Pipecolic esters as minimized templates for proteasome inhibition

Article abstract of DOI:10.1039/C9OB00122K

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic “proteolytic machine” such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.

Full text of DOI:10.1039/C9OB00122K

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Pipecolic Esters as Minimized Templates for Proteasome
Inhibition
a
b
a
b
a
Matthew B. Giletto, Pawel A. Osmulski, Corey L. Jones, Maria E. Gaczynska * and Jetze J. Tepe *
Received 00th January 20xx,
Accepted 00th January 20xx
Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is
also the case for proteasome, a major intracellular protease and a target of anti‐cancer drugs. All clinically used proteasome
inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable
resistance associated with this type of inhibition drives the search for non‐competitive agents. The multisubunit and
multicatalytic “proteolytic machine” such as the proteasome is occasionally found to be affected by agents with other
primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the
proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel
proteasome‐targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome
inhibition even without the macrocyclic context of the parent compound. By subsequent structure‐activity relationship
studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors.
Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar
efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into
one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a
docking area for proteasome‐regulating protein modules and the gate is critical for controlling access to catalytic chamber.
Thus, the pipecolic ester template represents a new and attractive mechanism for proteasome inhibition distinct from
classical competitive drugs.
DOI: 10.1039/x0xx00000x
www.rsc.org/
including changes in specificity or stability.13 Such effects extending
well beyond the activity suppression offered by the common
competitive inhibitors provide an attractive opportunity to
Introduction
The proteasome is an essential protease that regulates intracellular
processes and maintains biological homeostasis through the
proteolytic degradation of misfolded and redundant proteins.1
Inhibition of this essential proteasomal service induces apoptosis, a
overcome the resistance to competitive drugs or to fine‐tuning the
1
4
physiological performance of the proteasome.
Thus, these
unexpected secondary target compounds may provide templates
for design of new proteasome regulators.1 Here, we present the
case of using a pharmacophore derived from rapamycin.
3,15
2
trait especially pronounced in rapidly proliferating cancer cells. Not
surprisingly, proteasome inhibitors became successful drugs for
blood cancers, most notably multiple myeloma and mantle cell
As we reported before, the natural product and established
immunosuppressive drug rapamycin allosterically inhibits the
3
‐7
lymphoma. All the clinically used proteasome inhibitors are small
molecules designed to specifically and competitively block the major
active center of this multi‐catalytic protease. However, it is not
uncommon for the proteasome to be affected by compounds with
non‐proteasomal primary targets. Notable examples include
1
2
human catalytic core of the proteasome. Rapamycin and its close
homologs (rapalogs) that are used as anti‐cancer drugs are
macrocyclic compounds sharing common “binding” and “effector”
1
6
domains. The former domain promotes dimerization of mTOR
mammalian/mechanistic target of rapamycin) and FKBP12 (FK‐
(
8
9
chloroquine, proline and arginine (PR) rich cathelicidin PR39,
binding protein 12), whereas the latter allosterically inhibits the
1
0
11
12
ritonavir, chlorpromazine and rapamycin. Established or
putative proteasome binding sites for these compounds are
positioned away from catalytic centers. The consequent
noncompetitive inhibition relies on allosteric mechanisms and
results in diverse effects on performance of the proteasome,
kinase activity of mTOR.1 The mTOR signaling pathway is one of
7,18
1
9
major regulators of intracellular homeostasis.
The anti‐
proteasome actions of rapamycin are exerted at much higher
concentrations than the mTOR inhibition and thus would have no
physiological relevance if not for some additional important
observations. Namely, we found that seco‐rapamycin, a primary
metabolite and the ester hydrolysis product of rapamycin, still
aDepartment of Chemistry, Michigan State University, East Lansing, Michigan
12
inhibits the proteasome while not affecting the mTOR pathway.
4
8824, United States
Thus, the opportunity arose to search for a novel proteasome‐
targeting pharmacophore.
The proteasome is a multi‐subunit, multi‐activity and modular
enzyme. The module responsible for actual cleavage of
polypeptides, the 20S core particle, is a barrel‐shaped structure built
b
Institute of Biotechnology, University of Texas Health Science Center at San
Antonio, 15355 Lambda Drive, San Antonio, Texas 78245, Unites States †
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Figure 1. Design of proteasome‐targeting pharmacophore of rapamycin. We found rapamycin to noncompetitively inhibit the core proteasome and
1
1
traced down the putative pharmacophore to the binding domain.
from four stacked pseudoheptameric rings. The inner β‐rings host
three pairs of active centers enclosed in a catalytic chamber and
specialized in cleaving after hydrophobic or branched amino acids
minimization, we replaced the hemi‐acetal moiety of the minimal
binding domainwith a cyclohexyl moiety (R ), as we investigated the
requirements of the R ‐moiety (Table 1). Prior work by Hauske
1
2
(
the “workhorse” chymotrypsin‐like activity; ChT‐L), basic (trypsin‐
illustrated the significance of the ketone as a critical recognition
moiety in FKBP binding, thus our first goal was to eliminate this
like; T‐L) and acidic or small neutral amino acids (caspase‐like or
20
29
post‐glutamyl peptide hydrolysing; PGPH).
The outer α‐rings
interaction. Replacement of the ketone with a large hydrophobic
provide gate regulating access to the catalytic chamber and also
form a binding interface (“‐face”) for additional protein modules.
The most common and physiologically important module is the
multisubunit 19S regulatory particle that can “cap” the core and
substitute is known to further provide high selectivity against
FKBP51, whereas smaller substituents led to loss of FKBP51 activity
and binding. To avoid a positive interaction with FKBP residues
3
0
known to interact with either the hemiketal hydroxyl groups of
2
1
31‐34
form the 26S proteasome assembly, 19S‐20S‐19S. The core
particle alone can degrade peptides and unfolded or poorly
structurally organized proteins. However, at least one 19S module
is required to allow the proteasome to recognize and process
substrates tagged for degradation by chains of small protein
FK506 or its urea’s analogues,
we focused primarily on
carbamate‐type pipecolic acids, including benzylcarbamate as these
Scheme 1a
2
2
ubiquitin.
proteins, deubiquitinates and unfolds them, and threads into the
The regulatory particle binds polyubiquitinated
2
0S core for cleavage. The 20S proteasome exists primarily as a
closed‐gate (inactive) conformation in its latent form.2
Conformational changes leading to gate opening can be induced by
docking an activator protein onto the ‐ring, by inserting the
3‐25
“
anchors” of 19S Rpt subunits (regulatory particle ATPases) into ‐
2
1,25‐
ring pockets and by allosteric signaling from the active centers.
2
7
Interestingly, rapamycin was found to allosterically modulate
proteolytic activity by putatively binding to the ‐face (the outer
1
2
surface of α rings) of the 20S proteasome. Since linear seco‐
rapamycin and single (binding) domain macrocycles FK506 and
pimecrolimus all displayed proteasome inhibiting actions, we
considered using the binding domain as a proteasome‐targeting
pharmacophore. We describe herein the development of small
molecules with a pharmacophore resembling the “binding domain”
of rapamycin, as novel scaffolds capable of allosteric regulation of
the proteasome. These studies extend our earlier work on a class of
B1 compounds derived from the binding domain, which we found to
aReagents and conditions: (a) R
TEA, DMAP and R OH.
2
1
2 3 2
‐Cl, Na CO , H O, dioxane; (b) EDCI,
were previously found to be depleted of FKBP activity.29 The
compounds were prepared starting with commercially available C2‐
or C3‐ substituted pipecolic acids. Acylation of pipecolic acid with
acyl chlorides or chloroformates followed by EDCI coupling of the
carboxylic acid with the respective alcohols yielded products 2‐11
(Scheme 1). Similarly, acylation of proline using various
benzylchloroformate followed by EDCI coupling of the
corresponding alcohols provided 12‐15.
inhibit the proteasome, attenuate proliferation of cultured cancer
cells and suppress tumor growth in mouse xenograft models.28
We evaluated the compounds using the human housekeeping
0S proteasome. It was activated with the Rpt5 derived 10‐residue
2
Results and discussion
peptide (KKKANLQYYA; “tail of Rpt5”; “tRpt5”) that contains the C‐
terminal Hb‐Y‐X (hydrophobic‐Tyr‐any AA) motif activating the
proteasome in trans and to some extent mimics the activation by
Our goal was to minimize the structure of rapamycin as a starting
point for analog design. We identified the piperidine ring in the
minimal domainof the parent pharmacophore (Figure 1), as an ideal
starting template for functionalization. For the purpose of substrate
1
2,35
the 19S regulatory particle.
