Cathepsin C inhibitors: Property optimization and identification of a clinical candidate

Article abstract of DOI:10.1021/jm401705g

A lead generation and optimization program delivered the highly selective and potent CatC inhibitor 10 as an in vivo tool compound and potential development candidate. Structural studies were undertaken to generate SAR understanding.

Full text of DOI:10.1021/jm401705g

Article
pubs.acs.org/jmc
Cathepsin C Inhibitors: Property Optimization and Identification of a
Clinical Candidate
Mark Furber,*,† Anna-Karin Tiden,‡ Philip Gardiner,† Antonio Mete,‡ Rhonan Ford,‡ Ian Millichip,‡
Linda Stein,‡ Andrew Mather,‡ Elizabeth Kinchin,‡ Christopher Luckhurst,‡ Simon Barber,‡ Peter Cage,‡
Hitesh Sanganee,§ Rupert Austin,‡ Kamaldeep Chohan,‡ Raj Beri,‡ Bob Thong,‡ Alan Wallace,‡
Victor Oreffo,‡ Ray Hutchinson,‡ Steve Harper,‡ Judit Debreczeni,§ Jason Breed,§ Lisa Wissler,†
and Karl Edman†
†AstraZeneca, Pepparedsleden 1, Molndal 431 83, Sweden
̈
‡AstraZeneca, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, United Kingdom
§AstraZeneca, Alderley Park, Cheshire, SK10 4TG, United Kingdom
ABSTRACT: A lead generation and optimization program delivered the highly
selective and potent CatC inhibitor 10 as an in vivo tool compound and potential
development candidate. Structural studies were undertaken to generate SAR
understanding.
target effects. Overcoming such issues is a serious hurdle, and
there can be little doubt that this is the principal reason why no
CatC inhibitor has progressed in clinical development so far.
A commonly used warhead in cysteine cathepsin inhibitors is
the nitrile group,8 and a number of nitrile inhibitors of CatC
have been described7 wherein the nitrile group traps a cysteine
residue in a covalent but reversible fashion. Three chemical
classes of nitriles have generally been used in such cysteine
protease inhibitors: (i) cyanamides, (ii) aryl or heteroaryl
nitriles, and (iii) amino- or amidoacetonitriles; these differ in
their electrophilic properties. Where electrophilicity is weak
(e.g., simple arylnitriles), protease inhibition is invariably also
weak. Where electrophilicity is too high (some cyanamides and
heterocyclic nitriles), poor selectivity and nonspecific tissue
binding can present a significant concern (e.g., irreversible
binding in human liver microsomes).9 Amidoacetonitrile
derivatives have, in some studies, shown a low potential for
such nonspecific covalent binding,9 and Combio was the first
company to describe amidoacetonitrile-based inhibitors of
CatC (e.g., compound 1 in 2004).10
INTRODUCTION
■
Cathepsin C (CatC or dipeptidyl peptidase I) is a lysosomal
cysteine protease that is important for intracellular protein
degradation and plays a key role in the activation of the
proinflammatory serine proteases neutrophil elastase (NE),
cathepsin G (CatG), and proteinase-3 (PR-3).1 In humans,
CatC mutations are associated with the autosomal recessive
̀
disorders Haim−Munck syndrome and Papillon−Lefevre
syndrome, both associated with almost complete loss of the
secondary enzymatic activities of all three serine proteases, NE,
CatG, and PR-3, in neutrophils and granzyme B in NK cells and
characterized by palmoplantar keratosis and severe childhood
periodontal disease.2,3 Knockout studies in mice and
pharmacological inhibition using CatC inhibitors have con-
firmed a central role for CatC in the activation of these granule
serine proteases and strongly support the therapeutic strategy
of targeting CatC inhibition in diseases such as COPD that
carry a high neutrophilic burden.4,5 At the same time, such
studies have demonstrated the need for a high degree of
inhibition to demonstrate therapeutically significant effects.5,6
The potential therapeutic utility and medicinal chemistry of
cathepsin C have been reviewed,1 as have attempts to develop
inhibitors of this enzyme.7 Most known CatC inhibitors are
believed to interact through covalent bond formation with the
active site cysteine (Cys-234), and the electrophilic and
sometimes peptidic nature of these molecules is frequently
associated with poor metabolic stability while also presenting a
potential safety concern because of poor selectivity and off-
Understanding Cell Potency and Inhibitor Binding. In
assessing amidoacetonitriles as potential start points for a
medicinal chemistry program targeting CatC, the potential for
lysosomotropism11 was also considered given that the basic
nature of some of these CatC inhibitors could result in
Received: November 5, 2013
Published: March 4, 2014
© 2014 American Chemical Society
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accumulation in the acidic lysosomal compartment. Although
achieving higher effective concentration at the target, which is
itself located within lysosomes, such effects can, in some cases,
result in a loss of selectivity.11 We therefore developed a cell
assay to monitor this effect and to profile compounds for cell
activity versus enzyme activity.12 Interestingly, in this cellular
assay, compound 1 was shown to be relatively poorly potent
(pIC50 7.2) despite high activity against the isolated enzyme
(pIC50 8.7). This is contrary to expectation for a basic
compound of this type, and it indicated a potential compound
stability or permeability issue. Indeed, on further investigation,
compound 1 and related examples from the series were shown
to be subject to rapid proteolysis in the cell assay medium,
indicating a level of instability that could not be carried forward.
Hydrolysis of the amide bond was shown to be enzymatic in
nature because the instability could be repeated in plasma (t1/2
rat plasma, 1.1 h; t1/2 human plasma, 3.1 h), whereas the
Figure 2. Initial lead structures.
having improved plasma stability, the compound had poor
metabolic stability, being rapidly turned over in the presence of
rat hepatocytes in vitro. In fact, poor metabolic stability is a
feature of both compounds 1 and 2, but, if anything, metabolic
stability had decreased compared to 1 (intrinsic clearance, Clint
>150 vs 91 μL/min/106 cells for 1 in rat hepatocytes). To
understand the high metabolic clearance of this compound, a
metabolite profile was determined. Compound 2 was shown by
LC−MS/MS analysis to be heavily oxidized on the two phenyl
rings of the biphenyl system in both rat (fresh) and human
(cryopreserved) hepatocytes. Although the precise location of
oxidation was not determined, it was reasoned that
incorporation of electron-withdrawing substituents would
reduce the propensity for oxidation. To this end, substituents
were incorporated into the biphenyl to determine their effect
on the ease of oxidation. This was most successful with
compound 3 (Table 1) bearing a simple nitrile substituent in
the 4-position of the outer phenyl ring. This simple substitution
gave a promising compound because the greatly increased
metabolic stability in vitro, accompanied by good in vivo PK in
rat (Cl, 16 mL/min/kg; Vss, 5.6 L/kg; t1/2, 4.5 h; and
bioavailability, 75%), was attended by a modest improvement in
potency in both the enzyme and cell assays.
compounds were chemically stable at pH 2, 7.4, and 10 (t1/2
>
100 h). Although plasma instability was viewed as problematic
for a lead compound, it was anticipated this could be resolved
by substitution around the amide bond, and this could form
part of an initial chemistry exploration.
Alongside such a chemistry program, we wished to generate
structural information to help understand SAR. The published
X-ray crystal structure of human CatC reveals a tetrameric
structure containing four identical subunits.13 Each subunit
comprises three chains: light and heavy chains forming the
catalytic domain, and a third chain constituting the so-called
exclusion domain that blocks the active site cleft beyond the S2
pocket and is responsible for the exopeptidase activity of CatC.
As reference point, the structure of compound 1 was
determined in the active site of full-length human CatC. The
structure of CatC in complex with compound 1 confirmed
covalent interaction of the nitrile group with Cys234 and,
consistent with the high enzyme activity of compound 1,
showed several putative hydrogen bonds to backbone atoms of
Gly277 and Asn380 as well as to the side chain of Asp1 (Figure
1). In addition, the primary amine makes an indirect interaction
to Thr379 via a water molecule in the S2 pocket.
The structure of CatC in complex with compound 3 (Figure
3) revealed that the piperidine ring occupies additional space in
the S2 pocket and displaces a bound water molecule from the
compound 1 structure. This displacement results in loss of the
indirect interaction to Thr379 and might explain the lower
enzyme potency observed for compounds 2 and 3.
Although the potency of 3 itself was not viewed as sufficient
for this particular compound to progress further, the compound
was viewed as providing a good start point for a lead
optimization program with an initial aim of regaining the
potency that had been sacrificed to achieve stability. Toward
this goal, a program was initiated to vary substitution on the
biphenyl further and also on the piperidine ring to seek a
suitable combination of metabolic stability and high potency.
Selected examples from this work are illustrated in Table 1,
where it can be seen that both highly potent and metabolically
stable compounds can be identified in this series.
Within Table 1, compounds 4 and 5 represent selected
examples where potency could be improved by further biphenyl
substitution. More interesting, however, are compounds 6 and
7, where incorporation of a 4(S)-hydroxyl into the piperidine
ring affords a log unit increase in enzyme potency. Although the
corresponding 4(R) isomer was never prepared, comparison of
compound 6 versus 3 and likewise isoindolone 7 versus 5
clearly illustrates the increase in potency in the isolated enzyme
assay resulting from introduction of this piperidine hydroxyl
group. Furthermore, the cocrystal structure of CatC complexed
with compound 6 reveals that the piperidine hydroxyl mimics
Figure 1. X-ray structure of compound 1 (magenta) bound in the
active site of CatC (PDB 4cdc). The exclusion domain, heavy chain,
and light chain of CatC are colored green, pink, and blue, respectively.
In an initial chemical exploration to improve plasma stability
of compound 1, piperidine derivative 2 was prepared (Figure
2). Although less potent than compound 1 in the isolated
enzyme assay, 2 proved to be resistant to plasma hydrolysis (no
evidence of hydrolysis in rat or human plasma after 10 h) and
showed no noticeable drop-off in potency in moving from the
isolated enzyme assay to the cell assay. Unfortunately, although
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Table 1. SAR of Piperidinecarboxamide Acetonitriles
a
b
pIC50 determined for rhCatC cleavage of fluorescent substrate H-Gly-Phe-AMC.12 pIC50 determined for CatC cleavage of fluorescent substrate H-
Gly-Phe-AFC in human THP1 cells.12 Intrinsic clearance, Clint, determined in human microsome and rat hepatocyte preparations.
c
d
Electrophysiology (Ephys) IonWorks IC50 versus hERG (human Ether-a-
̀
go-go-related gene) channels.
