Design and Synthesis of Amine Building Blocks and Protease Inhibitors Susana Ayesa Alvarez

Design and Synthesis of
Amine Building Blocks and
Protease Inhibitors
Susana Ayesa Alvarez
Stockholm University
© Susana Ayesa Alvarez, Stockholm 2008
ISBN 978-91-7155-690-5
Printed in Sweden by Universitetsservice US -AB, Stockholm 2008
Department of Organic Chemistry
Stockholm University
To my family
Abstract
The first part of this thesis addresses the design and synthesis of amine
building blocks accomplished by applying two different synthetic
procedures, both of which were developed using solid-phase chemistry.
Chapter 1 presents the first of these methods, entailing a practical solidphase parallel synthesis route to N-monoalkylated aminopiperidines and
aminopyrrolidines achieved by selective reductive alkylation of primary
and/or secondary amines. Solid-phase NMR spectroscopy was used to
monitor the reactions for which a new pulse sequence was developed.
The second method, reported in Chapter 2, involves a novel approach to
the synthesis of secondary amines starting from reactive alkyl halides and
azides. The convenient solid-phase protocol that was devised made use of
the Staudinger reaction in order to accomplish highly efficient alkylations of
N-alkyl phosphimines with reactive alkyl halides.
The second part of the thesis describes the design and synthesis of three
classes of protease inhibitors targeting the cysteine proteases cathepsins S
and K, and the serine protease hepatitis C virus (HCV) NS3 protease.
Chapter 4 covers the design, solid-phase synthesis, and structure-activity
relationships of 4-amidofurane-3-one P1-containing inhibitors of cathepsin S
and the effects of P3 sulfonamide groups on the potency and selectivity
towards related cathepsin proteases. This work resulted in the discovery of
highly potent and selective inhibitors of cathepsin S.
Two parallel solid-phase approaches to the synthesis of a series of
aminoethylamide inhibitors of cathepsin K are presented in Chapter 5.
Finally, Chapter 6 reports peptide-based HCV NS3 protease inhibitors
containing a non-electrophilic allylic alcohol moiety as P1 group and also
outlines efforts to incorporate this new template into low-molecular-weight
drug-like molecules.
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals:
I.
An Expeditious Library Synthesis of N-Monoalkylated
Aminopiperidines and –pyrrolidines
Susana Ayesa, Dimitris Argyopoulus, Tatiana Maltseva, Christian
Sund, and Bertil Samuelsson
Eur. J. Org. Chem., 2004, 2723-2737.
II.
A One-Pot, Solid-Phase Synthesis of Secondary Amines from
Reactive Alkyl Halides and an Alkyl Azide
Susana Ayesa, Bertil Samuelsson and Björn Classon
Synlett, 2008, 1, 97-99.
III.
Solid-Phase Synthesis and SAR of 4-Amidofurane-3-one Inhibitors
of Cathepsin S: the Effect of Sulfonamides at P3 on Potency and
Selectivity
Susana Ayesa, Charlotta Lindquist, Tatiana Agback, Kurt Benkestock,
Björn Classon, Ian Henderson, Ellen Hewitt, Katarina Jansson, Anders
Kallin, Dave Sheppard and Bertil Samuelsson
Submitted
IV. Preparation and Characterization of Aminoethylamide Inhibitors of
the Cysteine Proteinase Cathepsin K
Susana Ayesa, Jinq-May Chen, Björn Classon, Jose Gallego, Urszula
Grabowska, Ian Henderson, Narinder Heyer, Tony Johnson, Jussi
Kangasmetsä, Mark Liley, Magnus Nilsson, Kevin Parkes, Laszlo
Rakos, Matthew J. Tozer, and Michelle Wilson
Submitted
V.
Investigation of Allylic Alcohols in the P1 Position of Inhibitors of
Hepatitis C Virus NS3 Protease
Susana Ayesa, Tatiana Maltseva, Laszlo Rakos, Elizabeth Hamelink,
Björn Classon, and Bertil Samuelsson
Manuscript
I have also contributed to the following article, which is not included in this
thesis. This publication reports further development of the research
described in paper V.
VI.
Novel potent macrocyclic inhibitors of the hepatitis C virus NS3
protease: Use of cyclopentane and cyclopentene P2-motifs
Baeck, M.; Johansson, P.-O.; Waangsell, F.; Thorstensson, F.;
Kvarnstroem, I.; Ayesa, S.; Waehling, H.t; Pelcman, M.l; Jansson, K.;
Lindstroem, S.; Wallberg, H.; Classon, B.; Rydergaard, C.; Vrang, L.;
Hamelink, E.; Hallberg, A.; Rosenquist, A.; Samuelsson, B.
Bioorganic & Medicinal Chemistry, 2007, 15(22), 7184-7202
Papers I and II were reprinted with the kind permission from the publishers.
Abbreviations
Ac
APC
BAP
BMD
BOC, Boc
Cha
CPMG
COSY
DCM
DIC
DIEA
DMAP
DMF
DQF
EDC
ELSD
Fmoc
FTIR
h
HATU
HBTU
HCV
HIV
HLA
HMBC
HOBt
HRMAS
HSQC
HTS
HPLC
Ii
IL
acetyl
antigen-presenting cells
borane pyridine complex
bone mass density
t-butoxycarbonyl
β
-cyclohexylalanine
Carr-Purcell-Meiboom-Gill T 2-dependent spin-echo sequence
correlation spectroscopy
dichloromethane
diisopropylcarbodiimide
N,N-diisopropylethylamine
4-(dimethylamino)pyridine
dimethylformamide
Double-quantum filter
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride
evaporative light scattering detector
9-fluorenylmethyloxycarbonyl
fourier transformation infrared spectroscopy
hour(s)
N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-n-methylmethanaminium hexafluorophosphonate
N-oxide
N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide
hepatitis C virus
human immunodeficiency virus
human leukocyte antigen
heteronuclear multiple bond correlation
1-hydroxybenzotriazole
high-resolution magic-angle spinning
heteronuclear single-quantum correlation
high throughput screening
high pressure liquid chromatography
invariant chain
interleukin
Ki
LCMS
MeOH
MHC
NMM
NMP
NMR
NS
NS3
NTPase
Nva
PyBOP
RT
SAR
SLP
SPE
RAPiD
TES
TMOF
TMS
TNFa
TFA
TOCSY
inhibitory constant/dissociation constant for inhibitor binding
liquid chromatography mass spectroscopy
methanol
major histocompatibility complex
4-methylmorpholine
N-methylpyrrolidinone
nuclear magnetic resonance
non-structural
non-structural protein 3
nucleoside triphosphatase
norvaline
benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate
room temperature
structure-activity relationship
spin lock pulse
solid-phase extraction
rational approach to protease inhibitor design
triethylsilane
trimethylorthoformate
trimethylsilyl
tumor necrosis factor alpha
trifluoroacetic acid
total correlation spectroscopy
Table of Contents
Part I. Synthetic Methods for the Preparation of Amine
Building Blocks
1 Solid-Phase Parallel Synthesis of N-Monoalkylated
Aminopiperidines and Aminopyrrolidines (Paper I)
1.1 Introduction ............................................................................................... 1
1.1.1 Solid Phase Parallel Synthesis (SPPS) ............................................1
1.1.2 NMR Spectroscopy .......................................................................... 2
1.1.3 Aim of the Study................................................................................4
1.2 Solid-Phase Synthesis: Resin Selection .................................................. 4
1.3 Reductive Alkylation of Primary Amines ...................................................5
1.3.1 Solid-Phase Chemistry ..................................................................... 5
1.3.1.1 Coupling to the Solid Support .................................................. 6
1.3.1.2 CPMG-T2/diffusion-Filtered gHSQC Experiments ................10
1.4 Reductive Alkylation of Secondary Amines ............................................11
1.4.1 Solid-Supported Chemistry and HRMAS NMR ..............................11
1.5 Conclusions.............................................................................................13
2 A One-Pot, Solid-Phase Synthesis of Secondary Amines from
Reactive Alkyl Halides and an Alkyl Azide (Paper II)
2.1 Introduction .............................................................................................15
2.1.1 Preparation of Secondary Amines .................................................15
2.1.2 Utility of the Azide Moiety...............................................................15
2.1.3 Alkylation of Iminophosphoranes: Background .............................16
2.1.4 Aim of the Study.............................................................................17
2.2 Solid-Phase Alkylation of Iminophosphoranes .......................................17
2.2.1 Scope and Limitations....................................................................18
2.3 Conclusions .............................................................................................20
Part II. Design and Synthesis of Inhibitors Targeting the
Cysteine Proteases Cathepsins S and K and the Serine
HCV NS3 Protease
3 General introduction
3.1 Proteases: Hydrolysis of Peptide Bonds.................................................21
3.2 Cysteine and Serine Proteases: Mechanism of Action
and Specificity......................................................................................... 22
3.2.1 Catalytic Mechanism of Serine Proteases.....................................22
3.2.2 Catalytic Mechanism of Cathepsin Cysteine Proteases................23
3.2.3 Specificity of Proteases .................................................................24
3.3 Protease Inhibitors ..................................................................................25
3.3.1 Serine Protease Inhibitors .............................................................26
3.3.2 Cathepsin Cysteine Protease Inhibitors ........................................27
4 Solid-Phase Parallel Synthesis and SAR of 4-Amidofuran-3-one P1
Containing Inhibitors of Cathepsin S: Effect of Sulfonamides P3
Substituents on Potency and Selectivity (Paper III)
4.1 Introduction .............................................................................................29
4.1.1 Antigen Presentation and the Immune Response......................... 29
4.1.2 Cathepsin S as Potential Therapeutic Target ...............................32
4.1.3 Background Data and Aim of the Study ........................................33
4.2 Chemistry................................................................................................35
4.2.1 Solid-Phase Chemistry of Dihydro-2(3H)-5-ethyl Furanones........35
4.2.2 Synthesis of P3 acid Capping Groups...........................................37
4.3 Tables: Inhibitory Activity and Selectivity ................................................38
4.4 Structure-Activity Relationships and Modeling .......................................43
4.4.1 Enzyme Inhibitory Activity..............................................................44
4.4.2 Selectivity Profile ...........................................................................47
4.4.3 Cellular Potency ............................................................................48
4.5 Conclusions .............................................................................................49
5 Preparation and Characterization of Aminoethylamide Inhibitors of
the Cysteine Protease Cathepsin K (Paper IV)
5.1 Introduction .............................................................................................51
5.1.1 Cathepsin K and Osteoporosis......................................................51
5.1.2 Aminoethylamide Cathepsin K Inhibitors: Aim of the Study ..........55
5.2 Solid-Phase Synthesis of Aminoethylamides .........................................56
5.2.1 Aminoethylamide Template ...........................................................56
5.2.2 Aldehyde Resin-Based Synthesis .................................................56
5.2.3 Sulfonamide Resin-Based Synthesis ............................................57
5.3 Inhibitory Activity and Kinetic Studies ..................................................... 59
5.4 Photodegradation ....................................................................................63
5.5 Conclusions .............................................................................................66
6 Investigation of Allylic Alcohols in the P1 Position of Inhibitors of
Hepatitis C Virus NS3 Protease (Paper V)
6.1 Introduction .............................................................................................67
6.1.1 Hepatitis C Virus ............................................................................67
6.1.2 HCV NS3 Protease Inhibitors........................................................68
6.1.3 Aim of the Study ............................................................................69
6.2 Synthesis of P1 Nva Allylic Alcohols .......................................................70
6.3 Isomerically Pure Allylic Alcohols............................................................71
6.4 Synthesis of Hexapeptides .....................................................................72
6.5 Synthesis of Tetrapeptides .....................................................................74
6.6 Synthesis of Tripeptides..........................................................................75
6.7 Tables: Inhibitory Activity ........................................................................75
6.8 Structure-Activity Relationships ..............................................................77
6.9 Conclusions .............................................................................................77
References
Acknowledgments
Appendices
Part I:
Synthetic Methods for the Preparation of Amine
Building Blocks
1. Solid-Phase Parallel Synthesis of NMonoalkylated Aminopiperidines and
Aminopyrrolidines (Paper I)
1.1 Introduction
1.1.1 Solid-Phase Parallel Synthesis
In recent years, numerous reports have been published that describe the
developments in solid-phase synthesis1,2 and combinatorial chemistry
adapted to polymeric supports,3–5 technologies that are now widely applied
to generate drug-like libraries for high-throughput screening (HTS) against a
multitude of drug targets. The idea of such approach is to couple a scaffold
molecule to a polymer-based resin, after which the scaffold can be
chemically manipulated in several ways before it is cleaved off from the
resin. The reagents, which are in solution, can be removed after each
reaction step by simple filtration and washing of the resin. Accordingly,
workup and purification procedures are not required between steps, and the
reagents can be used in excess, which helps to drive the reactions to
completion. Unfortunately, in general solution-phase reaction conditions
must be modified and optimized to be used in the solid-phase approach, thus
method development in order to transfer solution-phase conditions to solidphase conditions is often required.
There are still several challenges that need to be addressed in the design
and synthesis of compound libraries. One such consideration is the choice of
linker, which has to be stable under the reaction conditions employed and
must be selectively cleaved under mild conditions after the synthesis
sequence is completed in order to yield pure product. The appropriateness of
a linker is often dictated by the functionality present in the specific class of
molecules of interest.
A major general drawback of solid-support reactions has been the
difficulties related to analysis of the reaction products on the solid phase. To
1
circumvent this problem, analytical control is frequently carried out in
solution after cleavage from the polymer, which is a time-consuming
strategy and results in loss of material. Consequently, recent developments
have focused on techniques for non-destructive on-bead analysis that are
adapted to solid-phase chemistry6 and utilize both single-bead FTIR and
high-resolution magic-angle spinning (HRMAS) NMR spectroscopy.7–9
Thus it is apparent that use of solid-phase chemistry can be tedious and
time consuming, but, once such a method is developed, this approach is very
efficient.
1.1.2 NMR Spectroscopy
More than fifty years have passed since the first observations of nuclear
magnetic resonance (NMR) were made. The seminal step occurred during
the early 1950s, when it was realized that the resonant frequency of a
nucleus is affected by its chemical environment, and that one nucleus can
influence the resonance of another through intervening chemical bonds.
NMR spectroscopy is now an important tool employed to determine the
structure of organic compounds. The molecule to be analyzed is dissolved in
a suitable solvent and placed in a tube, which is then subjected to a strong
magnetic field, and a target nucleus in the sample experiences this external
field. A range of one-dimensional (1D) experiments can be carried out to
assign the structure of the molecule of interest. However, when considering
large structures, the chemical shifts have a higher degree of overlap in the
1D spectrum, and hence in such cases it is necessary to perform advanced
1D and 2D hetero- and homonuclear experiments.
High-Resolution Magic-Angle Spinning (HRMAS) NMR
Spectroscopy
HRMAS NMR spectroscopy, is one of the most powerful nondestructive
analytical techniques used to monitor reactions on solid support.7–9 A
problem that can be encountered with 1D and 2D homo- and heteronuclear
HRMAS NMR experiments concerns the multitude of non-relevant signals
that arise from the resin, residual solvents, and from organics encapsulated in
the beads, which make interpretation of the spectra non trivial. There are
some reports describing the use of 1D techniques to remove such unwanted
signals. 10–14
An application of high-resolution MAS NMR spectroscopy to
compounds attached to Merrifield resin faces a challenge due to the short
length of the resin linker.15 The resulting slow mobility of the attached
compound and the very fast T2 -relaxation of the protons give rise to a
broadening of the signals in the spectrum.
2
CPMG-T2 /Diffusion-Filter gHSQC Experiments
Two approaches can be applied to remove the residual resin signals in highresolution NMR: (a) the use of presaturation to saturate the resin signals and
allow the signals of interest to appear; (b) the use of CPMG-T2 type filters to
simultaneously suppress all the resin signals. A disadvantage of the first
method is that only a small given number of resin peaks can be presaturated,
and the presaturation will also remove any useful signals that are hidden by
the resin signals. The use of multiple frequency presaturation with SLP type
pulses to remove all the resin signals does not solve this problem, because it
“erases” a greater region of the spectrum.
The basis of the second method is that the resin molecules, in essence
because they are in the solid phase, have much shorter T2 relaxation times.
Therefore, if the magnetization is retained for a while in the XY plane after
the initial excitation pulse, the intensity of the resin signals will decrease
much faster than the signals from the bound molecules, which are more or
less in the liquid state. In this way, all the resin signals will be attenuated,
allowing the signals of the bound species to appear. A CPMG-T 2 type filter
(delay–180 o–delay)n can be used to achieve simultaneous removal of the
residual resin signals in high-resolution NMR spectra. This approach
emanates from the differences in mobility exhibited by the resin part of the
molecule and the resin-bound moieties, which still retain reasonably free
rotational diffusion. 13 This means that a nucleus belonging to the resin part
should have a considerably shorter T2 relaxation time compared to the nuclei
of the resin-bound moieties. Filtering times of 20–80 ms are usually
employed. A few reports have described the use of such filters but mainly in
combination with 1D techniques.10–14 The T2 parameters must be carefully
selected, since the signals of interest are also attenuated, albeit to a lesser
degree. Another problem that can arise, particularly when using long T2
filtering times and many 180o pulses, is related to B1 inhomogeneity. The
pulses are not precisely 180 o over the entire spectral window, and therefore
the repeated use of them causes signals to lose intensity, especially in the
edges of the spectrum.
When moving from simple 1D techniques to modern 2D experiments,
difficulties can also occur due to J-evolution during the time spent in the XY
plane. That effect results in severely phase-distorted multiplets in the 1D
spectra and rather unpredictable behavior of the system with all 2D
techniques that use 1H decoupling in the F 2 dimension. The 2D experiments
most often applied are DQF-COSY, TOCSY, HSQC and HMBC. However,
in all such experiments, with the exception of when using HSQC, what is
known as the mixing or magnetization transfer time (i.e., the time it takes for
the system to obtain the desired properties to detect) is usually in the same
order of magnitude as the T2 relaxation times of the resin signals.
Accordingly, in these techniques, the solution is provided by the experiment
3
itself, as a side effect, and there is no need for a dedicated T2 filter. However,
when applying HSQC, the transfer times are very small, usually not more
than 10–15 ms, which is not sufficient for the resin signals to die out, and
thus in this case it would be appropriate to conduct a CPMG-T2 diffusionfiltered experiment.
1.1.3 Aim of the Study
N-Monosubstituted aminopiperidines and aminopyrrolidines are important
building blocks for the synthesis of small-molecule directed compound
libraries. The aim of the study reported in Paper I was to find a practical
solid-supported parallel synthesis route that would supply these key
components. It was also desired to achieve a facile monitoring of the
synthesis steps by use of non-destructive methodologies such as FTIR or
HRMAS NMR. Two synthetic routes (A and B, depicted schematically
below) were proposed and investigated that were intended to accomplish
derivatization of the primary or the secondary amino moiety by reductive
alkylation. The suggested routes can be readily applied to an extensive pool
of commercially available aromatic and aliphatic aldehydes for conversion
into the corresponding N-monoalkylated diamino templates. This procedure
nicely complements methodologies entailing amide reductions and reversed
reductive aminoalkylations. 16–18
X
X, Y= bond, CH 2
H N
Y
A
NH2
B
X
X
N
Y
H N
N
H
X
H N
Y
Y
NH2
R
R
X
N
N
H
Y
NH2
1.2 Solid-Phase Synthesis: Resin Selection
The p-nitrophenyl carbonate linker was chosen for development of the two
solid-supported synthetic strategies described above. Its functionality is ideal
for the attachment of amines onto the solid support. Specifically,
nucleophilic amines are able to form a carbamate bond with the solid
support, which displaces the stable p-nitrophenol from the resin and thereby
gives a characteristic yellow color to the reaction. The newly formed
carbamate bond is quite stable in relation to most reaction conditions, except
for acidic treatments. The acid liability of the resin is dependent on the
nature of the linker.
4
Merrifield- and Wang-type p-nitrophenylcarbonate resins were investigated
in the current study. No differences between the two were found in terms of
yields and purities when considering the first route (A), in which the
aminopiperidines are attached to the resin through the secondary amine
moiety. However, there were substantial differences in the yields obtained
by the second route (B), which involves attachment of the aminopiperidines
through the primary amine moiety. In this route, the amines used as starting
materials are Boc-protected at the secondary amine moiety. Once attached to
the solid support an acidic treatment is performed to remove the protecting
group, which, in the case of the Wang-type resin, causes cleavage of a large
proportion of the amines at this step, thus lowering the yields of the target
compounds. By comparison, the Merrifield-type resin is less acid labile, and
no cleavage of the amines is observed at the low acidic deprotection
treatment used; instead, the final cleavage of the amine products from the
resin requires a stronger acidic treatment.
