme 6.11 Selective nucleophilic aromatic substitution of 2-fluoro-4-iodopyridine using a deprotonated sulphone amide as nucleophile.
To begin with, 2-fluoro-4-iodopyridine and allylamine were heated (180−200 °C) using microwave irradiation, but the mono substituted product 75 was not observed. This may, at least partly, be due to selectivity problems. Allylamine was therefore transformed to tosyl amide 76 to get a more acidic NH proton, thereby facilitating the formation of a nitrogen anion. When 76 and 2-fluoro-4-iodopyridine were treated with KH in DMSO (140 °C, 30 minutes) using microwave irradiation, iodopyridine 77 was obtained in acceptable yield (63%) with the ‘anilinic’ nitrogen atom protected. When using other solvents (THF, DMF or H2O) or other bases (Cs2CO3 or NaH) no product was formed, suggesting that the anion of DMSO is the best choice of base to be employed in this type of SNAr reaction. Continuing with the Grignard exchange reaction using iso-propyl magnesium chloride at room temperature followed by addition of aldehyde 49 failed to give the desired product 78. Instead, the deiodinated starting material was observed as the major product in the reaction mixture (LCMS). Thus, it seemed like the preferred order to perform the substitutions on the pyridine scaffold still was to start with the Grignard reaction.
6.5 Completing the synthesis − A successful Boc strategy The ‘anilinic’ proton, present in several of the synthetic approaches described above, was believed to be responsible for the failures in completing the synthesis so far. Protection of the ‘anilinic’ nitrogen as a tosyl amide described in the preceding section lead us to the idea to use a Boc-protection strategy in the last steps of the synthetic sequence. Therefore
43
allylamine substituted pyridine 67 was treated with Boc2O and a catalytic amount of DMAP in CH2Cl2 which allowed 79 to be isolated in excellent yield (99%) (Scheme 6.12).79
N
NH
N
N
Boc
Boc2O
DMAP
99%
67 79
65%
O3NaOH
MeOHN
N
Boc80
OMe
O
Scheme 6.12 Protecting the ‘anilinic nitrogen allowed oxidation of the alkene.
To our delight, purging ozone through a CH2Cl2 solution containing 2 M methanolic NaOH and 79, now afforded 80 in acceptable yield (65%).77 As described previously oxidation of 67 in the same manner failed to give the desired ester.
With all parts in hand, the synthesis of the β-strand mimetic could now be brought to completion. This started with the SNAr reaction, which was performed by heating 60 to 17 bar (∼150 °C) using microwave irradiation in neat allylamine (Scheme 6.13). Excess allylamine was then removed and amine 65 was Boc-protected using excess Boc2O and a catalytic amount of DMAP in CH2Cl2 to afford 81 (86%, from 60).79 The olefin was treated with ozone in a CH2Cl2 solution containing methanolic NaOH to afford ester 82 (58%).77 Here an unwanted oxidation of the benzyl ether to a benzoyl ester was observed, but adjusting the reaction time carefully minimized this side reaction. Next, the benzyl ether was subjected to hydrogenation to afford alcohol 83 (75%). To eliminate the risk for epimerization in the following step, during oxidation of the alcohol, Dess-Martin periodinane was employed to afford ketone 84 (81%).71 Removal of the Boc-group was thereafter accomplished in neat formic acid to afford β-strand mimetic 85 (89%). Surprisingly, when 85 was analyzed by chiral chromatography it was found that a partial epimerization of the stereogenic center (60% ee). Fortunately, chiral chromatography of Boc-protected 84, the direct precursor of β-strand mimetic 85, showed no detectable epimerization which proved that the synthetic steps used to obtain 84 had not caused the epimerization. Evidently, the epimerization of 85 had occurred during the acidic conditions applied in the last step to remove the Boc-group.
44
N
FAcHN
OBn
N
NH
AcHN
OBn
N
NAcHN
OBn Boc
N
NAcHN
OBn Boc
OMe
O
N
NAcHN
OH Boc
OMe
O
N
NAcHN
O Boc
OMe
O
N
NH
AcHN
O
OMe
O
AllylamineBoc2ODMAP
86%, from 60
O3
NaOHMeOH
58%
H2
Pd/CMeOH
75%
Dess-Martinperiodinane
65 81
82 83
84 85
60
Formic acid
81%
89%
Scheme 6.13 Synthesis of the β-strand mimetic 85. Chiral chromatography of 85 revealed that partial epimerization had occurred in the last step.
Our first idea to obtain 85 as a single enantiomer was to reverse the order of the two last synthetic steps. Application of acidic conditions to alcohol 83, instead of to the enolizable ketone 84, would eliminate the risk for epimerization (Scheme 6.14). Consequently, alcohol 83 was treated with TFA (25%) in CH2Cl2 to liberate the amine affording 86, directly followed by oxidation using Dess-Martin periodinane for 3 minutes with a reductive workup. This gave β-strand mimetic 85 (66%, from 83) without any detectable epimerization. In this way the synthesis of β-strand mimetic 85 was completed over a twelve step synthetic sequence with an overall yield of 7%.
N
N
Boc
OMe
O
AcHN
OH
N
NH
OMe
O
AcHN
OH
N
NH
OMe
O
AcHN
O
TFACH2Cl2
Dess-Martinperiodinane
83 8586
66%, from 83
Scheme 6.14 By reversing the order of the two final steps, 85 was afforded as a single enantiomer.
45
6.6 Incorporation of a second chiral amino acid analogue and attempts to elongate the β-strand mimetic 6.6.1 Introducing a chiral amino acid analogue instead of glycine as C-terminus As presented above ethanolamine was successfully employed as nucleophile in the SNAr reaction with 2-fluoropyridine or 60 as electrophiles (Scheme 6.7 and 6.8). Therefore the idea to introduce a chiral aminoalcohol, generated from the corresponding amino acid, was explored. When excess S-leucinol was added to 60 in pyridine and heated to 200 °C using microwave irradiation for 1 h, only starting material was recovered. In an attempt to enhance the reactivity, pyridine was excluded from the reaction and 60 was heated in neat S-leucinol (18 equiv., 200 °C for 1.5 h). Somewhat surprising, but really encouraging, the high concentration conditions afforded substituted pyridine 87 in 86% yield (Scheme 6.15). To further challenge the SNAr reaction the β-branched iso-leucinol (31 equiv.) was also employed as nucleophile. Using similar conditions (200 °C for 1.5 h and 210 °C for 0.5 h) gave substituted pyridine 88 (80%).
N
FAcHN
OBn
60
S-leucinol
86%
N
NH
AcHN
OBn
OH
87
N
FAcHN
OBn
60
S-iso-leucinol
80%
N
NH
AcHN
OBn
OH
88
Scheme 6.15 Fluoropyridine 60 was successfully substituted with sterically hindered aminoalcohols.
In an attempt to convert 87, to a compound more similar to the designed β-strand mimetic, the primary alcohol was first protected as a silyl ether using TBDMSOTf and collidine (Scheme 6.16). Then Boc-protection with an excess of Boc2O in the presence of a catalytic amount of DMAP afforded 90. Next, the silyl group was intended to be removed to afford the primary alcohol by using TBAF. The alcohol was then planned to be oxidized either to the corresponding aldehyde or to a carboxylic acid/ester. Unfortunately, under the applied conditions the deprotected alcohol spontaneously attacked the carbonyl group in the Boc-group to give the five membered
46
oxazolidinone 91 (75%, from 87). Because of lack of time no further attempts to transform 87 into a β-strand mimetic were made.
N
NH
AcHN
OBn
OH
87
TBDMSOTfCollidine
N
NH
AcHN
OBn
OTBDMS
N
NAcHN
OBn
OTBDMS
Boc2O
DMAP
Boc
N
NAcHN
OBn
TBAFTHF
OO
89
90 91
75%, from 87
Scheme 6.16 Attempts to further transform 87 into a β-strand mimetic.
6.6.2 Attempts to elongate the β-strand mimetic The Leu-Gly-Gly β-strand mimetic 85, described in section 6.5, may be able to form β-sheets, but the propensity to form β-sheets would most likely be increased if the length of the mimetic was increased. Therefore, studies directed towards increasing the length of the β-strand mimetic were made. First connection of a pyridine moiety at the N-terminal amine was investigated (Scheme 6.17). Heating a large excess of 2-fluoropyridine and 59 in pyridine to 190 °C for 1 h using microwave irradiation did not lead to any consumption of the starting material. The temperature was therefore raised to 220 °C for another hour which resulted in a black reaction mixture. The elongated product 92 could still be isolated, but in a low yield (12%), despite complete consumption of the starting material. A base promoted method, by using excess n-BuLi and 2-fluoropyridine together with 59 (−78 °C → rt), also proved rather inefficient to generate target compound 92 (31%).
N
H2N
OBn
F
59
N
NH
OBn
N
F
92
heat
or
BuLi
12-31%
N
F
+
Scheme 6.17 Attempts to extend the β-strand mimetic at the N-terminal.
We reasoned that a way to improve this reaction could perhaps be achieved by activation of the pyridine ring as electrophile, prior to the SNAr reaction. The pyridine ring could of course be chosen to contain different electron withdrawing groups (EWG’s) (e.g. NO2, CN or SO2R), but transformation of the EWG to afford the target molecule 92 did not seem straightforward.