Proteolytic activity was monitored
2
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structure‐activity relationship studies focused on the S‐enantiomer.
n =
R
1
R
2
CT‐L
IC50 (M)
n
3
3
2
1
R
1
R
2
IC50
(
M)
B1
3
5.8
(
±)
1
2
23.8
2
(
±)
3
3
3
11.5
2.0
1
3
6
.6
3
4
14
19.2
15.2
1
5
33.5
5
6
7
3
2
1
>50
20.1
19.8
Table 2. Inhibition of 20S CT‐L activity by proline
derivatives.
Replacement of the benzyl carbamate with the aliphatic mimic
carbamate 4, resulted in a significant drop in activity (IC50 15.2 M).
Further reduction of size (methylcarbamate 5) completely
2
abrogated the inhibitory effect. In addition, shortening of the R ‐
chain length from n=3 to n=2 (compound 6) or to one methylene
unit (compound 7) also significantly reduced its effectiveness.
Fortunately, we found that the trimethoxy‐moieties did not
significantly impact the overall potency of the ester side chain and
that the free aryl group (compound 8) was found to be a potent 20S
proteasome inhibitor (IC50 2.6 M). The aryl moiety did seem to be
important for inhibition, since the alkyl ester 9 and carboxylic acid
8
9
3
3
0
3
2.6
>50
>50
14.7
H
H
1
0 were found to be inactive. Changes in the positioning of the
10
ester (C‐2 substitution versus C‐3 substitution) also resulted in a
drop in potency (compound 11, IC50 14.7 M).
Next, we investigated the ring size requirements by replacing
the pipecolic acid with proline. The proline analogues followed the
same trends as the pipecolic acid derivatives. The proline derivative
1
2 only had modest activity, whereas the derivative 13 exhibited
Rapamycin
NA
NA
NA
6.8
good potency (IC50 6.6 M). Reduction of the R ‐ester side chain
2
length (compounds 14 and 15) also decreased activity, consistent
with the trend seen with the pipecolic acids 7 and 8.
Table 1. Inhibition of ChT‐L activity of tRpt5‐activated 20S by pipecolic
acid derivatives. Rapamycin serves as a reference.
over time using a fluorogenic peptide substrate specific for the
chymotrypsin‐like active center (ChT‐L, Suc‐LLVY‐AMC) in the
presence of a concentration range of the inhibitors (Tables 1 and 2).
In this activity assay, hydrolysis of the substrate results in release of
the highly fluorescent aminomethylcoumarin fluorophore. We used
the linear portions of the fluorescence intensity plots to calculate
the rates of Suc‐LLVY‐AMC hydrolysis and then to determine the IC50
(
concentration of drug at which 50% of the maximum rate inhibition
occurs). Starting with the R ‐moiety, we first replaced the
1
Figure 2. Docking of scaffold 3 indicates preference for binding to the ‐
ring on the 20S proteasome. In particular, the compound enters deep
into the pocket between subunits 2 and 3. In the 26S proteasome
assembly this pocket provides a binding site for the C‐terminal fragment
of regulatory particle ATPase Rpt6.
cyclohexyl‐oxoacetamide of the lead agent B1, with a benzyl
carbamate to render our abridged scaffold, compound 2. The
racemate compound 2 reduced the ChT‐L activity of the proteasome
with an IC50 of 11.5 M, which provided a good starting point to
interrogate the structural requirements for activity of this new
template. Next, we prepared the enantiomer of 2, compounds (3S)
starting from the corresponding chiral pipecolic acids. Gratifyingly,
compound 3 was highly effective in reducing the ChT‐L peptidase
activity of the 20S with IC50 of 2.0 M. Considering the
stereochemical match with rapamycin and B1, we pursued our
We conducted unbiased in silico docking studies to gain insight
into a possible binding site of the compound in the 20S
1
1
proteasome. We used Autodock Vina run through PyRx to manage
3
6,37
the workflow.
For these studies, we examined several of our
most active and inactive agents. The compounds were geometry
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optimized with the MM2 force field. Autodock Vina identified
molecular conformations with the best fit and strongest binding
affinity (global minimums). These docking studies suggest that the
compounds bind into the /‐ intersubunit pockets of the rings,
as seen with endogenous Hb‐Y‐X ‐ tails of the regulatory particles
the Hb‐Y‐X containing anchor subunits Rpt5, Rpt3 and Rpt2 in
docking into the  face pockets and promoting an intermediate
conformation between latent and activated (open gate)
3
9
proteasomes. The C‐terminus of the Rpt6 subunit does not display
the Hb‐Y‐X motif and thus it is not expected to activate the 20S core
in trans. However, addition of the tRpt6 peptide to the 20S
proteasome before addition of compound 3 significantly attenuated
inhibition of the core (Fig. 4). No attenuation was observed when
compound 3 was pre‐incubated with the core before addition of
tRpt6 (Fig. 4). Importantly, pre‐incubation with tRpt6 did not affect
inhibition of the proteasomal ChT‐L peptidase by a competitive
inhibitor, Z‐LLL‐VS. Namely, treatment with 0.5 µM Z‐LLL‐VS lowered
the activity of the latent core to 54% ± 4%. The activity remained at
(
RPs or caps).21 Compound B1 and scaffold 3 indicated a preference
for binding deep into the hydrophobic binding pocket below the
2/3 intersubunit cavity in the ‐ring (Fig. 2), The pocket is used

as the binding site for the Rpt6 C‐terminal “tail” of the Rpt6 subunit
of the 19S regulatory particle. Compound 3 displayed preference for
the 2/3 intersubunit pockets (>0.6Kcal/mol favored over the

3/4 and 6/7 sites targeted), with 4 of the 9 top biding modes
residing within the 2/3 binding pocket.
5
4% ± 6% (n=3) when the proteasome was pre‐incubated with
Next, we tested the biochemical effects of the most potent
compound 3 in more detail. The leading ChT‐L activity of the 20S
proteasome was not the only core peptidase affected by compound
tRpt6, and at 58% ± 8% when tRpt6 was added last.
3
. The PGPH activity was inhibited as well, with IC50 2.6 μM (Fig. 3a).
Figure 4. The C‐terminal
peptide derived from Rpt6
subunit compromises
inhibition of the ChT‐L activity
of the latent core by the
compound 3, however only
To the contrary, the T‐L peptidase was not affected (Fig. 3a).
Importantly, the activation of 20S with the tRpt5 was not mandatory
to inhibit the ChT‐L peptidase by compound 3. As demonstrated in
Fig. 3b, inhibition of the latent core proceeded with a respectable
IC50 of 2.8 μM, however it plateaued at about 40% of activity left.
Neither PGPH nor T‐L peptidases were significantly affected, with
IC50>50 μM.
when
added
to
the
proteasome prior to the
inhibitor. The peptide alone
did not affect the proteasome
activity. 1 µM compound 3
and 2 µM tRpt6 were tested.
Mean ± SD; n= 3 to 6.
a
b
We interpret the result as a strong indication of competition
between the tRpt6 and compound 3 for binding to the core,
consistent with the outcome of in silico docking studies (Fig. 2). For
comparison, no competition between tRpt5, expected to occupy the

5/6 pocket, and compound 3 was detected. The tRpt5 peptide
was used at the maximal‐activation saturating concentration (10
μM), and the order of addition of tRpt5 and compound 3 did not
compromise the inhibition (see caption to Fig. 3).
Figure 3. Compound 3 inhibits selected peptidase activities of the core
0S proteasome with low micromolar IC50. (a) Not only ChT‐L but also
2
PGPH peptidase was inhibited with low‐micromolar IC50 by the
compound when the core was activated with ten‐residue C‐terminal
peptide tRpt5 derived from the 19S ATPase Rpt5. The maximal 10‐fold
activation was induced by the 10 μM tRpt5, and was used in all the
assays. tRpt5 was routinely added to the reaction buffer before the
inhibitor. Adding compound 3 before tRpt5 did not significantly affect
the proteasome response: the remaining ChT‐L activity was 56% ±4%
The putative binding site of compound 3 in the α ring pocket,
identified by molecular modeling (Fig. 2) and biochemical data (Fig.
