3-Oxo-hexahydro-1H-isoindole-4-carboxylic Acid as a Drug Chiral Bicyclic Scaffold: Structure-Based Design and Preparation of Conformationally Constrained Covalent and Noncovalent Prolyl Oligopeptidase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01296

Bicyclic chiral scaffolds are privileged motifs in medicinal chemistry. Over the years, we have reported covalent bicyclic prolyl oligopeptidase inhibitors that were highly selective for POP over a number of homologous proteins. Herein, we wish to report the structure-based design and synthesis of a novel class of POP inhibitors based on hexahydroisoindoles. A docking study guided the selection of structures for synthesis. The stereochemistry, decoration, and position within the molecule of the bicyclic scaffolds were assessed virtually. Following the synthesis of the best candidates, in vitro assays revealed that one member of this chemical series was more active than any of our previous inhibitors with a K_i of 1.0 nM. Additional assays also showed that the scaffold of this potent inhibitor, in contrast to one of our previously reported chemical series, is highly metabolically stable, despite the foreseen potential sites of metabolism. Interestingly, computer docking calculations accurately predicted the optimal features of the inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b01296

Article
pubs.acs.org/jmc
3‑Oxo-hexahydro‑1H‑isoindole-4-carboxylic Acid as a Drug Chiral
Bicyclic Scaffold: Structure-Based Design and Preparation of
Conformationally Constrained Covalent and Noncovalent Prolyl
Oligopeptidase Inhibitors
Gaelle Mariaule, Stephane De Cesco, Francesco Airaghi, Jerry Kurian, Paolo Schiavini, Sylvain Rocheleau,
́
̈
Igor Huskic, Karine Auclair, Anthony Mittermaier, and Nicolas Moitessier*
́
́ ́
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada
S
* Supporting Information
ABSTRACT: Bicyclic chiral scaffolds are privileged motifs in
medicinal chemistry. Over the years, we have reported
covalent bicyclic prolyl oligopeptidase inhibitors that were
highly selective for POP over a number of homologous
proteins. Herein, we wish to report the structure-based design
and synthesis of a novel class of POP inhibitors based on
hexahydroisoindoles. A docking study guided the selection of
structures for synthesis. The stereochemistry, decoration, and
position within the molecule of the bicyclic scaffolds were
assessed virtually. Following the synthesis of the best
candidates, in vitro assays revealed that one member of this
chemical series was more active than any of our previous inhibitors with a Ki of 1.0 nM. Additional assays also showed that the
scaffold of this potent inhibitor, in contrast to one of our previously reported chemical series, is highly metabolically stable,
despite the foreseen potential sites of metabolism. Interestingly, computer docking calculations accurately predicted the optimal
features of the inhibitors.
INTRODUCTION
■
Proline oligopeptidase (POP, sometimes referred to as PREP)
is a serine protease which cleaves short peptides at the α-
carbonyl of a proline residue. POP was discovered in the mid-
seventies, and its high concentration in the central nervous
system (CNS) immediately drew attention.1−3 Early studies
attributed POP protease activity to the cleavage of neuro-
peptides and peptide hormones, and inhibition of this activity
was first investigated with the discovery of the reversible
covalent inhibitor Cbz-Pro-Prolinal (1) over 30 years ago
(Figure 1).4 However, after significant medicinal chemistry
efforts and clinical trials, this interest reached a plateau with
covalent inhibitors such as JTP-4819 (2)5,6 and KYP-2047
(4),7,8 and noncovalent inhibitors such as S-17092 (3).9 More
recently, the potential of such inhibitors in the treatment of
Alzheimer’s disease and Parkinson’s disease has spurred a
second boost in the development of POP inhibitors.1−3,10
Among the important findings are the establishment of an
ability to disrupt protein−protein interactions,11 the significant
reduction of α-synuclein levels in vitro and in vivo by POP
inhibitors,12 and the colocation of POP with α-synuclein and β-
amyloid.13 Elevated levels of POP in cancer cells have also been
observed, and our previous work has demonstrated that
inhibitors can block the POP protease activity in various
cancer cell lines.14,15
Figure 1. Selected POP inhibitors.
In 2009, we disclosed a series of chiral bicyclic POP
inhibitors illustrated with 5 (Figure 2). These covalent
inhibitors were found to be cell-permeant and showed enzyme
inhibition in the high nanomolar range.16 Three years later, we
Special Issue: Computational Methods for Medicinal Chemistry
Received: August 21, 2015
© XXXX American Chemical Society
A
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specificity.19−24 Following this trend, 5 and 6 were designed to
be covalent inhibitors. However, unlike inhibitors such as 1 and
2 featuring a reactive carbonyl group, we focused our efforts on
nitrile derivatives that are significantly less reactive and are
likely to provide safer drugs.25
Design of Bicyclic Scaffolds. The promising activities of
both chiral 5 and achiral scaffold-based 6 led us to design
hybrid structures, i.e., chiral (as 5) structures mimicking 6.
Thus, the aromatic ring was modified (i.e., virtually reduced)
leading to several derivatives (7) built around scaffolds 8.
Docking studies confirmed that this class of scaffolds was
accommodated very nicely in the binding site of POP when the
stereochemistry was the one shown in 8a (Figure 2). We have
previously reported a synthetic strategy toward these bicyclic
scaffolds (Figure 2b).26 The use of a bicyclic scaffold is
expected to improve the metabolic stability, as we have shown
before, as well as the selectivity for POP. In addition, if the fit of
these molecules in the POP binding site is optimal, rigid-
ification is expected to reduce the entropic cost associated with
ligand binding. Although the shift from achiral to chiral
molecules may be perceived as a potential disadvantage, the
excellent predicted fit in the binding site led us to further
explore this chemical series. In addition, their synthesis is now
optimized to the point where no more than two steps are
necessary from either readily or commercially available
aldehydes.
We have previously reported the synthesis of two
diastereomeric members of this novel chemical series with R1
= Me and R2 = NH-Boc.26 The developed synthetic strategy
conveniently allows for diversification at R1, R2, and R3.
Structure-Based Design. In order to evaluate the potential
activity of 7 and other diversely functionalized and diastereo-
meric derivatives, we initiated a docking study using our in-
house developed program FITTED, which was previously
modified to account for covalent inhibition.15 To date, no
crystal structure of the human isoform of POP has been
reported (our biological evaluations are carried out on
recombinant hPOP), but the high sequence homology of the
binding site residues enables the use of porcine POP for
docking experiments (97% identity with the human form).27
Over 20 crystal structures of POP with or without an inhibitor
bound are available and were first superposed. The seven most
conformationally diverse structures (binding site RMSD greater
than 0.2 Å) were identified using our program MATCH-UP and
further investigated. As shown in Figure 3, the backbone
conformation does not vary significantly between structures,
and none of the side-chains adopts a different conformation.
Thus, although FITTED can be used in a flexible protein mode,
rigid protein docking was expected to provide reliable results. It
Figure 2. Designed series of potential POP inhibitors.
reported the discovery by virtual screening and docking-guided
optimization of a hit molecule, 6, which turned out to be five
times more active than 5.15 In addition, we found that the
introduction of bicyclic molecular scaffolds improved the
metabolic stability of our POP inhibitors.15 However, some
docking studies suggested that the potency of our current lead
could be further improved before proceeding with in vivo
testing, with the potency of 6 being 3 orders of magnitude
weaker than the reported potency of 2 or 4. We wish to report
herein our successful efforts in the development of a novel class
of POP inhibitors designed from 5 and 6, and based on a novel
bicyclic core.
RESULTS AND DISCUSSION
■
POP Inhibitors. A large fraction of the reported POP
inhibitors are covalent inhibitors, reacting with the catalytic
serine (Ser554). Covalent drugs can be extremely effective and
profitable pharmaceuticals, yet to date, they have been all but
ignored in structure-based drug design campaigns.17 Until
recently, concerns about their potential off-target reactivity and
toxicity have often been raised.18 Lately, however, a significant
shift in medicinal chemists’ opinion about covalent drugs has
been observed, with the advantages of covalent drugs becoming
increasingly recognized: these include extremely high potencies,
long residence times (slow off-rates), and high levels of
Figure 3. Seven most conformationally different POP crystal
structures. Left panel, global fold ribbon diagram; right panel, binding
site residues. Co-crystallized inhibitors are shown in orange.
B
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is worth noting that adding protein flexibility would have been
appropriate if our objective was to design inhibitors structurally
very different from the cocrystallized ligands as described in our
previous large evaluation study.28
focus should be on the key interactions mentioned above.
Selected docking data is summarized in Table 1 and Figure 5.
Through this docking study, we wished to focus our
synthetic efforts toward structures with a high likelihood of
being active. Analogues with diverse R1 and R2 groups were
drawn and docked. The selection of R1 and R2 groups
considered the synthetic feasibility. Figure 5a shows the binding
mode of Cbz-Pro-Pip-CN cocrystallized with POP.29 As with
other cocrystallized ligands, this structure revealed two key
hydrogen bonds with Arg643 and Trp595, a covalent bond with
Ser554 and hydrophobic interactions with Phe173. Our
compounds were docked and compared to this ligand
cocrystallized structure (Figure 5b−f).
Figure 5. Structure-based design of potential covalent POP inhibitors.
(a) Cocrystallized ligand (PDB code: 2xdw) together with known
inhibitor 4 docked; (b) 8a, R1 = Me, R2 = Bn; (c) 8a, R1 = H, R2 = Bn;
(d) 8b, R1 = Me, R2 = Bn; (e) 8a, R1 = H, R2 = CbzNH; (f) 8a, R1 = i-
Pr, R2 = Bn.
It was first envisaged that scaffold 8 could be decorated with
a (S)-cyano-pyrrolidine (R3 in Figure 2) to provide 7d, 7e, and
7a, as hybrids of 5 and 6, or alternatively converted into nitrile
analogues such as 9a.
Both the scores computed using RankScore (a force field-based
scoring function, the lower, the better) and MatchScore, which
evaluates the match between the ligand and protein functional
groups (the higher, the better), are given. MatchScore is
instrumental in automatically measuring the key interactions,
while RankScore evaluates other factors such as internal strain
energy.
Covalent Inhibition. The physical processes governing the
action of covalent bond forming drugs differ fundamentally
from those of noncovalent drugs. Covalent drugs inhibit their
targets in a two-step fashion (Figure 4).22 First, they bind
It is worth mentioning that a docking mode in which both
noncovalent and covalent binding were investigated concom-
itantly was used. The results shown in Table 1 were collected
for the lowest-in-energy poses, although in half of the cases,
both covalent and noncovalent poses were proposed by FITTED.
Care must be taken when analyzing the data as, in contrast to
noncovalent binding, kinetic and thermodynamic factors
control whether the binding will be covalent. If the lowest-
energy pose is covalent, it suggests that the fit of the compound
(first equilibrium in Figure 4) favorably positions the nitrile
group leading to covalent inhibition. This mode of binding is
optimal. If the lowest-energy pose is noncovalent but the
second lowest-energy pose is covalent and with a score within
1−2 kcal/mol of the best pose, we still considered targeting the
covalent molecule. In these cases, we anticipate that the
affinities of the covalent and noncovalent inhibitors are
comparable (uncertainties in our predicted binding energies
are on the order of a few kcal/mol); however, we expect the
Figure 4. Reaction scheme for covalent inhibition. E, enzyme; I,
inhibitor; E···I, noncovalent complex; k1, association rate constant; k−1,
dissociation rate constant.
noncovalently (E···I) and then react to form a chemical bond
(reversible in our present study) with their targets (E−I).
Consequently, whereas the potency and selectivity of conven-
tional noncovalent bond forming drugs are typically expressed
in terms of equilibrium binding affinity, it is essential to
consider the time-dependence of inhibition for covalent drugs
(i.e., IC50’s vary over time for reversible covalent inhibitors).30
Docking Covalent Inhibitors. We previously found that
our current scoring function could distinguish between inactive
and active inhibitors and can be used successfully in prospective
studies.15 However, it is not accurate enough to rank covalent
inhibitors that are within 2 orders of magnitude in Ki. When
optimizing our previous chemical series, we observed that the
Table 1. Selected Docking Data
entry
R1
R2
scaffold
RankScore
MatchScore
binding
1
Me
Me
Me
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
8a
−15.9
−7.4
−9.4
107.9
105.4
116.2
112.5
109.5
106.6
105.1
97.1
covalent
2
8b
noncovalent
noncovalent
covalent
3
ent-8a
8a
4
−16.2
−15.3
−11.2
−13.8
−10.3
−9.9
−16.0
−14.6
−16.2
−12.2
−10.2
−15.6
5
H
8b
covalent
6
H
ent-8a
8a
noncovalent
covalent
7
Et
8
iPr
Ph
H
8a
noncovalent
noncovalent
covalent
9
8a
104.2
69.9
10
11
12
13
14
15
CbzNH
BnO
8a
H
8a
94.0
covalent
H
BnCONH
8a
83.5
covalent
9a
113.4
64.0
noncovalent
covalent
ent-9a
4
107.6
covalent
C
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covalent molecule to have more favorable binding kinetics, i.e.,
longer bound lifetimes. The relatively high energy barrier to
breaking the covalent bond leads in general to lower values of
the dissociation constant, koff. Thus, when noncovalent binding
was suggested by the docking program, a close look at the other
proposed poses was required. In addition, when docking
potential covalent inhibitors, the reactivity of the warhead may
differ. In the present study, only nitrile derivatives with
expected similar reactivity were investigated.
methyl, selected four groups of different lengths for R2, and
focused on scaffold 8a, which is synthetically more accessible.