Figure 4. X-ray structure of compound 6 (magenta) bound in CatC
showing the interaction of the 4(S)-hydroxyl with Thr379 (PDB 4cdf).
The exclusion domain, heavy chain, and light chain of CatC are
colored green, pink, and blue, respectively.
Figure 3. X-ray structure of compound 3 (magenta) bound in CatC
(PDB 4cdd). The exclusion domain, heavy chain, and light chain of
CatC are colored green, pink, and blue, respectively.
the role of a bound water molecule in the compound 1
structure, making putative interactions to both the backbone
and side chain of Thr379 and thus returning a large portion of
the potency lost in unsubstituted piperidines 2 and 3 (Figure
4).
Initial concerns about the weakly basic nature of compounds
related to 3 and lysosomotropism proved largely unfounded
because the cell potencies of all but a few compounds were
generally consistent with their enzyme potencies. At the same
time, compounds were profiled for activity at the hERG
channel and for phospholipidosis effects in vitro. hERG data is
displayed in Table 1, and it can be observed that although
potentially problematic for compound 4 as well as for
compound 3 itself the LogD-lowering effect of either the
piperidine hydroxyl or the isoindolone substituent effectively
removes this unwanted activity. Likewise, phospholipidosis
effects were observed to be dependent on lipophilicity as
measured by LogD, as might be anticipated for weakly basic
amines. Safety profiling of compound 3 in an in vitro assay
measuring phospholipid accumulation in rat hepatoma cells
incubated with compound 3 (EC50 9 μM) translated into
histological findings in rat in vivo studies. Significant findings
were observed in liver (hypertrophy, cholangitis, vacuolation)
and lung (foci of foaming macrophages), consistent with a
phospholipidosis effect. In compounds 6 and 7, both basicity
and overall LogD was reduced by incorporation of a hydroxyl
group on the piperidine ring, and although these two
compounds themselves were not studied in vivo, the in vitro
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Table 2. Cathepsin Selectivity
compound
CatC Enz pIC50
CatK Enz pIC50
CatL Enz pIC50
CatS Enz pIC50
CatB Enz pIC50
3
4
5
6
7
7.4 0.3
8.4 0.2
7.6 0.1
8.3 0.1
8.4 0.1
6.0 0.3
6.4 0.1
6.6 0.1
5.2 0.1
5.1 0.1
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Table 3. SAR of Aminomethylenecarboxamide Acetonitriles
data for both compounds indicated a lower propensity to cause
phospholipidosis (EC50 75 and > 120 μM, respectively).
An additional observation with hydroxypiperidines 6 and 7
relates to cathepsin selectivity. Given the covalent reversible
nature of the interaction with CatC, we were initially concerned
about cathepsin selectivity. However, from structural informa-
tion, it was clear that a key interaction with CatC is the
hydrogen bond between the amino group of the ligand in the
S2 pocket and Asp1 of the size-exclusion domain. Because that
domain is exclusive to CatC, the scope for selectivity was
anticipated to be good. Indeed, as shown in Table 2, the
cathepsin selectivity was generally high, with the possible
exception of CatK. Introduction of the piperidine hydroxyl
significantly increases the separation between CatC and CatK
activity, which is in good agreement with the fact that in CatK
Thr379 is replaced by a leucine residue.
to reduce both basicity and lipophilicity, this time by
incorporation within the newly introduced ring in the form
of pyrans 10 and 11, gave an improved profile, much the same
effect as observed with the hydroxyl group in piperidine
examples 6 and 7. All of pyran compounds 9−12 displayed low
Cat K activity, with only compound 12 reaching 50% inhibition
at 10 μM (pIC50 5.1 for 12)
The structure of CatC in complex with compound 10
(Figure 5) shows that the tetrahydropyran ring is positioned
orthogonal to the corresponding piperidine of compound 6. As
a result, the tetrahydropyran oxygen is placed in an identical
position as the piperidine hydroxyl in compound 6. At the same
In parallel to this work, a second chemical approach was
undertaken to stabilize initial lead structure 1. Following the
same reasoning that instability of this compound arises because
of enzymatic hydrolysis of the peptidic bond, a series of
compounds was prepared wherein the α position was
incorporated within a ring. Initial compounds were prepared
with a cyclohexyl ring in this position (e.g., compound 8; Table
3), and such compounds were indeed found to be stable in the
cell assay and stable in plasma (both human and rat), but the
potential to reintroduce lipophilicity-driven hERG and
phospholipidosis effects is obvious, and some hERG activity
was indeed evident in compound 8 itself. In a similar fashion to
the first series of compounds, introduction of an oxygen atom
Figure 5. X-ray structure of compound 10 (magenta) bound to CatC
showing how this compound also forms favorable interactions with
Thr379 (PDB 4cde). The exclusion domain, heavy chain, and light
chain of CatC are colored green, pink, and blue, respectively.
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Scheme 1. Synthetic Scheme for the Preparation of Compounds 3 and 5
Scheme 2. Synthetic Route to 4(S)-Hydroxypiperidine Compounds 6 and 7
time, the primary amine is at the same position as the amine of
compound 1, donating hydrogens to interact with Asp1 and the
backbone of Gly277.
lysosomes. Although lysosomal accumulation of these com-
pounds is not generally believed to be significant, it is possible
that some concentration occurs for compound 8 but is absent
with compounds 9−12.
Chemistry. The preparation of compounds 3−12 was
achieved using the synthetic routes illustrated in Schemes 1−3.
Compounds 3 and 5 were prepared from the 4-iodophenyla-
lanine carboxamide derivative 1315 through either palladium-
catalyzed coupling of the aryl iodide with an appropriately
substituted arylboronate or through conversion of the
aryliodide itself to an arylboronate and reaction with the
appropriate aryl halide (Scheme 1). Compound 4 was prepared
by an analogous route to 3.
The synthetic route to 4-hydroxypiperidine compounds 6
and 7 starting from BOC-protected (S)-4-iodophenylalanine
carboxamide 1820 is illustrated in Scheme 2. As in Scheme 1,
the aryliodide is either coupled directly with an arylboronate in
the preparation of 6 or converted itself into an arylboronate
before coupling with the arylhalide; in this case, 5-bromo-2-
methylisoindolin-1-one for the preparation of 7. The
subsequent steps of BOC deprotection; coupling with
Importantly, for compounds 10 and 11, activity at the hERG
channel is also reduced, and no measurable phospholipidosis
effect could be observed in in vitro assays. Furthermore, early in
vivo safety profiling of compound 10 showed no observations
that were ascribed to a phospholipidosis effect. An interesting
observation with pyran compounds 9−12 compared to the
corresponding cyclohexyl compound 8 is the finding that they
display an activity drop in moving from the enzyme to the cell
assay. There are several possible explanations for this
observation, but one explanation is founded on the very low
basicity of the amine group in such pyran compounds. The
measured pKa for compound 10 is 5.8, which is extremely low
for an amine of this type compared to the measured value of 7.1
for cyclohexyl compound 8; this is undoubtedly due, in part, to
the electron-withdrawing effect of the ring oxygen. The effect of
this pKa reduction would be to reduce protonation greatly, even
in the acidic environment of lysosomes, thus avoiding effects
resulting from the concentration of compound within the
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Scheme 3. Synthetic Route to Compounds 8−12
temperature. Eleven liters of supernatant was collected, and CatC was
activated over the weekend by adding 47 mL of papain (2 U/mL) and
cysteamine to a final concentration of 5 mM, pH ∼6.5. The activation
was performed during slow mixing at ∼10 °C. Ammonium sulfate was
added to 1.8 M and EDTA to 5 mM. The pH was then adjusted to 4.5
by adding citric acid (1 M solution) followed by 20 min of stirring.
The precipitated media was centrifuged at 15 900g for ∼1.5 h (JLA
8.1000, 15 °C) followed by filtration. The sample was loaded onto a
HIC column (butyl sepharose FF, 200 mL XK 50) overnight. The
column was previously equilibrated with buffer (1.8 M (NH4)2SO4, 20
mM citric acid, 5 mM EDTA, 5 mM cysteamine, and 0.1 M NaCl, pH
4.5). The unbound sample was washed out from the HIC column until
the UV signal was less than 45 mAU (∼3 cv). A 0−100% buffer (20
mM citric acid, 5 mM EDTA, 5 mM cysteamine, and 0.1 M NaCl, pH
4.5) gradient during five column volumes was applied, and relevant
fractions were pooled according to activity analysis. The buffer was
exchanged on the pooled CatC fractions to 10 mM Tris-HCl, 1 mM
EDTA, and 5 mM cysteamine, pH 7.6. The buffer exchange was
performed on a Sephadex G-25 XK 50 column (column volume 200
mL) in two consecutive runs. The buffer-exchanged HIC eluate was
applied to 10 mL Q Sepharose FF (two 5 mL Hightrap columns). The
columns were previously equilibrated with 10 mM Tris-HCl, 1 mM
EDTA, and 5 mM cysteamine, pH 7.6. The unbound sample was
washed out from the IEX column during a washing step. A 0−50%
buffer (10 mM Tris-HCl, 1 mM EDTA, 5 mM cysteamine, and 1 M
NaCl, pH 7.6) gradient was applied followed by a step elution with
100% buffer before relevant fractions were collected for activity
analysis. The IEX pool was loaded onto a SEC column (Superdex 200
pg, 980 mL XK 50), which was previously equilibrated with 20 mM
Tris-HCl, 200 mM NaCl, and 5 mM cysteamine, pH 7.6. Relevant
fractions were collected for analysis. The SEC pool was concentrated
to 27 mL using a Centricon plus-80 (30 kDa cutoff). The protein
concentration on the concentrated SEC pool was measured by
Bradford assay to 2.5 mg/mL (67.5 mg protein). All purifications steps
were performed at room temperature.