47
More interesting was the corresponding N-oxide of 2-fluoropyridine which could be reduced after the SNAr reaction. This type of activation has previously been reported to enhance the reactivity in SNAr reactions using amines as nucleophiles.81,82 Therefore oxidation of 2-fluoropyridine to the corresponding N-oxide 93 was achieved by treatment with urea hydrogen peroxide (UHP) and trifluoroacetic anhydride (TFAA).83 The N-oxide of 2-fluoropyridine is highly unstable, it decomposes quickly upon standing, and should be stored in solution. When 59 was reacted with 93 under various conditions the highest yield of 94 was obtained (38%) using pyridine as solvent at 130 °C (microwave irradiation) for 30 minutes. To complete the synthetic route to 92 a reduction of the N-oxide in 94 remained. Use of the most common N-oxide reducing method, ammoniumformate and Pd/C in methanol, left the N-oxide intact. Instead, the reduction of N-oxide 94 was accomplished using a mixture of LiAlH4 and TiCl4 in THF and 92 was afforded in almost quantitative yield based on the NMR spectra of the crude product.84 A comparison of the substitution of 2-fluoropyridine with the N-oxide thereof indicated that the activation of 2-fluoropyridine as an N-oxide was successful, but not sufficient to afford the target molecule in high yields. Both methods suffered from low and variable yields and extension of the β-strand mimetic at the N-terminal was not explored further.
N
NH
OBn
N
F
N
H2N
OBn
F
59
ON
NH
OBn
N
F
94 92
Pyridineheat93
38%
LiAlH4TiCl4
>90%
N
F
UHPTFAA
44%
93
N
F
O
Scheme 6.18 Further attempts to extend the β-strand mimetic by activating 2-fluoropyridine as N-oxide prior to the SNAr reaction.
The focus was now set on elongation of the β-strand mimetic at the C-terminal. The first approach was based on reacting a C-terminal aldehyde, with a pyridine based Grignard reagent. Olefin 79 was chosen as a precursor of the desired model aldehyde 100 (Scheme 6.19). Attempts to prepare aldehyde 96 by acidic hydrolysis of the corresponding acetal 95, using both Lewis and BrØnstedt’s acids, have previously been found to be unsuccessful.67 The literature suggests that this may be due an intramolecular reaction where the pyridine nitrogen atom acts as a nucleophile to form a five membered ring, as in 99, which then undergoes further decomposition. It has, however, been reported that hydrolysis of acetals 97 can provide aldehydes 98, if these are isolated as their hydrochloride salts. In these cases it also seems to be crucial to have an electron withdrawing group directly connected to the pyridine ring in order to decrease the nucleophilicity of the
48
pyridine nitrogen atom.85 In our case the aldehyde was planned to be used as electrophile in a subsequent Grignard reaction and a hydrochloride salt could therefore not be used to prevent side reactions. Instead the use of a Boc-protection group at the amine functionality could maybe decrease the nucleophilicity of the pyridine nitrogen atom to avoid the undesired intramolecular ring closure. The Boc-protected aldehyde 100, or more complex analogues, would provide a useful intermediate in the C-terminal elongation strategy. Consequently, olefin 79 was treated with a stream of ozone and the ozonoide was quenched with PPh3 aiming for aldehyde 100. Unfortunately, no trace of the aldehyde was observed. Instead, 79 was oxidized to diol 101 (88%) using potassium osmate and NMO in a solvent mixture containing acetone, H2O and THF. Diol 101 was thereafter treated with Pb(OAc)4, but again no traces of aldehyde 100 were observed. The findings from the literature described above, together with our own experiences, indicated that aldehydes such as 100 are labile and decompose under the conditions applied for their preparation, despite the Boc-protection.
N
N
Boc
N
N
Boc
O
N
N
Boc
N
N
Boc
O
OH
OH
100
79
101
100
88%K-osmateNMO
Pb(OAc)4
Na2CO3
O3
reductivework-up
N
OOMe
OMeMe
I
N
OO
Me
I
various conditions
95 96
N
NH
ON
NH
OHCl
EWGEWG
N
NH
O
EWGO
HCl
98 99
HCl
97
Scheme 6.19 Hydrolysis of 95 was unsuccessful in a previous study even though several methods were tested. The problem could be explained by the published side reaction, where the pyridine nitrogen atom in 98 acts as a nucleophile that attacks the generated aldehyde moiety to form the five membered ring 99. In our attempts to prepare aldehyde 100 a Boc-protection group at the amine functionality was used in an attempt to prevent the undesired intramolecular ring closure.
49
N
FH2N
OBn
Allylamine
31%, from 49
N
NH
H2N
OBn
N
NH
NH
OBn
N
O
NHBoc
49 59
OBn
AcHN
102
103
heatN
FAcHN
OBn
60
+ 102
Scheme 6.20 A synthetic approach attempting to connect tripeptidomimetic 102 with dipeptidomimetic 60 using microwave irradiation without solvent.
Encouraged by the previously performed substitution of 60 with leucinol and iso-leucinol (Scheme 6.15), a tripeptidomimetic 102 was prepared (31% yield from aldehyde 49, Scheme 6.20). Fluoropyridine 60 was then heated (210 °C for 1.5 h) in 102 (4 equiv.) using microwave irradiation without any solvent. The reaction turned into a black solid which after aqueous workup and purification (silica-gel) revealed complete consumption/decomposition of both starting materials, unfortunately without any observed formation of pentapeptidomimetic 103 (LCMS, NMR).
6.6.3 Conclusions Elongation, both at the N- and C-terminal, of pyridine based di- and tri-β-strand mimetics to obtain a tetra- or penta-β-strand mimetic turned out to be problematic and could not be solved within the stipulated time. Still the chemistry developed to afford the tri-β-strand mimetic, could be used to produce a wide scope of peptidomimetics with interesting properties (see Chapter 7 and 8).
6.7 Summary A synthetic route to a tripeptide β-strand mimetic of Leu-Gly-Gly, based on a pyridine scaffold, has been developed. The amide bonds of the original peptide were replaced by a carbon-carbon bond connected at the 4-position and by an amine at the 2-position of the pyridine ring, respectively. The pyridine moiety mimics the central amino acid (Gly) in the chosen sequence and originates from 2-fluoro-4-iodopyridine, which was used as starting material in the synthetic sequence. The crucial connection of the N-terminal leucine moiety at position 4 of the pyridine ring was accomplished by using a Grignard exchange reaction with Boc-protected leucinal as electrophile. The other key transformation, an SNAr reaction of fluorine in 2-
50
fluoropyridine ring using allylamine as nucleophile, was accomplished by using elevated temperatures and a large excess of amine. Boc-protection of the 2-aminopyridine moiety proved essential or at least beneficial for many of the transformations attempted. Incorporation of more sterically demanding amino acid analogues (Leu and Ile) as C-terminal residues in the tripeptidomimetic was also accomplished in high yields by using aminoalcohol derivatives. Transformation of the generated compounds containing two chiral centers to more sophisticated β-strand mimetics remains to be accomplished.
Elongation of the β-strand mimetic, both at the N- and C-terminal, have also been attempted in order to increase the propensity of the mimetic to aggregate into β-sheets. Thus, the N-terminal was capped with an additional pyridine ring via an SNAr reaction using 2-fluoropyridine, or the N-oxide thereof, as electrophiles, but the desired product was unfortunately obtained in variable and low yields. Next, attempts to prepare a C-terminal aldehyde were made in efforts to enable a stepwise elongation, alternating between a Grignard reaction and an SNAr reaction. This strategy failed already during the synthesis of the aldehyde, which appeared to decompose under the oxidative conditions applied. Previous work on hydrolysis of an acetal to afford a closely related aldehyde has also proven unsuccessful. The third strategy to obtain an extended β-strand mimetic was to synthesize a dipeptidomimetic and a tripeptidomimetic and fuse them via an SNAr reaction at elevated temperatures without any solvent. The desired pentapeptidomimetic was not generated despite complete consumption of both starting materials.
51
7. Thrombin inhibitors
7.1 Biological action of thrombin In the developed countries blood coagulation disorders is the single most common cause of death. Abnormal blood coagulation (thrombosis) can lead to myocardial infarction and stroke. The sophisticated blood coagulation cascade is regulated by several enzymes, both by an intrinsic pathway, with all components present in the blood stream, and an extrinsic pathway that is initiated upon injury by release of thromboplastin. Thrombin acts as the key enzyme in the last step of both pathways (Figure 7.1).86 Thrombin hydrolyzes an amide bond between Arg and Gly in fibrinogen, by use of a catalytic triad involving the three amino acids Asp, His and Ser. In this way fibrinogen is converted into the insoluble fibrin which forms the matrix of a blood clot. Inhibition of the enzymatic activity of thrombin will reduce blood clotting and thereby decrease the risk to develop disorders related to excessive blood coagulation.
intrinsic and extrinsicregulation
factor X factor Xa
prothrombin (factor II) thrombin (factor IIa)
fibrinogen fibrin blood clot
Figure 7.1 The last part of the sophisticated blood coagulation cascade. Factor X is converted to factor Xa both by intrinsic and extrinsic regulation. Factor Xa converts prothrombin to thrombin, which converts fibrinogen to fibrin that forms blood clots.