4), inspired us to test whether the inhibitor induced conformational
changes in α face gate dynamics. For this purpose, we employed
imaging of native 20S proteasomes with our established method of
the atomic force microscopy (AFM). In this method, topography of
the α face of buffer‐submerged proteasomes that stand on their
opposite α face is rendered in real time by the ultra‐sharp probe. A
tip of the probe interacts with the molecules mostly by van der
(
tRpt5 last) versus 53% ±7% (tRpt5 1 first; n=3 in both cases). (b) ChT‐L
peptidase of the latent core was inhibited by low micromolar
concentrations of compound 3. The PGPH peptidase was not affected
even by 10 μM compound 3 (99% ± 14% of control activity; n=5); only 50
μM of compound 3 reduced this activity to 71% ± 11% of the control
(
n=3). In turn, the T‐L peptidase was weakly inhibited even at low
40
Waals forces. We routinely assess the distribution of conformation
concentrations (with 1 µM of compound 3: 77% ± 11% of the control;
n=3). However, under the assay conditions, the activity was never
inhibited more than 50% (with 50 µM of compound 3: 64% ± 12%; n=4).
For comparison, rapamycin inhibited the ChT‐L and PGPH peptidases of
the latent core with IC50 =1.9 μM and 0.4 μM, respectively, and activated
the T‐L peptidase nearly two‐fold.12
types of α face with sequential scans of fields of multiply randomly
distributed proteasomes. Alternatively, we collect consecutive
single scan lines representing the gate status of individual particles.
We classify the particles as closed‐gate if their α face is smooth and
regularly concave (Fig. 5). In contrast, the open‐gate particles display
a centrally placed “crater”. In intermediate conformers, the α face
forms a slant that it is not concave and lacks a local‐minimum (see
Experimental section for description of numerical particle
Since the 2/3 cavity has been demonstrated as the docking
1
3
site of the C‐terminal “tail” of the Rpt6 subunit of the regulatory
classification). We found previously that the native 20S core
constantly switches between these gate conformations. The
abundance of conformers depends on a type of the ligand bound to
the core. Namely, in the latent core in the absence of any ligands,
except water molecules, the closed‐gate state prevails (about
particle,3
8,39
we explored the potential competition between
compound 3 and the 10‐residue peptide derived from C‐terminal
fragment of Rpt6 (KNMSIKKLWK; tRpt6). According to the most
recent model of the 26S proteasome dynamic cycle, Rpt6 succeeds
4
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peptides (derivatives of PR39), imidazoline derivative TCH‐165 and
rapamycin all induced a strong decrease in contribution of the
1
2,13,43
closed forms in the 20S particles population.
The activity of the
core proteasome was inhibited or activated by these compounds,
however a common trait was destabilization of the 26S proteasome.
1
2,13,43
Weakening of this most physiologically important
proteasome assembly by compound 3 would be expected, as in fact
2
8
it is suggested by our preliminary tests.
The effects of compound 3 observed in vitro prompted us to test
its influence on the proliferation of cultured cancer cells, namely the
multiple myeloma RPMI 8226 line. The treatment did lower the
content of live cells with EC50 of 230 nM (Fig. 6 left). Such relatively
low EC50, as compared to in vitro determined IC50 values for
peptidase activities (2‐3 μM; Fig. 3) should come to no surprise. Even
a partial destabilization of proteasome assemblies would be
Figure 5. Compound 3 destabilizes the  face of proteasome. Left: The
partition of conformers is shifted toward lower representation of the
closed‐gate particles and higher of the intermediate and open‐gate 20S
proteasomes upon treatment with 10 µM of compound 3. 269 events for
control and 408 events for compound 3 – treated 20S from 3 independent
experiments were analysed. “Event” was a single run of the AFM probe
across the proteasome particle. The differences of abundance between
closed and intermediate forms were statistically significant, with p<0.0001
o
and p<0.0005 (T test). Right: AFM images (pseudo 3D, 20 tilt) of an α face
of typical closed, intermediate and an open particle (top to bottom). The
lighter shades of grey represent exposed (higher) portions of the  face of
top‐view particles.
Figure 6. The compound 3 inhibits proliferation of human multiple
myeloma RPMI 8226 cultured cells. However, it does not target
mTOR and FKBP12, the two proteins that rapamycin is binding. (a)
The cells were treated with compound 3 or the vehicle (DMSO) for
48 hours. The data are averages (±SD) of three‐to‐five independent
experiments. The calculated EC50 based on count of live cells was
230 nM. Under the same conditions treatment with 10 nM
bortezomib lowered the count to 60%±6%. (b) The Western blotting
of extracts from RPMI 8226 cells treated for 24 hours with
rapamycin (2 nM), a macrocyclic mimic of rapamycin binding
domain FK506 (10 µM), compound 3 (0.2 µM) or compound B1 (0.2
µM). Under no conditions the count of live cells fell below 50% of
the control. However, compound 3 and B1 inhibited the cells’
proliferation to 60%‐65% of control. Crude lysates prepared from
the harvested cells were separated by SDS‐PAGE, Western blotted
and probed with specific antibodies against the mTOR substrate S6,
the product of mTOR kinase PS6 (phosphorylated S6) and a “loading
control” housekeeping protein COX IV. Only rapamycin inhibited
phosphorylation of S6. In turn, FK506 in combination with
rapamycin suppressed the inhibition by competing for binding to
FKBP12. Compound 3 and B1 did not inhibit the S6 phosphorylation
and did not mimic the actions of FK506. The data are representative
of three independent experiments.
7
5%).41 Interestingly, the AFM‐detected partition of closed (most
stable), intermediate and open conformers in the free latent core
closely matches conformational landscape of the 19S‐bound core
recently revealed by cryoEM studies. Based on the AFM data, we
proposed a model of the allosteric positive feedback loop between
4
2
the active centers and the gate, with the catalytic act prompting
gate opening.25 Here, we found that presence of only 100 nM
compound 3 increased the abundance of closed‐gate conformers
and rendered the open‐gate forms nearly undetectable (Fig. 5). At 1
μM concentration of compound 3, the open‐gate proteasomes were
detectable again, but their contribution was only about a half of that
observed in the control particles. The landscape changed strikingly
when the proteasome particles were treated with 10 μM of
compound 3. Now, less than a half of the particles were in the
closed‐gate conformation, and the contribution of open‐gate and
intermediate forms increased about twice, comparing to the control
(Fig. 5). We interpret this continuous decline of contribution from
the closed particles in the inhibitor‐treated proteasomes as a result
of potential cooperativity between two identical allosteric sites
being progressively saturated with compound 3. It is plausible that
the higher ligand concentrations limit coordination between the
rings leading to a substantially larger representation of the
intermediate conformations. Although the proteasomes still could
flip between the gate conformations, their peptidolytic capabilities
were severely restricted at the highest tested concentration of
compound 3. We cannot completely exclude that the weak
attachment of α ring to a mica surface may limit ligand access to the
binding site or change the binding site structure or constrain its
dynamics effectively producing proteasomes with two binding sites
of different ligand affinity. However, we did not observe such
expected to significantly affect physiology of cancer cells. The in
vitro readout of single peptidase activities is a useful and convenient
measure of the catalytic prowess of proteasome. However,
especially in the case of allosteric regulators, it may not fully reflect
the important structural and functional effects. The anti‐
proliferative effects of compound 3 were not universal: the
monocyte‐like human U937 cells did not significantly respond to the
treatment with 1 µM or even 10 µM compound 3, with live cell count
of 93% and 89% of vehicle‐treated control, respectively (averages of
two experiments differing by <10%).
2
5,40
restrictions with other small ligands.
The presented results
strongly indicate a destabilization α face that interferes with the
allosteric network. This is not a surprising effect for the compound
that likely intercalates between the α subunits (Fig. 2). Such
disruption of the conformational equilibrium by allosteric regulators
has been observed before. For example, treatment with cathelicidin
Importantly, neither B1 nor compound 3 affected the activity of
mTOR. As demonstrated in Fig. 6 right, in the cells treated with B1
or compound
3 the canonical product of mTOR kinase,
phosphorylated ribosomal protein S6 (PS6) was readily detectable.