The synthesis of these scaffolds required the conversion of two
aldehydes into the corresponding hydrazides 12a−c, alkoxy-
amine 12d, and amines 12e and 12f (Table 2).
Table 2. Scaffold Synthesis
First, the docking indicated that the diastereomeric scaffold
8a with the absolute configuration shown in Figure 2 was
preferred over the other diastereomers including 8b and ent-8a
to form a covalent bond with the enzyme (Table 1, entries 1−3
and 4−6). The same trend was consistently observed when
docking was carried out with other R1 and R2 groups.
According to our prediction, increasing the size of R1 would
result in weaker binding when R1 exceeded one carbon and
even in noncovalent binding with R1 = iPr and Ph Table 1,
entries 1, 4, and 7−9). The data collected for R2 were more
ambiguous. For example, while RankScore suggested that R2 =
BnCONH with R1 = H should be optimal (entry 12),
MatchScore indicated that R2 = H should be favored (entry
4). As a reference, a highly active compound (4) was docked
and scored (Table 1). Interestingly, our best scoring designed
inhibitor was predicted to be as strong as this subnanomolar
compound (entries 4 and 15, Figure 5a).
As the key interactions were featured by the cocrystallized
compound shown in yellow in Figure 5a, this compound was
overlaid with our docked compounds, thus revealing whether
the key interactions and overall binding were retained. For
example, while the compounds shown in Figure 5b, c, and e
appeared to bind in a manner similar to that of the
cocrystallized ligand, the two compounds shown in Figure 5d
(built around scaffold 8b) or Figure 5f (with a large R1 group)
were not predicted to retain all of the key interactions with
POP. This predicted loss of interactions was more accurately
measured by MatchScore than by RankScore.
a
b
Yields are given in parentheses. Isolated yield for the first step (11 to
c
12a−f). Isolated yield of the major diastereomer from 12a−f to 13a−
f.
The initial formation of the unsaturated hydrazides or amines
was achieved by a solventless condensation (R1 = Me) or in
methanol (R1 = H). The chemoselective reduction was next
achieved with dimethylamino borane for products 12a−d and
with NaBH4 for products 12e−f, as reported previously.26 The
six intermediates were subsequently reacted with maleic
anhydride leading to mixtures of exo 13a−f and exo 13a−f
products, with the exo adducts being the major isomers.
During our optimization of the synthetic methodology,31 we
solved the difficult separation of the diastereomers through
diastereoselective precipitation. We were very pleased that this
stereoselective precipitation was also possible with the
proposed substitutions at position R2. Thus, treatment of the
diastereomeric mixture with a mixture of ether and hexane (or
dichloromethane) led to selective precipitation of the major exo
adduct in most cases (Figure 7). The relative stereoselectivity of
the scaffolds was determined by 1D NOE and 2D NOESY
experiments on the scaffolds and some derivatives as reported
previously (Figure 8).26 We were also pleased to obtain crystals
of 13d (Figure 8). Consequently, the selective precipitation of
the exo adduct was unambiguously confirmed by X-ray
crystallographic analysis.
CHEMISTRY
■
Synthetic Strategy. We have previously reported an
expedient synthesis of 10a and 10b26 (Figure 6) and more
Figure 6. Generic structure of potential POP inhibitors.
recently optimized the synthetic protocol and investigated an
observed solvent effect.31 This new protocol enabled the
selective precipitation of the major diastereomer, hence
removing the need for extensive chromatography. We
envisaged taking advantage of this synthetic strategy and
probing its application to other analogues by varying R1 and R2.
Synthesis of Bicyclic Scaffolds Varying R1 and R2.
According to the docking study, as long as R1 is small enough,
the potential inhibitor should fit in the POP binding site. These
computational investigations were also indicating that R2 of
different sizes should fit, although with the shorter group
preferred, and that the stereochemistry of the scaffold was
critical. Accordingly, we restricted R1 to only hydrogen and
This optimized strategy not only provided a single isolated
diastereomer but also eliminated the need for chromatographic
purification of the polar bicyclic scaffolds. In the case of 13c,
the precipitation was more problematic and was difficult to
reproduce. The mixture remaining in the mother liquor was not
further investigated.
D
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1
Figure 7. H NMR spectrum of (a) a mixture of endo and exo adducts 14d and 13d; (b) exo adduct 13d.
Figure 8. Stereochemistry of scaffolds 13b ascertained by NOESY and the structure of 13d determined by X-ray crystallography (oxygens in red,
nitrogen in blue, carbons in gray, and hydrogens in light gray).
Synthesis of Potential Inhibitors. The synthesis of
various derivatives of 7 is shown on Table 3. Following the
strategy described above, the scaffolds 13a−f with the exo
configuration were diastereopure yet racemic.26 Compound
13a was next converted into the corresponding nitrile derivative
9a by an amidation/dehydration sequence in reasonable yields.
As the modeling study suggested that this series should not lead
to significant activity, no other derivatives were prepared. The
potential covalent inhibitors 7a−f and 16a−f were accessed by
coupling with (S)-cyano-pyrrolidine, while their noncovalent
counterparts 15a−f were prepared as racemic mixtures through
coupling with pyrrolidine. Although the scaffold 8a was our
priority, ent-8a analogues were produced along with 8a
analogues. The other scaffolds (R1 = other than H and methyl)
were not synthesized since the docking study predicted that
increased steric bulk at that position would either affect the
potency (RankScore) or disrupt the covalent binding.
Extensive chromatographic work enabled the separation of
the diastereomers resulting from coupling the racemic scaffolds
(including rac-13d) to enantiopure (S)-cyano-pyrrolidine.
While the relative stereochemistry of rac-13d and other
scaffolds was assigned by X-ray crystallography and NMR
spectroscopy, determination of the stereochemistry of the
resulting pairs of diastereomers (such as enantiopure 7d and
16d from rac-13d) was difficult until we managed to crystallize
16d (Figure 9). While crystallography assigned the relative
stereochemistry, the absolute stereochemistry was derived
knowing the absolute stereochemistry of (S)-cyano-pyrrolidine,
which was coupled in this process. Interestingly, the NMR
spectra of 7d and 16d were significantly different revealing a
mixture of rotamers for 16d only. Similarly, 7c and 7e appeared
as a single averaged conformation on NMR spectra, while the
NMR signatures of 16c and 16e were more complex (Figure
9). Using the crystal structure as a starting point and the NMR
similarities, we were able to ascertain the relative and absolute
stereochemistries of these 12 compounds. This solved a
structural assignment challenge that was foreseen to be difficult.
BIOLOGICAL EVALUATIONS
■
Inhibition of POP. As discussed above, the binding is a
two-step process, including a fast binding and a slow covalent
bond formation. Thus, the inhibition potencies were measured
after equilibria were reached. With this in mind, the 19
synthesized compounds were assessed for their inhibitory
potency on recombinant human POP (Tables 4 and 5) and
their activities compared to our computational predictions
(Table 6). Gratifyingly, an excellent match between exper-
imental data and computational predictions was observed. In
order to summarize the information gained, we will examine the
most notable results of this study.
First, we noted that rigidifying the right side of the
cocrystallized structure (i.e., compound 9a) did not lead to
any noticeable activity and was, as predicted, detrimental to
binding. Second, the potentially covalent inhibitors (featuring a
nitrile group) were found to be more potent than their
noncovalent counterparts. For example, 15d is 3 orders of
magnitude less active than 7d. This difference is much more
pronounced than that in our previous chemical series for which
an order of magnitude was the norm. Third, the absolute
stereochemistry of the scaffold was a key factor, as
demonstrated with 16c, 16d, 16e, and 16f being micromolar
at best, while 7c, 7d, 7e, and 7f were nanomolar inhibitors with
E
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Table 3. Potential POP Inhibitor Synthesis
Figure 9. Structure of 16d (oxygens in red, nitrogens in blue, carbons
in gray, and hydrogens in light gray) as determined by X-ray
1
crystallography and H NMR spectra of the enantiopure compounds.
Table 4. Inhibition of POP
7e being the most active (Ki = 1 nM). There are cases (i.e,
fluoxetine, an antidepressant)32 in which the changes in
stereochemistry do not substantially affect the potency. In the
case of noncovalent drugs, their position and orientation within
the binding site can adjust to optimize interactions. However,
the formation of a covalent bond provides less opportunity for
the drug to adjust its binding. As a result, the stereochemistry
difference between 7e and 16e, although four bonds away from
the “covalent group”, was not tolerated. Fourth, the
introduction of an R1 group even as small as methyl was
detrimental to the activity (15e vs 15f, Table 4). This
observation was in line with the predictions. Fifth, the size of
the R2 group can be used to modulate the affinity. Our docking
study showed that a benzyl group (7e: Ki = 1.0 nM) would be
the optimal size, while longer chains would be tolerated, with
the best fit for benzyl and the worst with Cbz. The increasing
size of this appendage correlated with a predicted increase in
distance from the side chain of Phe173.
Our survey of the field revealed that the hydrophobic
interaction is not as critical for the binding as are the two
F
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activity (Ki = 1.0 nM), significantly lower than that of our
previous hit (6, Ki = 23.0 nM) and similar to the potencies of
POP inhibitors which have entered clinical trials such as KYP-
2047.1 In addition, this novel lead molecule 7e showed
improved metabolic stability over our previous leads. Thus, 7e
will now be moved to in vivo studies.
Interestingly, this chemical series was also used to assess our
computational predictions and confirmed that the current
version of FITTED can be used to guide the design of active
covalent inhibitors.
Table 5. Tight Binding Inhibition with 4 and 7e
inhibitor
Morrison Ki (tight binder) (nM)
regular Ki (nM)
4
0.92 0.02
0.75 0.02
0.95 0.15
1.33 0.15
7e
Table 6. Docking-Based Predictions vs Experiments
prediction
example
experiment
correct
scaffold on the left side > scaffold on the right 9 vs 7
side
This successful study together with our previous reports
improves our understanding of the geometrical requirements
for optimal POP inhibition as well as our comprehension of
covalent inhibition, in both computational and experimental
perspectives.
covalent more active than noncovalent
stereochemistry: 3S,4R,7R > 3R,4S,7S
R1 = H, Me equally tolerated
7 vs 15
7e vs 16e correct
15e, 15f Me tolerated
correct
but less
than H
R2 = Bn > OBn > C(O)CH2Ph > NHCbz
16a, 16c, correct
16d,
16e
EXPERIMENTAL SECTION
■
4 and 7e should be equally potent
4 vs 7e
correct
Protein Expression. E. coli BL21 competent cells were trans-
formed with pETM10 hPOP. A starter culture of LB medium (100
mL) containing kanamycin (50 mg mL−1) was inoculated with one
colony and was incubated overnight at 37 °C with shaking. After 16 h,
four cultures of LB (4 × 1000 mL) containing kanamycin (50 mg
mL−1) were inoculated with the overnight culture (20 mL). The
inoculated cultures were incubated at 37 °C and 220 rpm until the
OD600 was between 0.3 and 0.5 (3 h). The temperature was lowered
to 18 °C, IPTG was added (final concentration of 0.5 mM), and
induction was allowed to proceed for 5 h. Cells were harvested by
centrifugation (4000g, 15 min, 4 °C), and the pellet was resuspended
in suspension buffer (50 mL) [Tris-HCl (10 mM), NaCl (300 mM),
β-mercaptoethanol (5 mM), imidazole (1 mM), and 5% glycerol, pH
8] and sonicated for four cycles (2 min of sonication/2 min of rest;
pulse, 0.5 intensity; duty, 0.5), while the sample was kept on ice
(Branson sonifier 450, Emerson industrial automation, United States).
After sonication, the sample was centrifuged (40000g, 30 min, 4 °C),
and the supernatant was used immediately for POP purification. An
affinity column was used (10 mL, Toyopearl, AF-Chelate-650M) for
purification. The supernatant was applied at a flow rate of 0.5 mL
min−1 to a column previously equilibrated with 5 column volumes of
NiSO4 (0.2 M) followed by 5 column volumes of suspension buffer.
