Synthesis of β-Turn and Pyridine Based Peptidomimetics David Blomberg

Synthesis of β-Turn and Pyridine Based Peptidomimetics David Blomberg
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
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
1
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 Cterminus ................................................................................................... 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
2
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 Leuenkephalin. 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, 416423.
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.
3
4
2. List of Abbreviations
Bn
Boc
BSA
Cbz
DAMGO
DBU
DCC
DCE
DIBAL
DIC
DIPEA
DMAP
DPDPE
DTI
EWG
Fmoc
GPCR
HATU
HMDS
HOAt
HOBt
K-selectride
LCMS
LHRH
N,O-DMHA
NMO
NMR
NOE
NOESY
PAM
PMB
Q
rt
benzyl
tert-butoxycarbonyl
bovine serum albumin
benzyloxycarbonyl
[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
1,8-diazabicyclo[5.4.0]undec-7-ene
dicyclohexyl carbodiimide
1,2-dichloroethane
diisobutylaluminium hydride
N,N’-diisopropyl carbodiimid
diisopropylethylamine
N,N-dimethylaminopyridine
[3H] [D-Pen2, D-Pen5]enkephalin
direct thrombin inhibitor
electron withdrawing group
9-fluorenylmethyloxycarbonyl
G protein-coupled receptor
O-(7-azabenzotriazole-1-yl)-N, N,N’N’tetramethyluronium hexafluorophosphate
hexamethyldisilazane
1-hydroxy-7-azabenzotriazole
N-hydroxybenzotriazole
potassium tri-sec-butylborohydride
liquid chromatography mass spectrometry
luthenizing hormone releasing hormone
N,O-dimethylhydroxylamine
N-methyl morpholine N-oxide
nuclear magnetic resonance
nuclear overhauser enhancement
nuclear overhauser enhancement spectroscopy
4-(Hydroxymethyl)phenylacetamidomethyl
p-methoxybenzyl chloride
tetrabutylammonium
room temperature
5
TBAF
TBDMS
TEA
TMP
TFA
TFAA
TFE
TMS
Tris
UHP
3-D
6
tetrabutylammonium fluoride
tert-butyldimethyl silyl
triethylamine
2,4,6-trimethylpyridine
trifluoro acetic acid
trifluoro acetic anhydride
2,2,2-trifluoroethanol
trimethylsilyl
tris hydroxymethylaminoethane
urea hydrogen peroxide
three dimensional
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.
R1
N
H
O
Amino acid
residue
R3
O
H
N
R2
N
H
H
N
O
R4
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
7
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 αhelices 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
!
1
1
"
HN
O
O HN
H
N
O
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
8
O
H
N
HN
O
CO
H
N
O
O
H
N
O
N
H
O
H
N
N
H
O
H
N
N
H
H
N
N
H
O
O
H
N
O
N
H
O
OC
H
N
NH
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
9
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
Warfarin
OH
O
O3S
O
O
O
HO
O3S
AcHN
O
O
HO
O
O2C
O3S
O
Heparin
O
O
HO
NH O
O3S
HO
O2C
O
OH O
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
10
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.
O
O
H
N
HO
O
N
N
H
Melagatran
NH2
NH
O
O
H
N
O
O
N
N
H
H
N
Ximelagatran
OH
NH
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.
11
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.
12
•
Stabilized turn mimetics were to be synthesized in order to gain
further understanding of the bioactive conformation of the
endogenous endorphin Leu-enkephalin (H2 N-Tyr-Gly-Gly-PheLeu-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.
5. Peptidomimetics of Leu-enkephalin
5.1 Biological action and conformation of Leuenkephalin
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 Leuenkephalin 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.
OH
HN
O
O
H2N
HN
H
N
O
HO
O
O
NH
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 Leu13
enkephalin in the pseudo ten membered ring observed in the crystalline
form.33
Me
N
HO
O
OH
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.
14
HO
OH
H2N
O
H
N
O
N
H
O
O
O
OH
O
3
H2N
N
HO
OH
O
H2N
HN
O
O
H
N
O
OH
O
N
H
N
O
NH
O
O
H2N
H
N
O
O
O
N
H
OH
O
OH
1
2
4
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 Leuenkephalin
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.
