Inhibition of the human proteasome by imidazoline scaffolds

Article abstract of DOI:10.1021/jm400235r

The proteasome has emerged as the primary target for the treatment of multiple myeloma. Unfortunately, nearly all patients develop resistance to competitive-type proteasome inhibitors such as bortezomib. Herein, we describe the optimization of noncompetit

Full text of DOI:10.1021/jm400235r

Brief Article
pubs.acs.org/jmc
Inhibition of the Human Proteasome by Imidazoline Scaffolds
Lauren M. Azevedo, Theresa A. Lansdell, Jacob R. Ludwig, Robert A. Mosey, Daljinder K. Woloch,
Dillon P. Cogan, Gregory P. Patten, Michael R. Kuszpit, Jason S. Fisk, and Jetze J. Tepe*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States
S
* Supporting Information
ABSTRACT: The proteasome has emerged as the primary target for the treatment of multiple myeloma. Unfortunately, nearly
all patients develop resistance to competitive-type proteasome inhibitors such as bortezomib. Herein, we describe the
optimization of noncompetitive proteasome inhibitors to yield derivatives that exhibit nanomolar potency (compound 46, IC50
130 nM) toward proteasome inhibition and overcome bortezomib resistance. These studies illustrate the feasibility of the
development of noncompetitive proteasome inhibitors as additives and/or alternatives to competitive proteasome inhibitors.
potency would be a significant step toward the potential clinical
development of this new type of proteasome modulation.
Herein, we report the optimization of the imidazoline scaffold
as noncompetitive proteasome inhibitors with IC50 values in the
nanomolar range.
INTRODUCTION
■
Over the past decade, the proteasome has emerged as a clinical
target for the chemotherapeutic treatment of certain cancers,
and the first-of-its-class proteasome inhibitor bortezomib is
currently approved for the treatment of multiple myeloma
(MM) and relapsing mantle cell lymphoma (MCL).1 However,
all clinically relevant proteasome inhibitors, including bortezo-
mib and all second-generation proteasome inhibitors under
clinical evaluation, elicit their activity via the same mechanism,
which is the formation of a covalent bond to the N-terminal
threonine in the catalytic sites of the enzyme.2 In these cases,
the electrophilic headgroup of the inhibitor interacts with the
N-terminal threonine of the β1, β2, and β5 catalytic domains of
the enzyme.3 This type of binding blocks total proteasome-
dependent proteolysis, which subsequently results in cell death
and translates into potent anticancer efficacy.2a However, it has
also been well documented that, due to this particular
mechanism of binding, these agents exhibit permanent
abrogation of global protein degradation, lack of specificity,
low systemic tissue distribution, resistance, and severe off-target
effects.3,4 The use of these inhibitors in the clinic is therefore
limited to a few types of blood related cancers, and most of
those patients (>97%) become resistant or intolerant to the
treatment within a few years, after which survival is typically less
than one year.5
Noncompetitive modulation of enzymatic activity is
characterized by the interaction of a ligand with the enzyme
at a site distinct from the catalytic site, effectively modulating
the binding of some substrates to the enzyme’s catalytic
site(s).6 Aside from overcoming acquired resistance to active
site inhibitors, this type of modulation of enzyme activity may
limit off-target effects, thus limiting toxicity.6b,e,7 Examples of
small molecules that act as noncompetitive proteasome
inhibitors are very scarce and typically exhibit activity at high
concentrations and/or nonphysiologically relevant concentra-
tions.8,9
RESULTS AND DISCUSSION
■
The imidazoline scaffold 1 (TCH-013, Table 1) was first
reported to inhibit NF-κB-mediated gene transcription,12 and
Table 1. Inhibition of the Chymotryptic-Like Activity of
Purified Human 20S Proteasome by Compounds 1−5
more recent studies identified the human proteasome as its
molecular target.10 The mechanism of proteasome inhibition by
these imidazolines proceeded via noncompetitive kinetics,
which makes this class of compounds unique among
proteasome inhibitors.3,10 Our goal in this study was to modify
the imidazoline scaffold to increase its efficacy for proteasome