1.3 Reductive Alkylation of Primary Amines
1.3.1 Solid-Phase Synthesis
The synthetic route investigated is depicted in Scheme 1. The pnitrophenylcarbonate Merrifield- (0.8 mmol/g loading) and Wang-type (1
mmol/g loading) resins were used as polymeric supports. Attachment of the
starting amine to resin 1 (step a) was performed at 20 
C in DMF for 24 h,
and the products were analyzed by single-bead FTIR, the ninhydrin test,19,20
and HRMAS NMR.
O
a
O
1
b
O
NO 2
O
O
O
O
N
2
3
1
N
3
5
NH 2
c
d
O
O
4
1
N
3
6
5
N
H
HN
2
4
9
7
NH
2
6
4
9
7
R
N
8
R
5
R
8
Scheme 1. Reagents and conditions: (a) 4-(aminomethyl) piperidine (5 equiv. in dry DMF),
RT, overnight; (b) RCHO (21) (5 equiv. in TMOF; see scheme 3), RT, overnight; (c) BH3-py
complex (4 equiv.), DCM:MeOH:AcOH 2: 2: 1, RT, overnight; (d) 75% TFA in DCM, RT, 1
h.
5
Typically, during step a, the absorption bands at 1,760 and 1,350 cm-1 in the
FTIR spectra, corresponding to the carbonate group and the nitro group,
respectively, were transformed into a new band at 1,720 cm –1 , assigned to
the carbonyl group of the carbamate function. From the FTIR data, it was not
possible to determine whether the attachment to the resin occurred through
the secondary or the primary amino moiety, and neither could the absorption
band observed at 3,400 cm –1 (attributed to NH vibration10) be
unambiguously assigned to one or the other.
The primary amine 2 on solid phase (Scheme 1) was subjected to
alkylations with aldehydes (21) in solution by performing a two-step
procedure to minimize dialkylations. 21–22 The imine-forming step proceeded
readily with the amine on the solid phase and the aldehyde in solution.23
Trimethylorthoformate (TMOF) was employed as both solvent and
dehydrating agent,24–25 and the reaction was monitored by FTIR, which
showed a characteristic –N=C– absorption band at 1,645 cm–1 . Reduction of
the imine functionality of resins 3 to the corresponding amine moiety in
resins 4 was achieved using borane-pyridine complex26 (BAP) at room
temperature overnight. Final compounds 5 were obtained as bis
trifluoroacetate salts in good yields and purities after TFA treatment of resins
4.
1.3.1.1 Coupling to the Solid Support
Indirect Method
To investigate the attachment of aminopiperidines to the solid support and to
verify whether the coupling occurred through the primary or secondary
amino moiety, an indirect approach was developed at the beginning of this
study. It was envisaged that acylation of the remaining amino group
(Scheme 2, step b) would provide the required evidence of the selectivity
that was obtained. Experiments were performed using solution-phase NMR
analysis of the hydrolysis products 9 and 10 (Scheme 2).
To ensure that the reaction of unprotected 4-aminomethylpiperidine with
resin 1 had taken place through the secondary amine to give 2 and not 6, an
amide coupling experiment (Scheme 2) was performed. In short, 4azidobenzoic acid was coupled to the amine through a traditional PyBOP
peptide-coupling method, which could readily be followed by FTIR on the
resin through a characteristic azide absorption band at 2,120 cm –1 .
Nevertheless, FTIR could not provide information about the mode of
attachment to the resin (7 or 8).
6
O
O
N
O
a
O
6
or
b
NO 2
O
N
1
O
O
N
2
NH2
O
2
3
8
O
O
c
O
N
N
O
N3
N
N3
O
6
N3
5
7
9
or
N
7
N 9
4
H2N
O
or
1
1
11
3
4
6
8
12
2
HN
5
7
8
N3
13
H 10
N 9
14
15
O
10
Scheme 2. Reagents and conditions: (a) 4-(aminomethyl) piperidine (5 equiv. in dry DMF),
RT, overnight; (b) 4-azidobenzoic acid (4 equiv.), PyBOP (4 equiv.), DIEA (8 equiv.), dry
DMF, RT, overnight; (c) 75% TFA in DCM, RT, 1.5 h.
One of the options to explicitly establish that the reaction proceeded through
the secondary amine was to fully assign the NMR spectra of the cleaved
product. In the gCOSY spectrum of the cleaved product, the J-coupling
connectivity starting from the 8H proton and continuing to 7H2  4H 3H2 
2
H2 protons was identified that fits with both possible structures 9 and 10
(for atom numbering, see Scheme 2). The assignment of the 7 H2 , 3H2 , and
2
H2 protons of the methylene groups was confirmed by a gHSQC
experiment. The cross peaks between carbonyl carbon, 9 C  methylene
protons 7H2 and 9C  8H, were detected in the HMBC spectrum. This
observation supports that the cleaved product has structure 10 (Scheme 2),
which shows that the amide-forming reaction proceeded through the primary
amine 2 to give 8.
Direct Method: Use of HRMAS NMR Spectroscopy to Monitor the
Reactions
The ideal situation for the monitoring of the synthesis steps would be to use
a more direct method that does not require previous cleavage from the resin,
or alternatively to apply an indirect approach involving formation of
reference compounds, as described in the previous section (Indirect Method).
During the development time of this study, it was possible to find a solution
by conducting several HRMAS NMR experiments, and we explored the
possibility of applying CPMG-T2 type filters to remove all the polymer
signals and enhance the signals of interest. HRMAS NMR was used to
monitor the reductive alkylation steps on the resin (Scheme 1), and this is
7
exemplified by two aldehydes in Figure 1. It is noteworthy that the proton
signals of the piperidine fragment of compounds 3 were much broader than
those of the imine fragment, similar to amines 2 (Fig. 1, a, b, and d).
In the gHMBC spectrum of compound 3 (Fig. 2a), a cross peak was clearly
observed between the protons of the methylene group 7H2 and the carbon C9 ,
which further supports formation of the primary amine 2 in Scheme 1. The
full assignment of compound 3 was achieved through gHMBC, gHSQC, and
COSY experiments (Fig. 2, a–c). In the gHMBC spectrum (Fig. 2a) of
compound 3, the protons belonging to the piperidine and resin fragments did
not have any cross peaks with carbons two or three bonds away due to short
T2 relaxation. However, in the HSQC experiments the transfer times are very
short, usually not more than 10–15 ms, which is not enough time for the
resin signals to die out (Fig. 2b).
O
O
1
O
16
2
O
6
O
11
3
N
11
3
13
12
10
4
5
4 (Fi g. 1(c))
14
15
8
F
2
6
9
7
16
1
N
12
10
4
5
3 (Fig . 1(b))
N
O
F
N
13
9
15
NH
7
14
8
2 (Fig .1a )
NH2
O
O
16
N
2
o
3
6
5
3 (Fig. 1(d))
N
8
16
2
4 (Fig. 1(e))
o
3
6
13
14
15
5
11
12
10
4
7
17
13
9
NH
8
15
14
17
16
2b
6b 7
2a
6a
12
15
N
9
17
10
1
12
9
7
11
(e)
O
11
10
4
16
O
17
O
1
3a
5a
3b
5b
4
13
14
17
7
16
9
(d)
3a
5a
2b
6b
2a
6a
4
3b
5b
16
11
10
9
F
15
2b 7 16 3a
5a
6b
4
2a
6a
12
(c)
13
3b
5b
14
16
7
13
9
14
2a
6a
15
(b)
9
8
7
6
5
1
4
2b 7
6b
2a
6a
(a)
3a
5a
2b
6b
4
3
3a
5a 4
2
3b
5b
3b
5b
ppm
Figure 1. H spectra of the products presented in Scheme 1, obtained using T2 and diffusion
filters: (a) 2; (b) 3, and (c) 4,; (d) 3, and (e) 4.
8
O
O
3
1
N
2
3
6
5
4
9
R
N
8
7
16
10
14
13
(c)
11
16
F
12
R=
15
13
14
9
7
2a
6a
15
2b
6b
4
3a 3b
5a 5b
F2
(ppm)
3
4
5
6
7
8
(b)
8
7
6
5
F1
(ppm)
4
3 16
C
F1 (ppm)
2
1
5C
4C 3
C
2 C 6C
60
7C
80
100
120
140
13 C
14 C
15 C
9C
160
8
(a)
7
6
5
F1
(ppm)
60
80
140
160
3
2
1
4
3
2
1
4C
7C
100
120
4
5 3
F2 (ppm)C C
13 C
15C
11C
9C
12 C
8
7
6
5
F2 (ppm)
Figure 2. Spectral data of (a) gHMBC, (b) gHSQC, and (c) COSY experiments on imine 3.
This set of experiments clearly shows that the attachment of the unprotected
4-(aminomethyl) piperidine to the p-nitrophenyl carbonate resin occurred
exclusively through the secondary cyclic amino group.
9
1.3.1.2 CPMG-T2/Diffusion-Filtered gHSQC Experiments
CPMG-T2 type filters (delay–180 o –delay) n were used to remove the residual
resin signals from the high-resolution NMR spectra. The T2 filter parameters
have to be carefully selected, since the signals of interest are also attenuated,
although to a lesser degree. Additionally, a diffusion filter was used to
separate out the signals from the unbound molecules in the sample. 10,13
Figure 3 shows two HSQC spectra of compound 4 (Scheme 1) obtained
by a conventional gHSQC experiment (Fig. 3a) and a modified CPMGT2 /diffusion-filtered gHSQC experiment (Fig. 3b). With careful selection of
the T 2 filter parameters and diffusion parameters, most of the polymer
signals, as well as those belonging to the methylene fragments 2H2, 3H2, 5H2
and 6H2 of piperidine, were removed and gave rise to the signals of interest,
group R. The monitoring of the imine reduction was possible by detection of
new carbon and proton signals of the –N8 H-9CH 2 fragment of compound 4
(Fig. 3b) and disappearance of the –N8 = 9CH fragment of compound 3 (Fig.
2b). The proton chemical shifts of the methylene group 7H2 in compound 4
had moved upfield compared to the corresponding signal from compound 3
(i.e., from δ= 3.5 to 2.3ppm), and this was due to the reduction of the imine
moiety. Full data with proton and carbon assignment of compound 4 were
based on the gCOSY, gHMBC, gHSQC, and modified gHSQC experiments.
13
14
9
15
O
1
5
9
4
7
R
(ppm)
50
60
70
80
90
100
110
120
130
140
16
10
F
12
15
16 C
F1
NH
8
R=
3b
5b
(b)
3
6
11
3a
4
5a
2
N
4
2b 7 16
6b
2a
6a
O
13
14
(a)
4C
9
14C
C
7
13 C
15 C
7
6
5
4
3
F1
16
C
2
2
C 6C
7
C
9C
14
C
15
13
1
5C 3C
F2 (ppm)
(ppm)
50
60
70
80
90
100
110
120
130
140
C
4C
C
C
7
6
5
4
3
2
F2 (ppm)
Figure 3. 2D 1H-13C correlation experiments on compound 4 performed using (a)
conventional gHSQC and (b) modified CPMG-T 2/diffusion-filtered gHSQC.
10
1
1.4 Reductive Alkylation of Secondary Amines
1.4.1 Solid- Supported Synthesis and HRMAS NMR
The synthetic route investigated is depicted in Scheme 3. The primary
amines were attached to the solid support by using the corresponding Bocprotected secondary amines as starting material.
b
a
O
O
O
O
O
O
O
NO 2
HN Y
1 [a ]
HN Y
11
12
X
X
N
H
N
O
c
O
d
H 2N
O
X
N
Y
O
HN Y
13
x, y = b on d, CH2
R
14-19
X
N
R
20
O
N
H2 N
O
O
N
H 2N
N
b
21
c
18
14
H2N
N
N
O
O
12
13
10
H
15
11
12
12
O
12
14
N
10
10
9
g
10
H
9
12
11
Cl
m
O
13
H
18
15
13
13
11
9
15
14
9
10
H
O
N
13
10
k
12
11
l
16
O
16
14
O
9
15
13
O
f
12
O
14
17
o
n
11
j
11
12
13
O
10
9
O
9
16
H
e
12
10
10
11
15
H
9
O
H
14
F
12
O
15
12
H
14
S
16
i
O
Cl
13
f
O
11
10
d
15
h
14
9
O
10
14
12
O
H
11
13
12
11
12
13
15
11
H
13
O
11
10
13
c
b
O
14
10
a
11
9
H
11
H
9
H2N
e
16
10
9
O
16
O
9
9
O
d
O
O
19
17
O
H
N
H 2N
a
H
O H2N
O
O
O
O
O
H
O
O
10
17
11
H
12
10
11
p
12
13
13
15
14
q
r
O
13
O
15
H
N
9
H
10
s
11
10
14
16
9
12
13
N
H
11
12
t
Scheme 3. Reagents and conditions: (a) Boc-protected amines (20) (5 equiv. in anh. DMF),
RT, overnight; (b) selective Boc deprotection with 10% TFA in DCM, RT, 2 h; (c) RCHO
(21) (10 equiv. in TMOF:DMF/EtOH (3/1) 1:1, Borane-pyridine complex (BAP) 10 equiv.,
RT, 4 days ; (d) 75% TFA in DCM, RT, 4 h.
[a]
Merrifield-type resin gave better yields than Wang-type resin did.
11
Selective BOC group deprotection of compounds 11 was examined by
applying various acidic conditions (Fig. 4a). The best results were obtained
using 10% TFA in dichloromethane (DCM), 26 by which essentially
quantitative yields of compounds 12 (Fig. 4b) were achieved, as estimated
from the CPMG-T2 /diffusion-filtered 1D-1 H NMR-spectra (Fig. 4). After the
deprotection step, the resulting secondary amines were subjected to
reductive alkylation with aldehydes 21 (Scheme 3). This was achieved in
situ using BAP,27 which led to clean reactions and high yields of products.
Other reducing reagents examined, including NaBH3 CN and NaBH(OAc)3
were found to be less effective.28–30 As an illustration, alkylation products 13
(Scheme 3) were analyzed by HRMAS NMR shown in Figure 4 (c and d).
The assignment of resonances 8 and 16 of compound 13 with R 21e and
resonances 8, 16, and 17 of compound 13 with R 21r is based on CPMG-T2
and diffusion filters gHSQC experiments.
O
4
O
5
3
N
13
7
6
N
2
1
8
R
16
8
11
R=
(d)
16
F
10
12
15
13
14
16
17
17
O
11
10
R=
(c)
16
12
15
8
13
14
(b)
Boc
O
O
(a)
8
N
11
7
6
7
4
5
3
2
5
6
N
1
Boc
4
3
2
ppm
Figure 4. 1H spectra with T2 and diffusion filtering of the products of the reaction presented
in Scheme 3: (a) 11, (b) 12, (c) 13 [with R = 21r and amine 20e, Scheme 3], (d) 13 [with R =
21e and amine 20e, Scheme 3].
12
Compounds 14–19 were obtained as bis trifluoroacetate salts in good overall
yields and with high LCMS purities after cleavage from the solid support by
use of a solution of 75% TFA/DCM.
1.5 Conclusions
A practical solid-phase synthesis of N-monosubstituted aminopiperidines
and aminopyrrolidines was developed. Selective alkylation of the primary or
secondary amine moiety was accomplished by use of the two synthetic
routes outlined in this study. The products were isolated in good yields and
purities after simple filtration steps, which enabled easy parallel processing.
The non-destructive analytical methodologies solid-phase NMR and FTIR
were used to monitor the reactions. Beside the use of 1D techniques to
remove unwanted signals, an extension of these techniques for 2D, gHSQC
spectra was reported. Thus it seems that the use of 2D NMR CPMG-T2
filtered gHSQC experiments together with the present solid-phase
methodology aimed at procuring novel diamine building blocks will further
broaden the scope of solid-phase approaches and aid the preparation of novel
drug-like compounds.
13
14
2. A One-Pot, Solid-Phase Synthesis of Secondary
Amines from Reactive Alkyl Halides and an
Alkyl Azide (Paper II)
2.1 Introduction
2.1.1 Preparation of Secondary Amines
There are numerous methods reported in the literature describing the
preparation of secondary amines, and the majority of them entail either
reductive aminations or alkylations on primary amines. Although the yields
are usually good, it is generally difficult to avoid the over-alkylation that is
associated with the use of reactive alkyl halides, which leads to formation of
the corresponding tertiary amines and quaternary ammonium salts. Other
approaches to prepare secondary amines include reduction of the
corresponding amides and aza-Wittig procedures.31–33
Both solution-phase and solid-phase methods have been developed for
most of the indicated alternatives, but the solid-phase strategies offer the
important advantage of ease of purification by use of simple filtration steps.
However, the emergence of commercially available solid-supported reagents
and scavengers has increased the use of the solution-phase approaches by
facilitating the work-up procedures so that in many cases the use of
chromatography can be avoided.
2.1.2 Utility of the Azide Moiety
It is easy to synthesize alkyl azides starting materials by applying a number
of reported procedures, such as nucleophilic substitution of bromide with
sodium azide 34 or conversion from amines through diazo transfer
reactions.35,36 The use of Amberlite® azide exchange resins offers further
possibilities for the synthesis of azides.
Furthermore, organic azides have recently attracted considerable
interest, partly due to their popularity in the Cu I-catalyzed reactions between
azides and terminal alkynes (Scheme 4A),37a and use of the azide functional
group for synthesis of 1,2,3-triazoles is well known (Huisgen reaction).
As protecting groups, azides can help avoid many of the concerns
associated with amides and carbamates, and they provide the option of a
mild reduction (Staudinger reaction). The conversion of azides into amines is
a standard synthetic procedure for chemists. The Staudinger reaction
(Scheme 4B), 37b–d involves the treatment of an alkyl azide with
triphenylphosphine to produce a phosphoazide that rapidly eliminates
15
nitrogen to afford an iminophosphorane. This intermediate can be readily
hydrolyzed in a protic solvent to give the corresponding primary amine, a
process that has been transferred to polymer support. 38 Iminophosphoranes
are useful intermediates in synthesis of a variety of nitrogen-containing
compounds (Scheme 4B), including amines and carbamates, and in the azaWittig reactions. 39,40
(A) Huisgen reaction
R'
R
R- N3
CuI
N
R'
N N
(B) Staudinger reaction
R-N3
PPh 3
Ph
Ph
+
P N
Ph
R
N
N
N N
Ph P N
Ph Ph
R
-N 2
H2 O
Ph3 P=NR
R NHCO2 R' '
-Ph3PO
RNH2
R NHCO R' '
Scheme 4. Transformations of organic azides.
2.1.3 Alkylation of Iminophosphoranes: Background
The use of a phosphonium diaza-diylide or a phosphonium aza-yldiide as a
reagent for the synthesis of primary and secondary amines (Scheme 5) in a
stepwise solution-phase dialkylation of an aza-yldiide has been reported by
other investigators. 41 That approach led to good yields of N-alkyl
phosphimines or disubstituted aminophosphonium salts, which by hydrolysis
provided the corresponding primary or secondary amines. However, the
mentioned methodology requires the use of a strong base.
1
H 2N-R1
NH3
3 steps
Ph3P=N-Li
H N
HO-/ H2O
R1 I, THF, r.t.
Ph3 P=NR1
R
2
HO- /H2 O
R2 I, THF, 65 0C
Scheme 5. Synthesis of amines via alkylation of iminophosphoranes.
16
R
+
Ph3P
N
R
R
1
2
I
-
2.1.4 Aim of the Study
The aim of the study presented in Paper II was to develop a new method that
involves preparation of secondary amines through alkylation with reactive
halides in order to avoid the problem of over-alkylation. Such a method
should be easy to apply and, if possible, should not require chromatographic
purification.
There are reports describing procedures for conversion of azides into
secondary amines that do not involve iminophosphorane or imine
intermediates. 42 However, whilst methods via an intermediate
iminophosphorane have been developed, they entail aza-Wittig reactions
starting from azides and aldehydes. 31,43 Thus it seemed that an attractive new
methodology for the synthesis of secondary amines would be to use the
Staudinger reaction to accomplish alkylations of N-alkyl phosphimines or Naryl phosphimines with reactive alkyl halides, and that was the objective of
the present study.
2.2 Solid-Phase Alkylation of Iminophosphoranes
The investigation focused on combining the Staudinger reaction with the
alkylation of iminophoshoranes to achieve exclusive delivery of secondary
amines starting from azides and alkylating agents. A solid-phase approach
was undertaken to provide ease of purification. Scheme 6 depicts the solidphase protocol that was developed.