The column was then washed with suspension buffer until the
absorbance at 280 nm returned to basal level. The column was then
rinsed with 5 column volumes of washing buffer [Tris-HCl (20 mM),
NaCl (300 mM), β-mercaptoethanol (5 mM), imidazole (15 mM),
and 5% glycerol, pH 8]. The elution was then performed with 4
column volumes of elution buffer [Tris-HCl (20 mM), NaCl (300
mM), β-mercaptoethanol (5 mM), imidazole (500 mM), and 5%
glycerol, pH 8]. Fractions (4 mL) were collected during the entire
elution. Fractions testing positive for POP activity (REF) were
analyzed by SDS−PAGE and stained with phastgel blue R (GE
Healthcare, sweden). POP-containing fractions were combined and
subjected to size exclusion chromatography (HiLoad 16/60 Super-
dex75 prep grade on a Amersham Biosciences FPLC system) with
[Tris-HCl (20 mM), NaCl (150 mM), benzamidine (5 mM), EDTA
(1 mM), β-mercaptoethanol [5 mM], and 5% glycerol, pH 8] as the
running buffer. The purified enzyme was dialyzed into the appropriate
buffer. Recombinant hPOP was quantified by measuring the
absorbance at 280 nm using an extinction coefficient calculated by
the following equation (ε = nTrp*5000 + nTyr*1490 + nCys*125,
129090 L·mol−1·cm−1 for POP38). Aliquots of the recombinant
enzyme were prepared and immediately frozen with liquid nitrogen
and stored at −80 °C.
above-mentioned hydrogen bonds and the covalent bond.1 In
agreement with these previous observations, disrupting this
hydrophobic interaction led to decreased potency, although less
pronounced than breaking the covalent bond or the hydrogen
bonds.
In a previous report, Venalainen et al. reported a K of 0.023
̈
̈
i
nM for inhibitor 4,8 nearly 2 orders of magnitude more potent
than 7e (Ki = 1.0 nM), while our docking studies indicated
similar activities for these two compounds. In order to compare
the two inhibitors under the same conditions, we carried out a
second set of experiments with 7e and 4 following the
procedure of Venalainen et al., who computed the K using the
̈
̈
i
Morrison equation which considers tight binding.33 In our
experiments (Table 5), we observed that 4 and 7e were nearly
as potent.
Overall, this study identified a highly potent POP inhibitor
(7e, Ki = 1.0 nM), exhibiting potency on par with the most
active compounds reported to date (Figure 1) including those
moved to clinical trials.1
Metabolism Studies. One of the concerns that was raised
while this work was ongoing was the numerous possible sites of
metabolism on this class of scaffolds. In fact, our first chemical
series illustrated by 5 was terminated due to the high
complexity of its metabolism, leading to several potentially
toxic metabolites.34 The epoxidation of aromatic rings and
double bonds by cytochrome P450 enzymes present in the liver
is one of the major causes of drug withdrawal and therefore
should be carefully examined early in the drug discovery and
development process.35−37 In our lead compound 7e, both the
double bonds and the N-benzyl moiety may lead to reactive
metabolites and/or low metabolic stability (reduced half-life).
In fact, the former may be prone to epoxidation leading, while
the latter may undergo N-dealkylation. Unexpectedly, 7e was
found to be very stable in human liver microsomes (Clint = 4
μL/min/mg protein) and even more stable than our previous
lead 6. Under the experimental conditions used, no N-
debenzylated products were observed, and only trace amounts
of mono-oxidation metabolites were detected.
POP Activity Assays. Chemicals and Reagents. ZGP-pNA was
obtained from Bachem (Bubendorf, Switzerland).
CONCLUSIONS
■
IC50/Ki Measurement. The reactions were performed in microtiter
plates of 96 wells. For each reaction, the activity buffer (A.B.) (140 μL,
sodium phosphate (20 mM), NaCl (150 mM), β-mercaptoethanol (5
mM), EDTA (2 mM), 10% glycerol, and 0.5 mg/mL BSA, pH 8) was
preincubated for 30 min at 30 °C with hPOP (20 μL, 10 nM in A.B.)
The first use of 3-oxo-hexahydro-1H-isoindole-4-carboxylic acid
as a chiral drug template is presented. Structure-based guided
design and efficient synthesis led to the discovery of a highly
potent POP inhibitor (7e) exhibiting single digit nanomolar
G
DOI: 10.1021/acs.jmedchem.5b01296
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and with the corresponding inhibitor solution (20 μL) or activity
buffer (controls). Stock inhibitors were prepared in DMSO (100
mM); dilutions for inhibitor evaluation were prepared from the stock
in the activity buffer. A control experiment with the same DMSO
concentration was performed. After preincubation, ZGP-pNA (20 μL,
0.8 mM in A.B., final concentration of 80 μM) was added, and the
formation of the product was followed by absorbance at 405 nm every
30 s. Initial velocity was measured for each concentration of inhibitor
and compared to the initial velocity of reactions that did not contain
inhibitor. The IC50 value was defined as the inhibitor concentration
causing a 50% decrease in activity. The Ki was defined as IC50/
(1+([S]/Km)). The Km of the substrate has been measured by
monitoring the initial velocity of the enzymatic reaction of 1 nM of
hPOP with various concentrations of substrate. Data obtained were Km
= 74.6 μM; kcat = 20.56 s−1.
group P21/n and is not enantiopure. Compound 16d crystallizes in an
enantiomorphic space group P212121. There was insufficient
anomalous scattering from the crystal due to the crystal diffracting
poorly overall, even at a 100 K. Attempts to isolate a more suitable one
were either unsuccessful or resulted in a crystal of approximately the
same quality. The absolute structure of compound 16d was
determined in reference to a known chiral center which does not
change in the synthetic procedure. Structures have been deposited
with the Cambridge Structural Database, deposition codes CCDC
1430175-1430176.
Synthesis. All commercially available reagents were used without
further purification unless otherwise stated. The 4 Å molecular sieves
were dried at 100 °C prior to use. Optical rotations were measured on
a JASCO DIP 140 in a 1 dm cell at 20 °C. FTIR spectra were recorded
1
using a PerkinElmer Spectrum One FT-IR. H and 13C NMR spectra
Morrison Ki. For 7e, since the Ki was in the same range as the
enzyme concentration used in the assay, the Morrison equation was
used.39 The same protocol as the IC50 was used with the appropriate
concentration range. Initial velocities were determined in the presence
(vi) and the absence (v0) of the inhibitor, and the subsequent ratio (vi/
vo) was plotted against the inhibitor concentration and fitted to the
Morrison equation:
were recorded on Varian Mercury 400 MHz, 300 MHz, or Unity 500
spectrometers. Chemical shifts are reported in ppm using the residual
of deuterated solvents as internal standard. Thin layer chromatography
visualization was performed by UV or by development using KMnO4,
H2SO4/MeOH, or Mo/Ce solutions. Chromatography was performed
on silica gel 60 (230−40 mesh). Low resolution mass spectrometry
was performed by ESI using a Thermoquest Finnigan LCQ Duo. High
resolution mass spectrometry was performed by EI peak matching (70
eV) on a Kratos MS25 RFA double focusing mass spectrometer or by
ESI on a Ion Spec 7.0 T FTMS at McGill University. Prior to
biological testing, reversed phase HPLC was used to verify the purity
of compounds on an Agilent 1100 series instrument equipped with a
VWD-detector, a C18 reverse column (Agilent, Zorbax Eclipse XDB-
C18 150 mm 4.6 mm, 5 μm), and UV detection at 254 or 220 nm. All
tested compounds were at least 95% pure. All measured purities are
listed in Table 7.
(E + I + Kiapp) − (E + I + Kiapp)2 − 4EI
v
v0
i
= 1 −
2E
where E is the active enzyme concentration in the assay (1 nM here), I
is the inhibitor concentration, and Kai pp is the apparent dissociation
constant of the inhibitor. Similarly to that for the IC50, knowing the
Henri−Michaelis−Menten constant (Km = 74.6 μM) and the substrate
concentration used in the assay ([S] = 80 μM), it is possible to
compute the true dissociation constant following this equation:
Kiapp
Table 7. HPLC Analysis of Purity
Ki =
[S]
Km
a
compd
retention time (min)
purity (%)
1 +
(
)
b
9a
16.4
18.0
18.8
16.7
17.5
18.6
19.5
17.3
17.1
18.1
18.1
16.0
15.7
17.1
17.2
17.6
17.6
18.7
18.7
90.4
97.6
96.7
98.1
96.0
96.5
95.5
96.5
99.0
96.1
98.6
99.9
98.7
92.0
97.2
98.5
95.6
98.2
95.0
b
Metabolism. Analyses were performed on an Agilent 1100
modular system equipped with an autosampler, a quaternary pump
system, a photodiode array UV detector, a quadrupole MS detector,
and a ChemStation (for LC 3D A.09.03) data system. Separation was
achieved using a Zorbax Eclipse XDB-C18 150 mm × 4.6 mm, 5 μm.
Elution consisted of a gradient step from 99% mobile phase A (H2O)
and 1% mobile phase B (CH3CN), to 99% phase B over 20 min, at a
flow rate of 1 mL/min. The absorption was recorded at 220 nm.
Docking. For the docking study, we used our docking program
15a
15b
b
15c
15d
15e
15f
b
7a
b
16a
FITTED (FORECASTER platform41), which was previously modified to
40
b
7b
account for covalent inhibitors.15 The compounds were prepared for
docking using our program SMART and docked to a protein structure
prepared from the PDB file using PREPARE (PDB code: 2XDW) and
further processed for use in docking studies using PROCESS. The
docking was carried out with Ser554 identified as a catalytic residue
susceptible to forming a covalent bond with reactive groups. FITTED
uses a genetic algorithm as a conformational search algorithm. A
maximum number of generation of 200 and a convergence criterion
Diff_N_Best 0.25 (converged when the 20 lowest-in-energy
individuals were within 0.25 kcal/mol) were used. These parameters
ensured a more exhaustive conformation search. Default values were
used for all the other parameters.
b
16b
b
7c
b
16c
b
7d
16d
b
7e
16e
b
7f
b
16f
a
b
Conditions: (95% water, 5% methanol, and 1 mL/min). UV
detection at 220 nm.
Crystallography. Crystal and molecular structures of 13d and 16d
were determined by single crystal X-ray diffraction. Diffraction
measurements were made on a Bruker D8 APEX2 X-ray diffractometer
instrument using graphite-monochromated MoKα (λ = 0.71073 Å)
radiation. The X-ray diffraction data sets were collected using the ω
scan mode over the 2θ range up to 54° at 100 K. The structures were
solved by direct methods implemented in SHELXS and refined using
SHELXL.42 Structure refinement was performed on F2 using all data,
and hydrogen atoms were modeled with appropriate riding-hydrogen
models on the carbon centers. Calculations were performed, and the
drawings were prepared using the WINGX43 suite of crystallographic
programs. Compound 13d crystallizes in a centrosymmetric space
General Procedure for the Reductive Amination of
Aldehydes. To a solution of Cbz-hydrazide or amine (1 equiv) in
MeOH (concentration of 1 M) was added a solution of dienal (1
equiv) in DCM dropwise. The mixture was stirred for 30 min at room
temperature. The solution was cooled to 0 °C, and Me2NH·BH3 (1.5
equiv) was added slowly, followed by a solution of pTSA (6 equiv) in
MeOH (concentration 1 M). After stirring for another 2 h, a solution
of Na2CO3(aq) (2-fold dilution, 10% w/v) was added, and the mixture
was stirred for 2 h then concentrated under reduced pressure,
extracted with CH2Cl2, washed with brine, dried over Na2SO4, and
H
DOI: 10.1021/acs.jmedchem.5b01296
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
concentrated under reduced pressure. The crude product was purified
by flash chromatography with eluent Hex/EtOAc to afford the desired
product.
Benzyl 2-((2E)-penta-2,4-dienyl)hydrazinecarboxylate (12a).
Yield: 73%. IR (film) νmax (cm−1) 3314.85, 2972.36, 2891.14, 1686.76;
1H NMR (300 MHz, CDCl3) δ (ppm) 7.45−7.30 (m, 5H), 6.32 (dt, J
= 16.7, 10.2 Hz, 1H), 6.25−6.15 (m, 1H), 5.76−5.62 (m, 1H), 5.20 (d,
J = 16.5 Hz, 1H), 5.14 (s, 2H), 5.11−5.02 (m, 1H), 3.58−3.48 (m,
2H); 13C NMR (75 MHz, CDCl3) δ (ppm) 157.2, 136.3, 136.0, 134.5,
129.2, 128.6 128.3, 128.2, 117.5, 67.1, 53.5; HRMS (ESI+) for
C13H17N2O2 (M + H) calcd, 233.12900; found, 233.12770.