(2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypiperidine-2-carbox-
ylic acid, hydroxyl protection, dehydration, and deprotection,
are then common for the two target compounds.
The synthetic route to compounds 8−12 is illustrated in
Scheme 3 starting from BOC-protected 1-amino cyclo-
hexanecarboxylic acid or BOC-protected 4-aminotetrahydro-
pyran-4-carboxylic acid. For preparation of 8−11, coupling with
an appropriately substituted 4-arylphenylalanine carboxamide
affords intermediates 23−26, and after dehydration, 27−30.
Final deprotection with formic acid liberates the target
compounds. Compound 12 was prepared by the alternate
route of coupling BOC-protected 4-aminotetrahydropyran-4-
carboxylic acid with (S)-4-iodophenylalanine carboxamide to
afford 31 followed by conversion to arylboronate 32.
Palladium-catalyzed coupling with 5-bromo-2-methylisoindo-
lin-1-one then afforded 33, which was dehydrated and
deprotected to 12.
CONCLUSIONS
■
Compound 10 was selected as the most promising candidate as
well as a potential development candidate from the compounds
described for in vivo profiling on the basis of all of its measured
properties. Compound 10 has low hERG activity, low activity
in phospholipidosis assays (EC50 >100 μM), improved
selectivity versus CatK (pIC50 < 5), maintained selectivity
versus CatL, CatS, and CatB (pIC50 < 5), and a clean selectivity
profile in MDS Pharma screening (only a single activity >30%
in >170 assays tested at 10 μM: human LTB4 binding assay,
69% inhibition, IC50 2 μM). Compound 10 also displays low
clearance and high bioavailability in mouse, rat, and dog (Cl 9,
9, and 2 mL/min/kg; Vss 1.6, 1.9, and 1.4 L/kg; t1/2 2, 3.5, and
11.6 h; bioavailability 95, 92, and 72% respectively), satisfying
requirements for progression into more detailed in vivo
profiling studies.
Crystallization and Structure Determination. To aid crystal-
lization, protein buffer was exchanged to 20 mM sodium acetate, pH 5,
0.2 M sodium chloride, 0.5 mM EDTA, and 2 mM DTT with a
NAP10 column (GE Healthcare). The protein sample was
concentrated to 12 mg/mL. DTT and compound were added to a
final concentration of 2 mM, and the sample was incubated for 30 min.
Crystals were grown at 20 °C in sitting drops using 0.3 μL of protein
and 0.3 μL of well solution (21−27% PEG3350, 0.2 M ammonium
sulfate, and 0.1 M sodium acetate, pH 5.0). Crystals appeared after a
few hours but continued to grow for a few days. Crystals were frozen
in liquid nitrogen prior to data collection using 20% glycerol and well
EXPERIMENTAL SECTION
■
Protein Production. Sf21 cells were amplified in shaker bottles to
a cell density of 6.6 × 106/mL and diluted prior to infection to 2 ×
106/mL in 10 shaker bottles (3 L) at 1000 mL working volume. The
culture was infected using baculovirus stock expressing CatC (1−463)
to MOI = 2 and then cultivated at 27 °C. Ninety-six hours
postinfection, the culture was spun at 2500g for 15 min at room
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solution as cryo protectant. X-ray diffraction data were collected at the
European Synchrotron Radiation Facility (ESRF; Grenoble, France).
Data were integrated and processed using MOSFLM and SCALA of
the CCP4 suite.14 The crystals belonged to the space groups P3121 or
P31 with two or four molecules in the asymmetric unit. The structures
were solved by molecular replacement using Protein Data Bank (PDB)
entry 1K3B as the search model.13 The structures were refined using
the CCP4 suite and manual rebuilding using Coot.15 Figures 1, 3, 4,
and 5 were prepared using PyMOL.16 Crystallographic coordinates
and structure factor amplitudes for all complex structures were
deposited into the Protein Data Bank (accession codes 4cdc, 4cdd,
4cde, and 4cdf).
Fluorescence Assay Procedure Using Recombinant Human
(rh) CatC. The activity of CatC was determined by measuring the
enzymatic release of aminomethyl coumarin (AMC) from the peptide
substrate (H-Gly-Arg-AMC), which leads to an increase in
fluorescence intensity at λex = 350 nm and λem = 450 nm. The assay
was carried out in black 384−well plates in a final volume of 50 μL at
22 °C. The assay conditions contained the following: 25 mM
piperazine buffer, pH 5.0, 50 mM NaCI, 5 mM DTT, 0.01% (v/v)
Triton X100, 100 μM H-Gly-Arg-AMC, and rhCatC (∼50 pM).
Potential inhibitors were prepared in DMSO and then diluted in the
assay to give a final concentration not exceeding 1% (v/v) DMSO. A
ten-point half-log dilution series of the inhibitors (highest concen-
tration typically 10 μM) was tested, and the pIC50 was determined
using a four-paramater logistic equation in a non-fifteen linear curve
fitting routine. A standard vinyl sulfone CatC inhibitor12 was used as a
positive control in the assay. Routinely, inhibitors were preincubated
with rhCatC for 30 min prior to the addition of the peptide substrate
to start the reaction for a further 60 min at 22 °C. Afterward, the plates
were immediately read in a fluorescence plate reader using the above
emission and excitation wavelengths (modified from refs 17 and 18).
nitrogen in a cold water bath, and methyl chlorosulfonylcarbamate
(5.70 g, 32.86 mmol) in benzene (60 mL) was added dropwise. Once
addition was complete, the cold water bath was removed, and the
mixture was stirred at room temperature for 30 min. A solution of (S)-
tert-butyl 2-((S)-1-amino-3-(4-iodophenyl)-1-oxopropan-2-
ylcarbamoyl)piperidine-1-carboxylate (13)19 (6.59 g, 13.14 mmol) in
benzene (50 mL) was added dropwise, and the mixture was stirred at
room temperature for 2 h. Water was added to the reaction mixture,
and the organic phase was isolated, washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was purified by
flash silica chromatography eluting with 20% ethyl acetate in
isohexane. Pure fractions were evaporated to dryness to give
compound 14 (5.86 g, 92%) as a white solid.
(S)-tert-Butyl-2-((S)-1-cyano-2-(4′-cyanobiphenyl-4-yl)-
ethylcarbamoyl)piperidine-1-carboxylate (15). (S)-tert-Butyl 2-
((S)-1-cyano-2-(4-iodophenyl)ethylcarbamoyl)piperidine-1-carboxy-
late (14) (8.5 g, 17.59 mmol) and bis[1,2-bis(diphenylphosphino)-
ethane]palladium (0.159 g, 0.18 mmol) were stirred in dioxane, and to
the mixture was added 4-cyanophenylboronic acid (3.88 g, 26.38
mmol). The reaction mixture was stirred under nitrogen for 15 min,
and then 2 M aqueous potassium carbonate solution (17.59 mL, 35.17
mmol) was added. The reaction mixture was heated at 75 °C
overnight. LCMS showed that the reaction was complete. After
cooling to room temperature, the reaction mixture was split and
poured onto Chem Elut cartridges (Agilent), and the crude product
was washed through with CH2Cl2. The combined solvent was
removed in vacuo, and the crude material obtained was purified by
flash silica chromatography eluting with 25% ethyl acetate in isohexane
1
to give compound 15 (6.90 g, 86%) as a colorless solid. H NMR
(399.8 MHz, DMSO-d6) δ 8.64 (d, J = 7.7 Hz, 1H), 7.92 (dd, J = 6.5
and 1.9 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H),
7.44 (d, J = 8.2 Hz, 2H), 5.14−5.06 (m, 1H), 4.62−4.35 (m, 1H),
3.78−3.70 (m, 1H), 3.26−3.15 (m, 2H), 2.85 (td, J = 13.0 and 3.0 Hz,
1H), 2.00−1.87 (m, 1H), 1.55−1.16 (m, 13H), 1.06−0.94 (m, 1H).
(S)-N-((S)-1-Cyano-2-(4′-cyanobiphenyl-4-yl)ethyl)-
piperidine-2-carboxamide (3). (S)-tert-Butyl-2-((S)-1-cyano-2-(4′-
cyanobiphenyl-4-yl)ethylcarbamoyl)piperidine-1-carboxylate (15) (2.8
g, 6.11 mmol) was dissolved in formic acid (25 mL, 651.81 mmol),
and the solution was heated at 50 °C for 10 min. Solvent was removed
in vacuo, and the crude product was purified by preparative HPLC
using a 95−5% gradient of aqueous 0.1% trifluoroacetic acid in
acetonitrile as eluent. The fractions containing the desired compound
were concentrated in vacuo to afford compound 3 as the
SYNTHESIS
■
Reagents were obtained from commercial suppliers and used without
purification. Unless otherwise stated, reactions were carried out at
ambient temperature (18−25 °C) and under positive nitrogen
pressure with magnetic stirring. Flash chromatography was performed
on E. Merck 230−400 mesh silica gel 60 or by using Biotage KP-Sil
SNAP cartridges. Solvent mixtures used as eluants are volume/volume
ratios. Preparative HPLC purifications were performed on Waters
Symmetry, Novapak, or XTerra columns eluting with a gradient of
acetonitrile or methanol in aqueous ammonium acetate, ammonia, or
1
1
trifluoroacetic acid solution. Routine H and 13C NMR spectra were
trifluoroacetate salt (2.50 g, 87%). H NMR (399.8 MHz, DMSO-
d6) δ 8.51 (d, J = 7.6 Hz, 1H), 7.89 (m, 4H), 7.72 (d, J = 8.0 Hz, 2H),
7.44 (d, J = 8.0 Hz, 2H), 5.03 (m, 1H), 3.20 (m, 2H), 3.06 (m, 1H),
2.78 (m, 1H), 2.45 (m, 1H), 1.57 (m, 2H), 1.42−1.19 (m, 4H). 13C
NMR (125.8 MHz, DMSO-d6) δ 173.28, 144.15, 136.85, 136.40,
132.76, 130.14, 127.31, 126.91, 119.14, 118.77, 109.86, 58.91, 44.68,
36.63, 29.00, 25.53, 23.44. HRMS (ESI) m/z calcd for C22H22N4O (M
+ H)+, 359.1872; found, 359.1886.