Also, even if B1 and compound 3 resemble the binding domain of
rapamycin, they did not emulate actions of macrocyclic binding
9
PR39 induced a massive shift toward intermediate forms. PR
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DOI: 10.1039/C9OB00122K
ARTICLE
Organic and Bioorganic Chemistry
domain mimics such as FK506. FK506 lacks the effector domain
needed for mTOR inhibition, however it binds to FKBP12. When
used in excess together with rapamycin that utilizes both FKBP12
and mTOR binding, FK506 abolishes the kinase inhibition. No such
actions were detectable with B1 and compound 3 used at
concentrations nearing EC50 (Fig. 6).
3
(CDCl ) (amide rotamers) δ: 7.35 – 7.25 (5H, m), 6.39 – 6.36 (2H, m),
5.15 – 5.10 (2H, m), 4.97 – 4.85 (1H, m), 4.17 – 4.04 (3H, m), 3.83 –
3.82 (9H, m), 3.10 – 2.94 (1H, app dt), 2.55–2.40 (2H, m), 2.25‐2.20
(1H, m), 1.98 – 1.88 (2H, m), 1.71 – 1.62 (3H, m), 1.47 – 1.39 (1H, m),
1
3
1.29 – 1.21 (1H, m). C NMR (125 MHz) (CDCl
3
) (amide rotamers) δ:
171.6, 156.7, 153.1, 136.7, 136.5, 136.1, 128.4, 127.9, 127.7, 105.2,
6
2
4
7.3, 67.2, 64.2, 60.8, 56.0, 54.6, 54.4, 41.9, 32.4, 30.3, 26.7, 24.7,
4.5, 20.7, 20.6. HRMS calc’d for [M+Na] = 494.2155, observed =
94.2157.
Conclusions
In summary, by systematically investigating the requirements for
1‐((benzyloxy)carbonyl) (S)‐piperidine‐2‐carboxylic acid (10). To a
100 mL round bottom containing a stir bar was added (S)‐piperidine‐
2‐carboxylic acid (0.500 g, 3.8 mmol) followed by a 1:1 mixture of
1 2
activity of the R moiety and the R ‐ester side chain, we generated a
low micromolar inhibitor of the 20S proteasome (compound 3).
Molecular docking, atomic force microscopy and biochemical
studies suggest that compound 3 destabilizes the α face and the
gate area by inserting deep into the inter‐subunit pocket in the
proteasome α ring. Thus, the pipecolic ester scaffold emerges as an
attractive template for a new class of allosteric proteasome
inhibitors that destabilize the α ring and the gate of the core 20S
proteasome, and attenuate the growth of cancer cells.
water: dioxane (20 mL). Stirring commenced and sodium carbonate
was added (0.525 g, 5.0 mmol). Gas evolved; once the evolution had
ceased most of the solid had dissolved. To this stirring mixture was
added Cbz‐Cl (0.325 mL, 0.391 g, 2.3 mmol) dropwise. The reaction
mixture was allowed to stir overnight. The following day, the
reaction mixture was concentrated to approximately half‐volume on
a rotary evaporator. This mixture was transferred to a separatory
funnel and extracted with DCM (50 mL). This extract was discarded.
In the separatory funnel, the aqueous layer was acidified by addition
of concentrated HCl until the aqueous layer was acidic (pH ~ 2 by pH
paper). The acidified aqueous layer was extracted with DCM (3 x 50
mL). The organic layers were combined and dried over sodium
sulfate and concentrated in vacuo to yield the product (>95% purity)
as a clear oil (0.904 g, 89 %). Spectroscopic data matches that
reported for this compound and the racemate.4
Experimental
Synthetic procedures
1
‐((benzyloxy)carbonyl) piperidine‐2‐carboxylic acid. To a 100 mL
round bottom containing a stir bar was added piperidine‐2‐
carboxylic acid (1.29 g, 10.0 mmol) followed by a water: dioxane
mixture (40 mL of a 1:1 mixture). Stirring commenced and sodium
carbonate was added (2.41 g, 23.0 mmol). Gas evolved; once the
evolution had ceased most of the solid had dissolved. To this stirring
mixture was added Cbz‐Cl (1.7 mL, 2.04 g, 12.0 mmol) dropwise. The
reaction mixture was allowed to stir overnight. The following day,
the reaction mixture was concentrated to approximately half‐
volume in vacuo. This mixture was transferred to a separatory
funnel and extracted with DCM (50 mL). This extract was discarded.
In the separatory funnel, the aqueous layer was acidified by addition
of concentrated HCl until the aqueous layer was acidic (pH ~ 2 by pH
paper). The acidified aqueous layer was extracted 3 times with DCM
4
1‐benzyl 2‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (S)‐piperidine‐1,2‐
dicarboxylate (3). To a 100 mL round bottom flask containing a stir
bar was added (S)‐2‐piperidine carboxylic acid (0.250 g, 1.9 mmol),
followed by a water: dioxane mixture (10 mL of a 1:1 mixture).
Stirring commenced and sodium carbonate was added (0.630 g, 6.0
mmol), followed by Cbz‐Cl (0.328 mL, 0.391 g, 2.3 mmol). The
reaction mixture was stirred at room temperature for 48 hours.
After this time, the reaction mixture was transferred to a separatory
funnel and made acidic by the dropwise addition of concentrated
HCl (pH ~ 2 measured by pH paper). The acidified reaction mixture
was extracted with DCM (3 x 20 mL). The organic layers were
combined, dried over sodium sulfate, and concentrated to dryness
in vacuo to yield the crude product as a clear oil (0.432 g, 91 %). The
crude mixture was carried on to the following step.
To a 50 mL round bottom flask containing a stir bar was added
(
50 mL per extraction). The organic layers were combined and dried
over sodium sulfate and concentrated in vacuo to yield the pure
product as a clear oil (2.3 g, 88 %). Spectroscopic data matches that
1
reported for this compound.(41) H NMR (500 MHz) (CDCl
3
) δ: 10.08
(
4
1
1H, br s), 7.37 ‐7.31 (5H, m), 5.16 (2H, m), 5.01 – 4.90 (1H, app d),
.10 (1H, app d), 3.10 – 2.97 (1H, m), 2.25 (1H, dd, J = 27.5, 12.5 Hz),
1
3
3
.73 – 1.63 (3H, m), 1.47 – 1.23 (2H, m). C NMR (125 MHz) (CDCl )
δ: 177.1, 177.1, 156.8, 156.1, 136.6, 128.7, 128.6, 128.1, 127.9,
1
‐benzyl 2‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (S)‐piperidine‐1,2‐
1
2
27.1 67.6, 67.5, 67.2, 54.5, 54.4, 42.0, 41.9, 26.8, 26.7, 24.8, 24.6,
0.8, 20.7.
dicarboxylate (0.432 g, 1.7 mmol) was added DCM (10 mL) followed
by TEA (0.696 mL, 0.505 g, 5.0 mmol), EDCI (0.380 g, 2.0 mmol), 3‐
(
3,4,5‐trimethoxyphenyl)propan‐1‐ol (0.384 g, 1.7 mmol), and
1
‐benzyl
2‐(3‐(3,4,5‐trimethoxyphenyl)propyl)piperidine‐1,2‐
DMAP (0.024 g, 0.20 mmol). The reaction vessel was sealed, flushed
with nitrogen, and stirred overnight. The following day, the reaction
mixture was transferred to a separatory funnel and washed with
water (50 mL). The organic layer was separated, dried over sodium
sulfate, and concentrated in vacuo to yield the product as an oil. The
crude material was purified by silica gel chromatography (1:3 ethyl
dicarboxylate (2). To a 250 mL round bottom flask containing a stir
bar and 1‐((benzyloxy)carbonyl)piperidine‐2‐carboxylic acid (5.6 g,
2
0.0 mmol) was added DCM (100 mL). Stirring commenced and the
following reagents were added in the order listed: TEA (4.2 mL, 3.0
g, 30.0 mmol), EDCI (4.20 g, 22.0 mmol), DMAP (0.248 g, 2.0 mmol),
and 3‐(3,4,5‐trimethoxyphenyl)propan‐1‐ol (4.47 g, 19.8 mmol,
dissolved in 10 mL DCM). The reaction mixture was sealed with a
septum, flushed with nitrogen, and allowed to proceed overnight.