52
Thrombin is a trypsin like protease which belongs to the serine protease family. It was first crystallized in 1989 together with the covalently bound inhibitor PPACK (D-Phe-Pro-ArgCH2Cl).87,88 The active site is well characterized, and usually divided into three domains; the S1-, S2- (or P-pocket) and S3-pocket (or D-pocket, Figure 7.2). The S1-pocket is a hydrophobic channel with the acidic side chain of Asp in the bottom, which is known to bind strongly to basic moieties such as guanidine and amidine. The central S2-pocket, which is lined by the side chains of Tyr and Trp, has shown good binding to lipophilic structures containing 4-6 membered cyclic structures.86
O O
S1-pocket
Spacer
NH2H2N
Asp189
Trp60D
Tyr60AIle174
Trp215 Leu99
S2-pocket
S3-pocket
Figure 7.2 A schematic picture of the active site of thrombin occupied by a fictive thrombin inhibitor containing a charged amidine as an anchor to Asp189 in the S1-pocket.
The third domain of the active site, the S3-pocket, displays hydrofobic and aromatic amino acid side chains and is known to interact well to aromatic residues. A thrombin inhibitor should contain three structural fragments P1, P2 and P3 with correct functional groups, size and orientation to favor binding to the corresponding pockets in the active site of thrombin. The P1-residue is intended to bind to the S1-pocket and is often represented as a hydrophobic spacer with a terminal basic functionality. The P2 domain of the inhibitor often consists of a 4−6 membered cyclic structure which allows hydrophobic interactions in the S2-pocket. The remaining P3 residue of a thrombin inhibitor is often an aromatic group and interacts with the residues lining the S3-pocket.89 Several research groups and pharmaceutical
53
companies have put great efforts into the development of low molecular weight, orally active direct thrombin inhibitors. The progress has so far resulted in Exanta, which was launched to the market although then later withdrawn (see Chapter 3.4).90,91 One problem is related to poor passage over membranes, due to a strongly basic group in the P1 residue frequently used to achieve high potency inhibitors.
7.2 Structure based design As a part of our efforts to develop chemistry based on substituted pyridines we looked into the possibility to apply the chemistry outlined in Chapter 6 in the synthesis of potential thrombin inhibitors. Using the P1-P2-P3 nomenclature for the thrombin inhibitor, it was predicted that the pyridine ring could serve as a P2 scaffold. A para-amidino-benzylamine residue, known from many thrombin inhibitors, constituted a good P1 substituent with the basic characteristics to anchor in the S1 pocket.92-96 Various benzoyl groups containing no, or a small substituent, eg. o-OMe or m-Me, were chosen as P3 residues (Figure 7.5). Figure 7.3 shows the Glide docking of compound 130, with an interaction between the amidine and Asp 189, as well as a potential hydrogen bond between the benzoyl carbonyl group and the phenolic hydroxyl group in Tyr 60A.97
O
N
HN
NH2
NH
130
* AcOH
Figure 7.3 Compound 130 flexibly docked into the active site of thrombin (PDB code 1K22) using standard precision Glide with a rigid receptor. The molecular surface is colored by atom type, where red, blue, yellow and white denote oxygen, nitrogen, sulphur and carbon, respectively.
7.3 Retrosynthetic analysis of the thrombin inhibitors A retrosynthetic analysis of the designed thrombin inhibitors IX revealed that halogenated pyridine VII could be used as a central building block (Figure 7.4).
54
O
N
NH
R2
R1
N
FI
IX
VII
XXI
R1
H2N
R2n = 0,1O
n = 0,1
++
Figure 7.4 A retrosynthetic analysis of thrombin inhibitor IX suggests that 2-fluoro-4-iodopyridine (VII) could be used as scaffold.
Pyridine derivative VII was proposed to be substituted at position 4 via a Grignard exchange reaction, employing aldehydes X as electrophiles.64,66,67 R1 in aldehydes X was designed to be small and stable during the synthetic sequence leading to the final inhibitors. The next key step was planned to be an SNAr reaction at position 2 of the pyridine ring by using benzylamines XI as nucleophiles. The substituents (R2) in benzylamines XI should be a functional group, e.g. Br or CN which can be transformed to the desired amidine (Figure 7.5).98-100
O
N
HN
NH2
NH
132
O
N
HN
NH2
NH
131
O
N
HN
NH2
NH
130
OMe
Me
* AcOH* AcOH * AcOH
Figure 7.5 Three designed thrombin inhibitors to be synthesized.
7.4 Synthesis of thrombin inhibitors 7.4.1 Attempts to obtain thrombin inhibitors via a Grignard exchange reaction followed by an SNAr reaction using substituted benzylamines
First 2-fluoro-4-iodopyridine was treated with iso-propyl magnesium chloride to form a pyridine Grignard reagent. This was quenched with benzaldehyde to afford alcohol 104 (86%) or with phenylacetaldehyde to obtain alcohol 106, respectively (Scheme 7.1). The secondary alcohol in 106
55
was oxidized directly to ketone 107 (60%, from VII) to avoid elimination to the corresponding alkene.
N
FI
OH
N
F
OH
N
F
O
N
F
104
106 107
iPrMgClbenzaldehyde
86%
60%, from VII
iPrMgClphenylacetaldehyde
VII
Dess-Martinperiodinane
KHQIPMBCl
OPMB
N
F
105
37%
Scheme 7.1 Substitution of position 4 of the pyridine scaffold via a Grignard exchange reaction.
The secondary alcohol in 104 should be protected with a suitable protective group tolerating the harsh conditions in the subsequent SNAr reaction. Protection of the alcohol as a benzyl ether was ruled out because of the benzylic nature of the alcohol in 104. Protection with a silylether could be an option but it would probably be cleaved when applying high temperature and an excess amine in the SNAr reaction. Additionally, the fluoride ion generated in the SNAr reaction could contribute to cleavage of the silyl group. Therefore a strategy using a PMB ether which is deprotected by oxidative conditions (DDQ) was chosen. Attempts to prepare PMB ether 105 using PMBCl under phase transfer conditions resulted in multiple products. The PMB ether was instead obtained, but only in moderate yield (37%), by using KH and PMBCl in DMF at 0 °C.
The SNAr reaction was then attempted by heating 105 and an excess 4-bromobenzylamine (7.5 equiv.) in pyridine to 180 °C for 1 h using microwave irradiation. Unfortunately, only unreacted starting material 105 was recovered when applying these conditions. To reach higher temperatures, NMP (5%) was added as co-solvent to increase the microwave absorption (Scheme 7.2). The reaction mixture was thereafter heated to 220 °C for 1 h. Starting material 105 was still the major compound in the reaction mixture, but the desired benzylamine substituted pyridine 108 was formed in small amounts (<10% according to LCMS analysis) together with equimolar amounts of the byproduct 109 (LCMS analysis).
56
OPMB
N
F4-Br-benzylamine,pyridine, NMP
OPMB
N
HN
Br
105 108
+
OPMB
N
NH2
109
<10%
Scheme 7.2 An attempt to use 4-bromobenzylamine as nucleophile in the SNAr reaction.
In summary, the attempts to achieve 108 via PMB ether 105 were found to be sluggish and also suffered from low yields and formation of a byproduct. Further attempts to complete the synthetic route to the target thrombin inhibitor via PMB ether 105 were therefore not made.
OH
N
F
O
N
F
104
Dess-Martinperiodinane
88%
O
N
F
107
110
O
N
HN
Br
O
N
HN
CN
111
113
4-Br-benzylaminePyridineheat
4-CN-benzylaminePyridineheat
trace amounts
+
O
N
NH2
112
Scheme 7.3 Attempts to substitute fluoropyridines 110 and 107, using substituted benzylamines as nucleophiles.
The ketone attached to the pyridine ring at position 4, as in 110 and 107, decreases the electron density in the pyridine ring and should thereby increase the reactivity of the fluoropyridine in the SNAr reaction. Therefore alcohol 104 was oxidized to ketone 110 by using Dess-Martin periodinane (88%, Scheme 7.3). Displacement of the fluorine atom using a para-substituted benzylamine as nucleophile was thereafter attempted on both 110 and 107. 4-Cyanobenzylamine (3.3 equiv.) was heated together with 107 in pyridine using microwave irradiation (100 °C, 0.5 h). The reaction turned black under these relatively mild conditions and did not afford any product, but 107 could be recovered. Instead, fluoropyridine 110 and an excess 4-bromobenzylamine (3.3 equiv.) was heated in pyridine by microwave irradiation (130 °C, 0.5 h). The main part of starting material 110 remained unreacted but small amounts of 111 were also observed (LCMS). Unfortunately byproduct 112 was formed in equal amounts as the desired product 111. Thus, also with activation from the ketone connected to the
57
pyridine ring, the SNAr reaction using para-substituted benzylamines was unsuccessful.
7.4.2 A reductive amination approach Next, reductive amination was explored as an approach to the desired thrombin inhibitors. This approach was previously explored when developing the synthetic route to pyridine based β-strand mimetics (Chapter 6.4.2). First, the fluorine atom in 110 had to be transformed to the corresponding aminopyridine 112 (Scheme 7.4). The transformation was accomplished by heating 110 in 25% aqueous ammonia in a sealed steel cylinder as described earlier (Chapter 6, Scheme 6.10).80
O
N
F
110
O
N
NH2
NH
N
NH2
+
O
N
NH225% NH3 in H2Oheat citric acid 10% (aq.)