15
OH
OH
O
O
O
H2N
O
H2N
H2N
HN
O O
OH
I
O
III
HO
N
HO
O
O
O O
NH
NH2
HO
NH
1
II
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 CbzTyr(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
OtBu
O
OH
AllylMgBr
O
NO-DMHA
NHCbz
OtBu
81%
N
CbzHN
OMe
Me
5
O
74%
NHCbz
6
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.
16
OtBu
OtBu
K-selectride
O
OtBu
OH
+
OtBu
+
OH
H
H
NHCbz
NHCbz
NHCbz
6
7, 36%
8, 8%
HN
O
O
9, 14%
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
OtBu
OH
7.5 M KOH (aq)
MeOH
THF
H-1
H-2
NHCbz
HN
7
O
O
9
OtBu
OtBu
OH
7.5 M KOH (aq)
MeOH
THF
H-1
H-2
NHCbz
HN
8
O
O
10
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
17
with triflic anhydride, sodium azide and CuSO4 to afford azidoalcohol 12
(85%).46,47
OtBu
KOH
H2O/EtOH
7 or 9
OtBu
triflic anhydride
NaN3
CuSO4
OH
85%
80%
NH2
N3
11
OtBu
O
OtBu
O
OtBu
OsO4
NMO
O
73%, from 12
N3
13
12
OtBu
O
QHSO4
NaOH (aq)
t-Bu bromoacetate
OH
OtBu
Pb(OAc)4
Na2CO3
OH
OH
N3
14
O
OtBu
O
O
85%
N3
15
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 H2 O. 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, H2 N-Phe-Leu-OH was converted to H2 N-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
18
OtBu
OtBu
15 + 16
82%
O
N3
NH
H
N
MeO
85%
O
N3
N
H
N
MeO
O
99%
N
H
N
O
O
18
19
OH
N
H
N
O
56%, from 19
MeO
O
O
DBU
NHFmoc dioxan
reflux
N3
O
N3
N
H
N
O
O
SnCl2
PhSH
TEA
HN
O
20
N3
OPfp
O
DCC
PfpOH
EtOAc
O
MeO
OH
O
NHFmoc
formic acid
O
OH
OH
O
NHFmoc
17
MeO
O
O
O
O
OtBu
O
HATU
DIPEA
DMF
Fmoc-Gly-OH
O
Na(OAc)3BH
TEA
DCE
OH
OtBu
O
HN
O
O
R
NH2
N
H
N
O
O
O
21
LiOH
22 R = OMe
1 R = OH
57%, from 21
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 Leuenkephalin over a 15 step synthetic sequence with an overall yield of 3.2%.
19
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 Ntermini 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 βturns, 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.
20
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 Leuenkephalin, 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.
O
NHBoc
O
1. TFA/CH2Cl2
2. DIC
HOBt
Fmoc-Phe-OH
O
O
3. Piperidine
DMF
15
Na(OAc)3BH
TEA
NH2
H
N
O
23
tBuO
OH
O
N3
O
O
O
NH
H
N
O
N3
O
OtBu
O
TFA/CH2Cl2
O
NH
H
N
O
24
OH
O
OH
PfpOH
DIC
O
O
OPfp
NH
H
N
O
25
O
N3
O
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).
21
HO
26
TEA
dioxane
reflux
HO
NaOMe
MeOH
N3
O
O
N
H
N
O
HO
SnCl2
TEA
PhSH
N3
H2N
O
25%, over solid
phase synthesis
OMe
O
O
N
H
N
O
O
R
O
O
27
N
H
N
O
O
O
28
29 R = OMe
LiOH
2 R = OH
56%, from 28
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). βTurn 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 NaN 3 in a biphasic system
then afforded azidoalcohol 32 (72%).46,47
OtBu
OtBu
O
OtBu
iBu-Chloroformate
NaBH4
Tf2O
NaN3
Morpholine
90%
90%
OH
NHFmoc
OtBu
72%
OH
NHFmoc
30
OH
NH2
31
OH
N3
32
Scheme 5.8 The synthesis of azidoalcohol 32 from the corresponding Fmocprotected 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
22
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
OtBu
OtBu
Et-bromoacetate
KH
QI
EtOH
NaOH
89%
OH
OEt
O
N3
N3
32
80%
OH
O
O
N3
33
O
34
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
O
FmocHN
O
H
N
H2N
O
O
O
O
35
H2N
N
H
H
N
O
O
O
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, H2 O, 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.