(S)-N-((S)-1-Cyano-2-(4′-cyano-3′-(methylthio)biphenyl-4-
yl)ethyl)piperidine-2-carboxamide (4).19 1H NMR (399.8 MHz,
DMSO-d6) δ 9.29 (d, J = 7.2 Hz, 1H), 9.01−8.69 (m, 1H), 7.81 (d, J =
7.9 Hz, 1H), 7.78 (t, J = 7.8 Hz), 7.64−7.56 (m, 2H), 7.47 (d, J = 8.2
Hz, 2H), 5.07 (q, J = 7.4 Hz, 1H), 3.82−3.71 (m, 2H), 3.28−3.17 (m,
3H), 2.98−2.86 (m, 1H), 2.70 (s, 3H), 2.10-.00 (m, 1H), 1.82−1.74
(m, 1H), 1.73−1.65 (m, 1H), 1.64−1.42 (m, 2H). 13C NMR (125.8
MHz, DMSO-d6) δ 168.85, 144.66, 143.92, 137.15, 136.16, 134.06,
130.19, 127.34, 123.43, 123.30, 118.50, 116.88, 108.04, 56.48, 43.20,
41.80, 36.31, 26.67, 21.38, 14.34. HRMS (ESI) m/z calcd for
C23H24N4OS (M + H)+, 405.1749; found, 405.1761.
(S)-tert-Butyl-2-((S)-1-amino-1-oxo-3-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ylcarbamoyl)-
piperidine-1-carboxylate (16). A solution of (S)-tert-butyl-2-((S)-
1-amino-3-(4-iodophenyl)-1-oxopropan-2-ylcarbamoyl)piperidine-1-
carboxylate (13)19 (21.7 g, 43.28 mmol) in acetonitrile (250 mL) was
treated with potassium acetate (12.74 g, 129.85 mmol) and
4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (14.62 g,
57.57 mmol). Water (15 mL) was added followed by Pd-118 (30
recorded on a Varian UnityInova spectrometer at a proton frequency
of 300 or 400 MHz or a Bruker Avance III spectrometer at a proton
frequency of 500 MHz. Chemical shifts are expressed in ppm. Low-
resolution mass spectra were measured on an Agilent 1100 MSD
G1946D spectrometer using electrospray (ESI) ionization, atmos-
pheric pressure chemical ionization, or Agilent multimode ionization;
only ions that indicate the parent mass or a common fragment are
reported. High-resolution mass spectra were recorded on a Waters
LCTP time-of-flight instrument using ESI ionization. The purity of all
test compounds was determined by RPHPLC conducted on an Agilent
1100 LCMS system using UV detection and a gradient of 5−95%
acetonitrile in either aqueous ammonium acetate (0.1% w/v) or
trifluoroacetic acid (0.1% v/v) over 2.5 min on 2.1 mm × 5 mm
columns packed with Waters Symmetry C8 or Waters Symmetry C18
or on 2.0 mm × 50 mm columns packed with Phenomenex Max-RP.
All test compounds showed ≥95% purity.
(S)-N-((S)-1-Cyano-2-(biphenyl-4-yl)ethyl)piperidine-2-car-
boxamide (2).19 1H NMR (399.8 MHz, DMSO-d6) δ 9.23 (d, J =
10.3 Hz, 1H), 7.65−7.61 (m, 4H), 7.43 (m, 4H), 7.36 (m, 1H), 5.01
(q, J = 7.5 Hz, 1H), 4.29 (s, 1H), 3.80 (d, J = 13.6 Hz, 1H), 3.28−3.19
(m, 3H), 2.96−2.89 (m, 1H), 2.49 (t, J = 2.6 Hz, 2H), 2.09 (m, 1H),
1.80−1.45 (m, 3H). HRMS (ESI) m/z calcd for C21H23N3O (M +
H)+, 334.1919; found, 334.1945.
(S)-tert-Butyl-2-((S)-1-cyano-2-(4-iodophenyl)-
ethylcarbamoyl)piperidine-1-carboxylate (14). Triethylamine
(11.73 mL, 84.12 mmol) in benzene (20 mL) was stirred under
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mg, 0.05 mmol), and the mixture was stirred under nitrogen at 95 °C
for 17 h. The cooled reaction mixture was concentrated to low volume,
and the residue was partitioned between water (500 mL) and ether (2
× 500 mL). The ethereal phase was collected and dried over
magnesium sulfate and filtered. The filtrate was passed through a pad
of flash silica gel product eluting with diethyl ether then with ethyl
acetate. The ethyl acetate solution was concentrated to dryness to
ylcarbamate (18) (3.17 g, 8.12 mmol) and 4-cyanophenylboronic
acid (1.194 g, 8.12 mmol) in dioxane (5 mL) was treated with Pd-118
(0.080 g, 0.12 mmol), and the mixture was stirred at room
temperature for 15 min under nitrogen. An aqueous solution of
potassium carbonate (8.12 mL, 16.25 mmol) was added, and the
mixture was stirred for 18 h at 75 °C. LCMS demonstrated complete
reaction, and the product was extracted into ethyl acetate, dried, and
evaporated to afford (S)-tert-butyl-1-amino-3-(4′-cyanobiphenyl-4-yl)-
1-oxopropan-2-ylcarbamate (3.13 g), which was used without further
purification in the next step. 1H NMR (399.8 MHz, CDCl3) δ 7.72 (d,
J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H),
7.35 (d, J = 7.9 Hz, 2H), 5.90−5.83 (m, 1H), 5.42−5.34 (m, 1H),
5.10−5.01 (m, 1H), 4.48−4.37 (m, 1H), 3.21−3.07 (m, 2H), 1.42 (s,
9H).
1
afford compound 16 (20.5 g, 95%) as a colorless solid. H NMR
(399.8 MHz, CDCl3) δ 7.75 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 7.9 Hz,
2H), 6.48−6.39 (m, 1H), 5.30−5.23 (m, 1H), 4.72 (q, J = 7.6 Hz,
1H), 4.76−4.62 (m, 1H), 3.22−3.14 (m, 1H), 3.13−3.06 (m, 1H),
2.48−2.36 (m, 1H), 2.25−2.18 (m, 1H), 1.53−1.45 (m, 4H), 1.42 (s,
9H), 1.33 (s, 12H). MS [M-BOC + H]+ = 402.
(S)-tert-Butyl-2-((S)-1-amino-3-(4-(2-methyl-1-oxoisoindolin-
5-yl)phenyl)-1-oxopropan-2-ylcarbamoyl)piperidine-1-carbox-
ylate (17). A solution of (S)-tert-butyl-2-((S)-1-amino-1-oxo-3-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-
ylcarbamoyl)piperidine-1-carboxylate (16) (300 mg, 0.60 mmol),
potassium acetate (176 mg, 1.79 mmol), and 5-bromo-2-methyl-
isoindolin-1-one (271 mg, 1.20 mmol) in acetonitrile (30 mL) under a
nitrogen atmosphere was treated with Pd-118 (11.70 mg, 0.02 mmol),
and the mixture was heated at 80 °C for 3 h. The solvent was removed
in vacuo, and the residue was purified on silica gel eluting with ethyl
acetate followed by methanol to afford compound 17 (300 mg, 96%)
To a solution of (S)-tert-butyl-1-amino-3-(4′-cyanobiphenyl-4-yl)-1-
oxopropan-2-ylcarbamate (3.13 g, 8.57 mmol) in CH2Cl2 (30 mL) was
added TFA (1.32 mL, 17.13 mmol). The CH2Cl2 was distilled off on a
rotary evaporator at atmospheric pressure to leave a few milliliters of
solvent. The reaction was monitored by HPLC/MS, and when it was
complete, it was partitioned between water and CH2Cl2, dried, and
evaporated. The solid was purified on silica eluting with ethyl acetate
and then ethyl acetate containing 10% methanol to afford compound
1
19 (1.95 g, 86%). H NMR (399.8 MHz, CDCl3) δ 7.73 (dd, J = 6.7
and 2.1 Hz, 2H), 7.67 (dd, J = 6.4 and 2.1 Hz, 2H), 7.56 (dt, J = 8.0
and 1.7 Hz, 2H), 7.36 (dt, J = 8.4 and 2.1 Hz, 2H), 7.11 (s, 1H), 5.38
(s, 1H), 3.68 (dd, J = 9.1 and 4.2 Hz, 1H), 3.32 (dd, J = 13.8 and 4.1
Hz, 1H), 2.84 (dd, J = 13.8 and 9.2 Hz, 1H), 1.49 (s, 2H). MS (M +
H)+ = 266.
1
as a gum. H NMR (399.8 MHz, CDCl3) δ 7.88 (d, J = 7.7 Hz, 1H),
7.62 (dd, J = 7.9 and 1.5 Hz, 1H), 7.59 (s, 1H), 7.56 (dd, J = 6.5 and
1.7 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 6.54 (d, J = 7.7 Hz, 1H), 6.20−
6.05 (m, 1H), 5.51 (s, 1H), 4.80 (q, J = 7.8 Hz, 1H), 4.69−4.65 (m,
1H), 4.43 (s, 2H), 3.92−3.74 (m, 1H), 3.27−3.10 (m, 6H), 2.49−2.32
(m, 1H), 2.24−2.17 (m, 1H), 1.61−1.25 (m, 15H includes H2O peak).
MS [M − H]− = 520.