The following day, the reaction mixture was transferred to a
separatory funnel and extracted with water. The organic layer was
dried over sodium sulfate and concentrated in vacuo to yield the
crude product. This material was purified by silica gel
acetate: hexanes, R
clear oil (0.527 g, 66 % yield). H NMR (500 MHz) (CDCl
f
= 0.22) to obtain the product (>95% purity) as a
1
3
) (amide
rotamers) δ: 7.36 – 7.30 (5H, m), 6.43 – 6.37 (2H, m), 5.16 – 5.11
2H, m), 4.98 – 4.86 (1H, m), 4.18 – 4.05 (3H, m), 3.86 – 3.83 (9H, m),
(
3
1
(
(
.12 – 2.89 (1H, m), 2.65 – 2.56 (2H, m), 2.27 – 2.12 (1H, m), 2.07 –
.89 (2H, m), 1.72 – 1.63 (2H, m), 1.48 – 1.40 (1H, m), 1.31 – 1.22
1H, m). 13C NMR (125 MHz) (CDCl
) (amide rotamers) δ: 171.6, 156
3
chromatography (1:3 ethyl acetate: hexanes, R
product (>95% purity) as a clear oil (4.7 g, 50 %). H NMR (500 MHz)
f
= 0.22) to yield the
rotamers of carbamate carbon in base‐line), 153.1, 136.7, 136.5,
1
6
|
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DOI: 10.1039/C9OB00122K
Journal Name
ARTICLE
1
5
36.2, 128.4, 128.0, 127.7, 105.2, 67.3, 67.2, 64.3, 64.2, 60.8, 56.0,
4.7, 54.4, 41.9, 32.4, 30.3, 30.2, 26.7, 24.7, 24.5, 20.7, 20.6. HRMS
the crude product as a clear oil. The crude material was purified by
silica gel chromatography (1:1 ethyl acetate: hexanes) to yield the
pure produce as a clear oil (0.238 g, 66 %). Spectroscopic data
calc’d for [M+H] = 472.2335, observed = 472.2333.
4
5 1
matches that reported for this compound. H NMR (500 MHz)
(CDCl ) (amide rotamers) δ: 9.65 (1H, br s), 4.90 (1H, app d), 4.04
1
‐(isobutoxycarbonyl) (S)‐piperidine‐2‐carboxylic acid. To a 50 mL
round bottom flask containing a stir bar was added (S)‐pipecolic acid
0.250 g, 1.9 mmol) and water (5 mL) followed by 2N aqueous
3
(1H, app dd), 3.72 (3H, app d), 3.06 – 2.94 (1H, m), 2.25 (1H, br t, J =
(
15.0 Hz), 1.73 – 1.64 (3H, m), 1.44 – 1.27 (2H, m). 13C NMR (125 MHz)
sodium hydroxide (0.200 g, 5.0 mmol in 5 mL water). Stirring
commenced and isobutyl chloroformate was added (0.275 mL,
3
(CDCl ) (amide rotamers): 177.5, 177.4, 157.3, 156.6, 54.3, 54.0,
53.0, 41.8, 41.6, 26.7, 26.5, 24.6, 24.4, 20.6.
0
.285 g, 2.1 mmol). The reaction mixture was allowed to stir
overnight. The following day, the reaction was quenched by the
addition of concentrated HCl (until the mixture was pH ~ 2 by pH
paper) and extracted with ethyl acetate (2 x 25 mL). The organic
layers were combined, dried over sodium sulfate, and concentrated
in vacuo to yield the crude product as an oil. The crude material was
purified by filtering through a plug of silica gel (1:2 ethyl acetate:
hexanes) to yield the pure product as a clear oil which solidifies on
standing (0.302 g, 69 %). H NMR (500 MHz) (CDCl
rotamers) δ: 10.31 (1H, br s), 4.91 (1H, br d), 4.05 (1H, br d), 3.92 –
1‐methyl 2‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (S)‐piperidine‐1,2‐
dicarboxylate (5). To a 50 mL round bottom flask was added 1‐
(methoxycarbonyl) (S)‐piperidine‐2‐carboxylic acid (0.238 g, 1.32
mmol) followed by DCM (10 mL). Stirring commenced and to the
reaction mixture was added the following (in the order listed): EDCI
(0.304 g, 1.6 mmol) and DMAP (0.060 g, 0.5 mmol). The reaction
vessel was sealed with a septa and flushed with nitrogen. To the
stirring reaction mixture was added via syringe (in the order listed):
TEA (0.350 mL, 0.252 g, 2.5 mmol) followed by 3‐(3,4,5‐
trimethoxyphenyl)propan‐1‐ol (0.293 g, 1.3 mmol dissolved in 3.0
mL DCM). The reaction mixture was allowed to stir overnight. The
following day, the reaction was quenched by the addition of water
(10 mL) and partitioned in a separatory funnel. The organic layer was
dried over sodium sulfate and concentrated to dryness in vacuo to
yield the crude product as an oil. The crude material was purified by
1
3
) (amide
3
.82 (2H, m), 3.07 – 2.95 (1H, m), 2.25 (1H, br t, J = 14.0 Hz), 1.98 –
1.90 (1H, m), 1.74 – 1.65 (3H, m), 1.46 – 1.40 (1H, m), 1.36 – 1.28
1
3
(
(
5
1H, m), 0.92 (6H, dd, J = 21.0, 7.0 Hz). C NMR (125 MHz) (CDCl
amide rotamers) δ: 177.7, 177.5, 156.9, 156.2, 71.9, 71.8, 54.2,
4.0, 41.7, 41.5, 27.9, 26.7, 26.5, 24.6, 24.4, 20.7, 20.6, 19.0. HRMS
3
)
calc’d for [M – H] = 228.1241, observed = 228.1233. IR (NaCl, DCM):
3
o
095, 2960, 2874, 1744, 1704, 1667, 1470. M.P. = 67 C.
silica gel chromatography (1:2 ethyl acetate: hexanes, R
f
= 0.42) to
1
yield the product (>95% purity) as a clear oil (0.222 g, 44 %). H NMR
1‐isobutyl 2‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (S)‐piperidine‐1,2‐
3
(500 MHz) (CDCl ) (amide rotamers) δ: 6.39 (2H, s), 4.95 – 4.80 (1H,
dicarboxylate (4). To a 100 mL round bottom flask containing a stir
bar was added 1‐(isobutoxycarbonyl)piperidine‐2‐carboxylic acid
m), 4.17 – 4.11 (2H, m), 3.87 – 3.82 (9H, m), 3.72 – 3.70 (3H, m), 3.08
– 2.92 (1H, m), 2.62 (2H, br t, J = 7.0 Hz), 2.25 – 2.23 (1H, m), 1.96
(2H, quintet, J = 7.0 Hz), 1.72 – 1.63 (4H, m), 1.44 – 1.42 (1H, br m),
(
0.302 g, 1.32 mmol) followed by DCM (10 mL). To this solution was
13
added 3‐(3,4,5‐trimethoxyphenyl)propan‐1‐ol (0.316 g, 1.4 mmol
dissolved in 2 mL DCM). Stirring commenced and the following
reagents were added in the order listed: EDCI (0.304 g, 1.6 mmol),
TEA (0.350 mL, 0.252 g, 2.5 mmol) and DMAP (0.06 g, 0.50 mmol).
The reaction vessel was sealed, flushed with nitrogen, and allowed
to stir overnight. The following day, the reaction mixture was
transferred to a separatory funnel and washed with water (50 mL).
The organic layer was dried with sodium sulfate and concentrated
in vacuo to yield the crude product as an oil. This material was
3
1.29 – 1.21 (1H, m). C NMR (125 MHz) (CDCl ): δ: 171.7, 157.2 and
156.56 (carbamate rotamers), 153.2, 136.7, 136.2, 105.2, 64.2, 60.8,
56.0, 54.3, 52.8, 41.8, 32.4, 30.3, 26.7, 24.7, 24.5, 20.7. HRMS calc’d
for [M+H] = 396.2022, observed = 396.2019.