23
36
35
34
HATU
DIPEA
CH2Cl2
OtBu
O
N3
O
N3
O
H
N
N
H
34
HATU
DIPEA
CH2Cl2
O
H
N
O
O
O
38
SnCl2
TEA
PhSH
THF
OtBu
O
O
O
O
O
37
H2N
O
H
N
N
H
OtBu SnCl
2
TEA
PhSH
THF
N
H
H2N
O
H
N
O
H
N
O
O
O
O
H
N
N
H
O
O
O
39
40
OtBu
H2O
TFA
scavangers
62% over solid
phase synthesis
70% over solid
phase synthesis
H2O
TFA
scavangers
OH
O
H2N
O
N
H
H
N
H2N
O
OH
O
H
N
O
N
H
O
H
N
O
OH
O
O
4
OH
3
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).
24
HO
OH
H2N
O
H
N
O
N
H
O
O
O
OH
O
3
H2N
N
HO
OH
O
H2N
HN
O
O
H
N
O
OH
O
N
H
N
O
NH
O
O
H2N
H
N
O
O
O
N
H
OH
O
OH
1
2
4
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
25
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
1
2
3
4
Leu-enkephalin
DSLET
pIC50
<6
6.13 ± 0.12
7.86 ± 0.09
<5.5
6.81 ± 0.12
7.41 ± 0.07
IC50 (nM)
>1000
740
14
>1000
160
39
nh
0.90
0.80
0.66
0.74
± 0.24
± 0.12
± 0.12
± 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 µreceptor (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.
26
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
1
2
3
4
Leu-enkephalin
DSLET
pIC50
<6
6.79 ± 0.12
8.88 ± 0.08
6.07 ± 0.14
8.44 ± 0.15
8.83 ± 0.16
IC50 (nM)
>1000
160
1.3
370
3.6
1.5
nh
0.98
0.92
1.03
0.67
0.58
±
±
±
±
±
0.26
0.14
0.37
0.15
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-PheLeu) 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
27
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.
28
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
H
O
N
H
H
N
O
H
H
N
N
O
O
N
O
N
N
O
H
O
N
N
O
H
N
H
O
H
H
O
O
H
N
O
N
H
H
N
O
N
H
O
O
H
N
O
N
H
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 βstrand, 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
29
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.
O
O
R
N
N
H
O
R
N
O
N
H
H
N
O
R HN
O
R HN
O
R HN
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 ProLeu-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).
30
R1
N
H
O
R3
O
H
N
R
N
H
2
R1
O
R3
N
H2N
OH
X
O
R
2
O
Va X = NH
Vb X = O
IV
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 Nacetylated 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-AlaAla-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
R1
N
H
R2
O
O
R2
H
N
R3
R3
N
N
N
H
R1
O
O
H
N
R1
N
H
R2
H
N
R3
N
H
R2
O
O
R3
N
O
O
O
N
H
H
N
R1
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 βsheets. Therefore nonpolar side chains were selected for the R1- and R3groups 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-4iodopyridine (VII) as starting material (Figure 6.5).
31
R1
H2N
O
R1
R3
O
H
N
N
H
IV
R3
N
H2N
N
H
O
CO2H
Va
OH
O
R3
R1
N
H
PgHN
O
VI
OH
H2N
I
F
VII
O
VIII
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
32
protection of the alcohol moiety in 41 as a silyl ether using TBDMSCl and
imidazole in CH2Cl2 to afford 42 (90%).
O
TBDMSCl
Imidazole
CH2Cl2
i-Bu Chloroformate
NaBH4
OH
97%
NHBoc
OH
OTBDMS
90%
NHBoc
41
TBAF
THF
Boc2O
DMAP
dioxane
OTBDMS
66%
NHBoc
42
NBoc2
43
Oxidation
OH
O
NBoc2
NBoc2
44
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.
Tf2O
NaN3
Formic acid
OH
NHBoc
41
OH
80%
NH2
46
Oxidation
OH
64%
N3
47
O
N3
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 , Tf2 O 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
33
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.
iPrMgCl
THF
2F,4I pyridine
Dess-Martin
periodinane
OH
NHBoc
41
BnBr
QHSO4
50% NaOHaq
N
O
88%
NHBoc
49
BocHN
F
OH
50
41%, from 49
N
BocHN
F
OBn
51
Scheme 6.3 A successful strategy to connect leucinal at position 4 in 2-fluoro-4iodopyridine 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).