(2S,4S)-tert-Butyl-2-((S)-1-amino-3-(4′-cyanobiphenyl-4-yl)-
1-oxopropan-2-ylcarbamoyl)-4-(tert-butyldimethylsilyloxy) pi-
peridine-1-carboxylate (20). To a solution of (2S,4S)-1-(tert-
butoxycarbonyl)-4-hydroxypiperidine-2-carboxylic acid (19) (157 mg,
0.64 mmol) and (S)-2-amino-3-(4′-cyanobiphenyl-4-yl)propanamide
(S)-N-((S)-1-Cyano-2-(4-(2-methyl-1-oxoisoindolin-5-yl)-
phenyl)ethyl)piperidine-2-carboxamide (5). A solution of (S)-
tert-butyl-2-((S)-1-amino-3-(4-(2-methyl-1-oxoisoindolin-5-yl)-
phenyl)-1-oxopropan-2-ylcarbamoyl)piperidine-1-carboxylate (17)
(300 mg, 0.58 mmol) and methyl N-(triethylammoniumsulfonyl)-
carbamate (412 mg, 1.73 mmol) in CH2Cl2 (15 mL) was stirred at
room temperature for 20 h. The reaction mixture was placed on a flash
silica column, and the mixture eluted with 70% ether/isohexane to
afford a gum (200 mg) (MS [M + H-tBu]+ = 448, MS [M − H]− =
502), which was dissolved in formic acid (1 mL, 26.07 mmol) and
then stirred at room temperature for 4 h. The mixture was then
concentrated to dryness, and the residue was purified on reversed-
phase HPLC (aq 0.1% TFA/MeCN). The fractions were freeze-dried
to afford compound 5 (65.0 mg, 31.6%) as a colorless solid. 1H NMR
(399.8 MHz, DMSO-d6) δ 9.31 (d, J = 7.4 Hz, 1H), 9.01−8.92 (m,
1H), 8.81−8.68 (m, 1H), 7.87 (s, 1H), 7.79−7.69 (m, 4H), 7.45 (d, J
= 8.2 Hz, 2H), 5.07 (q, J = 7.5 Hz, 1H), 4.52 (s, 2H), 3.82−3.73 (m,
1H), 3.27−3.18 (m, 3H), 3.10 (s, 3H), 2.99−2.86 (m, 1H), 2.10−2.02
(m, 1H), 1.82−1.42 (m, 5H). 13C NMR (125.8 MHz, DMSO-d6) δ
168.85, 166.99, 142.60, 142.53, 138.48, 135.14, 131.56, 130.14, 127.09,
126.39, 123.04, 121.39, 56.48, 51.43, 43.20, 41.81, 36.30, 28.99, 26.67,
21.39, 21.14. HRMS (ESI) m/z calcd for C24H26N4O2 (M + H)+,
403.2134; found, 403.2142.
(S)-tert-Butyl-1-amino-3-(4-iodophenyl)-1-oxopropan-2-yl-
carbamate (18). To a solution of (S)-2-(tert-butoxycarbonylamino)-
3-(4-iodophenyl)propanoic acid20 (2.05 g, 5.24 mmol) in DMF (15
mL) was added N-ethylmorpholine (0.995 mL, 7.86 mmol) followed
by TBTU (1.683 g, 5.24 mmol). The mixture was stirred at room
temperature for 30 min and then cooled to 0 °C. Aqueous ammonia
(35%, 0.580 mL, 10.48 mmol) was added, and the mixture was allowed
to reach room temperature and stirred overnight. The reaction mixture
was poured into water (150 mL), and the resulting suspension was
filtered. The solid was dried in vacuo to afford compound 18 (1.910 g,
93%) as an off-white solid, which was used without further purification.
1H NMR (399.8 MHz, CDCl3) δ 7.63 (d, J = 8.5 Hz, 2H), 6.98 (d, J =
(170 mg, 0.64 mmol) in DMF (5 mL) was added Hunig’s base (0.279
̈
mL, 1.60 mmol) followed by TBTU (308 mg, 0.96 mmol). The
resulting solution was stirred at room temperature for 2 h and then
poured into water (50 mL) and extracted with CH2Cl2 (2 × 50 mL).
The organics were dried (magnesium sulfate) and evaporated in vacuo
to afford crude (2S,4S)-tert-butyl-2-((S)-1-amino-3-(4′-cyanobiphenyl-
4-yl)-1-oxopropan-2-ylcarbamoyl)-4-hydroxypiperidine-1-carboxylate
(310 mg, 0.63 mmol, 98%) as a brown gum. This gum was dissolved in
CH2Cl2 (10 mL), and imidazole (107 mg, 1.57 mmol) and tert-
butyldimethylsilyl chloride (104 mg, 0.69 mmol) were added under
nitrogen. The resulting solution was stirred at room temperature for
24 h. The reaction was diluted with further CH2Cl2 (50 mL) and
washed with water (2 × 50 mL). The organics were dried (magnesium
sulfate) and evaporated in vacuo, and the residue was purified by flash
silica chromatography eluting with 60% ethyl acetate in isohexane.
Pure fractions were evaporated to dryness to afford compound 20
(294 mg, 77%) as a white foam. 1H NMR (399.8 MHz, CDCl3) δ 7.73
(d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H),
7.33 (d, J = 8.4 Hz, 2H), 6.58 (d, J = 8.6 Hz, 1H), 6.11−5.92 (m, 1H),
5.44 (s, 1H), 4.80−4.71 (m, 2H), 3.91−3.66 (m, 2H), 3.24 (dd, J =
14.0 and 6.8 Hz, 1H), 3.09 (dd, J = 14.0 and 6.8 Hz, 1H), 2.32 (d, J =
13.5 Hz, 2H), 1.66 (s, 2H), 1.58 (d, J = 12.9 Hz, 1H), 1.43 (s, 9H),
0.85 (s, 9H), 0.03 (d, J = 6.4 Hz, 6H). MS [M − H]− = 605.
(2S,4S)-N-((S)-1-Cyano-2-(4′-cyanobiphenyl-4-yl)ethyl)-4-hy-
droxypiperidine-2-carboxamide (6). A solution of (2S,4S)-tert-
butyl 2-((S)-1-amino-3-(4′-cyanobiphenyl-4-yl)-1-oxopropan-2-ylcar-
bamoyl)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate (20)
(290 mg, 0.48 mmol) in CH2Cl2 (10 mL) was treated with methyl
N-(triethylammoniumsulfonyl)carbamate (148 mg, 0.62 mmol) and
stirred at room temperature overnight. The reaction was evaporated in
vacuo, and the residue was purified by flash silica chromatography
eluting with 20% ethyl acetate in isohexane. Pure fractions were
evaporated to dryness to afford (2S,4S)-tert-butyl-4-(tert-butyldime-
thylsilyloxy)-2-((S)-1-cyano-2-(4′-cyanobiphenyl-4-yl)-
ethylcarbamoyl)piperidine-1-carboxylate (272 mg, 97%) as a colorless
gum. 1H NMR (399.8 MHz, CDCl3) δ 7.74 (d, J = 8.1 Hz, 2H), 7.66
(d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H),
8.5 Hz, 2H), 5.89 (s, 1H), 5.45 (s, 1H), 5.02 (s, 1H), 4.34 (s, 1H),
3.05−2.98 (m, 2H), 1.41 (s, 9H).
(S)-2-Amino-3-(4′-cyanobiphenyl-4-yl)propanamide (19). A
solution of (S)-tert-butyl-1-amino-3-(4-iodophenyl)-1-oxopropan-2-
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5.17 (s, 1H), 4.78 (s, 1H), 4.02−3.83 (m, 1H), 3.19−3.08 (m, 2H),
2.56−2.24 (m, 2H), 1.66 (d, J = 14.3 Hz, 1H), 1.51−1.30 (m, 13H),
0.87 (s, 9H), 0.06 (d, J = 5.9 Hz, 6H).
boxylic acid (190 mg, 0.77 mmol) and (S)-2-amino-3-(4-(2-methyl-1-
oxoisoindolin-5-yl)phenyl)propanamide (22) (361 mg, 0.85 mmol) in
DMF (5 mL) was added Hunig’s base (0.474 mL, 2.71 mmol)
̈
A solution of (2S,4S)-tert-butyl-4-(tert-butyldimethylsilyloxy)-2-
((S)-1-cyano-2-(4′-cyanobiphenyl-4-yl)ethylcarbamoyl)piperidine-1-
carboxylate (250 mg, 0.42 mmol) in formic acid (5 mL) was stirred at
50 °C for 30 min. The reaction was poured onto ice (50 mL), basified
to pH 9 with concentrated aqueous ammonia, and extracted with
CH2Cl2 (2 × 50 mL). The organics were dried (sodium sulfate) and
evaporated in vacuo. The residue was dissolved in dry tetrahydrofuran
(5 mL) and treated with TBAF (1 M in THF) (0.425 mL, 0.42
mmol). The reaction was stirred at room temperature overnight,
diluted with water (100 mL), and extracted with CH2Cl2 (3 × 100
mL). The organics were dried (sodium sulfate) and evaporated in
vacuo, and the residue was purified by preparative HPLC using a 95−
5% gradient of aqueous 0.1% trifluoroacetic acid in methanol as eluent.
Fractions containing the desired compound were combined, and the
methanol was removed in vacuo. The aqueous residue was basified
with saturated sodium bicarbonate solution and extracted with ethyl
acetate (2 × 50 mL). The organics were dried (sodium sulfate) and
evaporated in vacuo to afford compound 6 (88 mg, 55%) as a white
solid. 1H NMR (399.8 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 2H), 7.67
(d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.59 (d, J = 8.1 Hz, 2H), 7.40 (d, J =
8.1 Hz, 2H), 5.18 (q, J = 8.0 Hz, 1H), 3.87−3.80 (m, 1H), 3.70 (dd, J
= 7.2 Hz, J = 4.4 Hz, 1H), 3.29−3.22 (m, 1H), 3.18 (d, J = 7.2 Hz,
2H), 3.05−2.97 (m, 1H), 2.58−2.49 (m, 1H), 2.08−1.99 (m, 1H),
1.73−1.54 (m, 2H), 1.48−1.39 (m, 2H). HRMS (ESI) m/z calcd for
C22H22N4O2 (M + H)+, 375.1821; found, 375.1812.
(S)-tert-Butyl-1-amino-1-oxo-3-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)propan-2-ylcarbamate (21). A mix-
ture of (S)-tert-butyl-1-amino-3-(4-iodophenyl)-1-oxopropan-2-ylcar-
bamate (18) (1 g, 2.56 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-
bi(1,3,2-dioxaborolane) (0.781 g, 3.08 mmol), potassium acetate
(0.503 g, 5.13 mmol), and Pd-118 (0.05 g, 0.08 mmol) in aqueous
actonitrile (30 mL, 1:1) under nitrogen was heated at reflux for 11 h.