Benzyl 2-((2E,4E)-hexa-2,4-dienyl)hydrazinecarboxylate
(12b). Yield: 94%. IR (film) νmax (cm−1) 3274.60, 3019.77, 2912.85,
1702.27; 1H NMR (300 MHz, CDCl3) δ (ppm) 7.31 (s, 5H), 6.72 (bs,
1H), 6.06 (m, 2H), 5.64 (dq, J = 6.7, 13.5 Hz, 1H), 5.51 (dd, J = 7.1,
14.6 Hz, 1H), 5.10 (s, 2H), 4.25 (bs, 1H), 3.45 (d, J = 5.4 Hz, 2H),
1.72 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm) 157.2,
136.2, 134.3, 130.9, 129.8, 128.6, 128.2, 128.2, 125.8, 67.0, 25.3, 18.1;
HRMS (ESI+) for C14H18N2O2Na (M + Na) calcd, 269.1266; found,
269.1265.
rac-(3aS,4S,7aS)-2-(((benzyloxy)carbonyl)amino)-3-oxo-
2,3,3a,4,5,7a-hexahydro-1H-isoindole-4-carboxylic Acid (13a).
White solid. Yield: 99% (for all isomers) exo adduct: 65%. IR (film)
νmax (cm−1) 3030, 2941, 2922, 2853, 1739, 1720, 1709; 1H NMR (400
MHz, CDCl3) δ (ppm) 7.40−7.20 (m, 5H), 5.77 (bd, J = 10.0 Hz,
1H), 5.67 (bd, J = 6.9 Hz, 1H), 5.14 (s, 2H), 3.58 (dd, J = 7.5, 7.4 Hz,
1H), 3.50−3.41 (m, 1H), 3.40−3.31 (m, 1H), 3.10−3.00 (m, 1H),
2.68 (bd, J = 20.0 Hz, 1H), 2.45−2.40 (m, 1H); 13C NMR (125 MHz,
CDCl3) δ (ppm) 175.4 (2C), 155.3, 135.5, 128.6, 128.5 (2C), 128.3
(2C), 127.8, 125.1, 67.8, 45.1, 40.3, 35.8, 32.7, 28.1; HRMS (ESI-) for
C17H17N2O5 (M − H) calcd, 329.11430; found, 329.11429.
rac-(3aS,4R,5S,7aR)-2-(((Benzyloxy)carbonyl)amino)-5-meth-
yl-3-oxo-2,3,3a,4,5,7a-hexahydro-1H-isoindole-4-carboxylic
Acid (13b). White solid. Yield: 55%. IR (film) νmax (cm−1) 3251,
1
3024, 2960, 1706; H NMR (400 MHz, CDCl3) δ (ppm) 7.38−7.30
(m, 5H), 7.23 (brs, NH), 5.76 (d, J = 9.6 Hz, 1H), 5.60 (d, J = 9.6 Hz,
1H), 5.19−5.12 (m, 2H), 3.56 (t, J = 7.2 Hz, 1H), 3.46−3.33 (m, 2H),
2.99−2.92 (m, 3H), 2.42 (d, J = 7.2 Hz, 1H), 1.15 (d, J = 7.2 Hz, 3H);
13C NMR (125 MHz, CDCl3) δ (ppm) 175.8, 174.6, 155.5, 135.7,
134.1, 128.9, 128.7, 128.5, 124.4, 67.9, 42.9, 42.5, 33.3, 32.9, 21.8;
HRMS (ESI+) for C18H20N2O5Na (M + Na) calcd, 367.1269; found,
367.1275.
(E)-N′-(Penta-2,4-dien-1-yl)-2-phenylacetohydrazide (12c).
White solid. Rf = 0.15 (Hex/EtOAc, 5:5). Yield: 55%. IR (film) νmax
1
(cm−1) 3275, 3083, 2924, 1645, 1454, 1348, 1005; H NMR (400
rac-(3aS,4R,7aR)-3-oxo-2-(2-Phenylacetamido)-2,3,3a,4,5,7a-
hexahydro-1H-isoindole-4-carboxylic Acid (13c) and rac-
(3aS,4R,7aS)-3-oxo-2-(2-Phenylacetamido)-2,3,3a,4,5,7a-hexa-
hydro-1H-isoindole-4-carboxylic Acid (14c). White solid. Yield:
MHz, CDCl3) δ (ppm) 7.45−7.07 (m, 5H), 6.26 (dt, J = 16.8, 10.2
Hz, 1H), 6.08 (dd, J = 15.2, 10.5 Hz, 1H), 5.66−5.50 (m, 1H), 5.14 (d,
J = 16.6 Hz, 1H), 5.05 (d, J = 10.1 Hz, 1H), 4.29 (bs, 1H), 3.49 (s,
2H), 3.38 (bd, J = 5.6 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ (ppm)
170.5, 136.3, 134.8, 134.2, 129.4 (2C), 129.2, 129.0 (2C), 127.5, 117.6,
53.6, 42.1; HRMS (ESI + ) for C13H17N2O (M + H) calcd, 217.1335;
found, 217.1337.
(E)-O-Benzyl-N-(penta-2,4-dien-1-yl)hydroxylamine (12d).
Colorless oil. Yield: 31%. Rf = 0.36 (Hex/EtOAc, 9:1); IR (film)
υmax (cm−1) 3265, 3087, 1603; 1H NMR (500 MHz, CDCl3) δ (ppm)
7.37−7.28 (m, 5H), 6.34 (dt, J = 15.0, 10.0 Hz, 1H), 6.25−6.20 (m,
1H), 5.77 (dt, J = 15.0 Hz, 5.0 Hz, 1H), 5.19 (d, J = 15.0 Hz, 1H), 5.08
(d, J = 10 Hz, 1H), 4.72 (s, 2H), 3.58 (d, J = 5.0 Hz, 2H); 13C NMR
(125 MHz, CDCl3) δ (ppm) 137.9, 136.6, 134.2, 129.6, 128.6, 128.5,
128.0, 117.3, 76.4, 54.1; HRMS (ESI+) for C12H16NO (M + H) calcd,
190.1226; found, 190.1201.
(E)-N-Benzylpenta-2,4-dien-1-amine (12e). A solution of dienal
(800 mg, 9.74 mmol) and benzylamine (1.04 g, 9.74 mmol) was
stirred 2 h at room temperature. Then, the reaction mixture was
cooled to 0 °C and allowed to stir for an additional 30 min. To the
mixture was added NaBH4 (1.47 g, 38.96 mmol), and the resulting
mixture was allowed to stir for 2 h. The reaction mixture was quenched
with H2O and extracted with DCM. The combined organic layer was
washed with 10% NaOH, brine, dried over Na2SO4, filtered, and
evaporated. The crude product was chromatographed on silica gel with
eluent Hex/EtOAc 9:1, to give a colorless oil. Yield: 500 mg (31%). Rf
= 0.23 (Hex/EtOAc 7:3). Spectral data were consistent with data
reported in the literature.44
(2E,4E)-N-Benzylhexa-2,4-dien-1-amine (12f). After stirring a
solution of hexadienal (2.07 g, 21.6 mmol) and benzylamine (2.35 mL,
21.6 mmol) in MeOH (18 mL) for 2 h at room temperature, the
mixture was cooled to 0 °C and allowed to stir for an additional 30
min. To the mixture was added NaBH4 (1,6 g, 43.2 mmol), and the
resulting mixture was allowed to stir for an additional hour. The
reaction mixture was quenched with H2O and extracted with DCM.
The combined organic layer was washed with NaOH 10%, brine, dried
over Na2SO4, filtered, and evaporated. The crude product was
chromatographed on silica gel gel with eluent Hex/EtOAc 9:1, to give
a yellow oil. Yield: 2,6 g (64%). Rf = 0.35 (Hex/EtOAc 5:5). Spectral
data were consistent with data reported in the literature.44
48%. Rf = 0.24 (DCM/MeOH, 97:3). IR (film) ν
(cm−1) 3214,
max
1
3027, 2924, 1716, 1670, 1661, 1195; H NMR (500 MHz, CDCl3) δ
8.31 (s, 0.5H), 7.84 (s, 0.5H), 7.41−7.27 (m, 5H), 5.98−5.83 (m,
0.5H), 5.78 (d, J = 10.0 Hz, 0.5H), 5.68 (dd, J = 10.0, 3.4 Hz, 0.5H),
5.57 (d, J = 9.4 Hz, 0.5H), 3.94 (dd, J = 15.2, 8.3 Hz, 0.5H), 3.61 (dd, J
= 10.6, 6.7 Hz, 2H), 3.56−3.44 (m, 1.5H), 3.41 (dd, J = 7.5, 3.4 Hz,
0.5H), 3.33 (dd, J = 7.9, 3.4 Hz, 0.5H), 3.20 (d, J = 9.1 Hz, 0.5H), 3.07
(bs, 0.5H), 2.94 (bs, 0.5H), 2.87−2.76 (m, 0.5H), 2.70 (d, J = 18.8 Hz,
0.5H), 2.56−2.39 (m, 1H), 2.38−2.22 (m, 0.5H). 13C NMR (125
MHz, CDCl3) δ 176.0, 175.9, 174.0, 170.5, 170.1, 167.5, 133.9, 133.5,
129.5, 129.47, 129.2, 129.0, 128.7, 128.0, 127.7, 127.5, 126.9, 125.2,
53.8, 52.4, 45.1, 41.4, 41.3, 40.5, 39.2, 36.0, 33.1, 32.7, 28.2, 23.7;
HRMS (ESI−) for C17H17N2O4 (M − H) calcd, 313.1194; found,
313.1193.
rac-(3aR,4S,7aS)-2-(Benzyloxy)-3-oxo-2,3,3a,4,5,7a-hexahy-
dro-1H-isoindole-4-carboxylic Acid (13d). White solid. Yield:
59%. Rf = 0.24 (Hex/EtOAc 7:3). IR (film) νmax (cm−1) 3030, 1734,
1
1709; H NMR (400 MHz, CDCl3) δ (ppm) 7.44−7.42 (m, 2H),
7.39−7.36 (m, 3H), 5.71−5.64 (m, 2H), 5.00 (s, 2H), 3.39 (dd, J =
8.0, 3.6 Hz, 1H), 3.22 (t, J = 7.2 Hz, 1H), 3.04 (dd, J = 10.8, 7.6 Hz,
1H), 2.82−2.71 (m, 2H), 2.49−2.41 (m, 1H), 2.27 (dd, J = 12.4, 3.6
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ (ppm) 176.0, 170.4, 135.3,
129.9 (2C), 129.1, 128.7 (2C), 127.9, 125.0, 77.5, 51.4, 44.9, 35.9,
31.9, 28.0; HRMS (ESI+) for C16H17NO4Na (M + Na) calcd,
310.1055; found, 310.1049.
rac-(3aR,4S,7aS)-2-Benzyl-3-oxo-2,3,3a,4,5,7a-hexahydro-
1H-isoindole-4-carboxylic Acid (13e). White solid. Yield: 65%. Rf=
0.29 (Hex/AcOEt, 4:6). IR (film) ν
(cm−1) 3028, 1722, 1697,
max
1
1650; H NMR (500 MHz, CDCl3) δ (ppm) 9.15 (brs, OH), 7.36−
7.24 (m, 5H), 5.79 (dd, J = 10, 1.5 Hz, 1H), 5.72−5.69 (m, 1H), 4.53
(s, 2H), 3.45 (dd, J = 8.0, 3.5 Hz, 1H), 3.34 (dd, J = 8.5, 6.5 Hz, 1H),
3.01 (t, J = 10.5 Hz, 1H), 2.94 (brs, 1H), 2.80 (d, J = 19.0 Hz, 1H),
2.49−2.44 (m, 2H); 13C NMR (125 MHz, CDCl3) δ (ppm) 175.3,
174.7, 136.3, 128.9 (2C), 128.2, 128.1 (2C), 127.8, 125.3, 49.6, 47.1,
47.0, 36.5, 34.8, 28.2; HRMS (ESI+) for C16H17NO3Na (M + Na)
calcd, 294.1106; found, 294.1096.
rac-(3aS,4R,5S,7aR)-2-Benzyl-5-methyl-3-oxo-2,3,3a,4,5,7a-
hexahydro-1H-isoindole-4-carboxylic Acid (13f). White solid.
Yield: 51%. Rf = 0.46 (Hex/AcOEt, 3:7); 1H NMR (400 MHz,
CDCl3) δ (ppm) 7.35−7.22 (m, 5H), 5.73 (d, J = 10.0 Hz, 1H), 5.61−
5.59 (m, 1H), 4.51 (s, 2H), 3.32 (t, J = 8.4 Hz, 1H), 3.05 (d, J = 3.6
Hz, 1H), 3.02−2.97 (m, 2H), 2.88−2.81 (m, 1H), 2.42 (d, J = 12.8
Hz, 1H), 1.16 (d, J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
(ppm) 175.9, 174.9, 136.4, 134.2, 128.9 (2C), 128.1 (2C), 127.7,
General Procedure for Lactam Formation/Diels−Alder
Reaction. To a solution of diene (1 equiv) in CHCl3 (to a
concentration of 0.25M) was added maleic anhydride (1 equiv). After
stirring for 15 h, the solution was concentrated under reduced
pressure, and the two diastereomers were separable by precipitation in
a mixture of ether/DCM or by flash chromatography (Hex/EtOAc) to
afford the exo product.