Ph
R1 N3
PP h2
22
N R1
P
23
R2X
25
Ph
24
(a)
R1
Ph
P
N
Ph
26
Ph
X
2
R
R1
P N
X
Ph
2
R
26
H
N
(b )
1
R
R2
27
Scheme 6. Reagents and conditions: (a) filtration and washing of the resin; (b) KOH-MeOH,
65 C̊; filtration.
The method thus proceeded via a Staudinger reaction employing polymersupported triphenylphosphine (22)44 and an azide (23) to give the
corresponding iminophosphorane (24). The resulting suspension was
agitated at room temperature for 3–4 h. During development of the protocol,
the reactions were conveniently monitored by using FTIR to follow the
disappearance of the signal corresponding to the azide in the solution phase
(at 2,045 cm –1 ) as a new P=N absorption band in the solid phase (at 1,112
cm –1 ). Iminophosphorane 24 was subsequently alkylated in situ with alkyl,
17
allyl, or benzyl halides (25) to afford the corresponding disubstituted
aminophophonium salts (26). The best results were obtained when the
alkylation was allowed to proceed for 16 h at ambient temperature.
Prolonged reaction times or heating did not improve the overall yields. After
washing the resin, hydrolytic cleavage of the secondary amines from the
solid support was performed using one equivalent of methanolic KOH. The
cleavage time was optimized to 3–4 h at 65 C̊ to furnish the corresponding
secondary amine (27) in solution and resin-bound triphenylphosphine oxide,
which was removed by filtration. Microwave heating was attempted for 10–
40 minutes at temperatures ranging from 100 to 140 0 C without any
significant improvement of the outcome. After concentration, the resulting
secondary amines were isolated by using dichloromethane and ethyl acetate
for extraction of the aqueous phase containing the potassium salts. The
polymer-supported triphenylphosphine can be regenerated by reducing the
polymer-supported phosphine oxide with trichlorosilane.45
Table 1 shows the azides and alkylating agents used, as well as the isolated
yields of products and the purities. All products were characterized by
LCMS, NMR, and HRMS. No primary amine byproducts from incomplete
alkylation or any other byproducts were observed. The advantage of
avoiding dialkylation with this new method is most clearly observed in the
cases where MeI was used (entries 1 and 6), which, in contrast to other
methylation techniques, did not result in any dimethylated products.
2.2.1 Scope and Limitations
The yields obtained (Table 1) were generally good (78–87%), except for the
phenylazide (entry 5), and offered excellent purities (> 95%). An
explanation for the low yield of entry 5 might be due to slower reaction
kinetics between the phenylazide and the polymer-supported
triphenylphosphine, as has previously been observed in a study concerning
the reduction of phenylazide to aniline. 46
A similar finding was made when using 3,4-dichloro phenylazide as
starting azide and 4-tert-butyl benzyl bromide as alkylating agent (Fig. 5). In
that case, the expected product could not be found.
N3
N
+
Cl
Br
Cl
Cl
Cl
Figure 5
Although not part of the objective of this study, attempts were made to use
alkylating agents that are less reactive than the ones shown in Table 1 in
18
order to evaluate the scope of the method developed. For example, branched
halides and tosylates (Fig. 6) were examined in combination with benzyl
azide, but without success.
Azide 23:
O
O
R2 X 25:
Fm ocNH
O
N3
S
I
Figure 6
Table 1. Preparation of secondary amines 27 from azides and reactive halides
Entry
2
Azide 23
1
R X 25
N3
Product 27
MeI
Yield(%)
NH
a
Purity (%)
81
95/95
82
95/95
87
94/95
78
97/95
21
97/95
85
97/94
82
96/95
b
CN
2
Br
N3
NH
NC
Br
N3
3
NH
Br
4
N3
H
N
N3
5
Br
H
N
O
O
O
O
O
O
O
6
O
MeI
HN
O
HN
N3
Br
O
O
O
7
HN
HN
N
H
N3
a
N
H
Yield of pure compound fully characterized by LCMS, NMR, and HRMS with respect to the
initial loading of the resin.
b
Purity was determined by LCMS/NMR, except for entry 6, which was determined by
ELSD/NMR.
19
2.3 Conclusions
In summary, a convenient and efficient polymer-supported protocol for the
synthesis of secondary amines was developed that affords the target
molecules in good overall yields and purities from the corresponding azides
and reactive alkyl halides without the necessity of isolation or purification of
any intermediates. This procedure complements existing methods of amine
alkylations, reductive alkylations, and aza-Wittig procedures. Moreover, the
present results indicate that the new methodology developed works very well
when using alkyl azides and benzyl azides as starting materials in
combination with reactive halides, as shown in Table 1. Regarding the
limitations of the procedure, it can be deduced that it is generally not suitable
for use with phenyl azides and that the alkylating agent should be reactive.
To broaden the scope of this approach, further work can be envisioned
which would allow the use of phenyl azides as starting materials by adapting
the procedure to solution-phase parallel synthesis employing a fluoroustethered PPh3 in combination with FluoroFlashTM SPE.
20
Part II:
Design and Synthesis of Inhibitors Targeting the
Cysteine Proteases Cathepsins S and K and the
HCV NS3 Protease
3. General Introduction
3.1 Proteases: Hydrolysis of Peptide Bonds
The enzymes known as proteases (protein hydrolases) catalyze amide
(peptide) bond hydrolysis in protein or peptide substrates inside and outside
living cells, and they are involved in the control of dynamics of protein
turnover. Protease-catalyzed rates of hydrolysis are in the range of up to 10billion-fold faster than non-enzymatic rates. Hydrolysis is an energetically
favorable reaction, and therefore the induced proteolysis is an irreversible
process that must be stringently controlled.
R
N
H
C
H
O
C
R'
H
N
O
R
O
C
H
C
R'
O
C
H
C
H2O
C
H
C
N
H
N
H
O
+
H3 N
N
H
amine
acid
Proteases can be classified in two ways: (1) as exopeptidases
(aminopeptidases and carboxypeptidases) or endopeptidases based on their
site of action; (2) as serine/threonine, cysteine, aspartyl, or zinc (metallo)
proteases by considering their reaction mechanisms and the nature of the
active-site residues involved in the catalytic cleavage. The side chains of the
substrate/inhibitor recognized by the protease are designated P3, P2, P1, P1',
P2' and P3' and the corresponding subsites of the protease S3 , S2, S 1, S1 ', S2 '
and S3 ' as depicted in Fig. 7.47
Nonprime subsite
Enzyme
S2
N terminus
N
H
P3
S3
S 3'
S 1'
P2
O
Prime subsite
O
P1'
O
H
N
N
H
P1
O
S 1 Scissile
peptide bond
P3'
O
H
N
N
H
C terminus
P2'
S 2'
Figure 7. Prime and nonprime substrate peptidyl residues (P and P') and enzyme subsites (S
and S') oriented according to the peptide bond cleaved within the substrate.
21
Proteases play important roles in many physiological processes, such as
growth, signal transduction, cell proliferation, cell death, immune defense,
coagulation, fertilization, and wound healing.48 These catalytic proteins are
also involved in many pathological conditions and disorders, including
cancers,49–51cardiovascular diseases,52 autoimmune diseases, 53 osteoporosis,
Alzheimer’s disease and infectious diseases, e.g. malaria, HIV and HCV.54–56
Obviously, this means that manipulation of the activity (in most cases
inhibition) of proteases can provide an opportunity for therapeutic
intervention.
3.2. Cysteine and Serine Proteases: Mechanism of
Action and Specificity
Serine and cysteine proteases have nucleophilic moieties that act directly on
the substrate to generate a covalent acyl-enzyme intermediate. Aspartyl and
metalloproteases operate indirectly by activating water molecules as the
nucleophilic species attacking the peptide bond (Fig. 8).
Acyl-Enz
Enz-X
R'NH2
X-Enz
X-Enz
O
R
R
R
O
O
R
R
H2O
O
NHR '
X-Enz
NHR '
OH
RCOOH
O
RCOOH + NH 2R'
N HR'
OH
Tetrahedral
adduct
Figure 8. Activation mechanisms of proteases.
3.2.1 Catalytic Mechanism of Serine Proteases
Serine proteases constitute the largest class of proteases, and their catalytic
triad is characteristically composed of His57, Ser195, and Asp102. Ser195 is
the active nucleophile that attacks the peptide bond as a serine alkoxide
equivalent; His57 acts in sequential steps, first as a base and then an acid
catalyst for proton transfer; Asp102 orients the His57 side chain to form an
H-bond with Ser195. The backbone amide NH groups of Ser195 and Gly193
form the oxyanion hole. In this way, the protease accelerates peptide bond
hydrolysis by 10 9 -fold. All steps occur under the promotion of general
acid/base catalysis of His57 and Asp102, and through stabilization of the
negatively charged tetrahedral intermediates by the oxyanion hole.
22
The mechanism is divided in two half reactions: enzyme acylation and
enzyme deacylation (Fig. 9). In enzyme acylation, Ser195 attacks first the
peptide bond in the bound substrate to form a tetrahedral adduct, and
thereafter an acyl-enzyme bond resulting in the release of the amine product
(Fig. 9a). In enzyme deacylation, attack of an OH – on the acyl enzyme leads
to formation of a tetrahedral adduct which collapses delivering the acid
product (Fig. 9b).
(a)
(b)
Figure 9. The first (a) and second (b) steps of the catalytic mechanism of serine proteases.
3.2.2 Catalytic Mechanism of Cathepsin Cysteine Proteases
The name cathepsin (from the Greek kathepsein, to digest) was initially used
to describe any group of intracellular peptide hydrolases, although it was
later discovered that several proteins also have extracellular functions.
Cathepsins are further defined as being cysteine, aspartic, or serine
proteases, according to their catalytic mechanism. Cathepsins have been
identified in a wide array of organisms, ranging from viruses, bacteria, and
plants to mammals. To date, 11 human cathepsins are known. Lysosomal
cysteine proteases belong to a subgroup of the cathepsin family that is
essential for normal cellular functions, such as antigen processing, bone
remodeling, and general protein turnover.
23
The same fundamental mechanism of serine proteases is valid for cysteine
proteases, in which the cysteine thiolate replaces the serine alkoxide as the
active nucleophile, and the acyl-enzyme intermediate is peptidyl-S-enzyme.
The active triad comprises Cys25, His159, and Asn175 (papain numbering
system), and it shows optimal activity at a pH of ~5.5, which is the pH in
lysosomes. Loss of activity at pH down to 3.8 or a neutral pH can be due to
structural instability of the active enzymes, with the exception of cathepsin
S, which maintains its activity in a neutral pH range. 57-58
The enzymatic mechanism of the cysteine cathepsins involves a
nucleophilic thiol and general acid-base catalysis (Fig. 10).
His
N
H
Cys
His
Cys
His
+
NH
S
-
N
H
N
H
+
NH
S
R'
S
N
H
O
Cys
R
N
H
R
N
H
O
R'
NHR''
NHR''
R'NH 2
Cys
Cys
His
His
-S
His
+
NH
N
O
N
H
HO
R
N
H
S
Cys
O
N
R
HO
NHR''
NHR''
N
H
O
H
S O
R
H
NHR''
Figure 10. Catalytic mechanism of lysosomal cysteine proteases.
The negatively charged Cys25 nucleophile is stabilized by the positive
His159 and by the positive dipole of the central helix in which it resides. 59
The thiazole-imidazolium ion pair is characterized by an unusually low pKa
value of the reactive thiolate group (~2.5–3.5).59 The cysteine thiol group
attacks the carbonyl of the target peptide bond to form a tetrahedral
intermediate that collapses to the thiol ester, resulting in the release of the
first peptide product. A second tetrahedral intermediate is formed upon
water-catalyzed hydrolysis of the thiol ester, which then collapses to release
the second peptide product and thereby regenerate the active site.
3.2.3 Specificity of Proteases
Given that the catalytic triad inventory of all serine and cysteine proteases
exhibit conserved composition and a common mechanism of action, the
question arises as to how these enzymes achieve their specificity. This is
achieved through the differences manifest in the subpockets, specificity
pockets, of the protease. Many proteases have extensive specificity subsites
for recognizing n+1, n+2, and/or n-1, n-2 residue side chains on protein
substrates. For example, the Asp-His-Ser catalytic triad in chymotrypsin,
24
trypsin, and elastase is essentially identical but the specificity pockets they
contain differ in that they accommodate a hydrophobic aryl side chain, a
cationic side chain, or a small aliphatic side chain respectively (Fig. 11).
Figure 11. Specificity pockets of chymotrypsin, trypsin, and elastase.
In general, the cathepsins show broad specificity for their peptide substrates,
although some do prefer certain amino acids over others in the target
sequence, and S 2, S1 , and S1' substrate binding sites confer the greatest
degree of specificity. 47,60 For example, cathepsin K prefers a hydrophobic
amino acid/Pro at P2, whereas cathepsin S favors Leu/Val at P2 and
cathepsin L a hydrophobic residue at P1 and P3.
3.3. Protease Inhibitors
There are several types of protease inhibitors, and three main methods have
been used to create new inhibitors: (1) rational design of peptidomimetic
inhibitors using substrates as starting points; (2) screening of natural
products and corporate compound libraries as starting points and subsequent
refinement of the substances that are identified; (3) if accessible, use of data
on the three-dimensional structure of the active site of the enzyme (obtained
by NMR studies or X-ray crystallography) to apply rational design for the
development of new protease inhibitors.
Inhibitors and substrates generally adopt an extended β-strand
conformation in which the linear extended β
-strand backbone gives
maximum exposure of its side chains to the binding pockets of the protease.
This conformation precludes intramolecular hydrogen bonding in the peptide
and exposes the peptide bonds to catalytic hydrolysis.
25
3.3.1 Serine Protease Inhibitors
The serine protease inhibitors can be divided into two categories: those that
are covalent (reversible and irreversible) and those that are non-covalent.
One of the categories comprises inhibitors derived from the nonprime side of
the peptide substrate, wherein the scissile amide bond is replaced by an
electrophile that interacts with the catalytic serine residue. 48,61–63 However,
there are also several potent prime-side inhibitors, which indicates that
interaction with the prime sites of the enzyme can enhance the binding
affinity of an inhibitor. 61,64,65 The main reason for using an electrophilic
group to achieve inhibition is to increase the affinity through formation of a
covalent tetrahedral intermediate with the catalytic residue, thus resembling
the transition state of a normal substrate hydrolysis (Fig. 12). 66,67
Gly193
Ser195
P2
N
H
H
N
O
N
H
O
P2
Serine protease
X
N
H
P1
Serine-trap inhibitor
O
H
N
O
N
H
O
X
P1
Inhibited serine protease
Ser195
X= H, CF3 , C 2F 5, CONHR, COOR, COR, Heterocycle
Figure 12. Examples of electrophilic groups that can serve as serine trap inhibitors by
forming a tetrahedral intermediate with the enzyme and thus inhibiting the enzyme from
further activity.
Typical electrophiles are nitriles, trifluoromethyl ketones, pentafluoroethyl
ketones, aldehydes, α-ketoamides, α
-ketoester, α-diketones, α-heterocycles,
organoboranic acids, and organophosphonate esters.
Examples of inhibitors that interact with the enzyme via an irreversible
covalent bond are alkylating agents such as monohalomethylketones or
epoxides, or acylating agents such as β-lactams. 61,62,68,69a There is a potential
disadvantage in using covalent inhibitors, since reactivity of the electrophilic
moiety with endogenous proteins or off-targets may result in immune
activation and other adverse events. Another potential issue is low chemical
stability of the inhibitors. For these reasons, non-covalent inhibitors are often
preferred over covalent inhibitors.63 Hydrogen bonds, hydrophobic,
electrostatic, and/or van der Waals interactions deliver the binding affinity of
non-covalent inhibitors.
26
3.3.2 Cathepsin Cysteine Protease Inhibitors
The mode of action of enzyme inhibitors can be subdivided into distinct
categories. 68 The earliest reported cathepsin inhibitors were irreversible
inhibitors, and they included compounds such as those based on the epoxide
E-64, halomethylketones, and azapeptides, as well as those emanating from
Michael acceptors (e.g., the vinyl sulfone LHVS), which were particularly
useful in elucidation of the pharmacology of cathepsin S inhibition (Fig.
13). 55 These compounds react with the free thiol of the cysteine in the
cathepsin active site and bind in an irreversible manner to inhibit the
proteolytic activity of the enzyme.
R'
O
H
N
RN H
O
NH2 +
F
H2N
R''
N
H
CO2 H
E-64
O
N
O
N
H
O
Halomethyl ketone-based inhibitors
O
O
H
N
N
H
O
O
H
N
S
Ph
O
Ph
Vinyl sulphone: LHVS
Figure 13. Classical irreversible cysteine protease inhibitors.
Later, chemists developed inhibitors that form a reversible covalent bond
with the enzyme, most of which have an electron-deficient carbonyl group
and low steric hindrance. Various ‘warheads’ that form such a bond with the
active site cysteine have been described, including peptide aldehydes,
nitriles, α-ketones, and α-ketoamides, as well as cyclic ketones, β-lactams,
azepanones, and non-covalent aminoethylamides (Fig. 14).69
O
R'
O
H
N
RNH
O
X
R
n X
R''
R'
X= H, CO 2Me, CONHR
Nitriles
R'
R''
R'
Aminoethyl amides
Non-covalent inhibitors
O
R
N
CN
R
H
N
N
H
Cyclic ketones
n =1-3
R
R'
O
R
O
NH
N R'
O
Azepanones
B-Lactams
Figure 14. Examples of reversible cysteine protease inhibitors.
27
28
4. Solid-Phase Parallel Synthesis and SAR of 4Amidofuran-3-one P1-Containing Inhibitors of
Cathepsin S: Effect of Sulfonamides P3
Substituents on Potency and Selectivity
(Paper III)
4.1 Introduction
4.1.1 Antigen Presentation and the Immune Response
Antigen-presenting cells (APCs) degrade foreign proteins to small peptides
and display these peptides bound to a major histocompatibility complex
(MHC) protein on the cell surface, and this process is known as antigen
processing and presentation (Fig. 15). 70 APCs, in particular primarily
dendritic cells, B-lymphocytes, macrophages, and microglial cells take up
antigens from the extracellular environment. The internalized protein
antigens are processed by use of endosomal or lysosomal proteases to
generate peptides that become associated with MHC class II protein.
Peptide-loaded MHC-II complexes are subsequently transported to the cell
surface to be displayed to CD4+ T-cells. The biosynthesis of mature MHC-II
is controlled by interaction with the protein called invariant chain (Ii). In
short, Ii prevents loading of endogenous peptides onto newly synthesized
MHC-II within the endoplasmic reticulum and facilitates the sorting of the
MHC-II-Ii complex into the endosomal system. Once in the lysosomes, a
consortium of proteases degrades Ii to enable the peptide loading .
Antigen
CD4 T-cell
Denaturation
Peptide/MHC
Complex
Proteolysis
Peptide
Loading
MHC
Class II
Cathepsin S
Invariant
Chain
Figure. 15. Antigen processing and presentation.
29
Ii is processed in several steps (Fig.16) by several proteases, the exact
identity and number of which are unknown. Nevertheless, it is clear that, in
dendritic cells and B-cells, cathepsin S is the enzyme responsible for the
final proteolytic step where it degrades the remaining Ii p10 oligomer
fragment into a small peptide called CLIP. Finally, the CLIP peptide is
removed from MHC-II through an interaction with endosomal/lysosomal
resident protein HLA to permit binding of antigenic peptides.
Leu-Ile Met-Leu
Ii
80
103
C
N
Cytoplasm
Transmembrane
CLIP
Lumen
MHC Class II
Ii associated
Ii p23 N
CLIP
Ii p10 N
Cathepsin S
MHC Class II
Peptide complex
CLIP
Figure 16. Processing of the invariant chain.
Recognition of the MHC-II-peptide complexes triggers the activation of
antigen-specific CD4+ T lymphocytes, 71a which in turn stimulate other
components of the immune system, such as B-cells, macrophages, and CD8+
T-cells (Fig. 17). These cellular reactions are a crucial part of the body’s
response to pathogens, but they are also responsible for the development and
symptoms of allergies and autoimmune diseases.
The purpose of an immune response is to attack a foreign target, and in
that context it is extremely important to be able to distinguish ‘self’ from
‘non-self,’ because failure of the immune system to recognize ‘self’ will
result in an autoimmune disease. Examples of current therapies for such
disorders include TNFa blockers (e.g., Enbrel, Remicade and Humira) for
the treatment of rheumatoid arthritis (RA); interferon β
, glatiramer acetate,
mitoxantrone hydrochloride, and corticosteroids, which are often
accompanied by undesirable effects.71b Other immuno-based treatments like
cyclosporine and cytokine inhibitors target the effector molecules in
inflammatory reactions, and hence it may be possible to provide a novel
30
form of immunotherapy by focusing on the antigen presentation pathway
responsible for triggering autoimmune diseases. 71c
CD4 T cell
Activated
CD4 T cell
B cell
Cytokines
Disease
Pathology
MHC Class II
APC
Macrophage
CD8 T cell
Figure 17. Role of T-cells in the immune response.