(R)‐1‐((benzyloxy)carbonyl)piperidine‐3‐carboxylic acid. To a 100
mL round bottom flask containing a stir bar was added (R)‐3‐
pipecolic acid (1.0 g, 7.7 mmol) followed by 1,4‐dioxane (10 mL) and
water (10 mL). Stirring commenced and sodium carbonate was
added (2.1 g, 20.0 mmol) followed by Cbz‐Cl (1.20 mL, 1.44 g, 8.5
mmol). The reaction mixture was stirred overnight. The following
day the reaction was quenched by the addition of HCl (36 mmol HCl
10 mL of a 3.6 M solution). The reaction mixture was transferred to
a separatory funnel and extracted with ethyl acetate (3 x 20 mL per
extraction). The organic layers were combined, dried over sodium
sulfate, and concentrated in vacuo to yield the crude product as an
oil. This material was dissolved in a 1:1 mixture of ethyl acetate:
hexanes and passed through a plug of silica gel to yield the crude
purified by silica gel chromatography (1:3 ethyl acetate: hexanes, R
f
=
%
0.32) to yield the product (>95% purity) as a clear oil (0.301 g, 52
yield). H NMR (500 MHz) (CDCl ) (amide rotamers) δ: 6.39 (2H, s),
3
1
4
–
–
.95 – 4.83 (1H, br d), 4.17 – 3.91 (4H, m), 3.90 – 3.82 (9 H, m), 3.09
2.93 (1H, br dt), 2.62 (2H, br t, J = 7.0 Hz), 2.25 – 2.22 (1H, m), 1.99
1.89 (3H, m), 1.74 – 1.64 (4H, m), 1.45 – 1.43 (1H, m), 1.30 – 1.22
1
3
(
1H, m), 0.94 – 0.88 (6H, m). C NMR (125 MHz) (CDCl
3
) (amide
rotamers) δ: 171.8, 171.8, 156.8, 156.2, 153.1, 136.7, 136.2, 105.2,
7
2
=
1.7, 71.6, 64.3, 64.2, 60.8, 56.0, 54.5, 54.3, 41.7, 32.4, 30.3, 30.3,
7.9, 26.8, 26.7, 24.7, 24.5, 20.8, 20.6, 19.0. HRMS calc’d for [M+H]
438.2492, observed = 438.2494.
product as an oil that on standing turns into a waxy, white solid (1.74
1
g, 84 %). H NMR (500 MHz) (CDCl
3
) (amide rotamers) δ: 7.38 – 7.29
(
5H, m), 5.17 – 5.10 (2H, m), 4.27 – 4.15 (1H, m), 3.97 (1H, dt, J = 9.5,
1
‐(methoxycarbonyl) (S)‐piperidine‐2‐carboxylic acid. To a 50 mL
3.5, 3.5 Hz), 3.17 – 3.06 (1H, m), 2.96 – 2.90 (1H, m), 2.51 (1H, br s),
2.10 – 2.07 (1H, m), 1.74 – 1.65 (2H, m) 1.50 (1H, br s). C NMR (125
MHz) (CDCl ) (amide rotamers): 178.8, 155.2, 136.6, 128.5, 128.0,
1
3
round bottom flask containing a stir bar was added (S)‐pipecolic acid
0.250 g, 1.9 mmol) and water (5 mL) followed by 2N aqueous
(
3
sodium hydroxide (0.200 g, 5.0 mmol in 5 mL water). Stirring
commenced and methyl chloroformate was added (0.160 mL, 0.198
g, 2.1 mmol). The reaction mixture was allowed to stir overnight.
The following day the reaction was quenched with the addition of
concentrated HCl (until pH ~ 2 by pH paper) and extracted twice with
ethyl acetate (30 mL per extraction). The organic layers were dried
over sodium sulfate and concentrated to dryness in vacuo to yield
127.9, 67.2, 45.4, 44.1, 40.9, 27.0, 24.0. HRMS calc’d for [M+H] =
264. 1236, observed = 264.1259; calc’d for [M+Na] = 286. 1055,
o
observed = 286. 1058. M.P. = 97 C.
1‐benzyl
2‐(3,4,5‐trimethoxyphenethyl)
(S)‐piperidine‐1,2‐
dicarboxylate (6). To a 500 mL round bottom flask containing a stir
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ARTICLE
Organic and Bioorganic Chemistry
bar and (S)‐1‐((benzyloxy)carbonyl)piperidine‐2‐carboxylic acid
0.386 g, 1.5 mmol) was added DCM (10 mL). Stirring commenced,
ethyl acetate: hexanes, R
as a clear oil (0.276 g, 38 %). H NMR (500 MHz) (CDCl
rotamers) δ: 7.35 – 7.13 (10H, m), 5.16 – 5.13 (2H, m), 4.97 – 4.85
(1H, m), 4.15 – 4.05 (3H, m), 3.10 – 2.94 (1H, m), 2.68 – 2.61 (2H, m),
2.27 – 2.19 (1H, m), 1.98 – 1.89 (2H, m), 1.71 – 1.62 (3H, m), 1.47 –
f
= 0.30) to yield the product (>95% purity)
1
(
3
) (amide
and in the order listed was added TEA (0.417 mL, 0.303 g, 3.0 mmol),
EDCI (0.323 g, 1.7 mmol), DMAP (0.060 g, 0.50 mmol), and 2‐(3,4,5‐
trimethoxyphenyl)ethan‐1‐ol (0.318 g, 1.5 mmol). The reaction
vessel was sealed, flushed with nitrogen, and allowed to stir
overnight. The following day, the reaction mixture was quenched
with water (10 mL) and partitioned in a separatory funnel. The
organic layer was dried over sodium sulfate and concentrated in
vacuo to a yellow oil. The crude product was purified by silica gel
1
3
3
1.41 (1H, m), 1.29 – 1.21 (1H, m). C NMR (125 MHz) (CDCl ) (amide
rotamers) δ: 171.6, 156.5, 155.4, 141.0, 136.6, 128.4, 128.4, 127.9,
127.8, 126.0, 67.3, 67.2, 64.3, 54.6, 54.4, 41.9, 41.8, 32.0, 30.0, 26.8,
26.7, 24.7, 24.5, 20.7, 20.6. HRMS calc’d for [M+H] = 382.2018,
observed = 382.2044.
chromatography (1:3 ethyl acetate: hexanes) to yield the product
1
(
(
5
3
3
–
(
1
5
>95% purity) as a clear oil (0.145 g, 21 %). H NMR (500 MHz) (CDCl
3
)
1‐benzyl 2‐propyl (S)‐piperidine‐1,2‐dicarboxylate (9). To a 50 mL
round bottom flask containing a stir bar was added (S)‐2‐
(propoxycarbonyl)piperidin‐1‐ium chloride (0.220 g, 1.06 mmol)
followed by dioxane (2 mL) and water (2 mL). Stirring commenced
and sodium carbonate was added as a solid (0.315 g, 3.0 mmol),
followed by Cbz‐Cl (0.170 mL, 0.204 g, 1.2 mmol). The reaction
mixture was stirred at room temperature overnight. The following
day the reaction was quenched by the addition of HCl (24.0 mmol, 2
mL conc. HCl and 8 mL distilled water). The reaction mixture was
transferred to a separatory funnel and extracted with ethyl acetate
amide rotamers) δ: 7.37 – 7.29 (5H, m), 6.41 (2H, d, J = 14.0 Hz),
.16 – 5.10 (2H, m), 4.95 – 4.82 (1H, m), 4.38 – 4.29 (2H, m), 4.09 –
.99 (1H, m), 3.84 (6H, app d, J = 3.5 Hz), 3.81 (3H, app d, J = 5.0 Hz),
.00 – 2.80 (3H, m), 2.17 (1H, t, J = 16.0 Hz), 1.67 – 1.56 (3H, m), 1.43
1.38 (1H, m), 1.55 – 1.10 (1H, m). 1 C NMR (125 MHz) (CDCl
3
)
3
amide rotamers) δ: 171.6, 171.6, 156.5, 155.9, 153.2, 136.6, 136.6,
33.2, 133.1, 128.4, 128.0, 127.8, 105.7, 67.3, 67.1, 65.5, 65.4, 60.8,
6.0, 54.6, 54.4, 41.8, 41.7, 35.4, 35.3, 26.7, 24.6, 24.4, 20.6, 20.5.
HRMS calc’d for [M+Na] = 480.1998, observed = 480.2011.