The reaction mixture was diluted with water (100 mL), and the
products were extracted into ethyl acetate (100 mL). The extracts
were dried over magnesium sulfate and concentrated to an oil. The
crude product was purified on silica gel eluting with ethyl acetate to
give compound 21 (850 mg, 85%) as a gum, which slowly solidified.
1H NMR (399.8 MHz, CDCl3) δ 7.72 (d, J = 8.0 Hz, 2H), 7.22 (d, J =
followed by TBTU (298 mg, 0.93 mmol). The resulting solution was
stirred at room temperature for 72 h, poured into water (50 mL), and
extracted with CH2Cl2 (2 × 50 mL). The organics were dried
(magnesium sulfate), evaporated in vacuo, and purified on silica gel
using 85% ethyl acetate/methanol as eluant to afford (2S,4S)-tert-
butyl-2-((S)-1-amino-3-(4-(2-methyl-1-oxoisoindolin-5-yl)phenyl)-1-
oxopropan-2-ylcarbamoyl)-4-hydroxypiperidine-1-carboxylate (530
mg) as a gum, which was progressed directly into the next reaction
without further characterization. A solution of (2S,4S)-tert-butyl-2-
((S)-1-amino-3-(4-(2-methyl-1-oxoisoindolin-5-yl)phenyl)-1-oxopro-
pan-2-ylcarbamoyl)-4-hydroxypiperidine-1-carboxylate (530 mg, 0.99
mmol) in CH2Cl2 (25 mL) was treated with imidazole (67.2 mg, 0.99
mmol) and TBDMS-Cl (149 mg, 0.99 mmol), and the mixture was
stirred overnight. LC/MS showed complete conversion to silylated
product, and the mixture was concentrated to dryness. The residue
was purified on silica gel by initial elution with ether and then ethyl
acetate to afford a low-melting solid; 500 mg was progressed directly
into the next reaction. MS (M + H-BOC)+ = 511. A solution of
(2S,4S)-tert-butyl-2-((S)-1-amino-3-(4-(2-methyl-1-oxoisoindolin-5-
yl)phenyl)-1-oxopropan-2-ylcarbamoyl)-4-(tert-butyldimethylsilyloxy)-
piperidine-1-carboxylate (500 mg, 0.77 mmol) in CH2Cl2 (20 mL) was
stirred with methyl N-(triethylammoniumsulfonyl)carbamate (366 mg,
1.54 mmol) overnight. The solvent was removed in vacuo, and the
residue was treated with formic acid (10 mL, 260.73 mmol). After 4 h
at room temperature, the reaction mixture was concentrated to
dryness. The residue was basified with a saturated aqueous sodium
bicarbonate solution, and the precipitated product was extracted into
ethyl acetate (2 × 100 mL). The combined extracts were dried over
magnesium sulfate and concentrated to dryness to afford a gum, which
was purified by HPLC (0.1% aq ammonia/MeOH, 9505, X-Bridge) to
give compound 7 (60 mg, 19% yield over four chemical steps) as a
1
colorless solid. H NMR (500.3 MHz, CDCl3) δ 7.89 (d, J = 7.9 Hz,
1H), 7.69−7.55 (m, 4H), 7.42−7.34 (m, 2H), 5.18 (t, J = 7.0 Hz, 1H),
4.43 (s, 2H), 3.88−3.81 (m, 1H), 3.71−3.65 (m, 1H), 3.47 (s, 1H),
3.25−3.13 (m, 5H), 3.03−2.95 (m, 1H), 2.57−2.49 (m, 1H), 2.02−
1.95 (m, 1H), 1.72−1.61 (m, 2H), 1.48−1.40 (m, 1H). HRMS (ESI)
m/z calcd for C24H26N4O3 (M + H)+, 419.2083; found, 419.2076.
(S)-tert-Butyl-1-(1-amino-3-(4′-cyanobiphenyl-4yl)-1-oxo-
propan-2-ylcarbamoyl)cyclohexylcarbamate (23). To a solution
of (S)-2-amino-3-(4′-cyanobiphenyl-4-yl)propanamide20 (300 mg,
1.13 mmol) and 1-(tert-butoxycarbonylamino)cyclohexanecarboxylic
acid (275 mg, 1.13 mmol) in DMF (5 mL) was added triethylamine
(0.394 mL, 2.83 mmol) followed by TBTU (545 mg, 1.70 mmol). The
mixture was stirred at room temperature for 2 h. The mixture was
poured into water and diethyl ether (20 mL), and the mixture was
separated. The aqueous layer was further extracted with diethyl ether,
and the combined organic extracts were dried over magnesium sulfate.
The residue after evaporation was purified by chromatography on silica
eluting with methanol/CH2Cl2/Et3N 10:90:1 to give compound 23
(561 mg, 100%). MS [M + H]+ = 491, [M + H-BOC]+ 391.6.
(S)-tert-Butyl-1-(1-cyano-2-(4′-cyanobiphenyl-4-yl)-
ethylcarbamoyl)cyclohexylcarbamate (27). To a solution of (S)-
tert-butyl-1-(1-amino-3-(4′-cyanobiphenyl-4-yl)-1-oxopropan-2-
ylcarbamoyl)cyclohexylcarbamate (23) (550 mg, 1.12 mmol) in
CH2Cl2 (3 mL) was added methyl N-(triethylammoniumsulfonyl)-
carbamate (321 mg, 1.35 mmol), and the mixture was stirred
overnight. The mixture was poured into water (10 mL) and extracted
with CH2Cl2 (2 × 5 mL). The combined organic layers were dried
(Mg2SO4) and evaporated to dryness. Recrystallization from ethyl
acetate/isohexane afforded compound 27 (185 mg, 34.9%) as a white
8.0 Hz, 2H), 5.66 (m, 1H), 5.30 (m, 1H), 5.04 (m, 1H), 4.33 (m, 1H),
5.18 (q, J = 8.0 Hz, 1H), 3.14−3.09 (m, 1H), 3.09−2.98 (m, 1H), 1.59
(s, 6H), 1.31 (s, 9H), 1.21 (s, 6H). MS (M + H-BOC)+ = 291.
(S)-2-Amino-3-(4-(2-methyl-1-oxoisoindolin-5-yl)phenyl)-
propanamide (22). A mixture of (S)-tert-butyl-1-amino-1-oxo-3-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ylcarba-
mate (21) (850 mg, 2.18 mmol), potassium acetate (427 mg, 4.36
mmol), 5-bromo-2-methylisoindolin-1-one (650 mg, 2.88 mmol), and
Pd-118 (42.6 mg, 0.07 mmol) in a mixture of water (15 mL) and
acetonitrile (25 mL) under a nitrogen atmosphere was stirred and
heated under reflux for 6 h. The reaction mixture was concentrated to
dryness, and the residue was treated with TFA (10 mL, 129.80 mmol)
and heated at 60 °C for 15 min. Excess TFA was removed in vacuo,
and the residue was quenched with a saturated aqueous sodium
bicarbonate solution. The mixture was extracted with ethyl acetate (2
× 100 mL), which only extracted out unreacted starting materials. The
aqueous phase was repeatedly extracted with n-butanol until no
additional product could be extracted from the aqueous phase (∼200
mL). The butanol extract was concentrated to dryness, and the residue
was triturated with methanol (20 mL). The suspension was
concentrated to a viscous oil, which was purified by reversed-phase
HPLC (70−10% 0.1% aqueous TFA/methanol) to afford compound
1
1
22 (TFA salt, 500 mg, 54.2%) as colorless solid. H NMR (399.8
solid, which was collected by filtration and dried in vacuo. H NMR
MHz, DMSO-d6) δ 8.19−8.08 (m, 3H), 7.91−7.86 (m, 2H), 7.79−
7.71 (m, 4H), 7.61 (s, 1H), 7.40 (d, J = 8.2 Hz, 2H), 4.52 (s, 2H), 4.00
(s, 1H), 3.19−2.98 (m, 5H). MS (M + H)+ = 310.
(2S,4S)-N-((S)-1-Cyano-2-(4-(2-methyl-1-oxoisoindolin-5-yl)-
phenyl)ethyl)-4-hydroxypiperidine-2-carboxamide (7). To a
solution of (2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypiperidine-2-car-
(399.8 MHz, DMSO-d6) δ 8.04 (d, J = 8.2 Hz, 1H), 7.90−7.81 (m,
4H), 7.68 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 6.18 (s, 1H),
5.01 (q, J = 7.9 Hz, 1H), 3.24−3.09 (m, 2H), 1.86−1.40 (m, 8H), 1.37
(s, 9H), 1.31−1.11 (m, 2H).