94%
112
112
114
Scheme 7.4 Preparation of aminopyridine 112 for use in a reductive amination approach.
All starting material was consumed when the reaction was run at ∼150 °C for 10 h, but TLC revealed formation of two products. Analyzing the two compounds by NMR spectroscopy suggested very similar structures. The assumption that 112 was accompanied by the relatively stable imine 114 was made, and therefore the crude ketone/imine mixture was treated with 10% citric acid (aq.) to hydrolyze the imine. After 2 h only one compound was detected by TLC, and the desired aminopyridine 112 was obtained in excellent yield (94%). The reductive amination should then be performed between aminopyridine 112 and 4-cyanobenzaldehyde, where the cyano group was intended to serve as a masked amidine (Scheme 7.5). Several reaction conditions, with variations of reducing agents (Na(OAc)3BH or NaCNBH3), solvents (CH2Cl2, MeOH or mixtures thereof), drying agents (molecular sieves) and pH adjustments (acetic acid or TEA) were explored but without sufficient success.101,102 At best benzyl substituted aminopyridine 115 was generated in a moderate 12% yield using the conditions outlined previously (TEA and Na(OAc)3BH in CH2Cl2, Scheme 6.9).49 Since the SNAr approach to the thrombin inhibitors had failed, it was critical to find a route based on reductive amination. Therefore preformation of the imine, using Dean-Stark conditions, followed by application of reducing conditions was investigated.103 The imine was formed by reaction of 112, excess 4-
58
cyanobenzaldehyde (1.3 equiv.) and TEA in refluxing toluene for 10 h. The solvent was then removed and CH2Cl2 was added along with Na(OAc)3BH and the reaction was stirred for another 3 h. It was found that three repetitions employing the Dean-Stark method followed by reduction generated 115 in high yields. The product was heavily contaminated by 4-cyanobenzyl alcohol despite attempted purification on silica gel and was Boc-protected79 by using excess Boc2O and catalytic amount of DMAP to afford 116 in a satisfying 77% yield from 112. When the same strategy was applied to ketone 107 the SNAr reaction with NH3 failed, probably due to aldol condensation of 107. The synthetic route to thrombin inhibitors based on 107 was therefore not investigated further.
O
N
NH2
112
reductive
amination
O
N
HN
CN
115
Boc2O
DMAP
CH2Cl2
77% from 112
O
N
N
CN
116
Boc
O
N
F
25% NH3 in H2O
heat
O
N
NH2
107 117
Scheme 7.5 Aminopyridine 112 was subjected to several reductive amination strategies and 116 was finally obtained in high yield.
7.4.3 Conversion of the cyano group to the desired benzamidines With all structural motifs in place, conversion of the cyano group to the desired amidine remained to be accomplished. When Pinnerer conditions was applied to 116, followed by addition of NH3 in MeOH, only cleavage of the Boc-protection group was observed.100 Treatment of the cyano group with LiHMDS resulted in formation of multiple products. However, reacting 116 with hydroxylamine in the presence of DIPEA in refluxing ethanol, followed by O-acetylation afforded 119 (Scheme 7.6).98 The labile nitrogen−oxygen bond was then cleaved by hydrogenation at normal pressure over Pd/C in acetic acid. Protection of the crude product as a tert-butyl carbamate revealed that three products had been formed. These were alcohol 121, which was obtained in minor amounts, deoxygenated 122, obtained as the main product and amine 123, formed in trace amounts.
59
O
N
N
CN
116
Boc O
N
N
Boc
O
N
N
Boc OH
N
N
Boc
NH
NH
OH
NH
NH
OAcNH2
NH
OH
N
N
Boc NH
NH
Boc
N N
NH2
+ +
118
119 120
122 123
121, 22% from 116
hydoxylamineDIPEAEtOHreflux
AcOHAc2O
H2
Pd/C
Boc2OCH2Cl2
Scheme 7.6 An unreliable synthetic sequence to Boc-protected thrombin inhibitor 121.
To suppress formation of the unwanted 122 in the hydrogenation step the affect of the solvent was investigated. First THF was tested but despite hydrogenation for two hours only unreacted starting material (119) was recovered. In contrast the labile nitrogen−oxygen bond was cleaved quite rapidly in methanol. The ketone in 119 was still reduced to the corresponding alcohol, but deoxygenation to compound 122 was suppressed and the protected thrombin inhibitor 121 could be obtained in 22% yield after Boc-protection. However, the reaction outcome was not consistent between different batches and the yield varied between 0 and 22%. In order to find a more robust synthetic sequence alternative pathways were sought for. Since the problems seemed to originate from the ketone present in 116, it was decided to replace the ketone with a protected alcohol. A protected alcohol would avoid oxime formation in the first step when employing hydroxylamine as nucleophile. Therefore, ketone 116 was reduced to the corresponding alcohol by using NaBH4, followed by protection as a silylether using TBDMSOTf, to afford 124 (82% from 116, Scheme 7.7). Thereafter the conversion of the cyano group to an amidine was performed in the same manner as described above to afford 128.98 Satisfyingly, the silylether proved considerably more stable towards hydrogenation than the ketone and the deoxygenated byproduct was only detected in trace amounts (LCMS). The formed amidine 127 was Boc-protected followed by purification on silica gel to afford 128 (66% from 116).
60
O
N
N
CN
116
Boc
OTBDMS
N
N
Boc
N
N
Boc
OTBDMS
N
N
Boc
NH
NH
OHNH
NH
OAc
NH2
NH
OTBDMS
N
N
Boc NH
NH
Boc
125 126
127
128, 66% from 124
AcOHAc2O
H2
Pd/CMeOH
Boc2OCH2Cl2
OTBDMS
N
N
CNBoc
hydoxylamineDIPEAEtOHreflux
2. CollidineTBDMSOTf
1. NaBH4
82%
124
OTBDMS
Scheme 7.7 A reliable synthetic procedure to afford protected thrombin inhibitor 128.
The silylether was then cleaved from 128 using TBAF in THF to afford 121, followed by oxidation employing Dess-Martin periodinane to give ketone 129 (52% from 128, Scheme 7.8).71 The remaining transformation, i.e. removal of the Boc-protection group, was achieved by treating 129 with 75% TFA in CH2Cl2 for 35 minutes. The crude product was purified on reversed phase HPLC to give the target thrombin inhibitor 130 in 63% yield as an acetate salt.
OTBDMS
N
N
Boc NH
NH
Boc
128
OH
N
N
Boc NH
NH
Boc
O
N
N
Boc NH
NH
BocO
N
HN
NH2
NH
121
129 130
52% from 128 63%
AcOH
TBAFTHF
Dess-martinpeiodinane
TFACH2Cl2
*
Scheme 7.8 Completing the synthesis of thrombin inhibitor 130.
The previously designed thrombin inhibitors 131 and 132, containing substituents in the benzene ring, were prepared according to the same
61
synthetic protocol as 130 (Figure 7.5, for details see paper IV). Thrombin inhibitors 130, 131 and 132 were obtained over a 14 step synthetic sequence with an overall yield ranging from 10 to 14%.
7.5 Biological evaluation Compounds 130, 131 and 132 were evaluated as thrombin inhibitors in an enzymatic assay (Table 7.1).104 The results show that binding is influenced by the substitution pattern of the benzoyl group, i. e. the P3 residue. The ortho-methoxy group in 131 decreases the binding affinity while the meta-methyl group in 132 results in an equally active inhibitor as unsubstituted 130. It is possible that the proposed hydrogen bond between the carbonyl group of the inhibitors and the hydroxyl group of Tyr 60A in the binding pocket is disrupted by the ortho-substituent. However, all compounds in the series showed only modest inhibition of thrombin.
Table 7.1 Evaluation of compounds 130, 131 and 132 as thrombin inhibitors in a competitive enzyme assay.
O
N
HN
NH2
NHAcOH*
R Compound IC50 (µM) 130 R=H 15 131 R=o-OMe > 44.4* 132 R = m-Me 11 * The highest concentration tested in the assay.
7.6 Crystal structure Even though the affinity was moderate, a crystal structure of 132 in the active site of thrombin could be obtained (Figure 7.6a). As expected, the amidine group was found to anchor 132 in the S1 pocket of thrombin by binding to Asp 189. A comparison between 132 and the crystal structure of the potent thrombin inhibitor melagatran (PDB code 1k22) reveals a quite good shape match between the two inhibitors (Figure 7.6b). However, melagatran forms three hydrogen bonds to the backbone of the enzyme in the S2-S3 region, in addition to its interaction with Asp189 in the S1 pocket (Figure 7.6c). Such hydrogen bonds are absent for 132 (Figure 7.6a) and this difference most likely explains the relatively low potency of 132. In general
62
it can be concluded that the hydrogen bonds in the S2-S3 region of thrombin are of great importance and should be considered as a high priority in the design of thrombin inhibitors.