Cathepsin S is a 24-kD monomeric cysteine protease that belongs to the
papain super family, and it is expressed in many professional APCs as well
as ‘non-professional’ APCs (e.g., epithelial cells).72 Cathepsin S was
originally identified in bovine lymph nodes in 1975,73 and the human form of
the protein was cloned in 1992.74-75
The most well-characterized function of cathepsin S is related to its role
in antigen presentation to CD4+ T-cells, which involves participation in the
proteolysis of the Ii chaperon molecules that is associated with the MHC-II
complex, 71a, 76a-b a crucial step in the immune response mediated by CD4+ Tcells. Therefore, a selective inhibition of cathepsin S will block the
degradation of the p10 invariant chain (Ii-p10) and disrupt the presentation
of antigens, and thereby act in an immunosuppressive manner. Such
selective inhibition of cathepsin S represents a potential for modulating
autoantigen-driven immune responses in MHC-II-restricted autoimmune
diseases.
While the role of cathepsin S in MHC-II-mediated antigen presentation
has received the most interest, the potent elastinolytic and collagenolytic
activity of secreted cathepsin S (by macrophages and microglia) additionally
makes the inhibitors of this protein as attractive alternatives for modulating
connective tissue matrix degradation.
The crystal structure of cathepsin S is highly similar to the structures of
other cysteine proteases in the papain super family, especially cathepsins K
and L. 76c The substantial sequence homology between these three cathepsins
offers considerable challenges in designing selective cathepsin S inhibitors.
The tertiary structure of cathepsin S comprises an N-terminal domain that
consists primarily of α-helices and a C-terminal domain that contains βsheets. Between these two domains lies the catalytic triad formed by Cys25,
31
His164, and Asn184 (corresponding to Cys25, His159, and Asn175 in
papain), from which a substrate-binding site extends out on both sides. 77
Unlike most other cathepsins, which are inactive at neutral pH, human
cathepsin S achieves its optimal activity at pH 6.5 and remains active from
pH 4.5 to 7.8, consequently increasing its range of activity from acidic
lysosomes to neutral or slightly alkaline extracellular environments.78
4.1.2 Cathepsin S as a Potential Therapeutic Target
Cathepsin S is a potential target for the treatment of autoimmune diseases.
Substances that achieve selective inhibition of CD4+ T-cells should provide
a therapeutic advantage over non-selective immunosuppressive compounds,
since the function of CD8+ T-cells would remain largely intact. Deletion or
inhibition of cathepsin S in some disease-relevant animal models has been
shown to be efficacious. An example of this is a study of collagen-induced
arthritis in cathepsin S knockout mice, 79a in which it was found that the
severity of the disease was diminished relative to that seen in wild-type
mice. The available animal model data and knowledge regarding the
involvement of CD4+ T-cells in the disease pathogenesis, as well as the
medical need for effective treatment options, have made rheumatoid arthritis
a lead indication for therapy using cathepsin S inhibitors. In the case of
rheumatoid arthritis, inhibition of cathepsin-S-mediated degradation of
extracellular matrix might also be beneficial. Cathepsin S might also
represent a new pain-relief strategy as reported in a study concerning the
inhibition of spinal microglial cathespin S for the reversal of neuropathic
pain. 79b-c
In addition to rheumatoid arthritis, it seems that cathepsin S may be a
suitable target for the treatment of atherosclerosis and Sjögren’s syndrome,
based on evidence provided by animal models using inhibitors and knockout
mice. Moreover, it is possible that cathepsin S inhibitors can be beneficial in
a number of autoimmune and allergic conditions in which CD4+ T-cells are
important factors in the pathogenesis, ranging from type I diabetes to
multiple sclerosis. Cathepsin S has been found to be a key enzyme in the
processing of human myelin basic protein (MBP) in vitro 80 where MBP
appear to function as an autoantigen in the development of multiple sclerosis
(MS). Cathepsin S inhibition might thus be a viable therapeutic approach for
MS disease.
Involvement of cathepsin S in the pathogenesis of psoriasis has also
been explored.80b-81 Since the majority of psoriasis patients have elevated
interferon-gamma-producing T-cells in their epidermis, it was speculated
that upregulation of cathepsin S could be a factor in induction of the disease.
Furthermore, a study has shown that cathepsin-S-positive neurons are
present in the brains of patients with Alzheimer’s disease (AD) and Down’s
32
syndrome,82 and the presence of Aβplaques and vascular deposits in the
brain are considered to be characteristics of AD. Cathepsin S has also been
found to facilitate the formation of Aβfrom its precursor protein in a kidney
239 cell line, which suggests that cathepsin S can be a target for AD therapy.
Finally, cathepsin S has even been implicated in some cancers, for instance
substantial expression of this protein has been observed in prostate
carcinoma. 83
4.1.3 Background Information and Aim of the Study
We have previously described novel 4-amidofuran-3-ones as potent
cathepsin S inhibitors, 84 represented by compound 28, which has a Ki value
of 31 nM. The analogs of this series are equipotent against mouse and rat
cathepsin S, enabling proof-of-principle studies to be carried out in a rodent
model of human disease, such as multiple sclerosis (MS), 76c,85 and
rheumatoid arthritis (RA). The distal region of the S 3 subsite of cathespin S
is defined by backbone Gly62 and the side chains of Lys64 and Phe70 (using
the numbering from the PDB entry 1MS6 for cathepsin S). Analysis of
structural data from cathepsin S in complex with 28 implied that there were
potential for hydrogen bond interactions in the S3 subpocket, and it seemed
that the carbonyls of Gly62 and Asn67 were especially likely candidates as
hydrogen bond acceptors. Lys64 was also a possible hydrogen bond donor,
but with less certainty due to the flexibility of the side chain (Fig. 18).
H
N
S
O
O
O
N
H
O
28
Figure 18. Compound 28 modeled in the active site of cathepsin S. The subpockets are
denoted S1, S2, and S3.
Some recent studies have indicated that structural differences observed in the
S3 pockets of cathepsins S, K, and L give different preferences for the P3
substituents.77,86,87 The reduced size of the S3 pocket in cathepsin S has
previously been recognized from the crystal structures of cathepsin S
inhibitor complexes, 88 and the preference for a larger P3 substituent in S 3 of
cathepsin K has also been established. 89 However, in other experimental
33
endeavors, 86 receptor optimization calculations revealed that a unique Lys64
residue residing in the cathepsin S S3 pocket can re-orient its side chain to
accommodate an extended P3 moiety of the inhibitor. This feature provides
new opportunities for targeting selectivity when considered in combination
with the selectivity requirements of the S2 pocket, which, in cathepsin S,
accepts significantly larger groups compared to the S2 pocket in cathepsin K.
The use of structure-based drug design led us to the design of compound 29
(Fig. 19) with methyl cyclopentylalanine as preferred P2 side chain and a
sulfonamide hydrogen donating P3 moiety and as an extended P3 motif.
K64
G62
S3
O
O
S
G69
H
N
H
O
O
N
N
H
O
O
S1
29
S2
Figure 19. Schematic representation of the predicted binding mode of inhibitor 29 within the
active site of human cathepsin S. The sulfonamide moiety is assumed to bind in the S3 pocket,
the cyclopentyl group within the hydrophobic S2 binding pocket and the C5-ethyl fuarone in
the S 1 site.
Compound 29 showed very good inhibitory activity against cathepsin S (K i =
2.6 nM), with a 10-fold increase in potency compared to compound 28,
although with both P2 and P3 groups altered (Table 2), as well as acceptable
selectivity over cathepsin K (K i = 290 nM) and good selectivity towards
cathepsin L (Ki = 12,000 nM). These encouraging properties of compound 29
supported a more detailed exploration of this class of inhibitors, focusing in
particular on the S3 pocket of cathepsin S.
The aim of the study presented in Paper III was to explore the S 3 pocket of
cathepsin S and to investigate the scope of the sulfonamide moiety at the P3
residue of the C5-ethyl furanones. The intention was to consider the
indicated moiety as an extended P3 substituent, while keeping the P2 and P1
residues constant (Fig. 20), in order to see how it affects the potency and the
selectivity profile.
P1
R3
O
O S N
R2
O
O
N
R1
N
O
O
P3
P2
Figure 20. Schematic representation of the intended P3 variations (R1, R 2 and R3) in the C5ethyl furanone inhibitors of cathepsin S.
34
4.2 Chemistry
4.2.1 Solid-Phase Chemistry of Dihydro-2(3H)-5-ethyl
Furanones
Scheme 7 shows the solid-phase parallel synthesis of dihydro-2(3H)-5-ethyl
furanones 29 and 37–81 containing sulfonamide moieties as P3 motifs (see
structures in Tables 2–5).
O
H2N NH
Fmoc HN
O
xT FA
O
O
COOH
a, b
C
NH
FmocHN
HN NH
O
30
O
31
32
c, d
OO H
S N
R1
X
R2
O
C
O
O
HN
HN
O
NH
NH
HN NH
O
C
O
c, e
NH
O
NH
Fm oc HN
NH
HN NH
NH
NH
O
X= C, N
h, i
36
35:
X= C, N
OO H
S N
R1
X
O
H
2 R2
R1
N
3
S
O
O X
34
OH
O
O
R2
O
O
HN
O
c, f-h
O H
N
S
R1
HN
O
O
O
HN
O
N
H
O
X= C, N
43, 68-70
29, 37-42, 44-67, 71-81
Scheme 7. Solid-phase synthesis of dihydro-2(3H)-5-ethyl furanone inhibitors.
Reagents and conditions: (a) MeOH, 70 o C 2 h, then RT 2 h, 85%; (b) amino resin,91 HBTU,
HOBt, NMM, DMF, 16 h; (c) 20% piperidine in DMF, 1 h; (d) Fmoc-(methylcyclopentyl)alanine-OH (33),92 HBTU, HOBt, NMM, DMF, 16 h; (e) P3 acid (35), HBTU, HOBt, NMM,
DMF; (f) 4-amino benzoic acid, HBTU, HOBt, NMM, DMF, 16 h; (g) R1SO 2Cl, DMAP, Py,
DCM, 16 h; (h) 95%TFA/H 2O, 1 h; (i) additional step for comp 65: H2, Pd/C, MeOH, 2 h,
91%.
Dihydro-(4S-amino-[N-Fmoc])-5S-ethyl-3(2H)-furanone (30) synthesized as
previously reported 84 was treated with acid 3190 in MeOH. The resulting
semicarbazone acid linker was obtained in 85% yield. This linkage not only
temporarily blocks the reactive carbonyl functionality, but also serves as an
anchor for construction of the inhibitor via parallel synthesis. Subsequently,
the P1 resin 32 was obtained by treating the linker with the aminomethyl
functionalized polymer support 91 (0.24 mmol/g) in the presence of N-[(1Hbenzotriazole-1-yl)-(dimethylamino) methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HBTU), 1-hydroxybenzotriazole (HOBt), and
N-methyl morpholine (NMM) in DMF. Deprotection of resin 32 with 20%
35
piperidine in DMF followed by standard amino acid coupling of the Fmocprotected methyl cyclopentane alanine amino acid92 33 by use of HBTU,
HOBt, and NMM in DMF provided P2-P1 resin 34. After standard removal
of the Fmoc group, the P3 acids 35 (Fig. 21), which had been prepared
previously were coupled using the same coupling conditions as described
above to furnish P3-P2-P1 resins 36.
A slightly different procedure was employed for target furanones 43
(Table 3) and 68–70 (Table 5), for which coupling of the P3 acid residue was
performed in two steps: attachment of 4-amino benzoic acid onto deprotected resin 34 under standard amino acid coupling conditions (HBTU,
HOBt, NMM), and subsequent acylation with the corresponding sulfonyl
chloride using DMAP and pyridine in DCM.
Final acidic hydrolysis with 95% aqueous TFA cleaved the target
ketones 29 and 37–81 (Tables 2-5) from the Webb-linker-bound resin in 75–
80% yields. Catalytic hydrogenation using H2 in the presence of Pd/C was
required to provide target 65 (Table 5).
O
O
O S
HN
O S
HN
O
O S
HN
OH
35a
OH
O
35b
O
O S
HN
H
N
S
N
H
O S
HN
OH
O
35i
35h
35j
O
O
O
O S
HN
O
OH
OH
OH
O
35l
O
35k
35g O
O
O S
HN
OH
OH
O
OH
OH
O
O S
HN
OH
S
O
O S
HN
35f
35e
O
O S
N
O
O
NH
OH
O
O
35d
O
N
S
S
OH
O
35c
O
O
OH
O
O
O
O
O S
HN
35m
O
F
O
O
O S
HN
O
O S
HN
Cl
O
OH
35o
O
O S
HN
OH
O
35p
35q
N
HN
O
O S
HN
OH
O
35w
O S
HN
N
O
O S
HN
N
35ac
O
O
O
O S
HN
OH
35ak
O
O
OH
O
35ag
O
O
O S
HN
S
OH
O
35ah
N
O
S
O S
HN
O
35an
O
OH
O
35ai
O
N
O
S
O S
HN
N
S
OH
OH
OH
35am
O
O
O S
HN
O S
HN
OH
35af
35ab
O
N
O
O S
HN
OH
35al
OH
35aa
OH
O
O S
HN
N
O
O S
HN
O
OH
O
35z
N
N
OH
O
O
35ae
O S
HN
35aj
N
OH
N
O S
HN
Cl
O
O S
HN
O
35ad
O
Cl
OH
OH
O
OH
35u
O
O
O S
HN
OH
O
35y
O
NO 2
O
O
O S
HN
35t
O
O S
HN
OH
35x
O
OH
O
35s
O
OH
35v
O
O S
HN
O
F
O
O S
HN
O
O S
N
35r
F
OH
O
OH
O
O
O S
HN
O S
HN
F
OH
O
O
O S
HN
O
O S
HN
O
35n
OH
35ao
O
35ap
O
Figure 21. Structures of the commercially and non-commercially available P3 acids (35) that
were utilized in the synthesis.
36
4.2.2 Synthesis of P3 acid Capping Groups
Scheme 8 illustrates the general procedure for the synthesis of the noncommercially available P3 acids 35. Briefly, 4-amino benzoic acid methyl
ester derivatives were reacted with diverse sulfonyl chlorides using pyridine
and a catalytic amount of DMAP in DCM to provide the corresponding P3
capping group methyl esters. Basic hydrolysis of the methyl ester function
with 2.5M LiOH in THF:MeOH for 30 min at 110 o C in a microwave cavity
furnished the P3 acids in 3–98% overall yields.
2
H 2N
R2
R1
S Cl
3
X=C,N
a
o o
o
X
R1
S
H
N
o o
o
2
R2
b
3
R1
H
N
o o
o
X
S
o
2
R2
3
X
35
OH
o
Scheme 8. General procedure for the synthesis of P3 acids 35 (Fig. 21). Reagents and
conditions: (a) Py, DMAP, DCM; (b) LiOH (2.5M), THF:MeOH 2:1, microwave 110 oC, 30
min.
Scheme 9 shows an alternative approach93a to the synthesis of the pyridine
P3 acids 35 (35ai, 35aj, 35ak, and 35al) (Fig. 21) that were utilized to
generate target compounds 71–74 (Table 5). Trifluoromethane sulfonic acid
anhydride was reacted with polymer-supported triphenylphosphine oxide93b
in DCM followed by addition of the cold pyridinium salt of the
corresponding pyridine sulfonic acid and subsequent addition of the
corresponding 4-amino benzoic acid derivative to afford the corresponding
pyridine P3 capping group methyl esters. Basic hydrolysis with LiOH as
described above furnished the pyridine P3 acids 35ai–35al in 13–25%
overall yields.
N
O
S
a
O
R
O
Ar3P=O
O
O O
b
O
O
S N
H
N
R
N
H2N
O
O
c
O O
N
OH
S N
H
R
35ai-35al
R = H, Me
Scheme 9. Synthesis of P3 acids 35ai–35al (Fig. 21). Reagents and conditions: (a)
(CF3SO 2)2O, DCM, 1 h (b) 16 h; (c) LiOH (2.5M), THF:MeOH 2:1, microwave 110 oC, 30
min.
37
4.3 Tables: Inhibitory Activity and Selectivity
Table 2. Inhibition constant (Ki) values against the cathepsin S protease
Cmpd
Structure
Ki (nM)
Cath S
O
O
H
N
S
28
31
N
H
O
O
O
O S
HN
29
O
O
H
N
2.6
N
H
O
O
O
37
O
S
S
O
38
N
H
H
N
N
H
O
N
H
N
O
N
H
O
O
S
HN
H
N
39
H
N
O
H
N
S
O
O
110
O
O
O
N
H
O
12
O
O
O
O
40
O
O
140
O
O
O
N
H
190
O
38
Table 3. Effect of R1 variations
O
O H
N
S
O
O
N
R1
N
O
O
Cmpd
P3 acid
R1
Ki (nM)
Cath S
41
35ag
Et
1.9
42
35ah
n-But
3.4
F
43
8.3
F
F
44
35m
2.9
45
35q
2.4
46
35a
180
47
35i
48
35b
0.82
49
35c
31
5.3
N
39
Table 4. Effect of variations at the central ring
O
O H
N
S
R1
2
R2
O
O
3
N
X
N
O
O
Compd
P3 acid
R1
R2
X
Ki (nM)
Cath S
50
35j
Me
2-OMe
C
26
51
35p
Me
3-OMe
C
330
52
35l
Me
2-Me
C
4.9
53
35n
Me
3-Me
C
30
54
35o
Me
2-Cl
C
5.6
55
35r
Me
2-F
C
16
56
35s
Me 2-COMe
C
57
57
35y
Ph
2-Me
C
4
58
35z
Ph
2-Cl
C
12
59
35ac
Ph
H
N
56
60
35ad
Me
H
N
63
40
Table 5. Effect of R1: aromatic substituents and heteroaromatic rings
O H
O S N
N
O
O
R1
Cmpd P3 acid
61
O
O
N
R1
R1
Ki (nM) Cmpd P3 acid
Cath S
35u
2
72
35ai
3.4
73
35aj
3.8
74
35al
3.9
75
35af
1.3
76
35d
N
Ki (nM)
Cath S
2.1
F
62
35t
63
35x
F
N
N
2.3
0.93
F
Cl
64
35w
65
35ae
H 2N
5.9
N
2.3
S
N
66
35ab
67
35aa
O
O
3.3
77
35v
0.99
78
35an
4.5
N
N
13
S
68
1.1
79
35ap
N
NC
NC
69
5.7
S
0.64
80
35am
N
0.69
S
N
0.79
70
81
CN
71
35ak
2.7
N
41
35ao
8.6
S
Table 6. Selectivity assay: comparison of inhibitory activity of the compounds in human
cathepsin S, K and L
Cmpd
Cath S
K i (nM)
Cath K
Ki (nM)
Cath L
K i (nM)
Cath K/Cath S
28
29
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
31
2.6
12
110
140
190
1.9
3.4
8.3
2.9
2.4
180
5.3
0.83
31
26
330
4.9
30
5.6
16
57
4
12
56
63
2
3.4
3.8
3.9
1.3
3.3
0.99
1.1
0.64
0.79
2.7
2.1
2.3
0.93
5.9
2.3
4.5
13
5.7
0.69
8.6
318
290
1500
98
420
23000
470
1500
1200
500
440
950
300
430
1700
560
5000
240
1900
840
700
750
730
1600
2200
1200
370
520
980
550
950
500
270
950
730
820
1100
570
450
260
800
1000
640
3000
1700
1800
2300
10000
12000
2500
11000
27000
15000
17000
21000
15000
15000
12000
64000
16000
11000
15000
7200
29000
2000
23000
970
7800
9500
2300
1100
31000
14000
5900
7800
11000
8400
9100
11000
14000
7900
6800
8500
13000
14000
13000
8700
23000
13000
24000
27000
9100
16000
17000
10
110
120
1
3
120
250
440
140
170
180
5
60
520
50
20
10
50
60
150
40
10
180
130
40
20
180
150
260
140
730
150
270
860
1140
1040
400
270
190
280
130
430
140
230
300
2610
270
42
Table 7. Cellular Potency: effect R 1 substituents in the Ii p10 cell based assay
O
O H
N
S
O
O
N
R1
N
O
O
1
Cmpd
R
29
Me
48
K i (nM)
Cath S
Ii p10
IC50 (µM)
2.6
1.7
0.82
0.12
3.4
0.15
0.99
0.062
1.1
0.98
0.64
1.4
0.79
8
0.93
>20
0.69
0.31
F
62
O
67
68
NC
NC
69
70
CN
74
80
N
N
S
4.4 Structure-Activity Relationships and Modeling
The target compounds summarized in Tables 2–5 were screened against
human cathepsins S, K, and L to determine Ki values. The distal region of
the S3 subsite of cathepsin S is defined by backbone Gly62, the side chain of
Phe70 and Lys64.