(S)-1-Amino-N-(1-cyano-2-(4′-cyanobiphenyl-4-yl)ethyl)-
cyclohexanecarboxamide (8). To (S)-tert-butyl-1-(1-cyano-2-(4′-
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cyanobiphenyl-4-yl)ethylcarbamoyl)cyclohexylcarbamate (27) (160
mg, 0.34 mmol) was added formic acid (1.5 mL, 39.11 mmol), and
the mixture was stirred for 2 h. The mixture was poured into water and
neutralized with 880 ammonia. The aqueous phase was extracted with
diethyl ether (3 × 10 mL), and the combined organics were dried over
magnesium sulfate. The crude oil was diluted with methanol (2 mL)
and purified by preparative chromatography eluting with 25−85%
acetonitrile containing 0.1% TFA to give compound 8 (60.0 mg,
36.4%) as a white solid. 1H NMR (399.8 MHz, DMSO-d6) δ 9.14 (d, J
= 7.9 Hz, 1H), 8.17 (s, 2H), 7.93 (d, J = 9.4 Hz, 2H), 7.87 (d, J = 8.3
Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 5.16−5.08
(m, 1H), 3.32−3.18 (m, 2H), 1.99−1.87 (m, 1H), 1.85−1.75 (m, 1H),
1.75−1.65 (m, 1H), 1.62−1.40 (m, 6H), 1.34−1.19 (m, 1H), 3.76 (s,
1H). 13C NMR (125.8 MHz, DMSO-d6) δ 171.33, 144.12, 137.06,
136.11, 132.85, 130.29, 127.38, 126.99, 118.82, 118.80, 110.00, 59.52,
41.78, 36.50, 30.69, 30.29, 23.59, 19.67. HRMS (ESI) m/z calcd. for
C23H24N4O (M + H)+, 373.2028; found, 373.2016.
by reverse-phase HPLC eluting with 25−85% acetonitrile containing
0.1% TFA. Fractions containing product were concentrated in vacuo
to remove acetonitrile, neutralized with a saturated sodium
bicarbonate solution, and extracted with CH2Cl2, which was then
dried and concentrated in vacuo to give compound 10 (110 mg) as a
colorless solid. Anal. Calcd for C22H22N4O2: C, 70.57; H, 5.92; N,
1
14.96. Found: C, 70.32; H, 5.99; N, 14.87. H NMR (500.3 MHz,
DMSO-d6) δ 7.92 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.71
(d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 5.02 (t, J = 7.8 Hz, 1H),
3.65−3.54 (m, 3H), 3.45 (dt, J = 11.9 and 4.3 Hz, 1H), 3.32 (s, 2H),
3.26−3.16 (m, 2H), 1.89 (ddd, J = 13.4, 10.5, and 4.8 Hz, 1H), 1.73
(ddd, J = 13.4, 10.8, and 4.5 Hz, 1H), 1.21 (dq, J = 13.4 and 2.7 Hz,
1H), 1.12 (dq, J = 13.4 and 2.4 Hz, 1H). 13C NMR (125.8 MHz,
DMSO-d6) δ 176.91, 144.14, 136.84, 136.38, 132.76, 130.20, 127.31,
126.89, 119.23, 118.77, 109.86, 62.38, 62.22, 54.29, 41.09, 36.71,
34.56, 34.45. EE determination by chiral HPLC purity 98.2% at 275
nm. HRMS (ESI) m/z calcd for C22H22N4O2 (M + H)+, 375.1821;
found, 375.1841.
(S)-4-Amino-N-(1-cyano-2-(3′-(methylsulfonyl)biphenyl-4-
yl)ethyl)tetrahydro-2H-pyran-4-carboxamide (11).20 1H NMR
(399.8 MHz, DMSO-d6) δ 9.24 (d, J = 7.9 Hz, 1H), 8.44 (s, 3H), 8.13
(t, J = 1.8 Hz, 1H), 8.02 (dt, J = 8.0 and 1.4 Hz, 1H), 7.92 (dt, J = 8.0
and 1.2 Hz, 1H), 7.77−7.72 (m, 3H), 7.47 (d, J = 8.4 Hz, 2H), 5.19−
5.12 (m, 1H), 3.71−3.63 (m, 2H), 3.63−3.55 (m, 2H), 3.29 (s, 3H),
3.33−3.18 (m, 2H), 2.24−2.14 (m, 1H), 2.09−1.99 (m, 1H), 1.69 (d, J
= 14.1 Hz, 1H), 1.48 (d, J = 13.8 Hz, 1H). 13C NMR (125.8 MHz,
DMSO-d6) δ 176.08, 141.65, 140.82137.30, 135.61, 131.55, 130.23,
130.11, 126.96, 124.74, 61.35, 56.99, 43.41, 41.87, 30.46, 30.92, 36.43.
HRMS (ESI) m/z calcd for C22H25N3O4S (M + H)+, 428.1644; found,
428.1680.
(S)-4-Amino-N-(2-(biphenyl-4-yl)-1-cyanoethyl)tetrahydro-
1
2H-pyran-4-carboxamide (9). H NMR (500.3 MHz, CDCl3) δ
8.15 (d, J = 9.0 Hz, 1H), 7.53−7.49 (m, 4H), 7.40−7.35 (m, 2H),
7.31−7.25 (m, 3H), 5.06 (dt, J = 9.0 Hz, J = 6.8 Hz, 1H), 3.87−3.76
(m, 2H), 3.52 (dtd, J = 13.6, 11.5, and 2.3 Hz, 2H), 3.08 (q, J = 7.0 Hz,
2H), 2.22 (ddd, J = 13.9, 11.1, and 4.7 Hz, 1H), 2.10 (ddd, J = 13.8,
11.1, and 4.7 Hz, 1H), 1.24−1.19 (m, 1H), 1.13−1.08 (m, 1H). 13C
NMR (125.8 MHz, DMSO-d6) δ 176.96, 139.75, 138.87, 134.88,
130.02, 128.89, 127.36, 126.55, 126.52, 119.33, 62.46, 62.31, 54.37,
41.29, 36.81, 34.63, 34.53. HRMS (ESI) m/z calcd for C21H23N3O2
(M + H)+, 350.1868; found, 350.1875.
(S)-tert-Butyl-4-(1-amino-3-(4′-cyanobiphenyl-4-yl)-1-oxo-
propan-2-ylcarbamoyl)tetrahydro-2H-pyran-4-ylcarboxamate
(25). To a solution of 4-(tert-butoxycarbonylamino)tetrahydro-2H-
pyran-4-carboxylic acid (374 mg, 1.52 mmol), (S)-2-amino-3-(4′-
cyanobiphenyl-4-yl)propanamide,20 (405 mg, 1.52 mmol) and N-
ethyl-N-isopropylpropan-2-amine (0.664 mL, 3.81 mmol) in DMF (10
mL) was added TBTU (734 mg, 2.29 mmol), and the reaction mixture
was stirred at room temperature for 48 h. The reaction mixture was
evaporated to dryness, dissolved in CH2Cl2 (20 mL), and evaporated
onto silica. The silica was placed on the top of a silica column and
eluted with 20% ethyl acetate in isohexane, with 50% ethyl acetate in
isohexane, and then with 100% ethyl acetate to afford, after
concentration in vacuo, compound 25 (700 mg, 93%) as a colorless
(S)-tert-Butyl-4-(1-amino-3-(4-iodophenyl)-1-oxopropan-2-
ylcarbamoyl)tetrahydro-2H-pyran-4-ylcarbamate (31). 4-(tert-
Butoxycarbonylamino)tetrahydro-2H-pyran-4-carboxylic acid (3.14 g,
12.79 mmol), (S)-2-amino-3-(4-iodophenyl)propanamide (3.71 g,
12.79 mmol), and N-ethyl-N-isopropylpropan-2-amine (5.57 mL,
31.97 mmol) were dissolved in DMF (10 mL), and to the solution was
added TBTU (5.34 g, 16.63 mmol). The reaction mixture was stirred
overnight at room temperature. The reaction mixture was evaporated
to dryness, dissolved in CH2Cl2 (40 mL), and evaporated onto silica.
The silica was placed on the top of a silica column and eluted with
50% ethyl acetate in isohexane and then with 100% ethyl acetate to
1
1
solid. H NMR (399.8 MHz, CDCl3) δ 7.73 (dt, J = 8.5 and 1.7 Hz,
give compound 31 (6.33 g, 96%). H NMR (399.8 MHz, CDCl3) δ
2H), 7.67 (dt, J = 8.4 and 1.7 Hz, 2H), 7.58 (dt, J = 8.4 and 2.0 Hz,
2H), 7.42 (d, J = 8.2 Hz, 2H), 5.14 (dd, J = 14.4 and 7.2 Hz, 1H), 4.87
(s, 1H), 3.74−3.57 (m, 2H), 3.21−3.11 (m, 8H), 1.84 (s, 0.67H), 1.80
(s, 0.33H), 1.43 (s, 6H), 1.43 (s, 3H). [M + H-BOC]+ = 393.
(S)-tert-Butyl-4-(1-cyano-2-(4′-cyanobiphenyl-4-yl)-
ethylcarbamoyl)tetrahydro-2H-pyran-4-ylcarboxamate (29).
To a solution of (S)-tert-butyl-4-(1-amino-3-(4′-cyanobiphenyl-4-yl)-
1-oxopropan-2-ylcarbamoyl)tetrahydro-2H-pyran-4-ylcarboxamate
(25) (0.70 g) in CH2Cl2 (15 mL) was added methyl N-
(triethylammoniumsulfonyl)carbamate (0.356 g), and the mixture
was stirred at room temperature for 6 h. Additional methyl N-
(triethylammoniumsulfonyl)carbamate (0.15 g) was added, and the
mixture was stirred overnight. The mixture was absorbed onto silica
and purified by chromatography on silica eluting with 33% ethyl
acetate in isohexane and then with 100% ethyl acetate to afford
7.62 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.88 (s, 1H), 6.51
(d, J = 6.7 Hz, 1H), 5.38 (s, 1H), 5.00 (s, 1H), 4.73 (dd, J = 14.7 and
6.5 Hz, 1H), 3.87 (dt, J = 11.8 and 4.4 Hz, 1H), 3.70 (dt, J = 11.9 and
4.2 Hz, 1H), 3.56 (t, J = 10.7 Hz, 2H), 3.21−3.08 (m, 2H), 2.28 (ddd,
J = 14.1, 10.0, and 4.2 Hz, 1H), 1.89 (ddd, J = 13.7, 9.9, and 4.0 Hz,
1H), 1.81 (d, J = 14.1 Hz, 1H), 1.58 (d, J = 13.6 Hz, 1H), 1.36 (s, 9H).
[M + 2H-tBu]+ = 418.0.
(S)-tert-Butyl-4-(1-amino-1-oxo-3-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ylcarbamoyl)-
tetrahydro-2H-pyran-4-ylcarbamate (32). A mixture of
4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.576 g,
6.21 mmol), (S)-tert-butyl-4-(1-amino-3-(4-iodophenyl)-1-oxopro-
pan-2-ylcarbamoyl)tetrahydro-2H-pyran-4-ylcarbamate (31) (2.47 g,
4.77 mmol), and potassium acetate (1.406 g, 14.32 mmol) in
acetonitrile (50 mL) and water (8 mL) was treated with Pd-118 (50
mg, 0.08 mmol) under a nitrogen atmosphere, and the mixture was
stirred and heated under reflux for 24 h. Further potassium acetate
(0.352 g, 3.58 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-
dioxaborolane) (0.394 g, 1.55 mmol), and Pd-118 (50 mg, 0.08
mmol) were added, and heating was continued for an additional 24 h.