Figure 7.6 a) The crystal structure of thrombin inhibitor 132 in the active site of thrombin. b) An overlay of melagatran (green) and 132 (blue) when bound by thrombin. c) Melagatran bound in the active site of thrombin with the hydrogen bonding network in the S2 and S3 pockets displayed (red dashed lines). In the S2-S3 region are hydrogen bonds formed between two of the amide nitrogen atoms and one of the carbonyl oxygen atoms and the backbone of the enzyme.
A comparison of the crystal structure with results from Glide dockings show that docked 130 fill the S2 pocket in a better way than was borne out in practice (cf Figures 7.3 and 7.6a). The hydrogen bond between the benzoyl carbonyl group and the phenolic hydroxyl group in Tyr 60A, also suggested from dockings, was not formed in the cocrystal. It is possible that elongation of the benzylamine moiety to a phenylethylamine could give a compound with a better fit to the enzyme, e.g. filling out more of the S2 and S3 pockets and also reaching the important hydrogen bonds.
7.7 Summary A structure based design of a class of potential thrombin inhibitors containing a 2,4-disubstituted pyridine as a central fragment (P2 residue) has been made. The P1 residue, connected to the pyridine ring in position 2, was designed to contain a para-amidinobenzylamine moiety which could anchor to Asp 189 in the S1 pocket of thrombin. Position 4 in the pyridine ring was
63
planned to be substituted with various benzoyl groups, constituting the P3 residue of the inhibitor. Substitution in position 4 of the pyridine ring was achieved via a magnesium-iodide exchange of 2-fluoro-4-iodopyridine using iso-propyl magnesium chloride. The Grignard reagent was then quenched with various benzaldehydes. The P1 residue was introduced at position 2 in the pyridine ring using ammonia as nucleophile in an SNAr reaction, followed by a reductive amination accomplished by preformation of an imine in a Dean-Stark apparatus followed by addition of reducing agent. Other methods to carry out the reductive amination of the 2-aminopyridine moiety were unsuccessful. The desired benzamidine was obtained from a cyano group in four steps including a catalytic hydrogenation which suffered from selectivity problems. The selectivity issue was overcome by careful adjustment of the reaction conditions and by proper protection of the functionality that was sensitive to hydrogenation.
The synthesized compounds were evaluated as thrombin inhibitors in an enzymatic assay. Unfortunately, none of the compounds showed high affinity for thrombin, with an IC50 value of 11 µM as the best result.
64
8. Thrombin inhibitors with reduced basicity
8.1 Introduction Guanidine and amidine moieties are known to interact with the side chain of Asp 189 located at the bottom of the S1-pocket in the active site of thrombin (see Chapter 7.1).86,89 These strong bases are also known to make the passage over membranes problematic due to a positive charge at physiological pH. Therefore, compounds containing such strongly basic functionalities are generally considered unsuitable in drugs intended to be orally administered because of poor bioavailability. An interesting but challenging task is to prepare anticoagulants with good bioavailability and pharmacokinetics. One major task required for success is to replace the strongly basic moiety with less basic structures without losing the thrombin inhibiting potency.105-107
8.2 Structure based design and retrosynthetic analysis A structure based design was performed in the same way as described in Chapter 7.2 and revealed compounds XV as potential thrombin inhibitors.97 The basic moiety, often needed to obtain high potency thrombin inhibitors, has been replaced by less basic functionalities, i.e. a primary amine, a pyridine or a chlorophenyl group, which would provide better pharmacokinetic profiles (Figure 8.1). The aryl amide serves as P1 residue while the 2-aminopyridine was designed to function as a P3 residue. The central P2 fragment of the inhibitors provides possibilities for hydrogen bond formation with the enzyme. In addition, a flexible P2 region increases the conformational freedom for the terminal residues, P1 and P3, thereby allowing them to enter their respective parts of the active site. The structure based design revealed that a large R2 substituent would be involved in a steric clash within the active site of the enzyme and R2 was therefore chosen to be hydrogen or methyl (for specific structures see Figure 8.3).
65
OH
N
Nn = 0,1
NH
R2XR1
O
XV
P1 P2 P3
Figure 8.1 The designed thrombin inhibitors XV. X = C or N; R1 = Cl or CH2NH2 if X = C; R2 = H or Me; The cyclic amine in the P3 moiety was designed to be pyrrolidine or morpholine.
A retrosynthetic analysis, based on earlier experience, lead to a synthetic route starting with 2-fluoro-4-iodopyridine (VII) which was to be sequentially substituted in positions 4 and 2 (Figure 8.2). Protected aminoaldehydes XII were to be incorporated at position 4 of the pyridine ring via a Grignard exchange reaction.64,67 The cyclic amines XIII were intended to be introduced via an SNAr reaction by displacement of the fluorine atom in position 2 of the pyridine ring.108 Completion of the synthesis was to be accomplished via an amide bond formation between the amino group of aldehydes XII, and carboxylic acids XIV.
OH
N
N
N
FI
VII
X
n = 0,1NH
R2XR1
OH
OR1
n = 0,1
O
HN
PgHN
O
R2
XII
XIII
XIV
XV
Figure 8.2 A retrosynthetic analysis of potential thrombin inhibitors XV.
8.3 Synthesis of thrombin inhibitors 8.3.1 Synthesis of Boc-protected alaninal and glycinal To begin with, syntheses of glycinal and alaninal were attempted from Boc-protected ethanolamine 133 or from racemic Boc-protected alaninol 134, respectively (Scheme 8.1). First, Dess-Martin periodinane was employed in the oxidation of Boc-protected ethanolamine 133. The reaction was
66
monitored by TLC, which already after a few minutes showed complete conversion of the starting material. After standard reductive work-up using sodium bisulfite, multiple products were detected by TLC. The crude product was analyzed by NMR spectroscopy and was found to contain only small amounts of aldehyde 135. Somewhat surprisingly also other oxidation methods attempted such as Swern oxidation,70 TEMPO oxidation68 and ozonolytic cleavage of Boc-protected allylamine failed to generate the desired aldehyde.
H2NOH
BocHNOH
BocHNO
Boc2OCH2Cl2
Dess-Martinperiodinane
66%, fromethanolamine
H2NOH
BocHNOH
BocHNO
Boc2OCH2Cl2
Dess-Martinperiodinane
73%, fromalaninol
Me Me Me
133
134 136
135
Scheme 8.1 Aldehydes 135 and 136 were afforded in high yields and purity, but a modified work-up procedure was required after the Dess-Martin periodinane oxidation.
There are reports in the literature that support difficulties to afford Boc-protected glycinal, with 2,5-diketopiperazine formed as the major byproduct.109 To avoid this dimerization and overoxidation, the procedure for the Dess-Martin periodinane oxidation was altered. The concentration of the starting material was kept low during the reaction and 2-propanol was used for reductive work-up followed by filtration through silica gel. This afforded aldehyde 135 in high purity and good yield (66%) as a colorless crystalline compound. The same method was also applied to 134 which smoothly generated aldehyde 136 (73%).
8.3.2 A Grignard reaction and nucleophilic aromatic substitution with cyclic amines 2-Fluoro-4-iodopyridine (VII) was treated with iso-propyl magnesium chloride to afford an iodo-magnesium exchange (Scheme 8.2).
67
BocHN
R
O
N
F
OTBDMS
BocHN
R
N
F
OTBDMS
H2N
R
N
N
OTBDMS
H2N
R
N
N
OTBDMS
H2N
R
O
i-PrMgClVII
THF
TBDMSOTfCollidineCH2Cl2
Formicacid
135 R = H136 R = Me
139 R = H140 R = Me
141 R = H, 41% from 135142 R = Me, 45 % from 136
143 R = H, 76% 144 R = Me, 69%
145 R = H, 71%146 R = Me, 79%
N
F
OH
BocHN
R
137 R = H138 R = Me
Pyrrolidineheat
morpholineheat
Scheme 8.2 The synthetic route to the P2-P3 residues to be coupled with the P1 residues via an amide bond formation.
The Grignard reagent was quenched by addition of glycinal or racemic alaninal to afford substituted pyridines 137 and 138 followed by protection of the crude material as benzyl ethers as earlier described (cf. paper III, Chapter 6.3).74 However, deprotection of the benzyl ethers by hydrogenation at a later stage of the synthetic sequence proved unsuccessful. Therefore the use of a silyl ether as protection group was explored (earlier this was avoided due to anticipated cleavage during the SNAr reaction). Thus, alcohols 137 and 138 were treated with TBDMSOTf and collidine in CH2Cl2 to afford silyl ethers 139 and 140. The Boc-protection group was removed to give amines 141 and 142 in good yields over three steps (141 41% and 142 45%).
The SNAr reaction108 was now performed on 141 using pyrrolidine as solvent (microwave assisted heating at 130 °C, 40 minutes), but substituted pyridine 143 was only obtained in moderate 38% yield. Not surprisingly, LCMS revealed a partial cleavage of the silyl protecting group. Therefore, re−silylation using the same conditions as above was applied to the crude product before aqueous work-up. This method afforded 143 in a satisfying 76% yield.
With the Grignard reaction, the SNAr reaction and the protection group strategy outlined, 144, 146 and 145 were also prepared in good overall yields (when using morpholine as nucleophile in the SNAr reaction the temperature was raised to 165 °C for 1 h).