Variations at P3 were introduced as an extended sulfonamide motif (see
Fig. 20) with compound 29 (Table 2) as starting point for the SAR.
Investigation of the SAR of the P3 substituent was conducted to improve the
affinity for cathepsin S by utilizing interactions in the S3 pocket, and also to
achieve higher selectivity over cathepsin K and to maintain the good
43
selectivity towards cathepsin L. Modeling was done to rationalize the SAR
observed for the various P3 extensions and to explain the selectivity over
cathepsin K.
4.4.1 Enzyme Inhibitory Activity
Table 2 shows the SAR around the initial lead compound 29. In compound
37, the sulfonamide moiety was placed at the meta position in the P3 phenyl
ring, which resulted in a slight drop in activity (K i = 12 nM), indicating
preference for the substitution at the para position. Methylation of the
nitrogen of the sulfonamide 38 was detrimental to enzyme activity (50-fold
drop), as was also the case for compounds 39 and 40 with reversed
sulfonamides. These results indicate the importance of the NH group, the
phenyl substitution pattern and the orientation of the sulfonyl group for the
interactions in the S3 pocket.
Compound 29 was modeled in the active site of cathepsin S (Fig. 22)
using an in-house crystal structure as the starting point. This inhibitor binds
through a covalent reversible interaction with the side chain of Cys25 and a
hydrogen bond with His164. The backbone hydrogen bonds of the inhibitor
are between the P1 NH and the main chain carbonyl of Asn165, and between
the carbonyl and the NH of the P2 and the Gly69 main chain NH and
carbonyl, respectively. The P3 carbonyl interacts with solvent only, whereas
the P2 side chain is in close interaction with Phe70, Met71, Gly137, and
Val162. The P3 phenyl ring has an edge on aromatic interaction with Phe70
and is in close contact with Gly68 and Gly69 in the S3 subpocket. The
sulfonamide adds stability and activity to the binding of this series through a
hydrogen bond from the NH to the carbonyl of Gly62 and via hydrogen
bonds from the sulfone group to the side chain of Lys64.
Figure 22. Compound 29 modeled in the active site of cathepsin S. The binding mode of the
sulfonamide in particular gives this series increased activities against cathepsin S. The
subpockets are denoted S1, S2, and S3.
44
The slight drop in activity exhibited by compound 37, which has the
sulfonamide in meta position, depends on a different hydrogen bond pattern.
The NH of the sulfonamide participates in a hydrogen bond with the
backbone of Asn67 instead of Gly62, whereas the sulfone still interacts with
Lys64. The methylation of the nitrogen as in compound 38 makes the
hydrogen bond with Gly62 impossible, and hence there is a large drop in
activity. The reversed sulfonamides in compounds 39 and 40 lead to less
optimized interactions with Gly62 and Lys64 and therefore to lower potency.
We also investigated the effect of the R1 substituent on the sulfonamide
moiety, as shown in Table 3. Analysis of the data indicate a preference for
the phenyl group (48), with a considerable increase in enzyme activity (Ki =
0.82 nM). Small unbranched alkyl chains (41, 42) and small branched alkyl
chains and rings (44, 45) were well tolerated, whereas larger rings (46) had a
detrimental effect. A slight drop in activity was observed for other groups
(43, 47). Elongation of the phenyl substituent (49) led to substantial
reduction in activity compared to compound 48. From these observations, it
can be concluded that the phenyl substituent at R1 was the preferred one
(48), with a threefold increase in potency over the parent compound 29, due
to aromatic stacking between Phe70 and both P3 phenyls, while maintaining
good hydrogen bonds with Gly62 and Lys64 (Fig. 23).
Figure 23. Compound 48 modeled in the active site of cathepsin S. The additional aromatic
stacking with Phe70, and maintained optimal interactions with the sulfonamide, increased the
activity of compound 48 compared to compound 29.
Table 4 shows the SAR around the variations at the central phenyl ring.
Diverse R 2 substituents at the 2- and 3- positions of the ring and the
exchange of the central phenyl for a pyridine ring were explored, keeping R 1
as methyl or phenyl. A loss of inhibitory activity was observed for all these
compounds. Substitutions at the 2 position of the phenyl ring were better
45
tolerated than at the 3 position (50 vs. 51, 52 vs. 53). Small substituents like
a methyl group in targets 52 and 57 (K i values 4.9 and 4 nM, respectively)
were better tolerated than larger groups (50, Ki = 26 nM; 56, Ki = 57 nM).
Despite the fact that the 2 position was the best tolerated in terms of
substitution pattern, in general, decorations of the central phenyl ring by
introduction of R2 substituents did not have a favorable effect in terms of
potency, the corresponding unsubstituted compounds 29 (Table 2) and 48
(Table 3) were more potent (K i values 2.7 and 0.82 nM, respectively).
Pyridine compounds 59 and 60 were considerably less potent (Ki values 56
and 63 nM, respectively) than their phenyl analogs 29 and 48. Most
substitutions alter the position of the central phenyl ring, which is important
for the aromatic stacking with Phe70 and for allowing the optimal hydrogen
bond pattern of the sulfonamide with Gly62 and Lys64 residues on the S3
pocket of cathepsin S.
The encouraging results obtained for compound 48 (Table 3) prompted us to
further explore the development of the SAR around this new lead. Table 5
shows the changes that were made at the distal phenyl ring of the
sulfonamide. Substitutions were carried out at the different positions around
the phenyl ring using electron-withdrawing groups (61–63, 68–70), electrondonating groups (64, 66, 67), and an amino group (65). Replacement of the
distal phenyl ring with heterocycles such as pyridines (71–75), thiophene
(76), imidazole (77), and thiazoles (78–81) was also approached in order to
further extend the SAR.
The majority of these inhibitors showed good affinity for cathepsin S,
with Ki values in the same range as the parent lead compound 48. From these
findings, it can be deduced that a cyano substituent in the meta position at
the distal phenyl ring afforded the most potent compound (69), resulting in a
K i value of 0.64 nM, which represented a further increase in potency (> 4 x)
compared to initial compound 29 and an increase of 48 times respect to
compound 28 (Table 2). Pyridine rings were well tolerated (71–74, with K i
values of 0.93 to 5.9 nM). However, chloro-substituted pyridine 75 led to a
drop in activity (Ki = 5.9 nM), and methyl-substituted pyridine 74 was the
preferred compound of this type (Ki = 0.93 nM). Replacement of the distal
phenyl ring with a thiophene (76) also provided a compound with good
inhibitory activity against cathepsin S (K i = 2.3 nM), whereas other
heteroaromatic rings such as imidazole (77) and 2,5-thiazoles (78, 79, 81)
led to a 5–10-fold decrease in activity compared to compound 48.
Surprisingly, the 2,4-thiazole 80 proved to be a highly potent compound,
with a Ki of 0.69 nM. The increased activity of the compounds with an
aromatic R1 group in P3 (Table 5) can be explained by the additional
aromatic stacking with Phe70 together with preservation of the favorable
binding mode of the sulfonamide, as described above for compound 48.
46
4.4.2 Selectivity Profile
A selectivity assay was performed to measure the Ki values for all of the
synthesized compounds in relation to human cathepsins K and L. As shown
in Table 6, all compounds were highly selective for cathepsin L (> 1000 x),
and they had affinity varying from 98 to > 20,000 nM for cathepsin K. The
starting compound 28 had poor selectivity over cathepsin K (10-fold), and
compound 29 showed a selectivity of 100-fold over cathepsin K, which is
within the limit of acceptance for our target minimum selectivity ratio. Two
of the target compounds (38, 39) displayed no selectivity between cathepsins
S and K, and several other targets (46, 47, 49–53, 55, 56, 59, and 60) had
lost selectivity (by more than 100-fold).
However, a very interesting finding was made when examining the data: the
enhanced activity attained in compound 48 (> threefold) was accompanied
by a fivefold increase in selectivity over cathepsin K (> 500-fold) compared
to initial compound 29. Moreover, the same trend was found for the cyanophenyl compounds 69 and 70 and the 2,4-thiazole 80, which showed a
further increase in affinity for cathepsin S (Ki values of 0.64, 0.79, and 0.69
nM, respectively) compared to the phenyl compound 48 (> fourfold increase
with respect to 29) and also exhibited a greater increase in selectivity over
cathepsin K (> 1,000-fold for 69 and 70, and > 2,600-fold for 80).
Compound 80 proved to be the most selective of all the compounds of this
serie, showing a 24-fold and 240-fold increase in selectivity over cathepsin
K compared to compounds 29 and 28, respectively, and fivefold
improvement in selectivity ratio over cathepsin L compared to compounds
28 and 29.
Besides the use of a cathepsin S favored P2 group, this selectivity was
mainly due to the lack of specific interactions with the sulfonamide in the S 3
subpocket of cathepsin K. The hydrogen bond that is formed with the
backbone carbonyl of Gly62 in cathepsin S does not exist with the
corresponding Glu59 in cathepsin K due to a different loop conformation.
The hydrogen bonds with the side chain of Lys64 in cathepsin S could not be
replaced by the corresponding Asp61 in cathepsin K.
Even more interesting was the increased selectivity of the inhibitors
displaying an aromatic substituent on the sulfonamide, a characteristic that
can be explained by both greater activity towards cathepsin S and decreased
activity towards cathepsin K. The aromatic group cannot form a similar
stable binding mode in cathepsin K due to the lack of anchoring points of the
sulfonamide, and therefore the additional aromatic group is flexible without
specific interactions.
47
A comparison of the active sites of cathepsins K and S is presented in Figure
24, which shows a model of compound 80 in the active site of cathepsin S.
Figure 24. Comparison of the active sites of cathepsins K (left) and S (right) using the
numbering from PDB entry 1MS6 for cathepsin S and entry 1BG0 for cathepsin K. A model
of compound 80 is shown in the active site of cathepsin S. The different shapes of primarily
S2 (purple) and S3 (orange) are visible. The most important interactions exhibited by this
series that are lost in cathepsin K are the hydrogen bonds with the sulfonamide.
4.4.3 Cellular Potency
Selected compounds were tested for cellular potency using the Ii-p10
cellular assay, which measures inhibition of the cleavage of Ii in (human)
9001 B-lymphocytes (Table 7). That method is comparable to the assay
described by Thurmond et al.,94 which determines the accumulation of the
p10-Ii fragment (a natural cathepsin S substrate).
Some of the compounds (48, 62, 67, 68, and 80) showed markedly
improved cellular potencies compared to compound 29 (IC 50 = 1.7 µM), as
indicated by their IC50 values in the range 0.32–0.062 µM. Despite the good
enzymatic inhibitory activity obtained for the cyano-phenyl compounds 69
and 70, we did not observe a corresponding increase in their cellular
potency, and indeed the cellular activity of the pyridine analog 74 was
abolished. The reasons for the decreased cellular potency of 70 as compared
to the isomers 68 and 69 and for the low cellular potency of 74 are not
readily apparent. Factors that might influence cellular activity include
overall cell permeability, access to subcellular compartments containing
cathepsin S, and protein binding.
Notably, compound 67, with a K i value of 0.99 nM for cathepsin S,
conferred the best cellular potency, showing an IC50 value of 62 nM, which
represents a 27-fold increase relative to compound 29.
48
We also tested the compounds in a pseudo-functional cellular assay that
measures T-cell activation by assessing the release of the cytokine
interleukin-2 (IL-2) into the tissue culture medium (data not shown). The
results correlated well with the findings of the Ii-p10 assay, which further
supports the findings and demonstrates highly efficient inhibition of
cathepsin S.
4.5 Conclusions
The solid-phase parallel synthesis and SAR of a series of inhibitors with a
sulfonamide moiety in the P3 position that are reported here resulted in the
discovery of highly potent and selective 4-amidofuran-3-one inhibitors of the
cysteine protease cathepsin S. The findings of this study show that it is
possible to improve the inhibitory enzyme activity and the cellular potency
of this class of compounds towards cathepsin S by inducing interactions in
the S 3 subsite of the enzyme. Furthermore, despite previous reports
indicating that the S3 pocket in cathepsin S is smaller than that in cathepsin
K, our results involving extended P3 sulfonamides show that inhibitors with
large P3 groups can be accommodated in the S 3 pocket of cathepsin S. The
potency of the synthesized inhibitors towards cathepsin S is due to the
hydrogen bond network between the sulfonamide and two residues in the
distal region of the S3 pocket.
Although the main selectivity determinant for cathepsins S and K resides in
the S2 pocket, in the present study improved selectivity was achieved
through favorable interactions between the inhibitor and the S3 pocket of
cathepsin S (Table 6). The incorporation of aromatic substituents on the
sulfonamide moiety proved to be useful for increasing the enzyme and
cellular inhibitory potency against cathepsin S and the selectivity over
cathepsin K. Several members of this series, notably one carrying the P3
thiazole sulfonamide moiety (80), display very low (single-digit) nanomolar
K i values against cathepsin S and also exhibit an excellent selectivity profile
over cathepsins K and L. In addition, compound 67 has an IC50 value of 62
nM, which represents a 27-fold increase in cellular potency compared to
initial compound 29.
49
50
5. Preparation and Characterization of
Aminoethylamide Inhibitors of the Cysteine
Protease Cathepsin K (Paper IV)
5.1 Introduction
5.1.1 Cathepsin K and Osteoporosis
Osteoporosis has a significant impact on the quality of life, morbidity, and
mortality of ageing populations, and is most commonly found in
postmenopausal women.95a -bThis condition involves skeletal deterioration
that leads to bone fractures, and it constitutes the second largest health
problem in the world, being a major cause of death, disability, and medical
expenses. Statistics show that one in two women and one in eight men will
suffer osteoporosis-related fractures. Indeed, 1.7 million hip fractures were
reported in 1990, and it has been estimated that that number will increase to
6.3 million by 2050.
The human skeleton consists of 80% cortical bone and 20% trabecular
bone. Cortical bone is the dense, compact outer part, and trabecular bone
makes up the inner meshwork. Bone is a form of connective tissue that
contains type I collagen fibrils and hydroxyapatite. Calcium phosphate
crystals in the form of hydroxyapatite [Ca 10(PO4 )6(OH)2 ] are deposited in the
osteoid (uncalcified bone matrix) and converted into hard bone. The bone
mass undergoes continual remodeling that is performed by osteoblasts and
osteoclasts. The osteoclasts are large multinucleated cells responsible for
bone resorption, whereas the role of osteoblasts is in rebuilding or formation
of bone (Fig. 25).
Figure 25. The process of bone remodeling.
51
A cycle of bone remodeling starts with recruitment of osteoclasts by
cytokines (e.g., IL-6). The osteoclasts adhere to and move along an area of
trabecular bone, digging a pit by secreting hydrogen ions and proteolytic
enzymes, and this gradually liberates factors such as IGF-1 that are
embedded in the bone. These released factors recruit and activate successive
teams of osteoblasts, which have developed from precursor cells upon
activation by parathyroid hormone and calcitriol. The osteoblasts invade the
site and synthesize and secrete the organic bone matrix (osteoid). The
stimulated osteoblasts secrete IGF-1 and eventually other cytokines such as
IL6, which in turn recruit osteoclasts and thereby in a very simplistic
description bring the cycle back to its beginning.95c-d
When bone resorption by osteoclasts outpaces bone formation by
osteoblasts, an overall fragility occurs in the microarchitecture of the bone.
This imbalance is clinically manifested as osteoporosis, a disease
characterized by an increased risk of bone fractures resulting from a
reduction in bone mass. Patients suffering from osteoporosis have very low
bone density and increased curvature of the spine (Fig. 26).
(A)
(B)
(C)
Figure 26. (A) Micrographs comparing normal (left) and osteoporotic (right) bone tissue. (B)
Graph showing bone mass in relation to age for women and men. (C) Radiographs showing
the development of osteoporosis in the spine.
Cathepsin K is a papain-like cysteine protease96–97 that shows 73%
homology to cathepsin S and 76% to cathepsin L, and it is highly expressed
during bone resorption. Also, it is preferentially expressed in osteoclasts,
which, together with its ability to degrade type I collagen, suggests that
52
cathepsin K plays a specific and pivotal role in bone resorption during the
course of remodeling. Osteoclastic degradation of bone involves two
processes: demineralization of the inorganic components of bone and
degradation of the organic matrix. These two functions occur sequentially by
two separate mechanisms (Fig. 27). 96–101
Figure 27. The two phases of osteoclastic bone resorption: demineralization (a) and
degradation (b), reprinted with the kind permission from Elsevier Science Ltd.
In the first phase of resorption (Fig. 27a), the bone matrix is demineralized
by the production of an acidic environment in the sealed compartment in the
osteoclast-bone interface. The acidic environment is generated by the active
transport of hydrogen atoms into the lacunae by vacuolar H +ATPase. The
hydrogen atoms are produced through the spontaneous dissociation of
H2 CO3 which, in turn, is generated by action of the enzyme carbonic
anhydrase II (CAII). The pH within the osteoclast is maintained by a passive
Cl –/HCO3– exchanger at the resorptive surface of the cell. The acidic
degradation leaves behind a demineralized region of bone beneath the
osteoclast, and then in the second phase the organic component (comprising
mainly collagen) is removed by cathepsin K protease-mediated digestion
(Fig. 27b). Cathepsin K is stored as a pro-enzyme in the lysosomes of the
osteoclast. Release of cathepsin K into the resorption lacunae stimulates
conversion of the enzyme to its mature form, a process that has been shown
to be autocatalytic under acidic conditions.
The most striking evidence of the involvement of cathepsin K in the bone
remodeling process and in bone disorders is seen in patients with
pycnodysostosis, a disorder that is associated with an inactivating mutation
in the coding sequence of the cathepsin K gene and is characterized by facial
hypoplasia, premature closure of long bone growth, and bones that are
brittle, dense, and long and show severe osteosclerosis. 102 A role for
53
cathepsin K in bone resorption is further exemplified by the observation that
mice deficient in this enzyme display an osteopetrotic phenotype. 103,104
Evaluation of osteoclast resorption sites in bones of -/- mice revealed
osteoclasts apposed to regions of demineralized bone, demonstrating that the
extracellular matrix had undergone normal demineralization, but the
degradation of the demineralized matrix was retarded.103 In contrast, it has
been found that transgenic mice that overexpress cathepsin K have lower
trabecular bone volume as a result of higher bone resorption. 105
Thus there is now overwhelming evidence of a critical role for cathepsin K
in bone resorption. Consequently, inhibition of this enzyme can no doubt
provide a therapeutic option in disorders such as osteoporosis that are caused
by an increase in bone turnover, normally at menopause. Cathepsin K
inhibitors developed elsewhere have shown efficacy in vitro and in vivo.105107
For example, it has been reported that peptide aldehyde inhibitors of
cathepsin K reduced bone resorption in a human osteoclast resorption assay
and in the ovariectomized (OVX) rat model. 106 Furthermore, azepanonebased inhibitors have been found to lower levels of C-telopeptide (CTx), a
biomarker of bone resorption in the serum and urine, by 77% in cynomolgus
monkeys. 106 Cathepsin K inhibitors have also shown efficacy against bone
resorption measured by BMD in Phase II clinical trials. 107
The treatments for osteoporosis that are currently available are associated
with adverse events and there is a need for additional anti-resorptives which
are safe and with a mode of action that is independent of calcitonin,
estrogen, selective estrogen receptor modulators (SERMs) and
bisphosphonates. Importantly, bisphosphonates are also known to reduce
bone formation, which does not seem to be the case for cathepsin K
inhibitors.108 Therefore, it is expected that cathepsin K inhibitors will
attenuate bone resorption, and, by also allowing bone formation to continue,
that they will cause re-equilibration of the bone remodeling process and
thereby lead to healthier bone.
Who is at greatest risk of osteoporosis? People who are slender or thin
boned, those with a fair complexion, persons with a family history of
osteoporosis, individuals with a diet low in calcium, those who do not
exercise regularly, smokers, adults who require long-term use of drugs like
cortisone, anti-seizure medications, or thyroid hormone, and finally adults
with excessive alcohol consumption.
54
5.1.2 Aminoethylamide Cathepsin K Inhibitors: Aim of the
Study
Most of the work on cathepsin K inhibitors have focused on reversible
covalent binding compounds containing reactive groups such as aldehydes,
69b-c
nitriles, ketones, or azepanones.
However, non-covalent inhibitors are
considered to represent the therapeutic golden standard, because they entail
the lowest risk of adverse effects related to aspects of selectivity or to an
immune response induced by covalent modification of a host protein.