I
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Journal of Medicinal Chemistry
Article
124.6, 49.5, 46.9, 44.4, 43.3, 34.6, 33.5, 22.0. Spectral data were
consistent with data reported in the literature.45
24.3, 22.4; HRMS (ESI+) for C22H28N3O4 (M+ H) calcd, 398.2074;
found, 398.2068.
rac-Benzyl ((3aR,7R,7aS)-7-Cyano-1-oxo-3,3a,7,7a-tetrahy-
dro-1H-isoindol-2(6H)-yl)carbamate (9a). Acid 13a (170 mg,
0.51 mmol) was dissolved, under argon atmosphere, in THF (6.4 mL),
then TEA (115 μL, 0.82 mmol) was added, and the reaction was
cooled at −15 °C. Then, EtCO2Cl was added, and the reaction was
stirred at −15 °C. After 15 min, a solution of NH4OH (30% in water)
was added, and the reaction was stirred 15 h at room temperature. The
reaction was extracted 3 times with EtOAc. The organic phases were
washed with water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The crude product was directly used in the
next reaction step. To a cooled solution of the amide (21 mg, 0.064
mmol) in THF (1 mL) was added TEA (27 μL, 0.191 mmol). After
stirring, TFAA (13.5 μL, 0.096 mmol) was added at 0 °C, and the
reaction mixture was stirred for 1 h at 0 °C. Then, the reaction was
quenched with water and extracted with CHCl3 (3×). The organic
phases were washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude product was purified
by flash chromatography (Hex/EtOAc 5:5), to give nitrile 9a as a
white solid (15 mg, 75%). Rf = 0.35 (1:1 Hex/AcOEt). IR (film) νmax
rac-N-((3aS,4R,7aR)-3-Oxo-4-(pyrrolidine-1-carbonyl)-
1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)-2-phenylacetamide
(15c). Acid 13c (200 mg, 0.636 mmol) was dissolved, under argon
atmosphere, in DMF (6 mL). The solution was cooled to 0 °C, and
TEA (443 μL, 3.18 mmol) was added followed by PivCl (110 μL,
0.890 mmol). After 1 h, pyrrolidine (282 μL, 3.18 mmol) was added,
and the mixture was stirred overnight at room temperature. The
reaction was quenched with H2O, extracted with EtOAc, and the
organic phases washed with 1 M HCl, saturated NaHCO3, and brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The exo compound was selectively precipitated using ether and
afforded the pure compound 15c as a white solid (70 mg, 30%). Rf =
0.13 (EtOAc). IR (film) υmax (cm−1) 3232, 3026, 2974, 2874, 1729,
1
1686, 1613, 1454, 1348; H NMR (400 MHz, CDCl3) δ (ppm) 7.77
(brs, NH), 7.35−7.27 (m, 5H), 5.85 (d, J = 10.0 Hz, 1H), 5.70−5.67
(m, 1H), 3.99−3.93 (m, 1H), 3.60 (s, 2H), 3.49−3.34 (m, 6H), 2.53−
2.46 (m, 2H), 2.31 (d, J = 18.4 Hz, 1H), 1.95−1.73 (m, 5H); 13C
NMR (125 MHz, CDCl3) δ (ppm) 173.4, 172.3, 169.8, 133.9, 129.5
(2C), 129.1 (2C), 127.5, 127.3, 126.1, 52.4, 47.0, 46.4, 46.0, 41.5, 33.3,
32.5, 28.4, 26.3, 24.4; HRMS (ESI+) for C21H25N3O3Na (M+ Na)
calcd, 390.1788; found, 390.1790.
1
(cm−1): 3278, 2923, 2241, 1754, 1738, 1711; H NMR (300 MHz,
CDCl3) δ (ppm) 7.45−7.29 (m, 5H), 6.94 (bs, 1H), 5.92 (d, 1H, J =
9.8 Hz), 5.74−5.65 (m, 1H), 5.26−5.06 (m, 2H), 3.70−3.42 (m, 3H),
3.21−2.99 (m, 1H), 2.63−2.54 (m, 2H), 2.46 (bd, J = 9.4 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ (ppm) 154.9, 135.3, 128.6 (2C), 128.5
(2C), 128.2, 126.0, 125.6, 119.4, 68.0, 51.9, 44.7, 33.5, 29.8, 23.6;
HRMS (ESI + ) for C17H17N3O3Na (M + Na) calcd, 334.1162; found,
334.1160.
rac-(3aR,7R,7aS)-2-(Benzyloxy)-7-(pyrrolidine-1-carbonyl)-
2,3,3a,6,7,7a-hexahydro-1H-isoindol-1-one (15d). Acid 13d
(150 mg, 0.522 mmol) was dissolved, under argon atmosphere, in
DMF (5 mL). The solution was cooled to 0 °C, and TEA (364 μL,
2.61 mmol) was added followed by PivCl (90 μL, 0.731 mmol). After
1 h, pyrrolidine (232 μL, 2.61 mmol) was added, and the mixture was
stirred overnight at room temperature. The reaction was quenched
with H2O, extracted with EtOAc, and the organic phases washed with
1 M HCl, saturated NaHCO3, brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
chromatographed on silica gel (Hex/EtOAc 2:8) to afford the product
15d as a white solid (90 mg, 51%). Rf = 0.25 (Hex/EtOAc 2:8). IR
rac-Benzyl((4R,7aR)-3-oxo-4-(pyrrolidine-1-carbonyl)-
1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)carbamate (15a).
Acid 13a (100 mg, 0.304 mmol) was dissolved, under argon
atmosphere, in DMF (3.5 mL). The solution was cooled to 0 °C,
and TEA (212 μL, 1.52 mmol) was added followed by PivCl (55 μL,
0.455 mmol). After 1 h, pyrrolidine (124 μL, 1.52 mmol) was added,
and the mixture was stirred overnight at room temperature. The
reaction was quenched with H2O and extracted with EtOAc, and the
organic phases washed with 1 M HCl, saturated NaHCO3, and brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was chromatographed on silica gel (Hex/EtOAc
2:8) to afford the product 15a as a white solid (19 mg, 16%). Rf = 0.29
(AcOEt). IR (film) υmax (cm−1) 3211, 2970, 2876, 1744, 1715, 1617,
1
(film) υmax (cm−1) 3480, 2951, 1705, 1634; H NMR (400 MHz,
CDCl3) δ (ppm) 7.40−7.37 (m, 2H), 7.35−7.32 (m, 3H), 5.75−5.71
(m, 1H), 5.53−5.49 (m, 1H), 4.93 (q, J = 8.8 Hz, 2H), 3.55−3.37 (m,
5H), 3.15−3.09 (m, 2H), 2.95 (dd, J = 6.8, 2.4 Hz, 1H), 2.89 (dd, J =
6.4, 3.6 Hz, 1H), 2.25−2.19 (m, 1H), 2.15−2.11 (m, 1H), 1.97−1.90
(m, 2H), 1.88−1.79 (m, 2H); 13C NMR (125 MHz, CDCl3) δ (ppm)
172.6, 170.9, 135.2, 129.8 (2C), 129.0, 128.6 (2C), 127.3, 125.9, 76.9,
52.1, 46.8, 46.0, 39.9, 35.7, 29.2, 26.3, 24.3, 24.1; HRMS (ESI+) for
C20H24N2O3Na (M+ Na) calcd, 363.1679; found, 363.1688.
1
1454, 1228; H NMR (500 MHz, CDCl3) δ (ppm) 7.37−7.32 (m,
5H), 6.85 (brs, NH), 5.87 (d, J = 10.0 Hz, 1H), 5.71 (d, J = 10.0 Hz,
1H), 5.15 (brs, 2H), 4.06−4.01 (m, 1H), 3.57−3.53 (m, 2H), 3.42−
3.39 (m, 4H), 2.50−2.46 (m, 1H), 2.35 (d, J = 20.0 Hz, 1H), 1.95−
1.90 (m, 2H), 1.88−1.74 (m, 2H), 1.25 (brs, 2H); 13C NMR (125
MHz, CDCl3) δ (ppm) 172.1, 155.1, 135.6, 128.7, 128.5, 127.5, 125.9,
68.0, 52.6, 47.0, 46.6, 46.0, 33.3, 32.3, 29.8, 28.5, 26.3, 24.4; HRMS
(ESI+) for C21H26N3O4 (M+ H) calcd, 384.1918; found, 384.1907.
rac-Benzyl((3aS,4R,5S,7aR)-5-methyl-3-oxo-4-(pyrrolidine-1-
carbonyl)-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)carbamate
(15b). Acid 13b (150 mg, 0.435 mmol) was dissolved, under argon
atmosphere, in DMF (4.2 mL). The solution was cooled to 0 °C, and
TEA (304 μL, 2.177 mmol) was added followed by PivCl (75 μL,
0.610 mmol). After 1 h, pyrrolidine (193 μL, 2.177 mmol) was added,
and the mixture was stirred overnight at room temperature. The
reaction was quenched with H2O, extracted with EtOAc, and the
organic phases washed with 1 M HCl, saturated NaHCO3, and brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was chromatographed on silica gel (Hex/EtOAc
75:25) to afford the product 15b as a white solid (105 mg, 61%). Rf =
0.19 (Hex/EtOAc 2:8). IR (film) υmax (cm−1) 3209, 2958, 2872, 1743,
1715, 1617, 1453, 1230; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.34−
7.32 (m, 5H), 6.74 (brs, NH), 5.84 (d, J = 10.0 Hz, 1H), 5.61 (d, J =
10.0 Hz, 1H), 5.18−5.11 (m, 2H), 4.06−4.01 (m, 1H), 3.57−3.40 (m,
6H), 3.00 (d, J = 4.0 Hz, 1H), 2.57−2.56 (m, 1H), 2.42 (brs, 1H),
1.95−1.91 (m, 2H), 1.89−1.74 (m, 2H), 1.16 (d, J = 7.0 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ (ppm) 173.8, 172.1, 155.2, 135.7, 133.8,
128.6, 128.4, 125.0, 67.7, 52.4, 47.0, 46.0, 44.3, 41.3, 33.9, 32.4, 26.2,
(3aR,7R,7aS)-2-Benzyl-7-(pyrrolidine-1-carbonyl)-
2,3,3a,6,7,7a-hexahydro-1H-isoindol-1-one (15e). Acid 13e (150
mg, 0.553 mmol) was dissolved, under argon atmosphere, in DMF
(5.3 mL). The solution was cooled to 0 °C, and TEA (385 μL, 2.764
mmol) was added followed by PivCl (95 μL, 0.774 mmol). After 1 h,
pyrrolidine (245 μL, 2.764 mmol) was added, and the mixture was
stirred 15 h at room temperature. The reaction was quenched with
H2O, extracted with EtOAc, and the organic phases washed with 1 M
HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
chromatographed on silica gel (Hex/EtOAc 2:8) to afford the product
15e as a white solid (105 mg, 58%). Rf = 0.16 (Hex/EtOAc 2:8). IR
(film) υmax (cm−1) 3211, 2970, 2876, 1744, 1715, 1617, 1454, 1228;
1H NMR (400 MHz, CDCl3) δ (ppm) 7.32−7.15 (m, 5H), 5.83 (dd, J
= 9.6, 1.6 Hz, 1H), 5.69−5.65 (m,1H), 4.59 (d, J = 15.0 Hz, 1H), 4.27
(d, J = 15.0 Hz, 1H), 4.21−4.15 (m, 1H), 3.51−3.42 (m, 5H), 3.32−
3.28 (m, 1H), 2.90 (dd, J = 10.8, 8.8 Hz, 1H), 2.50−2.40 (m, 1H),
2.36−2.32 (m, 2H), 2.03−1.75 (m, 4H); 13C NMR (125 MHz,
CDCl3) δ (ppm) 173.7, 172.5, 136.8, 128.8 (2C), 128.1 (2C), 127.6,
127.5, 126.4, 49.7, 48.5, 47.1, 46.9, 45.9, 34.0, 33.7, 29.1, 26.4, 24.5;
HRMS (ESI+) for C20H24N2O2Na (M+ Na) calcd, 347.1730; found,
347.1737.