8.3.3 Completing the synthesis The carboxylic acids 4-chlorophenylacetic acid, 4-pyridylacetic acid and 4-(Boc-aminomethyl)benzoic acid, selected in the structure based design, were coupled with amines 143, 145, 146 and 144, using HATU and DIPEA in
68
CH2Cl2,51 to generate a small library of eight compounds (Figure 8.3). Cleavage of the silyl ether, by using TBAF in THF, completed the synthesis of the library. To afford compound 154, the Boc-protection group was removed prior to cleavage of the silyl ether.
N
NNH
OH O
O
N
NNH
OH
O
Cl
Cl
N
NNH
OH O
O
N
NNH
OH
O
Cl
ClMe
Me
N
NNH
OH O
O
N
NNH
OH
O
N
N
N
NNH
OH O
ON Me
N
NNH
OH
O Me
H2N
147, 17% 148, 59%
149, 30% 150, 54%
154, 40%*153, 62%
151, 51% 152, 59%
Figure 8.3 The potential thrombin inhibitors lacking a strongly P1 fragment. Yields given in the figure are over the two last steps. * Yield over three steps.
8.4 Biological evaluation The eight compounds 147 → 154 were evaluated as thrombin inhibitors using the same assay as described in Chapter 7.3.104 Unfortunately all compounds (see Figure 8.3 for structures) were inactive at the highest concentration used (44.4 µM).
8.5 Summary A small library of eight compounds was designed, synthesized and evaluated as thrombin inhibitors. The synthetic route started with an oxidation of Boc-protected aminoalcohols to the corresponding aldehydes glycinal and alaninal. An alternative work-up procedure was applied and both aldehydes were obtained in high purities and acceptable yields. The aldehydes were used as electrophiles in a Grignard exchange reaction using 2-fluoro-4-
69
iodopyridine and iso-propylmagnesium chloride. The fluorine atom in position 2 of the pyridine ring was then displaced with cyclic amines in an SNAr reaction. To complete the synthesis, an amide bond was formed between three different carboxylic acids and the amino group originating from glycinal and alaninal, respectively. The compounds were evaluated as thrombin inhibitors in an enzyme assay but none of them showed any activity at the highest concentration used in the assay (44.4 µM).
70
9. Concluding remarks
The bioactive conformation of peptide hormones binding to membrane bound receptors is difficult to determine due to the lack of crystal structures for such complexes. One approach towards revealing the bioactive conformation in such cases is by design, synthesis and evaluation of conformationally restricted peptidomimetics (Chapter 5).
In an attempt to gain further understanding of the bioactive conformation of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu), a ten membered β-turn mimetic was designed and synthesized (Figure 9.1). A key step in the synthetic sequence was a lactamization to afford the ten membered ring, which was achieved under high dilution condition. The ten membered β-turn mimetic was then subjected to an NMR study, which revealed that the conformation in solution closely resembled the crystal structure of Leu-enkephalin and an ideal type II β-turn.
O
HN
N
O
O
H2N
OH
NH
OO
HO
1
H2N
N
O
OHN
O
O
OH
HO
2
Figure 9.1 Ten- and the seven membered turn mimetics incorporated in Leu-enkephalin.
Also a seven membered turn mimetic lacking one of the two glycine residues of Leu-Enkephalin was synthesized. In this mimetic the residues important for receptor binding would be oriented slightly different than in the ten membered mimetic. Several of the reaction steps to afford the seven membered turn mimetic were carried out on solid phase, including the lactamization. To probe the effect of cyclization the linear analouges of the ten- and the seven membered turn mimetics were also synthesized. The four compounds were evaluated in a receptor binding assay towards the µ and δ opiate receptors. The ten membered ring showed no affinity at any of the
71
two receptors, while its linear analogue was the most active compound at both receptor subtypes (IC50, µ = 14 nM, δ = 1.3 nM). In contrast, the shorter linear peptidomimetic showed no affinity at any of the two receptors while the seven membered turn mimetic had affinity at both receptors (IC50, µ = 740 nM, δ = 160 nM). Thus, the biological evaluation indicated that the bioactive conformation of Leu-enkephalin probably is a turn, but not the ten membered β-turn probed by our original design. In conclusion, the seven membered ring showed promising results, and has potential to be developed further to evaluate the properties needed for binding at the opiate receptors. One approach could be to investigate the influence of incorporation of different amino acids. Other structural modifications would need more complex synthetic work. A β-strand is a peptide sequence oriented in an extended conformation. This structural motif is often involved in protein-protein and protein-DNA interactions (Chapter 6). β-Strands and their mimetics have therapeutic potential in diseases associated with abnormal β-sheet formation, such as Alzheimer’s and Parkinson’s disease, or as inhibitors of proteolytic enzymes. The important functions of β-strands encouraged us to design and develop a synthetic route to a β-strand mimetic (Figure 9.2). The synthetic strategy was based on a pyridine scaffold, which was substituted in positions 2 and 4 with amino acid analogues. The mimetic was designed to attain an extended conformation with simultaneous replacement of the amide bonds by proteolytically more stable alternatives. The two key steps of the synthetic sequence was to attach the two amino acid analogues to the pyridine scaffold via a Grignard exchange reaction followed by an SNAr reaction. A β-strand mimetic of Leu-Gly-Gly was prepared, and initial results towards exchanging the C-terminal Gly with Leu and Ile were made.
N
NH
AcHN
OBn
OH
88
N
NH
AcHN
O
OMe
O
85
Figure 9.2 The synthesized β-strand mimetic of Leu-Gly-Gly, and the substitution product using iso-leucinol as nucleophile in the SNAr reaction.
Synthetic attempts towards elongated β-strand mimetics containing four and five amino acid residues were also made but only with moderate success. The synthesis of a pentamer β-strand mimetic based on the pyridine scaffold seems to be a significant synthetic challenge and today no clear way forward can be seen.
72
The experiences from the development of the chemistry towards the β-strand mimetics provided an opportunity to synthesize two different series of potential thrombin inhibitors, which were obtained from a structure based design. The first series included the pyridine ring as the central P2 residue, substituted with a para-amidinobenzylamine moiety as P1 residue and various benzoyl derivatives as P3 residues (Chapter 7). Three potential thrombin inhibitors (Figure 9.3) were synthesized and evaluated in an enzymatic assay, which revealed them to be only moderately potent (µM) inhibitors of thrombin. Still, a crystal structure of thrombin with one of the synthesized thrombin inhibitors in the active site was obtained. The moderate affinity for thrombin can be explained by the lack of hydrogen bonding network through the S2-S3 region, which is found in more potent inhibitors such as melagatran. Based on the co-crystal structure it is suggested that elongation of the benzylamine part of the inhibitor to a phenyl ethylamine moiety could improve binding.
O
N
HN
NH2
NHAcOH*
R
Figure 9.3 The three synthesized thrombin inhibitors; R = meta-methyl, ortho-methoxy or H.
The second structural class of potential thrombin inhibitors lack a strongly basic moiety in the P1 position and are therefore predicted to present an improved pharmacokinetic profile (Chapter 8). A small library of eight compounds were synthesized with two different aminopyridines as P3 residues (Figure 9.4). Unfortunately none of the less basic thrombin inhibitors showed any affinity for thrombin.
OH
N
Nn = 0,1
NH
R2XR1
O
XV
P1 P2 P3
Figure 9.4 The designed thrombin inhibitors XV. X = C or N; R1 = Cl or CH2NH2 if X = C; R2 = H or Me; The cyclic amine in the P3 moiety was designed to be pyrrolidine or morpholine.
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10. Acknowledgement
Först av allt så vill jag tacka Jan Kihlberg, en enastående handledare. Du tar dig tid, och hittar alltid en utväg trots att det ibland ser svårt ut. Det har varit lärorikt och mycket inspirerande att få jobba med dig.
Kay Brickmann, för all hjälp med korrekturläsning och för dina klarsynta kommentarer i våra kemidiskussioner. Paul Kreye, som vägledde mig under mina första trevande steg som kemist, jag hade väldigt roligt. Tomas Fex och Yafeng Xue, för ett gott samarbete med trombin inhibitorerna. Chris Fowler, för att jag fick nyttja farmakologins lokaler, Britt Jacobsson och Ingrid Persson för all hjälp under dessa månader. Mattias Hedenström, Zhong Quing Yuan och Ingmar Sethson för ett givande och trevligt samarbete.
Tomas Gustafsson, för syntes ideer, diskussioner, fisketurer, film, mat och kamratskap. Dan Johnels, Fredrik Almqvist, Mikael Elofsson, Ingmar Sethson, Anna Linusson, för att ni gör en suverän insats för avdelningen och håller stämningen på topp. Johan Eklund, Tobias Sundberg mina skidåkarkompisar som jag fortfarande hänger med efter alla dessa år. Andreas Larsson, så goda vänner växer inte på trän, tack för alla glada tillrop under årens lopp. Jon Gabrielsson, som ordnar dom bästa midsommarfesterna och erbjuder kost och logi i Umeå. Även ett tack för att du ställer upp som toastmaster.