(
3 x 15 mL per extraction). The crude material was concentrated in
1
(
1
‐benzyl 2‐(3,4,5‐trimethoxybenzyl) piperidine‐1,2‐dicarboxylate
7). To a 100 mL round bottom flask containing a stir bar was added
‐((benzyloxy)carbonyl)piperidine‐2‐carboxylic acid (0.435 g, 1.6
vacuo to yield an oil, which was purified by silica gel chromatography
(1:2 ethyl acetate: hexanes) to yield the product (>95% purity) as a
clear oil (0.266 g, 75 %). H NMR (500 MHz) (CDCl
1
3
) (amide rotamers)
mmol), followed by DCM 1 mL). The reaction mixture was stirred
and the following reagents were added (in the order listed): TEA
δ: 7.36 – 7.30 (5H, m), 5.17 – 5.09 (2H, m), 4.95 – 4.83 (1H, m), 4.13
– 4.03 (3H, m), 3.10 – 2.94 (1H, m), 2.27 – 2.19 (1H, m), 1.70 – 1.58
(5H, m), 1.46 – 1.40 (1H, m), 1.29 – 1.21 (1H, m), 0.94 – 0.88 (3H, m).
(
0.417 mL, 0.303 g, 3.0 mmol), EDCI (0.342 g, 1.8 mmol), and DMAP
1
3
(
0.060 g, 0.50 mmol). Stirring commenced and the reaction vessel
3
C NMR (125 MHz) (CDCl ) (amide rotamers): 171.7, 171.6, 156.5,
was sealed with a septa and flushed with nitrogen. To this mixture
was added 3,4,5‐trimethoxybenzyl alcohol (0.273 mL, 0.336 g, 1.7
mmol, dissolved in 2.0 mL DCM) via syringe. The reaction mixture
was allowed to stir overnight. The following day, the reaction
mixture was transferred to a separatory funnel and extracted with
water (50 mL) and brine (50 mL). The organic layer was dried over
sodium sulfate and concentrated in vacuo to yield the crude product
as a clear oil. The crude material was purified by silica gel
155.9, 136.6, 128.5, 128.4, 127.9, 127.7, 67.2, 67.1, 66.6, 54.6, 54.4,
41.8, 41.8, 26.8, 26.7, 24.7, 24.5, 21.9, 20.7, 20.6, 10.4. HRMS calc’d
for [M+H] = 306.1705, observed = 306.1708.
1‐benzyl 3‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (R)‐piperidine‐1,3‐
dicarboxylate (11). To a 100 mL round bottom flask containing (R)‐
1‐((benzyloxy)carbonyl)piperidine‐3‐carboxylic acid (0.542 g, 2.0
mmol) was added a stir bar followed by DCM (15 mL), EDCI (0.414 g,
2.2 mmol), TEA (0.696 mL, 0.505 g, 5.0 mmol), DMAP (0.060 g, 0.5
mmol), and 3‐(3,4,5‐trimethoxyphenyl)propan‐1‐ol (0.461 g, 2.0
mmol). The reaction mixture was stirred overnight. The reaction
mixture was transferred to a separatory funnel and washed with
dilute HCl (10 mL 5 % HCl v/v) followed by saturated aqueous sodium
bicarbonate (20 mL). The organic layer was dried over sodium
sulfate and concentrated in vacuo. The crude material was purified
by silica gel chromatography (1:2 ethyl acetate: hexanes) to yield the
chromatography (1:3 ethyl acetate: hexanes, R
final product (>95% purity) as a clear oil (0.371 g, 50 %). H NMR (500
f
= 0.28) to yield the
1
MHz) (CDCl
3
) (amide rotamers) δ: 7.35 – 7.25 (5H, m), 6.55 – 6.52
(
2H, m), 5.15 – 4.89 (5H, m), 4.14 – 4.03 (1H, m), 3.83 – 3.82 (9H, m),
3
1
1
1
4
4
.11 – 2.96 (1H, m), 2.28 – 2.21 (1H, m), 1.70 – 1.41 (4H, m), 1.28 –
.22 (1H, m). C NMR (125 MHz) (CDCl ) (amide rotamers) δ: 171.5,
3
56.4, 155.9, 153.3, 137.7, 136.5, 131.4, 131.2, 128.4, 128.4, 128.0,
27.7, 127.6, 104.9, 104.8, 67.3, 67.2, 66.8, 60.8, 56.1, 54.7, 54.4,
1.9, 41.8, 26.7, 24.7, 24.5, 20.7, 20.6. HRMS calc’d for [M+Na] =
66.1842, observed = 466.1845.
1
3
1
product (>95% purity) as a clear oil (0.453 g, 48 %). H NMR (500
MHz) (CDCl
3
) (amide rotamers) δ: 7.36 – 7.30 (5H, m), 6.39 (2H, s),
5
.16 – 5.11 (2H, m), 4.28 – 3.90 (4H, m), 3.84 – 3.82 (9H, m), 3.15 –
1‐benzyl 2‐(3‐phenylpropyl) (S)‐piperidine‐1,2‐dicarboxylate (8).
3.04 (1H, m), 2.95 – 2.90 (1H, m), 2.62 (2H, br t, J = 7.0 Hz), 2.50‐2.48
To a 50 mL round bottom flask containing a stir bar was added 1‐
(benzyloxy)carbonyl) (S)‐piperidine‐2‐carboxylic acid (0.523 g, 1.98
mmol), followed by DCM (20 mL). To the reaction vessel was added
in the order listed) EDCI (0.45 g, 2.5 mmol), TEA (0.696 mL, 0.505 g,
.0 mmol), and DMAP (0.060 g, 0.50 mmol). The reaction vessel was
sealed and flushed with nitrogen. Stirring commenced and 3‐phenyl‐
‐propanol was added via syringe (0.258 mL, 0.258 g, 1.9 mmol). The
reaction was allowed to proceed overnight. The following day, the
reaction was transferred to a separatory funnel in which it was
washed with water (10 mL), dilute HCl (10 mL 0.5 M HCl), saturated
aqueous sodium bicarbonate (20 mL) and brine. The crude product
was dried over sodium sulfate and concentrated to dryness in vacuo.
The crude product was purified by silica gel chromatography (1:4
(1H, bm), 2.08 – 2.05 (1H, m), 1.97 – 1.92 (2H, m), 1.72 – 1.62 (2H,
(
m), 1.50 (1H, br s). 13C NMR (125 MHz) (CDCl
3
) (amide rotamers):
173.2, 155.1, 153.1, 136.8, 136.7, 136.2, 128.5, 128.0, 127.8, 105.2,
67.1, 63.9, 60.8, 56.0, 45.7, 44.2, 41.3, 32.5, 30.2, 27.2, 24.3. HRMS
calc’d for [M+H] = 472.2335, observed = 472.2331.
(
5
1
1‐benzyl 2‐(3‐(3,4,5‐trimethoxyphenyl)propyl) (S)‐pyrrolidine‐1,2‐
dicarboxylate (12). To a 100 mL round bottom flask containing
((benzyloxy)carbonyl)‐L‐proline (1.59 g, 6.4 mmol) was added a stir
bar and DCM (50 mL). The reaction mixture was stirred and the
following reagents were added in the order listed: EDCI (1.33 g, 7.0
mmol), DMAP (0.122 g, 1.0 mmol), and TEA (1.01 g, 10.0 mmol). The
reaction vessel was sealed and flushed with nitrogen. To the stirring
8
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DOI: 10.1039/C9OB00122K
Journal Name
ARTICLE
mixture was added 3‐(3,4,5‐trimethoxyphenyl)propan‐1‐ol (1.44 g,
.4 mmol) via syringe dissolved in DCM (10 mL). The reaction was
allowed to stir overnight. The following day, the reaction was
quenched with water (50 mL), transferred to a separatory funnel
and partitioned. The organic layer was dried over sodium sulfate and
concentrated in vacuo. The crude material was purified by silica gel
3
(CDCl ) (amide rotamers): 172.7, 172.5, 154.8, 154.2, 137.6, 137.4,
6
136.7, 136.6, 128.9, 128.8, 128.5, 128.4, 128.4, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 126.6, 126.5, 67.0,
66.9, 66.9, 66.9, 66.8, 66.7, 65.4, 65.3, 59.2, 58.9, 46.9, 46.9, 46.4,
46.3, 35.0, 34.8, 30.9, 29.8, 24.3, 24.2, 23.5, 23.4. HRMS calc’d for
[M+Na] = 376.1525, observed = 376.1525.
chromatography (1:3 ethyl acetate: hexanes, R
product (>95% purity) as a clear oil (1.3 g, 42 % yield). H NMR (500
f
= 0.51) to yield the
1
dibenzyl (S)‐pyrrolidine‐1,2‐dicarboxylate (15). To a 100 mL round
bottom flask containing a stir bar was added benzyl‐L‐prolinate
(purchased from Alfa Aesar, 0.858 g, 4.4 mmol) followed by DCM (20
mL). The vessel was sealed with a septa and flushed with nitrogen.