Recently,
several
companies
have
independently
described
aminoethylamides as non-covalent inhibitors of cathepsin K. 109–114 In one of
110
those studies, Altmann et al. proposed the concept of binding of the
leucine side chain into the hydrophobic S2 pocket and the terminal amine
component extending into the S 1' groove (Figure 28). In such an inhibitor,
van der Waals interactions make up most of the binding interactions. The
binding affinity of this type of non-covalent inhibitors is partly achieved
through lipophilic P1' interactions of the aminoethylaniline moiety, where
114
electron-donating substituents on the aniline are required for potency.
H
N
O
S3
O
O
N
H
H
N
OMe
S2
S 1'
Figure 28. Schematic representation of the assumed binding mode of a model
aminoethylamide inhibitor within the active site of human cathepsin K. The Cbz group is
believed to bind in the S3 subsite, the isobutyl group of the leucine is within the hydrophobic
S2 pocket, and the 4-methoxy-phenyl ring extends into the S 1'site.
Smyht 115 and Marquis116 have suggested that aminoethylamides might act as
slow turnover substrates for cathespin K.
Accordingly, the aim of the study reported in Paper IV was to further
investigate the utility of aminoethylamides as potential non-electrophilic
cathepsin K inhibitors.
At the outset of Medivir’s cathepsin K inhibitor program, libraries were
designed to exploit information generated by screens performed using the
Rational Approach to Protease Inhibitor Design (RAPiD), which is a
Medivir platform technology for obtaining data on enzyme pocket
requirements. 117 These initial studies led us to believe that, unlike other
related cathepsins, cathepsin K selectivity and potency can be harnessed by
inhibitors spanning across the prime site of S 1 ' and S2 '. To explore this
55
hypothesis, synthesis of a small focused library of aminoethylamides was
undertaken on solid phase as part of a larger program aimed at developing
novel cathepsin K inhibitors. My task in this study was to design an
improved solid-support synthesis route to the aminoethylamides that would
be compatible with molecules containing groups sensitive to reduction and
acidic conditions. It was also important that the route could be applied to a
broad range of amides outside the aminoethylamide category in order to
achieve a more general synthetic route to this type of protease peptide
inhibitors.
5.2 Solid-Phase Synthesis of Aminoethylamides
5.2.1 Aminoethylamide Template
Representation of the aminoethylamide core structure:
R1
O
R2
N
H
H
N
R3
N
O
R4
From a retrosynthetic standpoint, one way to obtain the core structure might
be to divide the skeleton into three parts by breaking the amide bonds at the
edges of the central amino acid part of the skeleton, as follows:
R1
O
R2
N
H
H
N
O
R1
O
N
R3
R2
OH
+
OH
H2 N
O
R4
+
H2 N
R3
N
R4
This provides the three building blocks that are needed: an amino acid, an
acid, and a primary aminoethylamine. Different solid-phase synthetic
approaches can be designed, depending on which diversity group it is of
interest to vary.
5.2.2 Aldehyde Resin-Based Synthesis
The solid-supported synthetic route depicted in Scheme 10 was established
at the onset of the project. The route was extended to various amines other
than aminoethyl amines. Diversity at the P2 was achieved by using different
amino acids, variation at the P1-P1' was introduced by using diverse amines
(R3 NH2), and variation at the P3 was accomplished by introducing different
acids (R2COOH).
56
O Me
OMe
OMe
a
b
R1
CHO
R1
N
82
83
N
H
O
84
O
O
O
OMe
OMe
c
d
e,f,g
R1
R1
Fmoc
OH
86
O
O
R2
R1
NH
N
N
85
Fmoc
NHR3
F moc
O
NHR3
87
O
Scheme 10. Reagents and conditions: (a) TosHNCHR1COOAllyl, NaBH 3CN, DMF, 18 h,
RT; (b) Fmoc-Cl, NMM, DMF, 18 h, RT; (c) Tetrakis (triphenylphosphine)Pd(0), PhSiH 3,
DCM, 30 min, RT; (d) R 3NH2, PyBOP, HOBt, DIEA, DMF, 18 h, RT; (e) 20%
piperidine/DMF, 2 h, RT; (f) R2COOH, DIC, DCM, 1 h, 0 °C, then filtrate and addition of
resin from previous step, 24 h, RT; (g) TFA/TES 95/5, 2 h, RT.
Allylester-protected amino acids were coupled to the aldehyde-based resin
82 to yield 83. Fmoc protection of the amine of 83 yielded 84, and
deprotection of the acid of 84 yielded 85. Subsequently, coupling on the
appropriate amine gave 86. Deprotection of the amine of 86, coupling with
the appropriate acid, and acidic cleavage from the resin afforded the
corresponding products 87. Overall yields of 60–80% were obtained.
Although this route worked for most of the targeted compounds, it had some
drawbacks:
- It required two additional reactions (besides the loading and
cleavage standard reactions): protection and de-protection of the
amino functionality of the amino acid.
- It was not advisable for reducion sensitive functionalities and acid
labile functionalities in the molecule due to the acidic cleavage
procedure.
Thus, an improved solid-phase methodology was required to cope with the
requirements. A shorter synthetic pathway would be an additional asset.
5.2.3 Sulfonamide Resin-Based Synthesis
A sulfonamide resin would be a very suitable solid-phase support for the
synthesis of the targeted aminoethylamides. Acylation of the sulfonamide
support by the amino acid provides a support-bound N-acylsulfonamide that
is highly stable to strong bases as well as to strongly acidic conditions prior
to activation. After the activation step, the product is released from the solid
support by the attack of a nucleophile, in our case a primary amine, which is
particularly useful because additional diversity is introduced in the cleavage
step. Furthermore, no reducing agents or acidic conditions are employed in
57
this synthetic pathway. The synthetic route illustrated in Scheme 11 was
designed and investigated.
oo
O
R1
S
OH
Fmo cH N
+
N H2
1
R
H
N
Fm ocH N
O
88
a
89
O
O
N
H
S
O
90
O
R1
O
b, c
R2
N
H
H
N
O
O
d, e
N
H
R2
S
O
R1
O
91
N
H
H
N
R3
O
87
Scheme 11. Reagents and conditions: (a) PyBOP, HOBt, DIEA, resin 88, DCM, RT, 16 h; (b)
20% piperidine in DMF, 2 h, RT; (c) R 2COOH, PyBOP, HOBt, DIEA, DCM, RT, 24 h; (d)
ICH 2CN, DIEA, NMP, 24 h, RT; (e) R3NH2, THF, 12 h, RT.
Fmoc-protected amino acids 89 were coupled to resin 88 to yield 90. Initial
experiments were performed using phenylsulfonamide linker 92 and loading
of the amino acid by a preformation of the symmetrical anhydride of the
amino acid by DIC in DCM at 0 °C.118 However, production of the library
was done using 4-sulfamylbutyryl linker 93119 and a loading procedure
involving standard peptide coupling PyBOP conditions.
O
S
H
N
O
O
NH2
HN
92
O
O
(CH2) 3 S
NH2
O
93
Deprotection of the terminal amine of 90 and coupling of the appropriate
acid yielded 91. Activation of the sulfonamide provided a highly activated
linker, which could be readily cleaved by nucleophilic displacement with
commercially available amines (R3 NH2 , aminoethyl amines among them) to
afford the corresponding products 87.
Activation of the acylsulfonamide linker was more efficient when
achieved by iodoacetonitrile treatment than by treatment with TMSdiazomethane (TMS-CH2N2 ) or bromoacetonitrile. The reaction steps were
monitored by FTIR and the ninhydrin test. 19 Overall yields of 40–80% were
obtained. This route did not make use of any acidic treatment, and thus the
new sulfonamide solid-support route that was developed was more general
and shorter than the previous approach in order to synthesize a broad range
of aminoethylamides.
58
5.3 Inhibitory Activity and Kinetic Studies
From the initial screen of the library of synthesized compounds, it was
considered that 94a and 94b (produced according to Scheme 11), with K i
values of 2.5 and 0.65 M, respectively, against human cathepsin K (Table
8), were an encouraging start. This led to a larger program aimed at
developing the series and addressing issues such as the modest selectivity
over cathepsin S (Table 8).
Table 8. Examples of activity values for measurements of inhibition of human cathepsins K
and S by aminoethylamides.
Structure
94a
O
H
N
N
H
O
94b
H
N
N
H
O
O
H
N
N
H
H
N
N
H
2452
9
2597
17
39110
300
15392
180
662
N
O
O
O
12
N
H
O
O
94f
3200
N
H
O
O
650
O
H
N
O
94e
4592
O
N
H
94di
2500
N
O
94c
Cathepsin S
K i (nM)b
N
H
O
O
Cathepsin K
K i (nM)a
H
N
N
H
N
O
94gii
N
O
O
N
H
H
N
O
N
H
Inhibition of recombinant human cathepsin K activity in a fluorescence assay using 70 M (=
K M) D-Ala-Leu-Lys-AMC as substrate in 100 L of 100 mM NaH2PO 4, 1 mM EDTA, 1 mM
DTT, and 0.1% PEG400 at pH 6.5 in 96-well microtiter plates read in a Fluoroskan Ascent
instrument.
b
Inhibition of the activity of recombinant human cathepsin S in a fluorescence assay using
100 M Boc-Val-Leu-Lys-AMC as substrate in 100 mM NaH2PO4, 1 mM DTT, and 100 mM
NaCl at pH 6.5.
i
This compound is 7h in Altmann et al. (Ref. 109).
ii
This compound is 3 in Kim et al. (Ref. 111) and compound 4 in Kim et al. (Ref. 114).
a
59
It was felt that the poor selectivity against cathepsin S could be addressed by
modification of the P3-P2 left hand side of the molecule. Based on the initial
SAR that was generated, amino acid 89 was fixed as L-leucine. A variety of
solution-phase routes were developed, which gave us greater flexibility and
scaling-up possibilities, and also helped us avoid the risk of partial
racemization of R 1 that has been observed in the final step of similar
methodologies. 120 Scheme 2 in Paper IV shows representative results of
some of these approaches, which yielded compounds 94c–f. An example of
the solution-phase methods used is illustrated in Scheme 12. Products 94d
and 94g are literature reference compounds. The aminoethylamide 94g was
prepared using the route described by Kim et al. 111
H 2N
a
N
H
O
O
N
H
b, c
H
N
N
H
O
O
O
H
N
N
H
N
H
O
94a
Scheme 12. Reagents and conditions: (a) Boc-L-Leu, EDC, HOBt, TEA, DCM:DMF 2:1, 12
h, 80%; (b) 50% TFA/DCM, 1 h, then (c) 3-furoic acid, EDC, HOBt, TEA, DCM:DMF 2:1,
12 h, 85%.
However, as we developed the series, we discovered that the measured Ki
values were not reproducible. More specifically, at times there were
substantial differences between separate batches of the same sample and,
more importantly, between the same sample on different occasions. For
example we recorded activities ranging from 0.49 to 11 µM for compound
94b and from 26 nM to 25 µM for compound 94f. This was clearly a very
disturbing observation and may account for the difficulties we encountered
in trying to develop the SAR of the series.
To better understand what was happening, we undertook a kinetic study of
the inhibition by measuring the on and off rates, using aminoethylamide 94c
as an exemplar of this series (Fig. 29). Close inspection of the assay kinetics
indicated that this compound displayed time-dependent inhibition, as shown
in the progress curve from cleavage of the substrate D-Ala-Leu-Lys-AMC,
which revealed that at 1 µM 94c the initial native rate of cleavage declined
as the portion of uninhibited enzyme dropped (Fig. 29, trace b). However, in
the dilution experiment in which the inhibitor and the enzyme were preincubated at high concentration and then diluted into the enzyme, it was
expected that there would initially be low activity that would steadily
increase as the inhibitor dissociated, and end up at the same cleavage
equilibrium rate as is obtained when the enzyme and the inhibitor were
mixed at the start of the experiment. This was clearly not the case for this
inhibitor (Fig. 29, traces c and d). Rough calculations based on the observed
enzyme velocities and the second order rate constant at 1 µM inhibitor
60
(~5,300 M–1s –1 ) gave rise to an off rate of approximately 1 x 10–4 s–1 ,
regardless of the mechanism assumed. This allowed simulation of the
expected results of the dilution experiment (trace e) and highlighted the
unexpected nature of the observations in trace c in Figure 29.
Figure 29. The kinetics of inhibition of cathepsin K by 94c. The curves illustrate the
following: (a) no inhibitor control; (b) 1 µM 94c and 0.5 nM cathepsin K; (c) 10 nM 94c and
0.5 nM cathepsin K; (d) 1 µM 94c and 50 nM cathepsin K incubated for 1 h and then diluted
into the assay; (e) simulation of the expected results of the dilution experiment in (c). The
experiments were carried out in a volume of 0.5 mL in a quartz cuvette in a Hitachi F4500
fluorometer. The substrate was used at a concentration of 100 µM.
A plausible explanation for these results is the presence of a very minor
contaminant that irreversibly inhibited cathepsin K. We hypothesized that,
during the pre-incubation, the minor component inactivated some of the
enzyme, resulting in a reduced cleavage rate upon dilution compared to the
control. Additional support for this theory was gained from measurements of
the inhibitory activity of aminoethylamide 94c at a concentration of 100 nM
against varying concentrations of cathepsin K (Fig. 30).
N
N
O
O
N
N
O
O
95a
Figure 30. Titration of cathepsin K against buffer control (●), 10 nM furanone 95a (■), 100
nM 94c (▲) and 10 nM E64 (▼). The concentration of furanone 95a was selected to give
similar levels of inhibition as caused by 100 nM 94c. The substrate was used at a
concentration of 100 µM.
61
The data from this experiment clearly show an x-intercept at 2.5 nM
cathepsin K, as would be expected if a minor contaminant had inactivated
some of the enzyme during the pre-incubation. In the same experiment, we
found that E-64, an irreversible inhibitor of cathepsin K, and at an enzyme
concentration of 10 nM gave an x-intercept at 10 nM, whereas no such
intercept was seen when using the fully reversible inhibitor furanone 95a.
Therefore, knowing the concentration of inhibitor present and the level of
irreversible inhibition from the x-intercept, we can calculate that the
concentration of the contaminant in the aminoethylamide 94c was 2.5 mol%.
This experiment was performed on several compounds (94a, 94b, and 94c),
and the results revealed that those with a low K i value tended to have a high
level of the impurity.
These results seem to disagree with the findings of dialysis experiments
conducted by Altmann 110 and Kim114 and coworkers, which suggested that
inhibitors of this class act as reversible competitive inhibitors. However,
there is not necessarily a conflict between the observations. When a high
concentration of enzyme is incubated with a concentration of inhibitor that
should totally block the activity of the enzyme, there could still be an
insufficient amount of the minor component to completely inactivate the
enzyme. If that is the case, then dialysis and dilution will lead to recovery of
most of the activity. Since there is always some degree of self-degradation
associated with this class of enzymes, complete recovery of activity would
not be expected, and recovery of the majority of the activity would suggest
that we are dealing with a reversible inhibitor. It is perhaps pertinent to note
that, in our hands, fresh samples of 94g (the compound used in the kinetic
studies performed by Kim’s group) had a Ki of 1.5 μM and only approached
the reported 10 nM activity after irradiation (vide infra).
Smyth 115 and Marquis116 have also discussed the possibility that these
compounds act as slow-turnover substrates for cathepsin K. For that to be the
case, the leucine side chain would have to bind in the S1 subsite, which we
consider unlikely since we found that cathepsin K does not cleave the
substrates H-Leu-AMC and Z-Gly-Gly-Leu-AMC. Lack of binding of Leu
in S 1 is further corroborated by our RAPiD™ data.
62
5.4 Photodegradation
Thus it appears that the activity of these compounds can be attributed to an
impurity, but what is this contaminating substance, and where does it come
from? The fact that the problem was encountered in samples of
aminoethylamides that were prepared using distinct synthetic routes and
were purified according to different protocols (importantly 94g, was
prepared using the route described by Kim et al.), along with the fact that
other laboratories have reported activity for some of these compounds, 115,116
suggests that it is not a question of a synthetic impurity or by-product.
Since the active component was present at levels that were too low to
enable structure determination of the degradation products from either NMR
or MS studies of the parent inhibitors, we attempted to isolate the component
by reverse-phase gradient HPLC. Fractions collected from a semipreparative purification of aminoethylamide 94e were subjected to both
HPLC reanalysis and measurement of their rate of inactivation of cathepsin
K. Figure 31 shows that the rate of inactivation paralleled the amount of
parent compound present in each sample, suggesting that either the impurity
co-eluted or it was a degradation product generated directly from the parent
in each fraction.
Figure 31. Analysis of HPLC fractions of 94e. Each fraction was re-chromatographed, and
the amount of parent compound was measured (solid bars). Each fraction was also assayed
against cathepsin K, and the data were fitted to obtain kobs (solid circles). Substrate
concentration was 100 µM. The rates for the most active fractions, 7 and 8, were probably
underestimated, since they required 16-fold and 4-fold dilutions, respectively, to enable
determination.
We had noticed that there was a tendency for samples of aminoethylamides
to gain potency over time and also that assay stock solutions prepared
freshly from solid stock initially had rather low potency. This led us to
consider whether the active component was being formed by degradation or
photodegradation of the parent material. To test this possibility, we prepared
63
a fresh 10 mM solution of 94g in DMSO and divided it into three aliquots:
one was kept in a foil-wrapped clear glass vial (placed in the dark); one was
left on the laboratory bench in a clear glass vial (in ambient light); and the
third was placed in a quartz cuvette in a temperature-controlled cuvette
holder set at 25 °C and was illuminated with a 150W Xenon arc lamp. All
three portions were assayed at hourly intervals up to four hours, and the dark
and ambient light samples were also assessed after 96 hours (Fig. 32). The
difference of 1,000 nM that appeared between the 24 nM cathepsin K K i
value after four hours of irradiation and the activity of the dark control is
quite striking.
Ki nM
1000
100
10
0
1
2
3
4
5
96
time (hours)
dark
ambient light
UV irradiated
Figure 32. The effect of irradiation on the activity of 94g. Note the log scale of the Ki axis.
Straight lines drawn through data are NOT best fits. The data did not fit well with single
exponential decay, and no other attempt at fitting was made due to insufficient data.
Similar increases in potency were observed upon irradiation of compound
94f in both DMSO and ethanol. This is in strong contrast to the loss of
potency observed after irradiation of reference inhibitor 95b, which was
from 360 to 750 nM, as could be expected for photodegradation of an
inhibitor to a non-inhibitory product121 (Table 9).
Table 9. Effect of light irradiation on the Ki of aminoethylamides against cathepsin K
Compound
94f
94f (ethanol)
94g
95b
Initial Ki (nM)
300
270
1500
360
Irradiated K i (nM)
10
3
24
750
O
O
O
N
N
O
O
95b
Thus it appeared that the observed increase in potency was the result of
photodegradation of aminoethylamides either in direct sunlight or under
64
fluorescent light. When protected from light in amber vials, the degradation
occurred at a slower rate but nonetheless continued. Our initial hypothesis
for this increase of potency with the aminoethylamides was based on an N to
N’ acyl migration in aminoethylamides, as documented by Perillo et al.,122
but this was not linked to photodegradation. The proposed mechanism for
the migration of the acyl from 97 to 99 is shown in Scheme 13.
+ N 3
R1 N
R
R2
X-
96
+ OHR1
N
R2
O
N
H
R3
R N
R1
HN
N R
1
3
R2 OH
97
N
O
R3
R2
99
98
Scheme 13. N to N’acyl migration.
The cyclic hemiorthoamide 98 or some cationic species such as 96 could
potentially react irreversibly with the cysteinyl protease, which would
account for the irreversible inhibitor component in the aminoethyl amides.
However, neutral or alkaline medium is normally required for this
rearrangement to proceed, and so far we have seen no evidence of the
rearranged product, although such information might be difficult to obtain
with only a small amount of irreversible component present. Still, even
though it is possible for aminoethylamide 94a to undergo such
rearrangement, a trisubstituted nitrogen like that present in the other
aminoethylamides synthesized (94b and 94e) clearly cannot, which suggests
that some other mechanism is responsible for formation of the irreversible
impurity.
The only other information found in the literature concerns norfloxacin,
a broad spectrum antibacterial quinolone that contains an aminoethylamine
functionality. Norfloxacin has been shown to be a thermostable but
photosensitive drug, especially in solution, which leads to the formation of
aminoethylamine degradates.123,124 Nonetheless, the cited studies focused
more on the method of analysis than on the structural implications of the
photolysis.
O
F
COOH
Norfloxacin
N
N
C 2H 5
HN
65
5.5 Conclusions
Two solid-supported synthetic routes to afford aminoethylamide compounds
were developed. The initial aldehyde-resin-based route provided good results
but was not compatible with reducing sensitive groups and acid labile
functionalities inherent in some of the target molecules. In order to solve
these problems, an optimized sulfonamide-resin-based method was
developed to obtain a more general and shorter route to parallel synthesis of
the aminoethylamide compounds.