34
Table 6.1 Different reactions performed with benzylamine (1.2 equiv.) as
nucleophile in an SNAr reaction using 2-fluoropyridine as electrophile.
N
N
+
F
N
H
H2N
52
Entry
Solvent
Base/additive
1
2
3
4
5
6
7
8
9
NMP
NMP
NMP
Pyridine
CH3CN
CH3CN
Toluene
THF
THF
TEA
TEA
DIPEA
Ag2CO3
CuCO3
DIPEA/NaCl
n-BuLi
n-BuLi/Ag Zeolite
Temp.
°C
150
250
250
250
120
120
200
78→rt
78→rt
Time
Heating method
Yield %
48 h
30 min
60 min
60 min
10 min
10 min
20 min
3h
3h
Sealed cylinder
Microwave
Microwave
Microwave
Microwave
Microwave
Microwave
-
30
42
52
79
-
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.
BnBr
QHSO4
NaOHaq
PhCH3
iBu chloroformate
NaBH4
OH
BocHN
O
BocHN
53
OH
Formic acid
BocHN
OBn
54
85%, over 2 steps
91%
H2N
OBn
55
Scheme 6.4 Conversion of Boc-Ile-OH to benzyl protected aminoalcohol 55.
35
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
N
+
OBn
H2N
F
OBn
N
H
55
56
Entry
Solvent
Base/additive
Temp. ° C
Time
1
2
3
4
5
6
7
8
HMDS
Pyridine
Pyridine
NMP
THF
THF
THF
THF
TEA/TMSCl
175
190
230
250
78→ 0
Reflux
78→rt
78→reflux
1h
30 min
1h
30 min
3h
7h
5h
7h
pyridine
BuLi
MeMgCl
NaHMDS/TMSCl
NaHMDS/15crown-5
Heating
method
Microwave
Microwave
Microwave
Microwave
Yield %
7
Oil bath
40
38
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).
15-crown-5
NaHMDS
THF
reflux
N
+
F
OBn
H2N
68%
O
O
N
O
O
O
55
57
Scheme 6.5 Unexpected byproduct formed in an attempt to react 2-fluoropyridine
with 55.
36
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).
MeMgCl
THF
N
OBn
H2N
55
N
+
BocHN
F
OBn
51
BocHN
N
H
OBn
OBn
58
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 H2 N-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).
37
N Formic acid
A.
BocHN
F
Ac2O
CH2Cl2
N
H2N
F
OBn
AcHN
OBn
59
N
B.
F
+
5 equiv.
OBn
N
pyridine
OtBu
H2N
60 (86%, from 51)
AcHN
O
N
+
AcHN
F
OH
H2N
N
NaHCO3
AcHN
5 equiv. O
OBn
NH
OH
N
H
OBn
60
+
AcHN
O
N
AcHN
F
+
H2N
OH
pyridine
190 !C
1h
63, < 10%
N
AcHN
10 equiv.
OBn
O
OBn
62, < 10%
D.
OBn
N
H
OH
64, 54%
60
Allylamine
17 bar
2.5 h
N
E.
F
60
O
61, trace amounts
C.
OBn
OtBu
N
H
OBn
60
AcHN
F
OBn
51
AcHN
N
N
AcHN
OBn
N
H
65, > 90% crude
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 H2 N-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
38
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).
RuCl3
NaIO4
N
N
OH
N
H
66
N
H
NaIO4
Br2
MeOH
N
N
H
51%
67
N
OH
N
H
OH
69
N
H
OMe
O
70
O3
NaOH
MeOH
N
67
O
68
K-osmate
NMO
N
OH
N
H
N
N
H
OMe
O
70
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, H2 O 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
39
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 CH2 Cl2 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 tertbutyl ester of α-hydroxy acetic acid was formed as the major product.
Na(OAc)3BH
TEA
DCE
N
NH2
NH2
N
H
36%
Reductive
amination
N
N
71
OMe
N
N
H
68
N
OH
O
or
N
H
72
OtBu
O
Scheme 6.9 Attempts to afford glycine substituted pyridine via reductive amination
of 2-aminopyridine.