The reaction was evaporated in vacuo, and the residue was partitioned
between ethyl acetate (200 mL) and water (200 mL). The organics
were dried (magnesium sulfate) and evaporated in vacuo, and the
crude product was purified by flash silica chromatography eluting with
ethyl acetate. Pure fractions were evaporated to dryness to give
compound 32 (1.710 g, 69.2%) as a pale brown foam. 1H NMR (399.8
MHz, CDCl3) δ 7.74 (d, J = 7.7 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H),
6.82 (s, 1H), 6.43 (m, 1H), 5.32 (s, 1H), 4.92 (s, 1H), 4.73 (q, J = 7.2
1
compound 29 (0.42 g) as a colorless solid. H NMR (399.8 MHz,
CDCl3) δ 7.74 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.59 (d, J
= 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 5.14 (dd, J = 15.2 and 7.6 Hz,
1H), 4.70 (s, 1H), 3.83−3.76 (m, 1H), 3.75−3.66 (m, 1H), 3.66−3.56
(m, 2H), 3.17 (dd, J = 13.5 and 6.2 Hz, 1H), 3.12 (dd, J = 14.0 and 7.8
Hz, 1H), 2.28−1.86 (m, 4H), 1.85−1.77 (m, 1H), 1.44 (s, 9H). MS
[M − H]− = 473.
(S)-4-Amino-N-(1-cyano-2-(4′-cyanobiphenyl-4-yl)ethyl)-
tetrahydro-2H-pyran-4-carboxamide (10). To (S)-tert-butyl-4-(1-
cyano-2-(4′-cyanobiphenyl-4-yl)ethylcarbamoyl) tetrahydro-2H-
pyran-4-ylcarboxamate (29) (420 mg) was added formic acid (2
mL), and the mixture was heated to 50 °C for 10 min. The mixture
was evaporated to dryness, dissolved in methanol (4 mL), and purified
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Hz, 1H), 3.87 (dt, J = 12.4 and 4.6 Hz, 1H), 3.68 (dt, J = 12.4 and 4.6
Hz, 1H), 3.59−3.48 (m, 2H), 3.30−3.12 (m, 2H), 2.35−2.25 (m, 1H),
1.91−1.77 (m, 2H), 1.56−1.49 (m, 1H), 1.33 (d, J = 2.3 Hz, 12H),
1.24 (s, 9H).
(3) Pham, C. T.; Ivanovich, J. L.; Raptis, S. Z.; Zehnbauer, B.; Ley, T.
J. Papillon-Lefevre syndrome: Correlating the molecular, cellular and
̀
clinical consequences of cathepsin C deficiency in humans. J. Immunol.
2004, 173, 7277−7281.
(S)-tert-Butyl-4-(1-amino-3-(4-(2-methyl-3-oxoisoindolin-5-
yl)phenyl)-1-oxopropan-2-ylcarbamoyl)tetrahydro-2H-pyran-
4-ylcarbamate (33). (S)-tert-Butyl-4-(1-amino-1-oxo-3-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ylcarbamoyl)-
tetrahydro-2H-pyran-4-ylcarbamate (32) (300 mg, 0.58 mmol), 6-
bromo-2-methylisoindolin-1-one (131 mg, 0.58 mmol), and potassium
acetate (171 mg, 1.74 mmol) in a mixture of acetonitrile (15 mL) and
water (5 mL) were stirred under nitrogen at 90 °C with Pd-118 (378
mg, 0.58 mmol). After 4 h, the reaction was judged to be complete,
affording one major product. The reaction mixture was cooled to room
temperature and diluted with water (50 mL). The products were
extracted with ethyl acetate (3 × 50 mL), and the combined extracts
were dried and concentrated to give compound 33 as a gum (200 mg).
[M + H-BOC]+ = 436.9.
(4) Adkinson, A. M.; Raptis, S. Z.; Kelley, D. G.; Pham, C. T.
Dipeptidylpeptidase I activates neutrophil-derived serine proteases and
regulates the development of acute experimental arthritis. J. Clin. Invest.
2002, 109, 363−371.
(5) Methot, N.; Rubin, J.; Guay, D.; Beaulieu, C.; Ethier, D.;
Jagadeeswar R., T.; Reindeau, D.; Percival, M. D. Inhibition of the
activation of multiplie serine proteases with a cathepsin C inhibitor
requires sustained exposure to prevent proenzyme processing. J. Biol.
Chem. 2007, 282, 20836−20846.
(6) Methot, N.; Guay, D.; Rubin, J.; Ethier, D.; Ortega, K.; Wong, S.;
Normandin, D.; Beaulieu, C.; Reddy, T. J.; Reindeau, D.; Percival, M.
D. In vivo inhibition of serine protease processing requires a high
fractional inhibition of cathepsin C. Mol. Pharmacol. 2008, 73, 1857−
1865.
(S)-4-Amino-N-(1-cyano-2-(4-(2-methyl-3-oxoisoindolin-5-
yl)phenyl)ethyl)tetrahydro-2H-pyran-4-carboxamide (12). (S)-
tert-Butyl-4-(1-amino-3-(4-(2-methyl-3-oxoisoindolin-5-yl)phenyl)-1-
oxopropan-2-ylcarbamoyl)tetrahydro-2H-pyran-4-ylcarbamate (33)
(200 mg, 0.37 mmol) in CH2Cl2 (20 mL) was stirred at room
temperature with methyl N-(triethylammoniumsulfonyl)carbamate
(178 mg, 0.75 mmol) for 20 h. The reaction mixture was concentrated
to dryness, and the residue was stirred at room temperature in formic
acid (0.5 mL, 13.04 mmol) for 2 h to affect deprotection. The solution
was diluted with water (20 mL) and basified with 880 ammonia
solution. The products were extracted into ethyl acetate (100 mL),
and the extract was dried over magnesium sulfate. Concentration
afforded a gum, which was purified by reversed-phase chromatography
(0.1% aq TFA/methanol, 70−30%). The product-containing fractions
were freeze-dried to give compound 12 (TFA salt, 90 mg, 45.3%) as a
(7) Laine, D. I.; Busch-Petersen, J. Inhibitors of cathepsin C. Expert
Opin. Ther. Pat. 2010, 20, 497−506.
(8) Frizler, M.; Stirnberg, M.; Sisay, M. T.; Gutschow, M.
̈
Development of nitrile-based peptidic inhibitors of cysteine cathepsins.
Curr. Top. Med. Chem. 2010, 10, 294−322.
(9) Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.;
Crane, S.; Berthelette, C. A generally applicable method for assessing
the electrophilicity and reactivity of diverse nitrile compounds. Bioorg.
Med. Chem. Lett. 2007, 17, 998−1002.
(10) Bondebjerg, J.; Fuglsang, H.; Valeur, K. R.; Pedersen, J.;
Naeerum, L. Dipeptidyl nitriles as human dipeptidyl peptidase I
inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 3614−3617.
(11) Black, C. W.; Percival, M. D. The consequences of
lysosomotropism on the design of selective cathepsin K inhibitors.
ChemBioChem. 2006, 7, 1525−1535.
(12) Thong, B.; Pilling, J.; Ainscow, E.; Beri, R.; Unitt, J.
Development and validation of a simple cell-based fluorescence
assay for dipeptidyl peptidase I (DPP1) activity. J. Biomol. Screening
2011, 16, 36−43.
(13) Turk, D.; Janvic, I.; Stern, M.; Podobnik, D.; Lamba, D.; Dahl, S.
W.; Lauritzen, C.; Pedersen, J.; Turk, B. Structure of human dipeptidyl
peptidase I: Exclusion domain added to an endopeptidase framework
creates the machine for activation of granular serine proteases. EMBO
J. 2001, 20, 6570−6582.
1
colorless solid. H NMR (500.3 MHz, D2O) δ 7.60−7.56 (m, 2H),
7.44 (d, J = 10.2 Hz, 2H), 7.38 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 10.2
Hz, 2H), 5.17 (dd, J = 9.5 and 6.7 Hz, 1H), 4.25 (s, 2H), 3.70−3.53
(m, 3H), 3.43−3.37 (m, 1H), 3.31 (dd, J = 13.9 and 6.6 Hz, 1H), 3.16
(dd, J = 13.8 and 9.7 Hz, 1H), 2.99 (s, 3H), 2.20−2.12 (m, 1H), 2.05−
1.97 (m, 1H), 1.86−1.80 (m, 1H), 1.68−1.61 (m, 1H). HRMS (ESI)
m/z calcd for C24H27N4O3 (M + H)+, 419.2083; found, 419.2085.
AUTHOR INFORMATION
■
(14) Collaborative Computational Project.. The CCP4 suite:
Programs for protein crystallography. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 1994, 50, 760−763.
Corresponding Author
*E-mail: mark1.furber@astrazeneca.com.
Notes
(15) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of COOT. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2010, 66, 486−501.
The authors declare no competing financial interest.
(16) DeLano, W. L. The PyMOL molecular graphics system; DeLano
Scientific: San Carlos, CA, 2001.
ACKNOWLEDGMENTS
■
We thank Richard Weaver and Cathy MacDonald for providing
DMPK support, Theresa Humphries for synthesis support, and
Marie Castaldo for assistance with protein production methods.
(17) Kam, C. M.; Gotz, M. G.; Koot, G.; McGuire, M.; Thiele, D. L.;
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ABBREVIATIONS USED
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TBTU, N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate; Pd-118, 1,1-bis(di-tert-butylphosphino)-
ferrocene palladium dichloride; Cl, clearance; Vss, apparent
steady-state volume of distrubution; t1/2, half-life
(20) Ford, R.; Mete, A.; Mather, A.; Millichip, I. Substituted 1-
cyanoethylheterocyclic carboxamide compounds. Patent WO2010/
128324.
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