The results of the kinetic studies performed using several members of the
aminoethylamide inhibitor class suggest that the bulk of the observed
inhibition of human cathepsin K may be due to the presence of a very minor
component that irreversibly inhibits the enzyme. Furthermore, the inhibitory
activity was found to increase over time and was associated with a number
of structurally different aminoethylamides prepared by different routes and
purified by different techniques, which implies that the inhibitory component
is a degradation product and not a synthetic impurity. The hypothesis we
currently favor is that the active constituent is a photodegradation product of
the aminoethylamides formed either in direct sunlight or under fluorescent
light, because the rate of degradation was significantly lower in samples
protected from light in amber vials.
Without an isolated sample of the degradation product, it is difficult to
propose a mechanism of formation or to suggest how this weakness in the
series might be avoided. However, we believe that the results are still of
substantial interest to other researchers working in this area. As a
consequence of the mentioned findings, we decided that the
aminoethylamide template was unsuitable for further drug development.
66
6. Investigation of Allylic Alcohols in the P1
Position of Inhibitors of Hepatitis C Virus NS3
Protease (Paper V)
6.1 Introduction
6.1.1 Hepatitis C Virus
The hepatitis C virus (HCV) was first identified in 1989, and since then it
has been estimated that more than 170 million people are infected with HCV
worldwide.125 In the Western world, HCV infection is the main cause of
chronic hepatitis, a disease that is a major health problem that leads to
cirrhosis of the liver and hepatocellular carcinoma 126 and therefore also
constitutes the primary reason for liver transplantation. 127 Approved
therapies involve administration of interferon-α(IFN-α) or pegylated IFN-α,
either alone or in combination with ribavarin.128 Unfortunately, these
regimens are far from optimal,129–133 because they require prolonged
treatment that is associated with a generally modest sustained virological
response rate (~50%) and significant adverse side effects that result in poor
tolerance in some patient populations.134,135 Consequently, new and more
effective therapies are urgently needed. 128,136–140 In addition, there is
currently no vaccine available for HCV, since development of such an
approach has been hampered by the existence of different HCV genotypes,
the high replication rate and the low fidelity of the proofreading of the viral
RNA.136
HCV is a small, enveloped virus of the Flaviviridae family, and it contains a
single positive-strand RNA genome of approximately 9.6 kb, which carries
one long open-reading frame that encodes a polyprotein of about 3,000
amino acids. The HCV polyprotein is composed of structural proteins in its
N-terminal portion and of non-structural (NS) proteins in the remainder of
the molecule, and it is cleaved co- and post-translationally by cellular and
viral proteases into at least 10 different products. 141 Practically every step in
the life cycle of HCV—from entry into a host cell to the replication and
formation of the new virion particles—is a potential target for antiviral
drugs. The various prospective targets have been comprehensively
reviewed. 128,142,143
67
6.1.2 HCV NS3 Protease Inhibitors
One of the most promising molecular targets for HCV therapy is the nonstructural serine protease NS3, 143,144 which is one of the best-characterized
HCV enzymes. The structure of HCV NS3 consists of a serine protease
domain in the N-terminal third of the polyprotein and an RNA
helicase/NTPase domain in the C-terminal portion.145 NS3 protease mediates
proteolytic cleavage of the non-structural proteins NS3, NS4A, NS4B,
NS5A, and NS5B. The NS4A protein is an integral structural component of
NS3 and is required to enhance its serine protease activity,146–150 which is
essential for the viral life cycle and represents one of the primary targets for
the development of new treatments against HCV. 151–157
X-ray crystal structures of HCV NS3 protease have shown that the binding
site of the enzyme is remarkably shallow and solvent exposed, thus making
the design of small-molecule inhibitors a challenge.158,159 Despite this
obstacle, several NS3 protease inhibitors have been reported to date. 151–
153,160,161
One category of such compounds comprises the classical serine-trap
inhibitors, which are characterized by an electrophilic P1 carbonyl group that
forms a reversible covalent bond with the hydroxyl group of the serine in the
active site of the enzyme. Examples of these inhibitors include aldehydes, αketo amides, α-keto esters, α
-diketones, boronic acids, and α-keto acids.162–
176
A second category of non-covalent NS3 protease inhibitors incorporate
structures such as P1 amides,177 P1-P1’ acylsulfonamides,178 or P1
carboxylic acids. The P1 carboxylic acids are characterized as product-based
inhibitors,173,179–185 and they display higher specificity for the HCV NS3
protease than for other serine proteases. 161,186 As deduced from 3D structure
modeling, the carboxylate in the active site binds to the oxyanion cavity (NH
of Gly137 and Ser139) and to the catalytic histidine.187 The helicase domain
also appears to participate in the binding of the inhibitors in the native fulllength NS3 protein (protease-helicase/NTPase).188
Early but impressive clinical proof-of-concept data were recently disclosed
for BILN-2061 (Ciluprevir; Boehringer Ingelheim),152,153,161,189 which is a
macrocyclic non-covalent mimic of the peptide product inhibitor. BILN2061 was the first HCV protease inhibitor to enter clinical trials, and the
initial results were promising. However, subsequent clinical trials were
halted, apparently due to cardiac toxicity observed in rhesus monkeys given
high doses. At present, the two covalent product-based linear
peptidomimetics VX-950 (Telapravir) and SCH 503034 (Boceprevir) are
reported to be in Phase III and Phase IIb/III clinical trials, respectively190–196
and the non-covalent macrocyclic peptidomimetic TMC435350 is in Phase II
clinical trials. Medivir licensed its HCV PI program to JNJ/Tibotec
68
Pharmaceuticals Ltd in 2004 and TMC435350 was discovered through this
research collaboration.
Preliminary investigational work recently published by our group (Paper
VI) led to the discovery of a lead compound and subsequently selection of
TMC435350 as the clinical candidate drug. In a Phase 1 trial, it was found
that TMC435350 was well tolerated during five days of dosing and exhibited
strong and rapid antiviral activity in patients infected with HCV genotype 1.
The results of that trial, which included both healthy volunteers and HCV
patients, formed the basis for the Phase IIa trial that is now underway
throughout Europe. 197
H
O
N
H
N
N
H
MeO
H
N
O
O
H
N
H
N
O
O
M eO
O
N
N
N
N
O
N
VX-950
O
S
S
H
N
N
O
H
N
O
H
N
OH
O
O
N
O
N
O
NH
NH
NH
O
HN
O
O
O
O O
S
N
H
O
O
NH2
TMC43 5350
O
BIL N 2061
SCH 50303 4
6.1.3 Aim of the Study
Our group has previously described a P1 allylic alcohol representing a new
class of non-electrophilic inhibitors with a Ki value of 0.98 µM (100). 198
Ac-Asp-D-Glu-Leu-Ile-Cha
H
N
OH
100
We were intrigued by the discovery of this class of molecules, because it can
enable exploration of the S1 ’ pocket of the HCV NS3 protease, which,
178,179,199,200
according to recent studies,
appears to provide additional
favorable binding interactions with the enzyme. Moreover, it seems that NS3
protease is highly flexible in the mentioned region, which should also make
134,178,199–202
it possible to gain additional binding interactions.
The study presented in Paper V was conducted to explore P1 allylic
alcohols and their interactions with the S 1’ pocket in NS3 protease in order
to achieve affinity that would allow truncation at the amino terminus and
69
also to be able to move from the hexapeptides towards more drug-like
molecules.
The study had two main objectives:
1. To determine how the P1 allylic alcohol hexapeptides bind to the
active site of the HCV NS3 protease and to examine the importance
in that context of the alcohol and the double bond moieties.
Ac-Asp-D-Glu-Leu-Ile-Cha
OH
H
N
R
P1
2. To assess this new P1 series in shorter, more drug-like peptides. For
that purpose, two known sequences were selected: Ac-Cha-ValPro(quinoline)-OH (101)203 and Boc-Val-Pro(quinoline)-OH
(102).204
N
N
O
N
Ac-Cha-Val
O
O
OH
Boc-Val
O
N
OH
O
O
101
102
6.2 Synthesis of P1 Nva Allylic Alcohol
A model P1 allylic alcohol was synthesized from -norvaline (Nva) so that it
could be incorporated into a pentapeptide Ac-Asp(OtBu)-D-Glu(OtBu)-LeuIle-Cha-OH (103), a tripeptide Ac-Cha-Val-Pro(Quinoline)-OH (101), and a
dipeptide Boc-Val-Pro (quinoline)-OH (102).
O
BocHN
OMe
dimethylmethylphosphonate,
BuLi, THF
O
O OMe
P
OMe
Bo cHN
O
BocHN
85%
94%
104
NaBH4
PhCHO
LiOH, Ether
105
OH
BocHN
*
98%
107
ds 2:1
Scheme 14. Synthesis of P1 Nva allylic alcohol.
70
106
The synthesis was performed by reacting Boc-protected amino acid 104
(Boc-Nva-OMe) with 6 equivalents of the lithium salt of
dimethylmethylphosphonate at –78 C̊ in THF205 to give the phosphonate
105 in 94% yield (Scheme 14). The trans-enone 106 was obtained in 85%
yield using Horner-Wadsworth-Emmons conditions with powdered LiOH in
dry ether and benzylaldehyde. The allylic alcohol 107 was obtained in 98%
yield as a diastereomeric mixture (2:1) by using sodium borohydride or
polymer-bound borohydride to reduce the ketone.
To better understand the role of the alcohol in the binding to HCV NS3
protease, attempts were made to obtain the isomerically pure allylic alcohols.
One of the methods attempted was separation of the diasteromeric mixture
107 by HPLC. However, a clean separation could not be achieved, and
selective reduction of the ketone 106 by use of 9-BBN derivatives, R-Alpine
Borane, or DIP-Chloride also failed to deliver pure diastereomers.
6.3 Isomerically Pure Allylic Alcohols
To be able to isolate both isomers of alcohol 107, these were exposed to
basic conditions (KtOBu in dry THF) 206 to convert them into the
oxazolidinone mixture 108 (Scheme 15), which gave a 93% yield of a 2:1
mixture. The mixture 108 was subsequently protected using Boc-anhydride
in the presence of NaH in dry THF to provide the Boc-protected
oxazolidinones in 66% yield, which were then separated by column
chromatography to furnish a 1:2 ratio of trans 109 and cis 110. Basic
cleavage of oxazolidinones 109 and 110 using cesium carbonate in
methanol:water 5:1 afforded (S,S)-107 and (S,R)-107 in 88% and 93% yield,
O
O
respectively.
N
O
O
O
OH
O
N
O
HN
*
KtOBu/THF
107
O
1.NaH, Boc 2 O/THF
*
93%
2.Column chromatography
O
O
66%
108
2:1 ds
109
+
O
N
O
2:1 cis : trans ds
Cs2CO 3
MeOH/aq
109
88%
OH
O
O
OH
(S,S)-107
Cs2 CO 3
MeOH/aq
110
110
TFA/DCM/H2 O
100%
N
TFA x H2 N
*
OH
O
N
TFA/DCM/H2 O
100%
O
93%
111
2:1 ds
(S,R)-107
Scheme 15. Synthesis to afford isomerically pure allylic alcohols
71
This methodology constituted a convenient way to obtain isomerically pure
alcohols (S,S)-107 and (S,R)-107. However, epimerization occurred upon
cleavage of the Boc group under standard deprotection conditions
(TFA/DCM/H2 O or TFA/DCM), which resulted in the 2:1 mixture 111 when
starting from either alcohol (S,S)-107 or (S,R)-107. This indicates that the
allylic alcohol is acid labile and prone to isomerization under these
conditions (i.e., conditions used to achieve cleavage of the Boc-protecting
group).
Due to the unsuccessful results of the attempt to procure isomerically pure
P1 allylic alcohols, the diasteromeric mixture was used for the subsequent
coupling with the hexa-, tri-, and tetrapeptide models. Column
chromatography was carried out to separate the diasteromeric mixture of
hexapeptides but was not successful.
6.4 Synthesis of Hexapeptides
Synthesis of the pentapeptide Ac-Asp(OtBu)-DGlu(OtBu)-Leu-Ile-Cha-OH
(103)198 was performed using Fmoc solid-phase methodology.207–209 Boc
deprotection of 107 with TFA/DCM/H 2O (70:25:5) and subsequent coupling
of the amine with the pentapeptide 103 using N-[(dimethylamino)-1H-1,2,3triazolo-[4,5-b]pyridin-1-yl-methylene]-n-methylmethanaminium
hexafluorophosphonate N-oxide (HATU) and N,N-diisopropylethylamine
(DIEA) in DMF furnished hexapeptide 112 (89% yield, Scheme 16).
OH
1. TFA/DCM
2. 103,
HATU,
DIEA,DMF
107 DMF
89%
TFA/DCM/H 2O
100%
OH
R´HN
Ph
R´´HN
O
1.H 2, Pd/C
MeOH
O
R´HN
Ph
118
TFA/DCM
100%
Ph
117
H 2 , Pd/C
MeOH
2. TFA/DCM
R´´HN
100%
Ph
114
P h H 2 , Pd/C
MeOH
R´´HN
Ph
100%
115
Dess-Martin
reagent,DCM
25%
R´´HN
Ph
113
TFA/TES/DCM
100%
112
OH
R´´HN
116
O
R´´HN
Ph
119
100%
R´= Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-ChaR´´= Ac-Asp-D-Glu-Leu-Ile-Cha-
Scheme 16. Synthesis of hexapeptides.
Also, to be able to study the role of the alcohol and the double bond in the
interaction with the enzyme, the corresponding unsaturated ketone 118,
72
saturated ketone 119, and saturated alcohol 114 were synthesized from the
hexapeptide 112.
Oxidation of compound 112 to ketone 117 (25% yield, Scheme 16) was
performed using Dess-Martin periodinane in DCM.210 Cleavage of the tbutyl esters with TFA/DCM afforded compound 118 in a quantitative yield.
Hydrogenation of 117 over palladium on carbon in MeOH, followed by
cleavage of the ester functions, delivered ketone 119 in a quantitative yield.
As a reference compound, Ac-Asp-D-Glu-Leu-Ile-Cha-Nva-OH (120, Table
10) was synthesized by coupling of t-butyl L-α-aminopentyrate (H-NvaOtBu. HCl) with pentapeptide 103 using HATU and DIEA in DMF, which
gave the protected hexapeptide Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-ChaNva-OtBu in 52% yield. The t-butyl esters were cleaved using
TFA/DCM/Et3 SiH, furnishing compound 120 in a quantitative yield.
Deprotection of the t-butyl esters in compound 112 using
TFA/DCM/Et3 SiH211 afforded compound 115, not compound 113 as
anticipated. The target compound 113 was instead obtained by a nonreductive cleavage procedure using aqueous TFA/DCM. This reduction was
observed only when the double bond was in conjugation with an aromatic
system.
A proposed intermediate for the obtained result is a carbocation stabilized
through conjugation with the phenyl ring:
H
N
+
The same kind of intermediate could give rise to the epimerized product 111,
the reduced product 115, and product 113. Hydrogenation of the double
bonds in compounds 113 and 115 was achieved by use of palladium on
carbon in MeOH, which afforded compounds 114 and 116 (Scheme 16) in
quantitative yields.
73
6.5 Synthesis of Tetrapeptides
Synthesis of P2-P4 tripeptide Ac-Cha-Val-Pro(Quinoline)-OH (101) was
performed according to procedures described in the literature. 203 Boc
deprotection of 107 with TFA/DCM/H 2O (70:25:5) and subsequent coupling
of the amine with the tripeptide 107 using HATU and DIEA in DMF
furnished tetrapeptide 121 (46% yield, Scheme 17). The unsaturated alcohol
121 was hydrogenated over palladium on carbon in MeOH to deliver a
quantitative yield of the saturated alcohol 122, which was oxidized to ketone
124 (95% yield) using Dess-Martin periodinane in DCM. Treatment of
compound 121 with TFA/DCM/Et3SiH afforded compound 123 (Scheme 17)
in quantitative yield.
1.H 2 , Pd/C
MeOH
OH
1. 30%TFA/DCM
or HCl/EtOAc
RH N
107
2. 101, HATU,
DIEA, DMF
OH
RHN
Ph
Ph
100%
121 46%
122
Dess-Martin
reagent, DCM
95%
TFA/TES/DCM
100%
O
RHN
RHN
Ph
123
Ph
124
N
R=
N
O
O
O
O
OH
N
Ac-Cha-Val
N
Ac-Cha-Val
O
103
O
Scheme 17. Synthesis of tetrapeptides.
The reference compound Ac-Cha-Val-Pro(Quinoline)-OH-Nva-OH (125,
Table 11) was synthesized by coupling methylnorvalinate (H-Nva-OMe.
HCl) with tripeptide 101 using HATU and DIEA in DCM, which gave the
methyl ester tetrapeptide in 87% yield. Subsequent hydrolysis of the ester by
treatment with lithium hydroxide in methanol-water afforded the target
compound 125 (41%).
74
6.6 Synthesis of Tripeptides
Synthesis of the P2-P3 dipeptide Boc-Val-Pro (quinoline)-OH (102) was
performed according to procedures in the literature. 204 Boc deprotection of
107 with TFA/DCM/H2 O (70:25:5) and subsequent coupling of the amine
with the dipeptide 102 using HATU and DIEA in DMF furnished the model
tripeptide 126 (36% yield, Scheme 18).
N
N
1. 30%TFA/DCM
or HCl/EtOAc
O
O
107
2. 102, HATU,
DIEA, DMF
Boc-Val
N
O
O
OH
H
N
Boc-Val
O
OH
N
O
102
126 36%
Scheme 18. Synthesis of the tripeptides.
6.7 Tables: Inhibitory Activity
Table 10. Percent inhibition at 10μM and 1μM and Ki inhibition constant for hexapeptides
screened against full-length NS3 (protease-helicase/NTPase) protease
A c-A s p -D -G lu -L e u -Ile -C h a
H
N
R
P1
Cmpd
P1
R
% inh at
10 μM
% inh at
1 μM
K i (μM)
95
71
0.137
98
92
0.107
69
13
3.4
91
34
2.28
O
120
n-Pr
OH
O
118
n-Pr
119
n-Pr
113
n-Pr
114
n-Pr
27
9
>10
115
n-Pr
44
52
5.64
116
n-Pr
25
18
>10
100
Et
32.5
6.7
13.3 (0.98)*
O
OH
OH
*Published data
OH
198
75
Table 11. Percent inhibition at 10µM and 1µM and Ki inhibition constant for tetrapeptides
screened against full-length NS3 (protease-helicase/NTPase) protease.
N
O
O
N
Ac-Cha-Val
N
O
Cmpd
P1
125
n-Pr
R
P1
R
% inh at
10 µM
% inh at 1
µM
Ki (µ M)
70
24
0.48
21
0
nd
O
OH
OH
121
n-Pr
122
n-Pr
17
0
nd
123
n-Pr
13
0
nd
124
n-Pr
20
0
nd
OH
O
nd = not determined
Table 12. Percent inhibition at 10μ
M and 1μM and Ki inhibition constant for tripeptides
screened against full-length NS3 (protease-helicase/NTPase) protease
N
O
Boc-Val
O
N
N
O
Cmpd
P1
127*
n-Pr
126
n-Pr
R
R
P1
% inh at
10 μM
% inh at
1 μM
Ki (μM)
98
83
0.167
28
21
8.3
O
OH
OH
*Reference compound provided by the University of Uppsala and assessed in our system
76
2 12
6.8 Structure-Activity Relationships
Examination of the three classes of inhibitors presented in Table 10 indicated
that the most potent compound was the α
,β-unsaturated ketone 118, which
had a K i value of 0.107 μ
M, approximately equipotent with the reference
product inhibitor 120 (Ki value of 0.137 μM). The inhibitory activity of the
α,β-unsaturated alcohol 113 (Ki 2.28 μM) found in this study was lower than
that of the reference product inhibitor 120 but represented a 1.5-fold and a
5.8-fold increase compared to the activities of 119 and the reference allylic
alcohol compound 100, respectively. The inhibitory activity of compound
113 was dramatically decreased when the double bond was reduced, as seen
in compound 114 (Ki > 10 μM), or when the alcohol function was not
present, as in compounds 115 (K i = 5.64 μ
M) and 116 (K i >10 μ
M). It can be
concluded that both the alcohol function and the double bond in compound
113 are integral functional moieties that contribute to the binding affinity for
the enzyme.
In Table 11, the reference tetrapeptide compound 125 exhibited a 3.5-fold
decrease in activity compared to reference hexapeptide product inhibitor
120. However, relevant inhibitory activities against NS3 protease were not
found for the tetrapeptide derivatives 121, 122, and 123 (corresponding to
the hexapeptides 113, 114 and 115), or the saturated ketone 124. The
moderate activity found for the α
,β-unsaturated alcohol hexapeptide 113
could not be transferred to the tetrapeptide 121.