40
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
AcHN
F
OBn
60
60%
perchloric acid
glyoxal
N
25% NH3 in H2O
AcHN
NH2
OBn
73
N
AcHN
OBn
N
H
OMe
O
74
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.
41
allylamine
heat
N
I
N
F
I
75
TsCl + H N
2
2.5 equiv.
CH2Cl2
Ts
99%
N
H
DMSO
KH
heat
2-F,4-I-pyridine
N
H
76
63%
N
I
N
77
Ts
iPrMgCl
49
N
BocHN
N
OH
Ts
78
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
42
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
Boc2O
DMAP
N
N
H
67
O3
NaOH
MeOH
N
N
99%
79
Boc
N
OMe
N
65%
80
Boc
O
Scheme 6.12 Protecting the ‘anilinic nitrogen allowed oxidation of the alkene.
To our delight, purging ozone through a CH2 Cl2 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.
43
Allylamine
N
AcHN
AcHN
OBn
60
OBn
O3
NaOH
MeOH
N
H
AcHN
OMe
Boc
75%
N
O
Boc
84
Dess-Martin
periodinane
N
AcHN
OMe
N
O
OH
Boc
81%
O
83
N
Formic acid
OMe
Boc
81
82
N
N
OBn
H2
Pd/C
MeOH
N
OBn
AcHN
65
N
AcHN
N
86%, from 60
F
58%
Boc2O
DMAP
N
89%
AcHN
O
N
H
O
OMe
O
85
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
AcHN
TFA
OMe CH Cl
2 2
N
OH
Boc
83
O
N
AcHN
N
H
OH
86
Dess-Martin
OMe periodinane
O
66%, from 83
N
AcHN
N
H
O
85
Scheme 6.14 By reversing the order of the two final steps, 85 was afforded as a
single enantiomer.
44
OMe
O
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 Sleucinol 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
N
S-leucinol
AcHN
F
86%
AcHN
OBn
OBn
60
87
N
AcHN
OBn
N
S-iso-leucinol
F
60
OH
N
H
80%
AcHN
OBn
88
N
H
OH
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 βstrand 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 Boc2 O 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
45
oxazolidinone 91 (75%, from 87). Because of lack of time no further
attempts to transform 87 into a β-strand mimetic were made.
TBDMSOTf
Collidine
N
AcHN
OH
N
H
OBn
87
Boc2O
DMAP
N
AcHN
OTBDMS
N
H
OBn
89
TBAF
THF
N
AcHN
OTBDMS
N
OBn
N
75%, from 87
Boc
AcHN
N
OBn
90
O
91
O
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
+
F
heat
or
BuLi
N
H2N
F
OBn
59
12-31%
N
N
N
H
F
OBn
92
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.
46
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 2fluoropyridine 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 Noxide 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 βstrand mimetic at the N-terminal was not explored further.
UHP
TFAA
N
N
44%
F
O
F
93
Pyridine
heat
93
N
H2N
F
OBn
59
38%
N
O
LiAlH4
TiCl4
N
N
H
F
OBn
94
>90%
N
N
N
H
F
OBn
92
Scheme 6.18 Further attempts to extend the β-strand mimetic by activating 2fluoropyridine as N-oxide prior to the SNAr reaction.
The focus was now set on elongation of the β-strand mimetic at the Cterminal. 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
47
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 Bocprotection 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, H2 O 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 Bocprotection.
N
I
N
various conditions
O
OMe
O
Me
I
O
OMe
Me
95
96
Cl
N
EWG
N
H
N HCl
HCl
O
O
N
H
EWG
O
EWG
98
97
O3
reductive
work-up
N
N
O
N
Boc
K-osmate
NMO
N
H
99
N
79
OH
N
Boc
100
88%
Pb(OAc)4
Na2CO3
N
N
O
N
Boc
101
OH
OH
N
Boc
100
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.
48
N
O
F 31%, from 49
H2N
NHBoc
N
Allylamine
H2N
OBn
49
59
+
F
OBn
60
102
heat
N
AcHN
N
H
OBn
N
N
102
AcHN
N
H
OBn
OBn
N
H
103
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-βstrand 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 249
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.