(3aR,6S,7R,7aS)-2-Benzyl-6-methyl-7-(pyrrolidine-1-carbon-
yl)-2,3,3a,6,7,7a-hexahydro-1H-isoindol-1-one (15f). Acid 13f
(200 mg, 0.70 mmol) was dissolved, under argon atmosphere, in DMF
(6.7 mL). The solution was cooled to 0 °C, and TEA (488 μL, 3.50
J
DOI: 10.1021/acs.jmedchem.5b01296
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol) was added followed by PivCl (121 μL, 0.98 mmol). After 1 h,
pyrrolidine (311 μL, 3.50 mmol) was added, and the mixture was
stirred 15 h at room temperature. The reaction was quenched with
H2O, extracted with EtOAc, and the organic phases washed with 1 M
HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
chromatographed on silica gel (Hex/EtOAc 5:5) to afford the product
15f as a white solid (168 mg, 71%). Rf = 0.34 (Hex/EtOAc 3:7). IR
= 9.6 Hz, 1H), 5.64−5.61 (m, 1H), 5.18−5.11 (m, 2H), 4.67 (brs,
1H), 4.12−4.06 (m, 1H), 3.65−3.55 (m, 2H), 3.48−3.36 (m, 2H),
2.95 (d, J = 4.4 Hz, 1H), 2.65−2.60 (m, 1H), 2.46−2.38 (m, 1H),
2.22−2.09 (m, 4H), 1.18 (d, J = 7.6 Hz, 3H); 13C NMR (125 MHz,
CDCl3): δ (ppm) 173.8, 172.7, 155.1, 135.5, 133.7, 128.7 (3C), 128.6,
128.5, 125.0, 118.7, 68.0, 52.5, 46.9, 46.6, 44.3, 41.2, 33.7, 32.5, 30.0,
25.3, 22.4; HRMS (ESI+) for C23H26N4O4Na (M+ Na) calcd,
1
445.1846; found, 445.1836. Data for 16b: Rf = 0.70 (EtOAc); H
1
(film) υmax (cm−1) 2954, 2868, 1698, 1637, 1423, 1355, 1250; H
NMR (400 MHz, CDCl3): δ (ppm) 7.37−7.33 (m, 5H), 6.85 (brs,
0.4NH), 6.66 (brs, 0.6NH), 5.84 (d, J = 10.0 Hz, 1H), 5.73 (dd, J =
7.6, 1.2 Hz, 0.5H), 5.70−5.61 (m, 1H), 5.21−5.11 (m, 2H), 4.67−4.65
(m, 0.5H), 4.50−4.46 (m, 0.5H), 3.66−3.14 (m, 5H), 3.12−3.03 (m,
1H), 2.70−2.67 (m, 0.5H), 2.59−2.53 (m, 1H), 2.33−2.02 (m, 4H),
1.22−1.13 (m, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) 172.5,
172.4, 155.2, 154.9, 135.4, 134.5, 133.8, 128.8 (2C), 128.7 (2C), 128.4,
124.8, 124.2, 119.6, 118.5, 68.3, 68.1, 52.8, 52.5, 47.9, 46.7, 46.2, 42.0,
41.6, 34.1, 33.9, 32.6, 32.4, 32.2, 30.2, 25.5, 23.3, 22.1, 21.9; HRMS
(ESI+) for C23H26N4O4Na (M+ Na) calcd, 445.1846; found,
445.1839.
NMR (500 MHz, CDCl3) δ (ppm) 7.32−7.29 (m, 2H), 7.26−7.23
(m, 1H), 7.20−7.18 (m, 2H), 5.79 (dt, J = 10.0, 1.5 Hz, 1H), 5.57 (dt,
J = 10.0, 3.5 Hz, 1H), 4.62 (d, J = 15.0 Hz, 1H), 4.23 (d, J = 15.0 Hz,
1H), 4.19−4.14 (m, 1H), 3.54−3.43 (m, 4H), 3.29 (dd, J = 8.5, 7.5
Hz, 1H), 3.04 (d, J = 4.5 Hz, 1H), 2.89 (dt, J = 11.0, 8.5 Hz, 1H),
2.58−2.53 (m, 1H), 2.31 (dd, J = 13.0, 4.5 Hz, 1H), 2.01−1.76 (m,
4H), 1.14 (d, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ (ppm)
174.0, 172.3, 136.8, 133.8, 128.8 (2C), 128.1 (2C), 127.5, 125.5, 49.6,
47.1, 46.9, 46.1, 46.0, 41.6, 34.4, 34.2, 26.3, 24.4, 22.5; HRMS (ESI+)
for C21H27N2O2 (M+ H) calcd, 339.2067; found, 339.2062.
Benzyl ((3aS,4R,7aR)-4-((S)-2-Cyanopyrrolidine-1-carbonyl)-
3-oxo-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)carbamate
(7a) and Benzyl ((3aR,4S,7aS)-4-((S)-2-Cyanopyrrolidine-1-car-
bonyl)-3-oxo-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)-
carbamate (16a). Acid 13a (189 mg, 0.572 mmol) was dissolved,
under argon atmosphere, in DCM (4 mL). The solution was cooled to
0 °C, and TEA (400 μL, 2.86 mmol) was added followed by PivCl (70
μL, 0.572 mmol). After 1 h, a solution of (S)-cyano-pyrrolidine (184
mg, 0.686 mmol) and TEA (400 μL, 2.86 mmol) in DCM (2 mL) was
added, and the solution was stirred overnight at room temperature.
The reaction was quenched with H2O, extracted with EtOAc, and the
organic phases washed with 1 M HCl, saturated NaHCO3, and brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was chromatographed on silica gel (Hex/EtOAc
2:8) to afford the products 7a (20 mg) and 16a (50 mg) as white
solids (70 mg, 34%). IR (film) υmax (cm−1) 3269, 2980, 2956, 1742,
N-((3aS,4R,7aR)-4-((S)-2-Cyanopyrrolidine-1-carbonyl)-3-
oxo-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)-2-phenylaceta-
mide (7c) and N-((3aR,4S,7aS)-4-((S)-2-Cyanopyrrolidine-1-
carbonyl)-3-oxo-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)-2-
phenylacetamide (16c). The acid 13c (700 mg, 2.23 mmol) was
dissolved, under argon atmosphere, in DMF (15 mL). The solution
was cooled to 0 °C, and BOP (1.38 g, 3.12 mmol) was added, followed
by (S)-cyano-pyrrolidine (956 mg, 3.56 mmol) and TEA (1.5 mL,
11.13 mmol). The mixture was stirred 15 h at room temperature. The
reaction was quenched with H2O, extracted with EtOAc, and the
organic layer was washed with 1 M HCl, saturated NaHCO3, and
brine, dried over Na2SO4, filtered, and evaporated. The crude product
was chromatographed on silica gel (Hex/EtOAc 2:8 to EtOAc/MeOH
8:2) to afford the products 7c (70 mg) and 16c (59 mg) as white
solids (129 mg, 15%). IR (film) υmax (cm−1) 3271, 3027, 2926, 1727,
1
1680, 1649, 1428, 1345, 1188. Data for 7c: Rf = 0.15 (AcOEt); H
1
1714, 1651, 1431, 1312, 1232. Data for 7a: Rf = 0.42 (EtOAc); H
NMR (500 MHz, CDCl3): δ (ppm) 7.37−7.29 (m, 5H), 5.88 (dd, J =
9.6, 1.2 Hz, 1H), 5.73−5.69 (m, 1H), 4.68−4.65 (m, 1H), 4.06−4.00
(m, 1H), 3.64−3.59 (m, 3H), 3.49−3.32 (m, 4H), 2.57−2.49 (m, 2H),
2.38 (d, J = 18.0 Hz, 1H), 2.23−2.08 (m, 4H); 13C NMR (125 MHz,
CDCl3): δ (ppm) 173.1, 172.9, 169.7, 133.6, 129.5 (2C), 129.2 (2C),
127.8, 127.1, 126.0, 118.7, 52.5, 46.9, 46.5, 46.2, 41.7, 33.2, 32.4, 30.1,
28.0, 25.4; HRMS (ESI+) for C22H25N4O3 (M + H) calcd, 393.1921;
NMR (500 MHz, CDCl3): δ (ppm) 7.37−7.31 (m, 5H), 6.93 (brs,
NH), 5.88 (d, J = 10.0 Hz, 1H), 5.72 (d, J = 10.0 Hz, 1H), 5.14 (brs,
2H), 4.67 (brs, 1H), 4.10−4.06 (m, 1H), 3.60−3.56 (m, 2H), 3.43−
3.35 (m, 3H), 2.54−2.37 (m, 3H), 2.17−2.09 (m, 4H); 13C NMR
(125 MHz, CDCl3): δ (ppm) 173.5, 172.8, 155.1, 135.5, 128.7 (3C),
128.6, 128.4, 127.2, 125.9, 118.7, 68.0, 52.6, 46.9, 46.6, 46.4, 33.2, 32.3,
30.1, 28.1, 25.3; HRMS (ESI+) for C22H24N4O4Na (M+ Na) calcd,
431.1690; found, 431.1678. Data for 16a: Rf = 0.46 (EtOAc); 1H
NMR (400 MHz, CDCl3): δ (ppm) 7.38−7.31 (m, 5H), 6.87 (brs,
0.4NH), 6.69 (brs, 0.6NH), 5.87−5.84 (m, 1H), 5.80−5.70 (m, 1.6H),
5.20−5.11 (m, 2H), 4.68−4.66 (m, 0.5H), 4.52−4.47 (m, 0.5H),
3.66−3.54 (m, 1.7H), 3.51−3.34 (m, 3H), 3.30−3.14 (m, 1.3H), 2.86
(d, J = 10.0 Hz, 0.3H), 2.59−2.38 (m, 3H), 2.33−2.01 (m, 4.5H); 13C
NMR (125 MHz, CDCl3): δ (ppm) 172.6, 172.4, 155.1, 154.9, 135.6,
135.4, 128.8, 128.7, 128.6, 128.4, 128.1, 127.5, 125.6, 124.9, 119.5,
118.6, 68.2, 68.0, 52.8, 52.5, 47.8, 47.1, 46.7, 46.7, 46.6, 46.2, 34.2,
33.9, 32.5, 32.3, 30.1, 28.6, 28.5, 25.5, 23.2; HRMS (ESI+) for
C22H24N4O4Na (M+ Na) calcd, 431.1690; found, 431.1684.
1
found, 393.1924. Data for 16c: Rf = 0.25 (EtOAc); H NMR (500
MHz, CDCl3): δ (ppm) 8.04 (brs, 0.5NH), 7.66 (brs, 0.5NH), 7.35−
7.28 (m, 5H), 5.83 (td, J = 11.0, 1.0 Hz, 1H), 5.76−5.73 (m, 1H),
5.70−5.67 (m, 0.5H), 4.63 (d, J = 5.5 Hz, 0.5H), 4.38 (t, J = 7.0 Hz,
0.5H), 3.60−3.56 (m, 2.5H), 3.51−3.32 (m, 4H), 3.28−3.22 (m,
0.5H), 3.16−3.10 (m, 0.5H), 2.60 (td, J = 13.0, 5.0 Hz, 1H), 2.50−
2.42 (m, 1H), 2.33−2.00 (m, 5H); 13C NMR (125 MHz, CDCl3): δ
(ppm) 173.4, 172.9 (2C), 172.5, 169.9, 169.6, 133.9, 133.7, 129.5
(2C), 129.4 (2C), 129.1 (2C), 129.0 (2C), 127.9, 127.6, 127.5, 127.1,
125.8, 125.0, 119.4, 118.7, 52.6, 52.3, 47.7, 47.0, 46.7, 46.6, 46.3, 46.1,
41.4, 41.2, 34.1, 33.7, 32.5, 32.4, 32.2, 30.1, 28.5, 28.3, 25.4, 23.1;
HRMS (ESI+) for C22H25N4O3 (M + H) calcd, 393.1921; found,
393.1934.
(S)-1-((3aS,4R,7aR)-2-(Benzyloxy)-3-oxo-2,3,3a,4,5,7a-hexa-
hydro-1H-isoindole-4-carbonyl)pyrrolidine-2-carbonitrile (7d)
and (S)-1-((3aR,4S,7aS)-2-(Benzyloxy)-3-oxo-2,3,3a,4,5,7a-hex-
ahydro-1H-isoindole-4-carbonyl)pyrrolidine-2-carbonitrile
(16d). Acid 13d (215 mg, 0.748 mmol) was dissolved, under argon
atmosphere, in DMF (5 mL). The solution was cooled to 0 °C, and
BOP (463 mg, 1.047 mmol) was added, followed by (S)-cyano-
pyrrolidine (321 mg, 1.197 mmol) and TEA (521 μL, 3.741 mmol).
The mixture was stirred for 15 h at room temperature. The reaction
was quenched with H2O, extracted with EtOAc, and the organic layer
was washed with 1 M HCl, saturated NaHCO3, and brine, dried over
Na2SO4, filtered, and evaporated. The crude product was chromato-
graphed on silica gel (Hex/EtOAc 2:8) to afford the products 7d (100
mg) and 16d (80 mg) as white solids (180 mg, 66%). IR (film) υmax
(cm−1) 2926, 2880, 2238, 1708, 1648. Data for 7d: Rf = 0.25 (AcOEt);
1H NMR (400 MHz, CDCl3): δ (ppm) 7.42−7.36 (m, 5H), 5.78 (dd,
Benzyl ((3aS,4R,5S,7aR)-4-((S)-2-Cyanopyrrolidine-1-carbon-
yl)-5-methyl-3-oxo-1,3,3a,4,5,7a-hexahydro-2H-isoindol-2-yl)-
carbamate (7b) and Benzyl ((3aR,4S,5R,7aS)-4-((S)-2-cyanopyr-
rolidine-1-carbonyl)-5-methyl-3-oxo-1,3,3a,4,5,7a-hexahydro-
2H-isoindol-2-yl)carbamate (16b). Acid 13b (212 mg, 0.616
mmol) was dissolved, under argon atmosphere, in DMF (4.1 mL).