In Table 12, the corresponding α,β-unsaturated alcohol tripeptide 126
showed some inhibitory activity (Ki 8.3 μM), although the level represented
a 3.6-fold decrease compared to the activity of the α
,β-unsaturated alcohol
hexapeptide 113.
6.9 Conclusions
Considering the SARs of the hexapeptides (Table 10), the unsaturated ketone
118 can be seen as the most potent compound, and it is apparent that both
the alcohol moiety and the double bond were necessary for the inhibitory
activity exhibited by the α,β-unsaturated alcohol 113. Furthermore,
shortening of the α,β-unsaturated alcohol hexapeptide to a tetrapeptide and
tripeptide (121 and 126 respectively) resulted in loss of inhibitory activity.
Notwithstanding, even though this limited set of P1’ substituents did not
generate promising low molecular weight NS3 protease inhibitors, it cannot
be excluded that a broader collection of P1’ groups would result in
substantially more potent and promising inhibitors.
77
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Acknowledgements
The present investigations were carried out at Medivir AB in Huddinge,
Sweden.
I would like to express my sincere gratitude to all those who inspired,
motivated, and supported me in different ways over the years and made this
thesis possible.
Special thanks to the following people:
Professor Bertil Samuelsson for giving me the opportunity to embark on
Ph.D. studies, for enabling completion of this research and for providing
excellent working facilities.
Docent Björn Classon for his support, for sharing his knowledge with me
and for creating a positive working atmosphere.
Professor Stefan Oscarson for accepting me in his research group at
Stockholm University during my initial years of graduate studies, and my
university colleagues Jenny U., Jenny A., and Catta.
Dr. Ellen Hewitt and Dr. Urszula Grabowska for all their help, support, and
valuable discussions regarding the work on cathepsins S and K.
Dr. Tatiana Maltseva for all assistance with NMR spectroscopy, support and
for fruitful collaboration.
Katarina Jansson, for all the nice molecular models, help and
encouragement, and for being such a good colleague and friend.
My many co-authors for productive teamwork.
All the people at Medivir for spreading such friendliness and making the
daily work interesting and pleasant. Special thanks to the past and present
members of my group—Daniel, Karolina, Laszlo, Lotta, and Stina—, as
well as Oscar for their support, friendship, and interest in my research.
My former colleagues at Pharmacia, especially Ronny Lundin and my
previous manager Marianne Nilsson, for introducing me to the world of
pharmaceutical research, and for sharing their knowledge with me.
Patricia Ödman, for excellent revision of the English in this thesis.
I would also like to thank all of those who contributed indirectly to this work
by helping me on a personal level by giving me courage and support.
Above all, my family, to whom this book is dedicated: my husband Johan,
for his love and enthusiasm and our two daughters Mikaela and Annika, por
ser tan increiblemente maravillosas, mina änglar, ni är livets glädje! They
are my constant source of love, inspiration, encouragement, energy, insight,
pride and fun.
My parents José y Carmen and my brothers Rubén y Nacho: Por vuestro
cariño, apoyo, ilusión, esfuerzo y por hacerme sentir orgullosa. Os quiero,
siempre en mi corazón.
My in-laws Svante och Bibbi, and Kristina och Gösta, för allt stöd och
uppmuntran.
All my friends outside the world of chemistry, my “faraway” friends, Laura
and Geles, por una duradera y entrañable amistad allí en la distancia, my
Spanish girlfriends in Stockholm, in particular my “really close” friends
Encarni y María, por vuestro apoyo incondicional y vuestra amistad, and
mina trevliga grannar for their companionship and for all the good times we
have shared.
Appendix A
This appendix contains experimental details, and/or spectroscopic data of
compounds 8 and 10 described in chapter 1.
General. p-Nitrophenylcarbonate Merrifield resin (0.8mmol/g) was
purchased from Novabiochem. Reactions were performed in 4 and 8 ml
Wheaton vials with screw caps. IR spectra on solid support were recorded on
a Perkin-Elmer FTIR spectrophotometer. LCMS analysis was taken on a
Water-Micromass instrument, using Waters 2790 Alliance, Waters 996
photodiode array detector and Micromass ZMD (API ES pos and neg) mass
detector. Synergi MAX-RP 50x4.6 mm 80A, 4u from Phenomex was used as
reverse phase column for the LCMS analysis, and acetonitrile / water
0.1%TFA were used as mobile phases. Ninhydrin test was performed
following the procedure described by Kaiser 9,27 using aminomethylated
polystyrene resin as positive control. NMR spectra were acquired using a
Varian Unity Inova spectrometer at a magnetic field strength of 11.7 T
operating at 499.84 MHz for 1 H, and 125,67 MHz for 13C.
Resin 8. Amide coupling (Scheme 2, step b). Dry resin bound 4(aminomethyl) piperidine (250 mg, 0.2 mmol) obtained as described above
was suspended in anhydrous DMF (2 mL). A solution of 4-azidobenzoic
acid (130 mg, 0.8 mmol), PyBOP (416 mg, 0.8 mmol) and DIEA (280 uL,
1.6 mmol) dissolved in anhydrous DMF (3 mL) were added to the resin. The
reaction was performed in a screw capped 8mL vial placed in the shaker at
room temperature over 24 hours. The resin was filtered through a fritted
column at the VacMaster and washed with DMF (2 x 10 mL), DCM (2 x 10
mL), MeOH (2 x 10 mL), DCM (2 x 10mL) and MeOH (2 x 10 mL) and
dried at the vacuum oven. FTIR of the resin: 3347cm -1 broad (NH-), 2121cm1
sharp (N3), 1689cm-1 broad (O-CO-N- and –NH-CO-). In order to elucidate
if the resin obtained was 7 or 8, high resolution MAS NMR experiment were
performed and the structure confirmed with standard tube NMR results from
the cleaved product 10.
Compound 10. 4-azido-N-(4-piperidinylmethyl) benzamide. Resin 8 from
the previous step was cleaved using a solution of 75% TFA in DCM (3 mL).
The reaction was performed in a screw capped 8mL vial placed at the shaker
during 1.5hours. The resin was filtered and the cleaved solution was kept
together with the washes in DCM (2 x 2 mL). The solvent was evaporated at
the Chiron equipment. The residue was re-dissolved in acetonitrile / water
and then lyophilized to afford 10 (48.4 mg, 96% pure by LCMS, 90% yield).
1
H NMR (DMSO-d6 ), (for atom numbering see Scheme 2), δ8.56 (t, 1H)
(N)H-8, δ7.87 (2, J=8.2 Hz, 2H) H-11, H-15, δ7.16 (2, J=8.2 Hz, 2H) H-12,
H-14, δ3.24 (m, 2H) H-2’, H-6’, δ3.15 (t, 2H) H-7’, H-7”, δ2.82 (q, 2H) H2”, H-6”, δ1.80 (m, 3H) H-4, H-3’, H-5’, δ1.33 (m, 2H) H-3”, H-5”. 13C
NMR (DMSO-d 6) δ166.15 C-9, 142.91 C-10, 131.70 C-13, 129.75 C-11, C15, 119.48 C-12, C-14, 44.58 C-7, 43.63 C-2, C-6, 34.30 C-4, 26.99 C-3, C+
5. MS (ESI): m/z 259.9 
M + H
.
Appendix B
This appendix contains experimental details, and/or spectroscopic data of the
starting material azide 23 (entry 7, Table 1) described in chapter 2.
(S)-(-)-1,1-Dimethylethyl[1-(Azidomethyl)-3-methylbutyl]carbamate (23,
entry 7, Table 1). N-Boc-L-Leucinol (5 g, 23 mmol) was dissolved in DCM
(100 mL) and cooled to 0 C̊. DIEA (9.6 mL, 55 mmol) was added to the
solution followed by the addition of methanesulfonyl chloride (2.1 mL, 27.5
mmol). The resulting solution was stirred for 16h and the temperature was
slowly allowed to reach RT, and then treated with brine. The organic phase
was separated, dried, filtered and concentrated in vacuo to a brown sticky
solid (6.5 g, 22 mmol, 96% yield). The mesylate obtained was dissolved in
DMF (35 mL) and treated with sodium azide (4.28 g, 66 mmol). The
reaction mixture was stirred at 80 C̊ for 24h, cooled down to room
temperature, and diluted with water. The product was extracted with several
portions of ethyl ether, and the extracts were combined, dried, and filtered
and concentrated in vacuo. The crude product was purified using silica gel
(elution with hexane: DCM 10:1) to afford the titled compound as a white
solid (3.9 g, 16.1 mmol, 73% yield). 1H NMR (400 MHz, CDCl3) δ4.554.44 (m, 1H), 3.85-3.75 (m, 1H), 3.54-3.42 (m, 1H), 3.37-3.25 (m, 1H),
1.71-1.59 (m, 1H), 1.45 (s, 9H), 1.42-1.25 (m, 2H), 0.93 (d, J=6.6 Hz, 6H).
13
C NMR (100 MHz, CDCl 3) δ155.4, 79.3, 54.9, 48.5, 41.0, 28.2, 24.6, 22.9.
FTIR: azide absorption band at 2100 cm-1. LCMS (ESI): m/z calcd for
C 11H22N4 O2: 242.32; found: 243.19 (MH+).
Appendix C
This appendix contains experimental details, and/or spectroscopic data of
compounds 83, 84, 86, 90-91, and illustrative examples of the final
compounds 87 obtained by Scheme 10 and 11 (94a) described in chapter 5.
General. Aldehyde resin (0.9 mmol/g) was purchased from Novabiochem
(01-64-0384), 4-sulfamylbenzoyl AM resin (0.89 mmol/g) was purchased
from Novabiochem (01-64-0121) and 4-sulfamylbutyryl AM resin
(1.1mmol/g) was purchased from Novabiochem (01-64-0152). Reactions
were performed in 4 and 8 ml Wheaton vials with screw caps. IR spectra on
solid support were recorded on a Perkin-Elmer FTIR spectrophotometer.
LCMS analysis was taken on a Water-Micromass instrument, using Waters
2790 Alliance, Waters 996 photodiode array detector and Micromass ZMD
(API ES pos and neg) mass detector. Synergi MAX-RP 50x4.6 mm 80A, 4u
from Phenomex was used as reverse phase column for the LCMS analysis,
and acetonitrile / water 0.1%TFA were used as mobile phases. Ninhydrin
test was performed following the procedure described by Kaiser 9,27using
aminomethylated polystyrene resin as positive control. NMR spectra were
acquired using a Varian Unity Inova spectrometer at a magnetic field
strength of 11.7 T operating at 499.84 MHz for 1H, and at 125.67 MHz for
13
C.
Preparation of Resins 83. General procedure for the reductive
amination (Scheme 10, step a). Aldehyde resin (3g, 2.7 mmol) was
suspended in DMF (50 mL). Tosylate salt of aminoacid allyl ester (13.5
mmol, 4.64g of TosLeu-Oallyl as example), and sodium cyano borohydride
(NaBH3 CN) (848 mg, 13.5 mmol) were added to the resin. The reaction was
gentle stirred for 18h. and after that the resin was filtered , washed with
DMF (2 x 40mL), DCM (2x 40mL), and MeOH (2x 40 mL) and dried to
afford the yellow resin 83 (3.79g).
Preparation of Resins 84. General procedure (scheme 10, step b). Resin
83 (3.47g, 3.41 mmol) was suspended in DMF (15 mL). FmocChloroformate
(4.41 g, 17.35 mmol) dissolved in DMF (10 mL) was added. The reaction
was allowed to go for 15 min. under gentle stirring. NMM (1.72 mL, 17.35
mmol) was added dropwise. After gentle stirring for 18h., the resin was
filtered , washed with DMF (2 x 40mL), DCM (2x 40mL), and MeOH (2x
40 mL) and dried to afford yellow resin 84 (3.2 g, 57% loading calculated by
Fmoc spectrophotometrical method).
Preparation of Resins 86. General procedure (scheme 10, steps c-d).
Resin 84 (3.31g) was subjected to allyl removal by treatment with a solution
of Tetrakis (triphenylphosphine) Pd (0) ( 1.63g) and phenylsilane (5.6 g) in
DCM (25 mL) for 5 min without any stirring. The procedure was performed
twice. The resin was washed with DCM (2x 50 mL), then 10 times with a
solution of sodium diethyl-dithiocarbamate trihydrate 0.5% w/v and DIEA
0.5 % v/v in DMF and dried to afford resin 85. Resin 85 (150 mg, 0.135
mmol) was suspended in DMF (3 mL). A solution of PyBOP (281 mg, 0.54
mmol), HOBt (73 mg, 0.54 mmol) and amine R 3 NH2 (0.54 mmol) in DMF
(3 mL) followed by DIEA (188 uL, 1.08 mmol) was added. The suspension
was stirred for 18h at RT. The resin was filtered and washed with DMF (2 x
40mL), DCM (2x 40mL), and MeOH (2x 40 mL) and dried to afford resin
86.
Peptide coupling and cleavage from the resin to afford final compounds
87. General procedure (scheme 10, steps e-g). Resin 86 (150 mg, 0.135
mmol) was treated with a solution of piperidine/DMF (20%v/v) for 2 hr,
filtrated, washed several times with DMF, DCM and MeOH and dried. The
corresponding acid building block R 2COOH (2.7 mmol) was dissolved in a
solution of 20%DMF/DCM, and colled to 0°C. Diisopropylcarbidiimide
(DIC) was added (18.9 mmol). The reaction was quickly shaked and then
left for 45 min at O°C. The urea prcipitate was filtrered off and the soluion
added to the resins (150 mg). The suspentions were stirred for 48h. The
resins were filtered, washed several times with DMF (2 x 4 mL), DCM (2x 4
mL), and MeOH (2x 4 mL) and dried. The final compounds 87 were
obtained after treatment of the resins with a solution of 5%TES in TFA for
2h. After filtration of the resins, the solutions were concentrated and the
crudes purified by prep LCMS.
Example of compounds 87 produced by Scheme 10 as described above:
N
O O
N
H
O
N
H
Furan-3-carboxylic
acid
[3-methyl-1-(2-pyrrolidin-1-ylethylcarbamoyl)-butyl]-amide. Synthetic procedure as described above
(sheme 9) to afford the title compound (45mg, 54% yield). >95% LCMS
purity. MS (ESI): m/z mass calcd for C 17H27N3 O3, 321.42, found: 322.19
(MH +). 1 H NMR (500 MHz, CDCl3) δ8.02 (s, 1H), 7.45 (s, 1H), 6.75 (s,
1H), 4.62 (m, 1H), 3.5 (m, 2H), 3.38 (m, 2H), 2.7-2.8 (m, 4H), 1.75-1.8 (m,
4H), 1.6-1.7 (m, 3H), 0.98 (m, 6H).
OH
O O
O
N
H
N
H
Furan-3-carboxylic acid [1-(2-hydroxy-1-phenyl-ethylcarbamoyl)-3methyl-butyl]amide. Synthetic procedure as described above (sheme 9) to
afford the title compound (31mg, 75% yield). >95% LCMS purity. MS
(ESI): m/z mass calcd for C17H27N3 O3, 344.41, found: 345.09 (MH+), 367.05
(MNa +) . 1 H NMR (500 MHz, DMSO) δ8.5 (d, NH), 8.3 (s, 1H), 8.2 (d,
NH), 7.76 (s, 1H), 7.35-7.25 (m, 4H), 7.24-7.2(m, 1H), 6.97 (m, 1H), 4.9 (t,
OH), 4.82 (m, 1H), 4.62 (m, 1H), 3.58 (m, 2H), 1.6-1.5 (m, 3H), 0.96 (m,
6H). 13C NMR (500 MHz, DMSO), δ172.48, 162.15, 146.15, 144.59,
141.98, 128.81, 128.72, 127.66, 127.42, 123.21, 109.93, 79.88, 65.38, 55.73,
51.70, 41.45, 25.02, 23.73, 22.21.
Preparation of Resins 90. General procedure to amino acid attachement
onto resin (Scheme 11, steps a-b). 4-sulfamylbutyrryl AM resin 93 (8.5g,
9.35 mmol) was suspended in DMF (50 mL). A solution of the
corresponding Fmoc-aminoacid (46.7 mmol, 13.2g of L-LeuNHFmoc as in
the illustrative example 94a, PyBOP (19.5g, 46.7 mmol) and HOBt (5g, 46.7
mmol) in DMF (40 mL) was added followed by addition of DIEA (13 mL,
93.5 mmol). After the stirring of the suspention for 18h at RT the resin was
filtered, washed with DMF (2 x 40mL), DCM (2x 40mL), and MeOH (2x 40
mL) and dried to afford resin 90. Solid support FT-IR: intense absorption
band at 1713 cm-1corresponding to the carbamide from the Fmoc group.
Negative ninhydrin test (the starting resin 93 gave pale blue color beads,
while the produced resin 90 gave colorless beads). The resin was then treated
with a solution of 20% pip/ DMF for 2h at RT, washed, and dried to afford
Fmoc-deprotected resin 90 (9.84 g). Solid support FT-IR: absence of the
absorption band at 1705 cm -1. Positive ninhydrin test (the produced resin
gave dark blue beads).
Preparation of Resins 91. General procedure to peptide coupling
(Scheme 11, step c). Resin form previous step (9.84 g, 10.8 mmol) was
suspended in DMF (30mL). The corresponding acid R2 COOH (43.2 mmol,
3-furoic acid, 4.85g, as in the illustrative example 94a was added to the resin
followed by PyBOP (22.5 mg, 43.2 mmol), HOBt (5.8g, 43.2 mmol) nad
DIEA (15 ml, 86.4 mmol). The suspention was stirred for 18 h at RT. The
resin was filtrered, washed with DMF (2 x 40mL), DCM (2x 40mL), and
MeOH (2x 40 mL) and dried to afford resin 91 (8.49 g, 95% yield). Negative
ninhydrin test.
Activation of resins 91. General procedure (Scheme 11, step d). Resin 91
(8.49 g, 9.3 mmol) was suspended in NMP (70 mL). Iodoacteonitrile (31.2 g,
186 mmol) was added followed by DIEA (8 mL, 74.4 mmol). The
suspension was stirred for 18 h at RT. After filtration, the resin was washed
several times with NMP, followed by washes of THF and then dried to
afford highly activated resin 91 (8.87g).
Nucleophilic cleavage to afford compounds 87. General procedure
(Scheme 11, step e). Activated resin 91 from previous step (300 mg, 0.33
mmol) was suspended in anhydrous THF (4 mL). The corresponding amine
R 3NH2 (0.26 mmol, 36 mg of N-phenylethylenediamine as in the illustrative
example 94a in THF (2 mL) was added. The suspention was allowed to react
for 18h at RT. The resin was filtered and the solution concentrated. The
crude was dissolved in MeOH and then purified by prep LCMS to afford
compound 87.
Examples of compounds 87 produced by Scheme 11 as described above:
H
N
O O
O
N
H
N
H
Furan-3-carboxylic acid [3-methyl-1-(2-phenylamino-ethylcarbamoyl)butyl]-amide (94a). Synthetic procedure described above as an illustrative
example to afford compound 64 (53mg, 60% yield). >95% LCMS purity.
MS (ESI): m/z mass calcd for C19H25N3 O3, 343.43, found: 344.16 (MH +). 1 H
NMR (500 MHz, CDCl3) δ7.98 (s, 1H), 7.42 (s, 1H), 7,18 (m, 2H), 6.70 (m,
1H), 6.60-6.55(m, 3H), 6.27 (d, NH), 4.58 (m, 1H), 4.05 (broad s, NH), 3.5
(m, 2H), 3.28 (m, 2H), 1.78-1.6 (m, 3H), 0.98 (m, 6H).
H
N
O O
N
H
O
N
H
Benzofuran-2-carboxylic
acid
[3-methyl-1-(2-phenylaminoethylcarbamoyl)-butyl] amide. Synthetic procedure as described above to
afford the tittle compound (48mg, 65% yield). >95% LCMS purity. MS
(ESI): m/z mass calcd for C19H25N3O3, 393.49, found: 394.16 (MH+). 1 H
NMR (500 MHz, CDCl3) δ7.68, 7.66 (d, 1H), 7.52-7.51(d, 1H), 7.48-7.44
(m, 2H), 7.33-7.39 (m, 1H), 7.15-7.12 (m, 2H), 6.95, 6.93 (d, NH), 6.68-6.66
(m, 1H), 6.60-6.55 (m, 2H), 6.44 (m, NH), 4.63-4.58 (m, 1H), 3.54-3.49 (m,
2H), 3.32-3.29 (m, 2H), 1.83-1.75 (m, 3H), 0.98-0.96 (m, 6H).