50
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 extrinsic
regulation
factor X
factor Xa
thrombin (factor IIa)
prothrombin (factor II)
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.
51
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 Ppocket) 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
Trp215
Leu99
S3-pocket
S2-pocket
Tyr60A
Ile174
Spacer
Trp60D
S1-pocket
H2N
O
NH2
O
Asp189
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 S1pocket.
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 P1residue 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
52
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
NH
O
H
N
*
AcOH
NH2
N
130
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).
53
N
n = 0,1
N
H
O
R2
IX
R1
N
+
R1
n = 0,1
I
F
VII
O
+
H2N
R2
XI
X
Figure 7.4 A retrosynthetic analysis of thrombin inhibitor IX suggests that 2-fluoro4-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
NH
O
H
N
*
NH
AcOH
NH2
OMe O
N
130
H
N
*
NH
AcOH
NH2
O
N
H
N
*
AcOH
NH2
N
131
132
Me
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
54
was oxidized directly to ketone 107 (60%, from VII) to avoid elimination to
the corresponding alkene.
OH
N
I
F
iPrMgCl
benzaldehyde
F
F
37%
N
86%
VII
OPMB
KH
QI
PMBCl
N
104
105
iPrMgCl
phenylacetaldehyde
N
N
Dess-Martin
periodinane
F
F
60%, from VII
OH
106
O
107
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 4bromobenzylamine (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).
55
OPMB
Br
OPMB
F
H
N
4-Br-benzylamine,
pyridine, NMP
N
OPMB
NH2
+
N
<10%
105
N
108
109
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
F
N
104
Dess-Martin
periodinane
88%
4-Br-benzylamine
Pyridine
F heat
O
N
Br
O
trace amounts
H
N
107
N
111
F
N
NH2
+
N
110
O
O
4-CN-benzylamine
Pyridine
heat
112
CN
O
113
H
N
N
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 parasubstituted 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 4bromobenzylamine (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
56
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
NH2
O
F
N
25% NH3 in H2O
heat
112
N
O
+
citric acid 10% (aq.)
NH
94%
110
NH2
NH2
N
112
N
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 457
cyanobenzaldehyde (1.3 equiv.) and TEA in refluxing toluene for 10 h. The
solvent was then removed and CH2 Cl2 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 4cyanobenzyl 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
CN
O
NH2
H
N
reductive
amination
N
N
115
112
O
Boc2O
DMAP
CH2Cl2
N
N
77% from 112
116
O
F
107
CN
Boc
N
O
25% NH3 in H2O
heat
NH2
117
N
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 tertbutyl 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.
58
O
NH
hydoxylamine
CN DIPEA
EtOH
reflux
Boc
N
O
N
H
Boc
N
N
OH
AcOH
Ac2O
N
116
118
NH
O
NH
N
H
Boc
N
OAc
H2
Pd/C
OH
NH2
Boc
N
N
N
119
120
NH
Boc2O
CH2Cl2
OH
Boc
N
N
121, 22% from 116
N
H
Boc
NH2
+
N
122
+
N
123
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).
59
O
CN
Boc
1. NaBH4
OTBDMS
N
N
82%
N
CN
Boc
2. Collidine
TBDMSOTf
hydoxylamine
DIPEA
EtOH
reflux
N
116
124
NH
OTBDMS
Boc
NH
N
H
N
OH
OTBDMS
Boc
AcOH
Ac2O
N
N
OAc
N
H
N
125
126
NH
OTBDMS
H2
Pd/C
MeOH
Boc
NH
NH2
OTBDMS
Boc
Boc2O
CH2Cl2
N
N
H
N
N
Boc
N
127
128, 66% from 124
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.
NH
OTBDMS
NH
N
H
Boc
N
Boc
TBAF
THF
OH
N
H
Boc
N
N
N
128
121
NH
Dess-martin
peiodinane
Boc
O
Boc
N
NH
N
H
Boc
TFA
CH2Cl2
O
H
N
* AcOH
NH2
63%
52% from 128
N
129
N
130
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
60
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 metamethyl 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.
NH
O
H
N
* AcOH
NH2
N
R
Compound
130
131
132
R=H
R=o-OMe
R = m-Me
IC50 (µM)
15
> 44.4*
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
61
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
62
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.
63
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).