To the stirring reaction mixture was added TEA (0.835 mL, 0.606 g,
6.0 mmol) and Cbz‐Cl (0.580 mL, 0.697 g, 4.1 mmol). The reaction
mixture was allowed to stir overnight. The following day the
reaction mixture was transferred to a separatory funnel and
extracted with dilute HCl (20 mL of 5 % HCl v/v) and saturated
sodium bicarbonate solution (20 mL). The organic layer was dried
over sodium sulfate and concentrated in vacuo to yield the crude
product. The crude material was purified by silica gel
MHz) (CDCl
3
) (amide rotamers) δ: 7.36 – 7.24 (5H, m), 6.39 – 6.34
(
4
2H, app d), 5.19 – 5.06 (2H, m), 4.38 (1H, ddd, J = 19.5, 15.5, 3.5 Hz),
.19 – 4.00 (2H, m), 3.84 – 3.80 (9H, m), 3.66 – 3.47 (2H, m), 2.62
(
1H, t, J = 7.5 Hz), 2.51 (1H, t, J = 8.0 Hz), 2.28 – 2.20 (1H, m), 2.02 –
.80 (5H, m). C NMR (125 MHz) (CDCl )(amide rotamers) δ: 172.8,
3
1
3
1
1
1
6
3
=
72.7, 154.8, 154.2, 153.1, 153.1, 136.9, 136.7, 136.6, 136.5, 136.1,
36.1, 128.4, 128.3, 127.9, 127.8, 127.7, 105.2, 105.1, 66.9, 66.9,
4.3, 64.2, 60.8, 59.3, 58.9, 56.0, 46.9, 46.4, 32.3, 32.3, 31.0, 30.3,
0.1, 29.9, 24.3, 23.5. HRMS calc’d for [M+H] = 458.2179, observed
458.2177.
1
‐benzyl 2‐(3‐phenylpropyl) (S)‐pyrrolidine‐1,2‐dicarboxylate (13).
chromatography (1:2 ethyl acetate: hexanes, R
product (>95% purity) as a clear oil (0.724 g, 51 %). H NMR (500
MHz) (CDCl ) (amide rotamers) δ: 7.38 – 7.23 (10H, m), 5.24 – 5.12
3
f
= 0.4) to yield the
1
To a 250 mL oven dried round bottom flask containing a stir bar was
added ((benzyloxy)carbonyl)‐L‐proline (2.50 g, 10.0 mmol), followed
by DCM (100 mL). Stirring commenced, and to the reaction mixture
was added (in the order listed) TEA (1.7 mL, 1.21 g, 12.0 mmol), EDCI
(2H, m), 5.09 – 5.00 (2H, m), 4.48 – 4.38 (1H, m), 3.67 – 3.57 (1H, m),
3.55 – 3.46 (1H, m), 2.29 – 2.19 (1H, m), 2.05 – 1.86 (3H, m). 13C NMR
(
2.0 g, 11.0 mmol), DMAP (0.244 g, 0.2 mmol), and 3‐phenyl‐1‐
3
(125 MHz) (500 MHz) (CDCl ) (amide rotamers) δ: 172.6, 172.4,
propanol (1.49 g, 1.49 mL, 11.0 mmol). The reaction vessel was
sealed and flushed with nitrogen and allowed to react overnight.
The following day, the reaction mixture was transferred to a
separatory funnel and extracted with water (once, 100 mL). The
crude reaction mixture was concentrated on a rotary evaporator
and purified through silica gel chromatography (1:4 ethyl acetate:
154.9, 154.2, 136.7, 136.5, 135.7, 135.5, 128.5, 128.4, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 67.0, 66.9, 66.8,
66.7, 59.2, 58.9, 46.9, 46.4, 30.9, 29.9, 24.3, 23.5. HRMS for [M+H] =
340.1549, observed = 340.1549.
Determination of proteasome activity. Human housekeeping 20S
purified from erythrocytes was purchased from Enzo Life Sciences,
Inc. (Farmingdale, NY) or from Boston Biochem, Inc. (Cambridge,
MA) The 1 mg/ml stock 20S proteasome was diluted to 0.2 mg/ml
working solution in 50 mM Tris/HCl, pH 8 containing 20% glycerol
(dilution buffer). The peptidase activities were measured as
arbitrary intensity units of fluorescence emitted by 7‐amino‐4‐
methylcoumarin (AMC) released from the canonical model peptide
substrates: succinyl‐LeuLeuValTyr‐AMC (for the ChT‐L peptidase;
SucLLVY‐AMC; Bachem Bioscience Inc., Philadelphia, PA),
butoxycarbonyl‐LeuArgArg‐AMC (T‐L; Bachem Bioscience Inc.,
Philadelphia, PA) and carbobenzoxy‐ LeuLeuGlu‐AMC (PGPH; Enzo
Life Sciences Inc., Farmingdale, NY ), all used at concentration of 100
μM. Free AMC (Sigma‐Aldrich, St. Louis, MO) was used as the
standard. The tRpt5 and tRpt6 C‐terminal peptides were synthesized
(standard solid‐phase peptide chemistry) and purified to at least
98% purity by GenScript (Piscataway, NJ). The tRpt5 peptide and the
peptide substrates were dissolved in anhydrous dimethylsulfoxide
(DMSO; Sigma‐Aldrich, St. Louis, MO) and such stock solutions were
stored at ‐20oC. The total concentration of DMSO in final reaction
mixtures never exceeded 3% (vol/vol). The Trp‐containing tRpt6
f
= 0.33) to yield the product (>95% purity) as a clear oil
1
3
) (amide rotamers) δ: 7.39 –
C
3
o
peptide was dissolved in ultrapure water and stored at ‐20 C,
protected from light. The reaction was carried out in 96‐well plates,
o
in 100 μL of reaction mixture, for 1 hour at 37 C, with fluorescence
1
2
readout once per minute. The reaction mixture consisted of 45
mM Tris/HCl, pH 8, 100 mM KCl, 1 mM EDTA (reaction buffer) and
100 μM of a fluorogenic peptide substrate, to which 200 ng (nearly
0.3 nmol) of proteasome and desired concentration of tested
compounds in 0.5 μL of DMSO were added. If desired, the
proteasome was activated with 10 μM (final concentration) of the
tRpt5 peptide premixed in the reaction mixture. To test the
competition with tRpt6 peptide the 20S proteasome was pre‐
1
3
)
13
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ARTICLE
Organic and Bioorganic Chemistry
incubated in the reaction buffer for 10 min (RT) with compound 3 or
carbobenzoxy‐LeuLeuLeu‐vinyl sulfone (Z‐LLL‐VS; Enzo Life Sciences,
Inc), or tRpt6, or solvents, and then the compounds/solvents and
the substrate were added, as indicated. The reaction rates were
calculated from a linear segment of kinetic curves constructed from
measurements in 1‐minutes intervals (Fluoroskan Ascent plate
reader; Thermo Fisher Scientific Inc., Waltham, MA). Reaction rates
were calculated using a linear fit performed with the Slope Analyser
and Enzyme Kinetics applications launched within Origin Pro 2017
membrane in a semi‐dry system. The membranes were probed with
specific primary antibodies from Cell Signaling Technology (Danvers,
MA) (S6, PS6) and Li‐Cor (COX IV), and then with secondary
antibodies from Cell Signaling Technology. In each experiment
visualization of S6, PS6 and COX IV was performed on the same
membrane with Odyssey Infrared Imaging System (Cell Signalling
Technology).
(
OriginLab Corporation, Northampton, MA). Specific activity of the
Conflicts of interest
The authors have no conflicts of interest with this work.
control 20S proteasome activated with the Rpt5 peptide was in the
range of 1.4 to 2.3 nanomoles of AMC product released by 1 mg of
2
0S per second (1.95 ± 0.24; n=18). The activity of a latent
proteasome (no tRpt5 added) was 7 to 10 times lower. The data are
presented as mean ± SD or as a representative set from at least 3
independent experiments.
Acknowledgements
The work was supported by the William and Ella Owens Foundation
for Medical Research (MG & PAO).
Atomic force microscopy (AFM) imaging. The single molecule
imaging of the 20S proteasome was carried out as previously
described, with a scanner E of the Multimode Nanoscope IIIa (Bruker
Notes and references
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DOI: 10.1039/C9OB00122K