The solution was cooled to 0 °C, and BOP (381 mg, 0.862 mmol) was
added, followed by (S)-cyano-pyrrolidine (264 mg, 0.985 mmol) and
TEA (429 μL, 3.078 mmol). The mixture was stirred for 15 h at room
temperature. The reaction was quenched with H2O and extracted with
EtOAc, and the organic layer was washed with 1 M HCl, saturated
NaHCO3, and brine, dried over Na2SO4, filtered, and evaporated. The
crude product was chromatographed on silica gel (Hex/EtOAc 2:8) to
afford the products 7b (98 mg) and 16b (68 mg) as white solids (166
mg, 64%). IR (film) υmax (cm−1) 3256, 2959, 2875, 1708, 1645, 1498,
1
1427, 1301, 1231. Data for 7b: Rf = 0.58 (EtOAc); H NMR (400
MHz, CDCl3): δ (ppm) 7.38−7.31 (m, 5H), 6.73 (brs, NH), 5.86 (d, J
K
DOI: 10.1021/acs.jmedchem.5b01296
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 10.0, 2.0 Hz, 1H), 5.71−5.66 (m, 1H), 4.98−4.89 (m, 2H), 4.71−
4.68 (m, 1H), 4.19−4.13 (m, 1H), 3.65−3.59 (m, 1H), 3.41 (t, J = 7.2
Hz, 1H), 3.36−3.33 (m, 1H), 3.31−3.23 (m, 1H), 3.01 (dd, J = 10.8,
7.2 Hz, 1H), 2.52−2.36 (m, 2H), 2.27−2.09 (m, 5H); 13C NMR (125
MHz, CDCl3): δ (ppm) 172.7, 169.7, 135.3, 129.7 (2C), 129.0, 128.7
(2C), 127.3, 125.6, 118.7, 77.4, 51.2, 46.9, 46.6, 46.1, 33.2, 31.5, 30.1,
28.2, 25.3; HRMS (ESI+) for C21H23N3O3Na (M + Na) calcd,
388.1630; found, 388.1641. Data for 16d: Rf = 0.33 (Hex/EtOAc 2:8);
1H NMR (400 MHz, CDCl3): δ (ppm) 7.45−7.34 (m, 5H), 5.88 (d, J
= 6.8 Hz, 0.5H), 5.76−5.67 (m, 2H), 5.00−4.87 (m, 2H), 4.74 (dd, J =
7.2, 2.0 Hz, 0.5H), 4.62 (td, J = 8.8, 2.0 Hz, 0.5H), 3.68−3.62 (m,
0.5H), 3.48−3.34 (m, 2.5H), 3.19−3.13 (m, 0.5H), 3.05−2.97 (m,
2H), 2.46−2.03 (m, 7H); 13C NMR (125 MHz, CDCl3): δ (ppm)
172.6, 172.3, 169.7, 169.5, 135.5, 135.1, 129.9 (2C), 129.7 (2C), 129.1,
129.0, 128.8 (2C), 128.7 (2C), 128.0, 127.5, 125.4, 124.7, 119.6, 118.6,
77.9, 51.4, 51.1, 47.7, 47.2, 46.7, 46.5, 46.4, 46.1, 34.3, 34.1, 32.3, 31.8,
31.1, 30.2, 28.6, 28.6, 25.6, 23.1; HRMS (ESI+) for C21H23N3O3Na
(M + Na) calcd, 388.1632; found, 388.1634.
1351. Data for 7f: Rf = 0.54 (AcOEt); 1H NMR (400 MHz, CDCl3): δ
(ppm) 7.34−7.27 (m, 3H), 7.19−7.17 (m, 2H), 5.82 (dt, J = 10.0, 2.0
Hz, 1H), 5.59 (dt, J = 10.0, 2.8 Hz, 1H), 4.76−4.74 (m, 1H), 4.59 (d, J
= 14.8 Hz, 1H), 4.27−4.20 (m, 2H), 3.69−3.65 (m, 1H), 3.43−3.30
(m, 2H), 2.99 (d, J = 4.8 Hz, 1H), 2.93 (dd, J = 10.8, 8.8 Hz, 1H),
2.67−2.59 (m, 1H), 2.34 (dd, J = 12.4, 4.8 Hz, 1H), 2.27−2.14 (m,
4H), 1.18 (d, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm)
173.7, 173.1, 136.6, 133.7, 128.9 (2C), 128.0 (2C), 127.7, 125.4, 118.9,
49.7, 47.0, 47.0, 46.6, 46.1, 41.5, 34.3, 34.1, 30.2, 25.4, 22.5; HRMS
(ESI+) for C22H26N3O2 (M + H) calcd, 364.2019; found, 364.2015.
1
Data for 16f: Rf = 0.63 (EtOAc); H NMR (500 MHz, CDCl3): δ
(ppm) 7.33−7.25 (m, 3H), 7.20−7.15 (m, 2H), 5.99 (dd, J = 7.5, 1.5
Hz, 0.6H), 5.79 (dt, J = 10.0, 2.0 Hz, 1H), 5.65−5.59 (m, 1H), 4.73−
4.70 (m, 0.6H), 4.58 (d, J = 15.0 Hz, 0.6H), 4.52 (d, J = 15.0 Hz,
0.4H), 4.36 (d, J = 15.0 Hz, 0.4H), 4.22 (d, J = 15.0 Hz, 0.6H), 3.68−
3.64 (m, 0.6H), 3.49−3.39 (m, 1H), 3.34−3.30 (m, 1H), 3.20−3.07
(m, 2H), 2.98−2.92 (m, 1H), 2.71−2.66 (m, 0.6H), 2.61−2.57 (m,
0.4H), 2.43 (dd, J = 13.0, 5.0 Hz, 1H), 2.37−2.25 (m, 2H), 2.18−2.05
(m, 2H), 1.20 (d, J = 7.5 Hz, 1.6H), 1.16−1.08 (m, 1.4H); 13C NMR
(125 MHz, CDCl3): δ (ppm) 173.9, 173.2, 172.8, 136.5, 136.4, 134.4,
133.8, 128.9 (2C), 128.9 (2C), 128.0 (2C), 128.0 (2C), 127.9, 119.9,
118.6, 49.8, 49.3, 47.9, 47.2, 47.1, 46.8, 46.7, 46.4, 46.2, 45.9, 42.5,
42.1, 34.5, 34.4, 34.4, 34.3, 32.3, 30.2, 25.6, 23.3, 22.1, 22.0; HRMS
(ESI+) for C22H25N3O2Na (M + Na) calcd, 386.1839; found,
386.1822.
(S)-1-((3aS,4R,7aR)-2-Benzyl-3-oxo-2,3,3a,4,5,7a-hexahydro-
1H-isoindole-4-carbonyl)pyrrolidine-2-carbonitrile (7e) and
(S)-1-((3aR,4S,7aS)-2-Benzyl-3-oxo-2,3,3a,4,5,7a-hexahydro-
1H-isoindole-4-carbonyl)pyrrolidine-2-carbonitrile (16e). Acid
13e (158 mg, 0.582 mmol) was dissolved, under argon atmosphere, in
DMF (6 mL). The solution was cooled to 0 °C, and BOP (360 mg,
0.815 mmol) was added, followed by (S)-cyano-pyrrolidine (250 mg,
0.932 mmol) and TEA (406 μL, 2.910 mmol). The mixture was stirred
15 h at room temperature. The reaction was quenched with H2O and
extracted with EtOAc, and the organic layer was washed with 1 M
HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and
evaporated. The crude product was chromatographed on silica gel with
eluent (Hex/EtOAc 3:7) to afford the products 7e (43 mg) and 16e
(20 mg) as white solids (63 mg, 31%). IR (film) υmax (cm-1) 3476,
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01296.
1
2968, 2043, 1689, 1632. Data for 7e: Rf = 0.28 (AcOEt); H NMR
Experimental details (PDF)
Crystallographic data (CSV)
(500 MHz, CDCl3): δ (ppm) 7.36−7.28 (m, 3H), 7.22−7.20 (m, 2H),
5.89−5.86 (m, 1H), 5.74−5.70 (m, 1H), 4.79−4.77 (m, 1H), 4.60 (d, J
= 14.5 Hz, 1H), 4.31−4.25 (m, 2H), 3.71−3.67 (m, 1H), 3.47−3.39
(m, 2H), 3.35 (dd, J = 9.0, 7.5 Hz, 1H), 2.97 (dd, J = 11.0, 8.5 Hz,
1H), 2.56−2.49 (m, 1H), 2.46−2.44 (m, 1H), 2.40 (dd, J = 12.5, 4.5
Hz, 1H), 2.29−2.16 (m, 4H); 13C NMR (125 MHz, CDCl3): δ (ppm)
173.5, 173.2, 136.6, 128.9 (2C), 128.0 (2C), 127.7, 127.3, 126.3, 118.8,
49.8, 48.3, 47.0, 46.9, 46.6, 34.1, 33.6, 30.2, 28.6, 25.4; HRMS (ESI+)
for C21H23N3O2Na (M + Na) calcd, 372.1682; found, 372.1681. Data
for 16e: Rf = 0.44 (Hex/EtOAc 2:8); 1H NMR (400 MHz, CDCl3): δ
(ppm) 7.33−7.24 (m, 3H), 7.20−7.14 (m, 2H), 6.07 (dd, J = 7.2, 1.6
Hz, 0.5H), 5.84−5.80 (m, 1H), 5.76−5.67 (m, 1H), 4.77−4.71 (m,
0.5H), 4.55 (dd, J = 15.2, 6.4 Hz, 1H), 4.35 (d, J = 15.2 Hz, 0.5H),
4.24 (d, J = 14.8 Hz, 0.5H), 3.70−3.64 (m, 0.5H), 3.55−3.52 (m,
0.5H), 3.49−3.40 (m, 2H), 3.35−3.30 (m, 1H), 3.23−3.08 (m, 1H),
3.00−2.92 (m, 1H), 2.50−2.47 (m, 2H), 2.40−2.22 (m, 3H), 2.18−
2.05 (m, 2H); 13C NMR (125 MHz, CDCl3): δ (ppm) 173.6, 172.9,
172.8, 136.5, 136.4, 129.0, 128.9 (2C), 128.1, 128.0 (2C), 127.9, 127.8,
127.7, 127.6, 126.1, 125.4, 119.7, 118.6, 49.9, 49.4, 48.6, 48.6, 47.8,
47.2, 47.0, 46.8, 46.7, 46.2, 34.8, 34.5, 34.3, 34.2, 32.3, 30.2, 29.2, 29.0,
25.6, 23.2; HRMS (ESI+) for C21H23N3O2Na (M + Na) calcd,
372.1682; found, 372.1692.
(S)-1-((3aS,4R,5S,7aR)-2-Benzyl-5-methyl-3-oxo-
2,3,3a,4,5,7a-hexahydro-1H-isoindole-4-carbonyl)pyrrolidine-
2-carbonitrile (7f) and (S)-1-((3aR,4S,5R,7aS)-2-Benzyl-5-meth-
yl-3-oxo-2,3,3a,4,5,7a-hexahydro-1H-isoindole-4-carbonyl)-
pyrrolidine-2-carbonitrile (16f). Acid 13f (300 mg, 1.051 mmol)
was dissolved, under argon atmosphere, in DMF (7 mL). The solution
was cooled to 0 °C, and BOP (651 mg, 1.471 mmol) was added,
followed by (S)-cyano-pyrrolidine (451 mg, 1.680 mmol) and TEA
(733 μL, 5.256 mmol). The mixture was stirred 15 h at room
temperature. The reaction was quenched with H2O, extracted with
EtOAc, and the organic layer was washed with 1 M HCl, saturated
NaHCO3, and brine, dried over Na2SO4, filtered, and evaporated. The
crude product was chromatographed on silica gel (Hex/EtOAc 4:6) to
afford the products 7f (120 mg) and 16f (159 mg) as white solids (279
mg, 73%). IR (film) υmax (cm−1) 3027, 2960, 2873, 1690, 1647, 1425,
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 514-398-8543. Fax: 514-398-3797. E-mail, nicolas.
moitessier@mcgill.ca.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank CIHR for funding (operating grant). S.D.C. thanks
FQRNT for a scholarship. Natural Sciences and Engineering
Research Council of Canada (NSERC) for a grant to K.A., and
the FRQNT Center in Green Chemistry and Catalysis
(CGCC) at McGill University (Canada).