74
David Bergström, min träningskompis i alla väder och sporter, nu kommer jag tillbaka. Mickael Mogemark, Hans Emtenäs, för korrekturläsning av avhandlingen och fortsatt samarbete. Pär Jonsson, Susanne Johansson och Anna för badmintonmatcher, skidturer, fikapauser och andra roliga avbrott i den vardagliga lunken. Nils Pemberton, Hans G Andersson, Erik Chorell, ungdomarna som jag hade nöjet att dela lab och musik med det sista året. Carina Sandberg, Pelle Lundholm, Kenneth Österlund, Ann-Helen Waara, Bert Larsson, för hjälp med allehanda ting genom åren och för att ni verkligen är rätt man/kvinna på rätt plats. Ida Andersson, Sara Spjut, Anna Kauppi, Susanne Johansson, Veronica Åberg och alla andra doktorander, som gör att det är en fröjd att gå till skolan varje dag. Sara Spjut, Mattias Olsson, Sandra Lindberg, Johan Westerberg, mina gamla? X-jobbare. Mina föredetta klasskamrater, David E, David B, David G, Sven, Johan, Pelle, Erik S….. för fester, bio, fisketurer och massor med annat. Vindelälvsloppets kämpar som höll ut längre än loppet. Mamma, som alltid ställer upp trots avstånd och förkylda barn. Pappa, Gerd, för att ni funnits som en hjälpande hand, och för att ni kämpade så tappert under pappas sista år. Urban, Matilda, brorsan och syrran er kan jag alltid lita på. Bror, Barbro, Hanna, för att upprätthålla vår fasta punkt och tillflyktsort i tillvaron där jag hoppas kunna spendera mer tid framöver, Rendalen. Stina, Axel, Knut, familjen som kan få mig på bra humör än fast skidföret är dåligt och synteserna krånglar. Ni är det viktigaste jag har.
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Appendix
Experimental section for chapter 8.
General methods and materials: 1H- and 13C-NMR were recorded in CDCl3 at 305 K, residual CHCl3 (δ = 7.26) or CDCl3 (δ = 77.0), CD3OD residual CD2HOH (δ = 3.35) or CD3OD (δ = 48.0) as internal standards. For compounds containing an uneven mixtures of diastereomeres only the resonances from the major diastereomer is reported. All microwave irradiation were performed in a Smith Creator single node instrument using Emrys process vials (2-5 mL), with temperature measurement by infrared detection. Fixed hold time was used with a ramp time of 0.5−1 min. All organic extracts were dried over Na2SO4 unless otherwise stated.
The supporting information includes: 1H-NMR for all isolated compounds and 13C-NMR spectra for selected compounds. HPLC chromatograms run on a C18 column with eluents A and B (A = 0.1 M NH4OAc in H2O B = 5% A in CH3CN, for specific gradient see each chromatogram, or experimental section) for compounds 147 → 154. 2-(tert-Butyl-dimethyl-silanyloxy)-2-(2-fluoro-pyridin-4-yl)-ethylamine (141) and 2-(tert-Butyl-dimethyl-silanyloxy)-2-(2-fluoro-pyridin-4-yl)-1-methyl-ethylamine (142)
General procedure:
2-Fluoro-4-iodopyridine (VII, 2.1 equiv. 6.2 mmol) was dissolved in THF (1 mL) and treated with iso-propyl magnesium chloride (2 M solution in THF, 2.1 equiv.) for 1 h. Boc-protected alaninal 136 or glycinal 135 (1.0 equiv.) was dissolved in THF (3 mL) and added to the in situ formed Grignard reagent and heated to 40°C for 1 h and further stirred for 10 h at room temperature. The reaction was quenched with NH4Cl (aq., sat.) and the resulting slurry was portioned between NaHCO3 (aq., sat.) and EtOAc. The aqueous phase was extracted with EtOAc and the combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/heptane 1:1 to give secondary alcohols 137 or 138. The alcohol was dissolved in CH2Cl2 (20 mL) followed by addition
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of tert-butyldimethylsilyl trifluoromethanesulfonate (1.3 equiv.) and 2,4,6-trimethylpyridine (1.3 equiv.). The reaction was stirred for 30 minutes at room temperature and then quenched by addition of NaHCO3 (aq., sat.). The two phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers was dried and concentrated. The residue was treated with formic acid (3 mL) for 3 h. The formic acid was removed under reduced pressure and the residue was taken up in EtOAc and washed with NaHCO3 (aq., sat.). The aqueous phase was extracted with EtOAc and the combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/MeOH 1:0→9:1 to give fluoropyridine 141 (41%, from VII) or 142 (45%, from VII).
Compound 141: 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 5.2 Hz, 1H), 7.10-7.07 (m, 1H), 6.88-6.86 (m, 1H), 4.67 (dd, J = 6.0 and 4.2 Hz, 1H), 2.86 (dd, J = 13.3 and 4.2, 1H), 2.78 (dd, J = 13.3 and 6.0, 1H), 1.56 (s, 2H), 0.89 (s, 9H), 0.06 (s, 3H), -0.08 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.9 (d, JC-F = 240 Hz), 158.3 (d, JC-F = 7.3 Hz), 147.4 (d, JC-F = 14.7 Hz), 118.7 (d, JC-F = 3.8 Hz), 106.6 (d, JC-F = 38 Hz), 74.8 (d, JC-F = 2.3 Hz), 50.1, 25.6, 18.0, -4.8, -5.0.
Compound 142: 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 5.2 Hz, 1H), 7.10 (d, J = 5.2 Hz, 1H), 6.87 (s, 1H), 4.38 (d, J = 5.0 Hz, 1H), 2.99-2.91 (m, 1H), 1.54 (br s, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.91 (s, 9H), 0.07 (s, 3H), -0.15 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.9 (d, JC-F = 240 Hz), 158.2 (d, JC-F = 7.4 Hz), 147.3 (d, JC-F = 15 Hz), 119.5 (d, JC-F = 3.9 Hz), 107.4 (d, JC-F = 38 Hz), 78.8, 53.4, 25.7, 19.7, 18.3, -4.6, -5.1. 2-(tert-Butyl-dimethyl-silanyloxy)-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethylamine (143) and 2-(tert-Butyl-dimethyl-silanyloxy)-1-methyl-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethylamine (144)
General procedure:
Fluoropyridine 141 or 142 (1.0 equiv., 0.45 mmol) was dissolved in pyrrolidine (2.5 mL) and subjected to microwave irradiation (130 °C, 2500 s). The excess pyrrolidine was removed under reduced pressure and the residue was treated with tert-butyldimethylsilyl trifluoromethanesulfonate (1.3 equiv.) and 2,4,6-trimethylpyridine (1.3 equiv.) in CH2Cl2 (5 mL) for 30 minutes. The reaction was quenched by adding NaHCO3 (aq., sat.) and the aqueous phase was extracted with EtOAc. The combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/MeOH 1:0→7:3 to give aminopyridine 143 (76%) or 144 (69%).
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Compound 143: 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.3 Hz, 1H), 6.43 (dd, J = 5.3 and 1.1 Hz, 1H), 6.35 (s, 1H), 4.60-4.56 (m, 1H), 3.51-3.39 (m, 4H), 2.92-2.76 (m, 2H), 2.12 (br s, 2H), 2.04-1.98 (m, 4H), 0.93 (s, 9H), 0.08 (s, 3H), -0.02 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 157.4, 152.5, 147.9, 108.9, 103.6, 75.4, 50.2, 46.7, 25.8, 25.5, 18.2, -4.6, -5.0.
Compound 144: 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 5.3 Hz, 1H), 6.40 (d, J = 5.3 Hz, 1H), 6.28 (s, 1H), 4.25 (d, J = 5.1 Hz, 1H), 3.48-3.37 (m, 4H), 2.95-2.87 (m, 1H), 2.16 (br s, 2H), 2.03-1.95 (m, 4H), 1.02 (d, J = 6.7 Hz, 3H), 0.91 (s, 9H), 0.04 (s, 3H), -0.14 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 157.3, 152.5, 147.8, 109.6, 104.1, 79.2, 53.3, 46.6, 25.8, 25.5, 19.6, 18.1, -4.5, -5.1. 2-(tert-Butyl-dimethyl-silanyloxy)-2-(2-morpholin-4-yl-pyridin-4-yl)-ethylamine (145) and 2-(tert-Butyl-dimethyl-silanyloxy)-1-methyl-2-(2-morpholin-4-yl-pyridin-4-yl)-ethylamine (146)
General procedure:
Fluoropyridine 141 or 142 (1.0 equiv., 0.41 mmol) was dissolved in morpholine (3 mL) and subjected to microwave irradiation (165 °C, 1 h). The excess morpholine was removed under reduced pressure with toluene as azeotrope and the residue was treated with tert-butyldimethylsilyl trifluoromethanesulfonate (1.3 equiv.) and 2,4,6-trimethylpyridine (1.3 equiv.) in CH2Cl2 (5 mL) for 30 minutes. The reaction was quenched by adding NaHCO3 (aq., sat.) and the aqueous phase was extracted with EtOAc. The combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/MeOH 1:0→4:1 to give aminopyridine 145 (71%) or 146 (79%).