64
P1
R1
P2
X
P3
R2
O
N
n = 0,1 H
N
N
OH
XV
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.
R1
X
R2
O
N
n = 0,1 H
N
N
OH
XV
N
R2
I
PgHN
F
VII
O
XII
R1
X
HN
O
n = 0,1
OH
XIII
XIV
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 Bocprotected 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
65
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.
OH
H2N
Me
H2N
OH
Boc2O
CH2Cl2
BocHN
133
OH
66%, from
ethanolamine
Me
Boc2O
CH2Cl2
BocHN
134
Dess-Martin
periodinane
OH
O
BocHN
135
Me
Dess-Martin
periodinane
73%, from
alaninol
O
BocHN
136
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 Bocprotected 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).
66
i-PrMgCl
VII
THF
R
O
BocHN
BocHN
TBDMSOTf
Collidine
CH2Cl2
N
R
F
OH
135 R = H
136 R = Me
N
R
BocHN
F
OTBDMS
137 R = H
138 R = Me
139 R = H
140 R = Me
N
R
R
Formic
acid
Pyrrolidine
heat
N
H2N
H2N
N
OTBDMS
F
143 R = H, 76%
144 R = Me, 69%
OTBDMS
141 R = H, 41% from 135
142 R = Me, 45 % from 136
morpholine
heat
N
R
H2N
N
OTBDMS
O
145 R = H, 71%
146 R = Me, 79%
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
67
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.
Cl
O
Cl
N
N
H
O
OH
N
H
O
O
Cl
N
N
H
N
N
N
OH
N
Me
N
OH
O
O
152, 59%
O
N
N
H
O
N
H
N
OH
O
N
150, 54%
151, 51%
N
O
Me
N
H
OH
O
N
H
N
OH
O
N
149, 30%
N
N
148, 59%
147, 17%
Cl
Me
N
N
OH
153, 62%
H2N
Me
N
H
N
N
OH
154, 40%*
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 Bocprotected 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-468
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).
69
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.
HO
OH
O
O
H2N
HN
O
H2N
N
O
HO
O
OH
O
NH
1
N
H
N
O
O
O
2
Figure 9.1 Ten- and the seven membered turn mimetics incorporated in Leuenkephalin.
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
70
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
AcHN
N
N
H
O
85
OMe
O
AcHN
OBn
88
N
H
OH
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.
71
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.
NH
O
* AcOH
NH2
H
N
N
R
Figure 9.3 The three synthesized thrombin inhibitors; R = meta-methyl, orthomethoxy 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.
P1
R
P2
P3
1
X
R2
O
N
n = 0,1 H
N
N
OH
XV
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.
72
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.
73
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.
74
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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 CDCl 3 (δ = 77.0), CD3 OD
residual CD2HOH (δ = 3.35) or CD3 OD (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 NH4 OAc in H2 O 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)-1methyl-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 NH4 Cl (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
85
of tert-butyldimethylsilyl trifluoromethanesulfonate (1.3 equiv.) and 2,4,6trimethylpyridine (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-(2pyrrolidin-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%).
86
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-(2morpholin-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.
87
2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-2-(2-morpholin-4-yl-pyridin-4yl)-ethyl]-acetamide (147), 2-(4-Chloro-phenyl)-N-[(2S, R)-hydroxy-2-(2pyrrolidin-1-yl-pyridin-4-yl)-ethyl]-acetamide
(149),
2-(4-Chlorophenyl)-N-[(2S, R)-hydroxy-(1S, R)-methyl-2-(2-pyrrolidin-1-yl-pyridin4-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 CH2 Cl2/MeOH 19:1 and lyophilized again to afford 147
(17%), 149 (30%), 150 (54%) or 148 (59%) as colorless solids.
Compound 147: 1 H 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: 1 H 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: 1 H 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: 1 H 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 =
88
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-pyridin4-yl-acetamide (151), N-[(2S, R)-Hydroxy-2-(2-pyrrolidin-1-yl-pyridin4-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-ylacetamide (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: 1 H 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: 1 H 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), 3.563.39 (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.
89
4-Aminomethyl-N-[(2S, R)-hydroxy-(1S, R)-methyl-2-(2-pyrrolidin-1-ylpyridin-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-(7Azabenzotriazol-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: 1 H 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), 3.443.28 (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.
90
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