ABBREVIATIONS USED
■
BOP, benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium-
hexafluorophosphate; CNS, central nervous system; DCM,
dichloromethane; DMF, dimethylformamide; Piv, pivaloyl;
POP, prolyl oligopeptidase; TEA, trimethylamine; TFAA,
trifluoroacetic anhydride; THF, tetrahydrofuran
REFERENCES
■
(1) Lawandi, J.; Gerber-Lemaire, S.; Juillerat-Jeanneret, L.;
Moitessier, N. Inhibitors of prolyl oligopeptidases for the therapy of
human diseases: Defining diseases and inhibitors. J. Med. Chem. 2010,
53, 3423−3438.
(2) Lambeir, A. M. Translational research on prolyl oligopeptidase
inhibitors: The long road ahead. Expert Opin. Ther. Pat. 2011, 21,
977−981.
L
DOI: 10.1021/acs.jmedchem.5b01296
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Journal of Medicinal Chemistry
Article
(3) Lop
́
ez, A.; Tarrago,
́
T.; Giralt, E. Low molecular weight inhibitors
(19) Guterman, L. Covalent Drugs Form Long-Lived Ties. Chem.
Eng. News 2011, 89, 19−26.
of Prolyl Oligopeptidase: A review of compounds patented from 2003
to 2010. Expert Opin. Ther. Pat. 2011, 21, 1023−1044.
(4) Cunningham, D. F.; O’Connor, B. Identification and initial
characterisation of a N-benzyloxycarbonyl-prolyl-prolinal (Z-Pro-
prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-
methylcoumarin (Z-Gly-Bro-NH-Mec)-hydrolysing peptidase in bo-
vine serum. Eur. J. Biochem. 1997, 244, 900−903.
(20) Kalgutkar, A. S.; Dalvie, D. K. Drug discovery for a new
generation of covalent drugs. Expert Opin. Drug Discovery 2012, 7,
561−581.
(21) Robertson, J. G. Mechanistic basis of enzyme-targeted drugs.
Biochemistry 2005, 44, 5561−5571.
(22) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence
of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307−317.
(23) Potashman, M. H.; Duggan, M. E. Covalent modifiers: An
orthogonal approach to drug design. J. Med. Chem. 2009, 52, 1231−
1246.
(5) Toide, K.; Shinoda, M.; Iwamoto, Y.; Fujiwara, T.; Okamiya, K.;
Uemura, A. A novel propyl endopeptidase inhibitor, JTP-4819, with
potential for treating Alzheimer’s disease. Behav. Brain Res. 1997, 83,
147−151.
(6) Jarho, E. M.; Venalainen, J. I.; Huuskonen, J.; Christiaans, J. A.
̈
̈
(24) Smith, A. J. T.; Zhang, X.; Leach, A. G.; Houk, K. N. Beyond
picomolar affinities: Quantitative aspects of noncovalent and covalent
binding of drugs to proteins. J. Med. Chem. 2009, 52, 225−233.
(25) Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C.
Nitrile-containing pharmaceuticals: Efficacious roles of the nitrile
pharmacophore. J. Med. Chem. 2010, 53, 7902−7917.
(26) Huot, M.; Moitessier, N. Expedient synthesis of novel bicyclic
peptidomimetic scaffolds. Tetrahedron Lett. 2010, 51, 2820−2823.
M.; Garcia-Horsman, J. A.; Forsberg, M. M.; Jarvinen, T.; Gynther, J.;
Mannisto, P. T.; Wallen, E. A. A. A cyclopent-2-enecarbonyl group
́
̈
̈
mimics proline at the P2 position of prolyl oligopeptidase inhibitors. J.
Med. Chem. 2004, 47, 5605−5607.
(7) Jalkanen, A. J.; Hakkarainen, J. J.; Lehtonen, M.; Venalainen, T.;
̈
̈
Kaariainen, T. M.; Jarho, E.; Suhonen, M.; Forsberg, M. M. Brain
̈
̈
̈
pharmacokinetics of two prolyl oligopeptidase inhibitors, JTP-4819
and KYP-2047, in the rat. Basic Clin. Pharmacol. Toxicol. 2011, 109,
443−451.
́
(27) Szeltner, Z.; Rea, D.; Renner, V.; Fulop, V.; Polgar, L.
̈
̈
Electrostatic effects and binding determinants in the catalysis of prolyl
oligopeptidase: Site-specific mutagenesis at the oxyanion binding site.
J. Biol. Chem. 2002, 277, 42613−42622.
(8) Venalainen, J. I.; Garcia-Horsman, J. A.; Forsberg, M. M.;
̈
̈
Jalkanen, A.; Wallen
Gynther, J.; Mannisto, P. T. Binding kinetics and duration of in vivo
action of novel prolyl oligopeptidase inhibitors. Biochem. Pharmacol.
2006, 71, 683−692.
́
, E. A. A.; Jarho, E. M.; Christiaans, J. A. M.;
̈
̈
(28) Therrien, E.; Weill, N.; Tomberg, A.; Corbeil, C. R.; Lee, D.;
Moitessier, N. Docking ligands into flexible and solvated macro-
molecules. 7. Impact of protein flexibility and water molecules on
docking-based virtual screening accuracy. J. Chem. Inf. Model. 2014, 54,
3198−3210.
(9) Barelli, H.; Petit, A.; Hirsch, E.; Wilk, S.; De Nanteuil, G.;
Morain, P.; Checler, F. S 17092−1, a highly potent, specific and cell
permeant inhibitor of human proline endopeptidase. Biochem. Biophys.
Res. Commun. 1999, 257, 657−661.
(29) Racys, D. T.; Rea, D.; Fulop, V.; Wills, M. Inhibition of prolyl
̈
̈
oligopeptidase with a synthetic unnatural dipeptide. Bioorg. Med. Chem.
2010, 18, 4775−4782.
(10) Van Der Veken, P.; Fulop, V.; Rea, D.; Gerard, M.; Van Elzen,
̈
̈
R.; Joossens, J.; Cheng, J. D.; Baekelandt, V.; De Meester, I.; Lambeir,
A. M.; Augustyns, K. P2-substituted N-acylprolylpyrrolidine inhibitors
of prolyl oligopeptidase: Biochemical evaluation, binding mode
determination, and assessment in a cellular model of synucleinopathy.
J. Med. Chem. 2012, 55, 9856−9867.
(11) Lambeir, A. M. Interaction of prolyl oligopeptidase with α-
synuclein. CNS Neurol. Disord.: Drug Targets 2011, 10, 349−354.
(12) Myohanen, T. T.; Hannula, M. J.; Van Elzen, R.; Gerard, M.;
(30) Singh, T.; Biswas, D.; Jayaram, B. AADS - An automated active
site identification, docking, and scoring protocol for protein targets
based on physicochemical descriptors. J. Chem. Inf. Model. 2011, 51,
2515−2527.
(31) Tomberg, A.; De Cesco, S.; Huot, M.; Moitessier, N. Solvent
effect in diastereoselective intramolecular Diels−Alder reactions.
Tetrahedron Lett. 2015, 56, 6852−6856.
(32) Nguyen, L. A.; He, H.; Pham-Huy, C. Chiral drugs: An
overview. Int. J. Biomed. Sci. 2006, 2, 85−100.
(33) Morrison, J. F. Kinetics of the reversible inhibition of enzyme-
catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta
1969, 185, 269−286.
(34) Schiavini, P.; Pottel, J.; Moitessier, N.; Auclair, K. Metabolic
instability of cyanothiazolidine-based prolyl oligopeptidase inhibitors:
A structural assignment challenge and potential medicinal chemistry
implications. ChemMedChem 2015, 10, 1174−1183.
(35) Wong, Y. C.; Qian, S.; Zuo, Z. Regioselective biotransformation
of CNS drugs and its clinical impact on adverse drug reactions. Expert
Opin. Drug Metab. Toxicol. 2012, 8, 833−854.
(36) Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.;
Callegari, E.; Henne, K. R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.;
Nakai, Y.; O’Donnell, J. P.; Boer, J.; Harriman, S. P. A comprehensive
listing of bioactivation pathways of organic functional groups. Curr.
Drug Metab. 2005, 6, 161−225.
(37) Stepan, A. F.; Walker, D. P.; Bauman, J.; Price, D. A.; Baillie, T.
A.; Kalgutkar, A. S.; Aleo, M. D. Structural alert/reactive metabolite
concept as applied in medicinal chemistry to mitigate the risk of
idiosyncratic drug toxicity: A perspective based on the critical
examination of trends in the top 200 drugs marketed in the United
States. Chem. Res. Toxicol. 2011, 24, 1345−1410.
̈
̈
Van Der Veken, P.; García-Horsman, J. A.; Baekelandt, V.; Mannisto,
̈
̈
P. T.; Lambeir, A. M. A prolyl oligopeptidase inhibitor, KYP-2047,
reduces α-synuclein protein levels and aggregates in cellular and
animal models of Parkinson’s disease. Br. J. Pharmacol. 2012, 166,
1097−1113.
(13) Hannula, M. J.; Myohanen, T. T.; Tenorio-Laranga, J.;
̈
̈
Mannisto, P. T.; Garcia-Horsman, J. A. Prolyl oligopeptidase
̈
̈
colocalizes with α-synuclein, β-amyloid, tau protein and astroglia in
the post-mortem brain samples with Parkinson’s and Alzheimer’s
diseases. Neuroscience 2013, 242, 140−150.
(14) Lawandi, J.; Toumieux, S.; Seyer, V.; Campbell, P.; Thielges, S.;
Juillerat-Jeanneret, L.; Moitessier, N. Constrained peptidomimetics
reveal detailed geometric requirements of covalent prolyl oligopepti-
dase inhibitors. J. Med. Chem. 2009, 52, 6672−6684.
(15) De Cesco, S.; Deslandes, S.; Therrien, E.; Levan, D.; Cueto, M.;
Schmidt, R.; Cantin, L. D.; Mittermaier, A.; Juillerat-Jeanneret, L.;
Moitessier, N. Virtual screening and computational optimization for
the discovery of covalent prolyl oligopeptidase inhibitors with activity
in human cells. J. Med. Chem. 2012, 55, 6306−6315.
(16) Lawandi, J.; Toumieux, S.; Seyer, V.; Campbell, P.; Thielges, S.;
Juillerat-Jeanneret, L.; Moitessier, N. Constrained peptidomimetics
reveal detailed geometric requirements of covalent prolyl oligopepti-
dase inhibitors. J. Med. Chem. 2009, 52, 6672−6684.
(17) Johnson, D. S.; Weerapana, E.; Cravatt, B. F. Strategies for
discovering and derisking covalent, irreversible enzyme inhibitors.
Future Med. Chem. 2010, 2, 949−964.
(18) Zhou, S.; Chan, E.; Duan, W.; Huang, M.; Chen, Y. Z. Drug
bioactivation, covalent binding to target proteins and toxicity
relevance. Drug Metab. Rev. 2005, 37, 41−213.
(38) Gill, S. C.; von Hippel, P. H. Calculation of protein extinction
coefficients from amino acid sequence data. Anal. Biochem. 1989, 182,
319−326.
(39) Morrison, J. F. Kinetics of the reversible inhibition of enzyme-
catalysed reactions by tight-binding inhibitors. Biochimica et Biophysica
Acta (BBA) - Enzymology 1969, 185, 269−286.
M
DOI: 10.1021/acs.jmedchem.5b01296
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(40) Corbeil, C. R.; Englebienne, P.; Moitessier, N. Docking ligands
into flexible and solvated macromolecules. 1. Development and
validation of FITTED 1.0. J. Chem. Inf. Model. 2007, 47, 435−449.
(41) Therrien, E.; Englebienne, P.; Arrowsmith, A. G.; Mendoza-
Sanchez, R.; Corbeil, C. R.; Weill, N.; Campagna-Slater, V.; Moitessier,
N. Integrating medicinal chemistry, organic/combinatorial chemistry,
and computational chemistry for the discovery of selective estrogen
receptor modulatorswith FORECASTER, a novel platform for drug
discovery. J. Chem. Inf. Model. 2012, 52, 210−224.
(42) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 2008, 64, 112−122.
(43) Farrugia, L. J. WinGX suite for small-molecule single-crystal
crystallography. J. Appl. Crystallogr. 1999, 32, 837−838.
́ ́
(44) Gulías, M.; Duran, J.; Lopez, F.; Castedo, L.; Mascarenas, J. L.
̃
Palladium-Catalyzed [4 + 3] Intramolecular Cycloaddition of
Alkylidenecyclopropanes and Dienes. J. Am. Chem. Soc. 2007, 129,
11026−11027.
(45) Mellor, J. M.; Wagland, A. M. Synthesis of hydroisoindoles via
intramolecular Diels-Alder reactions of functionalised amido trienes. J.
Chem. Soc., Perkin Trans. 1 1989, 997−1005.
N
DOI: 10.1021/acs.jmedchem.5b01296
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