Compound 145: 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 5.2 Hz, 1H), 6.63 (s, 1H), 6.60 (dd, J = 5.2 and 0.8 Hz, 1H), 4.61 (dd, J = 6.1 and 4.1 Hz, 1H), 3.85-3.81 (m, 4H), 3,50 (dd, J = 5.7 and 4.3 Hz, 4H), 2.88 (dd, J = 13.2 and 4.1, 1H), 2.80 (dd, J = 13.2 and 6.1 Hz, 1H), 2.12 (br s, 2H), 0.93 (s, 9H), 0.09 (s. 3H), -0.03 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.8, 153.3, 147.8, 111.7, 104.1, 75.4, 66.7, 50.1, 45.6, 25.8, 18.2, -4.6, -4.9. Compound 146: 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 5.2 Hz, 1H), 6.59-6.54 (m, 2H), 4.25 (d, J = 5.1 Hz, 1H), 3.83-3.77 (m, 4H), 3.49-3.45 (m, 4H), 2.94-2.86 (m, 1H), 1.88 (br s, 2H), 1.00 (d, J = 6.5 Hz, 3H), 0.89 (s, 9H), 0.04 (s, 3H), -0.16 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.7, 153.3, 147.6, 112.4, 104.6, 79.3, 66.6, 53.4, 45.6, 25.8, 19.6, 18.1, -4.6, -5.1.
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2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-2-(2-morpholin-4-yl-pyridin-4-yl)-ethyl]-acetamide (147), 2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethyl]-acetamide (149), 2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-(1S, R)-methyl-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethyl]-acetamide (150) and 2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-(1S, R)-methyl-2-(2-morpholin-4-yl-pyridin-4-yl)-ethyl]-acetamide (148)
General procedure:
Amine 143, 145, 146 or 144 (1.0 equiv., 51 µmol) was dissolved in CH2Cl2 (2 mL) followed by addition of 4-chlorophenylacetic acid (1.3 equiv.), HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium hexafluorophosphate) (1.5 equiv.) and DIPEA (N,N-diisopropylethylamine) (3.5 equiv.). The reaction was stirred for 20 minutes and quenched by addition of sat. NaHCO3 (aq.) and CH2Cl2 and the two layers were separated. The aqueous phase was extracted with CH2Cl2 and the combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/heptane 3:2 to afford the amides, which was treated with TBAF (tetrabutyl ammonium fluoride) trihydrate (1.75 equiv.) in THF (2 mL) for 2 h. The solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC (C18) running from 0→100% B in A over 35 minutes. The fractions were collected and lyophilized. The remainder was purified by flash chromatography CH2Cl2/MeOH 19:1 and lyophilized again to afford 147 (17%), 149 (30%), 150 (54%) or 148 (59%) as colorless solids.
Compound 147: 1H NMR (360 MHz, MeOD) δ 8.01 (d, J = 5.1 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 6.78 (s, 1H) 6.68 (d, J = 5,1 Hz, 1H), 4.71-4.65 (m, 1H), 3.79-3.75 (m, 4H), 3.50-3.35 (m, 8H).
Compound 149: 1H NMR (360 MHz, MeOD) δ 7.88 (d, J = 5.4 Hz, 1H), 7.25 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 8.2 Hz, 2H) 6.54 (d, J = 5.4 Hz, 1H), 6.46 (s, 1H), 4.70-4.64 (m, 1H), 3.50-3.36 (m, 8H), 2.04-1.97 (m, 4H).
Compound 150: 1H NMR (360 MHz, MeOD) δ 7.80 (d, J = 5.6 Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.55 (d, J = 5.6 Hz, 1H), 6.49 (s, 1H), 4.60 (d, J = 3.2 Hz, 1H), 4.29-4.21 (m, 1H), 3.48-3.30 (m, 6H), 2.03-1.98 (m, 4H), 1.22 (d, J = 6.8 Hz, 3H); 13C NMR (90 MHz, MeOD) δ 171.8, 156.3, 154.6, 144.9, 134.9, 132.5, 130.5, 128.5, 109.8, 105.6, 74.2, 50.2, 47.0, 41.9, 25.4, 16.8.
Compound 148: 1H NMR (360 MHz, MeOD) δ 7.99 (d, J = 5.3 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.82 (s, 1H), 6.69 (d, J =
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5.3 Hz, 1H), 4.65 (d, J = 3.5 Hz, 1H), 4.31-4.23 (m, 1H), 3.82-3.78 (m, 4H), 3.51-3.37 (m, 6H), 1.24 (d, J = 6.8 Hz, 3H). N-[(2S, R)-Hydroxy-2-(2-morpholin-4-yl-pyridin-4-yl)-ethyl]-2-pyridin-4-yl-acetamide (151), N-[(2S, R)-Hydroxy-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethyl]-2-pyridin-4-yl-acetamide (153) and N-[(2S, R)-Hydroxy-(1S, R)-methyl-2-(2-morpholin-4-yl-pyridin-4-yl)-ethyl]-2-pyridin-4-yl-acetamide (152)
General procedure:
Amine 143, 145 or 146 (1.0 equiv., 51 µmol) was dissolved in CH2Cl2 (2 mL) and treated with 4-pyridylacetic acid hydrochloride (1.3 equiv.), HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium hexafluorophosphate) (1.5 equiv.) and DIPEA (N,N-diisopropylethylamine) (4.5 equiv.). The reaction was stirred for 20 minutes followed by addition of sat. NaHCO3 (aq.) and CH2Cl2. The two layers were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography eluted with EtOAc to afford the amides, which was treated with TBAF (tetrabutyl ammonium fluoride) trihydrate (1.75 equiv.) in THF (2 mL) for 2 h. The solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC (C18) running from 0→100% B in A over 60 minutes. The fractions were collected and lyophilized. The remainder was purified by flash chromatography CH2Cl2/MeOH 17:3 and lyophilized again to afford 151 (51%), 153 (62%) or 152 (59%) as colorless oils.
Compound 151: 1H NMR (360 MHz, MeOD) δ 8.42 (d, J = 4.8 Hz, 2H), 8.02 (d, J = 5.2 Hz, 1H), 7.28 (d, J = 4.8 Hz, 2H), 6.81 (s, 1H), 6.70 (d, J = 5.2 Hz, 1H), 4.73-4.67 (m, 1H), 3.79-3.74 (m, 4H), 3.56-3.27 (m, 8H).
Compound 153: 1H NMR (360 MHz, MeOD) δ 8.45 (bs, 2H), 7.93 (d, J = 5.3 Hz, 1H), 7.31 (d, J = 4.9 Hz, 2H), 6.59 (d, J = 5.3 Hz, 1H), 6.52 (s, 1H), 4.75-4.70 (m, 1H), 3.61-3.39 (m, 8H), 2.07-2.02 (m, 4H).
Compound 152: 1H NMR (400 MHz, MeOD) δ 8.39 (d, J = 5.7 Hz, 2H), 7.98 (d, J = 5.2 Hz, 1H), 7.15 (d, J = 5.7 Hz, 2H), 6.81 (s, 1H), 6.69 (d, J = 5.2 Hz, 1H), 4.63 (d, J = 3.7 Hz, 1H), 4.30-4.23 (m, 1H), 3.79 (m. 4H), 3.56-3.39 (m, 6H), 1.22 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, MeOD) δ 170.3, 160.2, 154.2, 148.9, 147.0, 146.7, 124.9, 112.1, 105.2, 74.4, 66.7, 50.5, 46.1, 41.8, 16.7.
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4-Aminomethyl-N-[(2S, R)-hydroxy-(1S, R)-methyl-2-(2-pyrrolidin-1-yl-pyridin-4-yl)-ethyl]-benzamide (154)
Amine 144 (40 mg, 0.12 mmol) was dissolved in CH2Cl2 (3 mL) and treated with 4-(Boc-aminomethyl)benzoic acid (39 mg, 0.16 mmol), HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium hexafluorophosphate) (68 mg, 0.18 mmol) and DIPEA (N,N-diisopropylethylamine) (80 µL, 0.46 mmol). The reaction was stirred for 20 minutes and then was sat. NaHCO3 (aq.) and CH2Cl2 added. The two layers were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers was dried and concentrated under reduced pressure. The residue was purified by flash chromatography EtOAc/heptane 3:2 to afford the amide, which was treated with formic acid for 3 h. The formic acid was then removed under reduced pressure and the residue was dissolved in EtOAc and washed with sat. NaHCO3 (aq.). The organic solvent was dried and removed under reduced pressure. The residue was treated with TBAF (tetrabutyl ammonium fluoride) trihydrate (66 mg, 0.21 mmol) in THF (2 mL) for 2 h. The solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC (C18) running from 0→100% B in A over 60 minutes. The fractions were collected and lyophilized. The remainder was purified by flash chromatography CH2Cl2/MeOH/NEt3 17:3:0.2 and lyophilized again to afford 154 (40%) as an off-white solid.
Compound 154: 1H NMR (360 MHz, MeOD) δ 7.88 (d, J = 5.5 Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 6.61 (d, J = 5.5 Hz, 1H), 6.53 (s, 1H), 4.70 (d, J = 4.5 Hz, 1H), 4.47-4.38 (m, 1H), 4.13 (s, 2H), 3.44-3.28 (m, 4H), 2.01-1.96 (m, 4H), 1.91 (s, 3H), 1.26 (d, J = 6.8 Hz, 3H); 13C NMR (90 MHz, MeOD) δ 178.0, 168.1, 157.2, 153.8, 146.5, 137.7, 135.3, 128.9, 128.1, 109.9, 105.3, 74.9, 51.2, 46.9, 42.9, 25.5, 22.4, 16.4.