inhibition. As the proteasome is required for the activation of
NF-κB mediated gene transcription, we first compared the
previously reported cellular EC50 values of NF-κB inhibition13
with the IC50 values of inhibition of the purified 20S
proteasome to validate their direct correlation.
We first investigated the requirements of the trans-aryl
moieties and their relative stereochemistry (Table 1) for
inhibition of both the proteasome and NF-κB activity.
Imidazoline scaffolds 1−5 were prepared as previously
We previously reported the activity of the noncompetitive
proteasome inhibitor, TCH-013, in purified protein systems,
cell culture, and in vivo models of multiple myeloma10 and
arthritis.11 Although these noncompetitive inhibitors exhibit
good in vivo efficacy, the discovery of agents with increased
Received: February 14, 2013
© XXXX American Chemical Society
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described,13a and their ability to inhibit the 20S proteasome was
determined in vitro using purified human 20S proteasome and
the fluorogenic peptide Suc-LLVY-AMC as substrate for
chymotryptic-like activity.14 The rates of hydrolysis were
monitored by fluorescence increase at 37 °C over 30 min,
and the linear portion of the curve was used to calculate the
IC50 values. The results from Table 1 clearly indicate the
requirement of the trans-aryl stereochemistry seen in
compounds 1 and 2, as the cis (compound 3) and dearylated
scaffold (compound 4) as well as the planar imidazole 5 were
all depleted of activity. This data corresponded well with our
previously reported NF-κB inhibition of these compounds in
cell culture as the inactive proteasome inhibitors were inactive
as NF-κB inhibitors.13 Interestingly, upon standing, imidazo-
lines 2 and 3, slowly isomerized and dehydrogenated to the
inactive aromatic imidazole 5, which compromised the further
development of the disubstituted trans scaffold 2.13b This cis−
trans isomerization has been described previously in the
literature and is conveniently blocked by the ester in compound
1, which was subsequently chosen as our lead compound for
optimization.13b,15
Table 2. Inhibition of the Chymotryptic-Like Activity of
Purified Human 20S Proteasome by Compounds 1, 6−10
a
a
N.D. = not determined.
order to optimize the potency of the imidazoline scaffold
toward proteasome inhibition.
The synthesis of the imidazolines is accomplished via a
dipolar cycloaddition reaction of an oxazol-5(4H)-one and a
subsequent esterification of the carboxylic acid (Scheme 1).
Our initial studies started by examining the requirement of
the R1-moiety in the scaffold (Scheme 1). The compounds 11−
25 were prepared according to the methods described
previously (Table 3). Hydrogenation of the R1-benzyl group
of compound 1 resulted in compound 11, which was depleted
of all activity. Acetylation, benzoylation, and tosylation of 11
yielded compounds 12, 13, and 14, respectively, which were
also all depleted of activity. Replacement of the benzyl group
Scheme 1. Synthesis of Imidazolines via a Dipolar
a
Cycloaddition Reaction
Table 3. Inhibition of the Chymotryptic-Like Activity of
Purified Human 20S Proteasome by Compounds 11−33
b
a
(a) Na2CO3, H2O/1,4-dioxane, R3COCl, rt; (b) TFAA, DCM, rt; (c)
imine, TMSCl, DCM, reflux; (d) EDCI·HCl, DMAP, EtOH, DCM, rt.
Acylation of phenylglycine with the appropriate acid chloride
followed by cyclic dehydration using trifluoroacetic anhydride
provided the oxazolone (I). The oxazolone can undergo a
munchnone-type cycloaddition reaction in the presence of
̈
Lewis acids, such as TMSCl, with imines to render the
functionalized imidazoline scaffold (II) as a single diaster-
eomer.16 The cycloaddition reaction places a carboxylic acid in
the C-4 position of the imidazoline scaffold (II). The free
carboxylic acid is metabolically unstable,13b thus efforts were
directed toward the functionalization of this group. Various
compounds including esters 7, 8, and 10 and the amide 9 were
prepared and tested for their ability to inhibit the proteasome in
vitro (Table 2). The formation of esters (III) was accomplished
using the carbodiimide EDCI and dimethylaminopyridine
(DMAP) to yield compounds 1, 7, and 8 and prevented the
decarboxylation and aromatization. The imidazolines were able
to tolerate multiple different ester groups (1, 7, and 8) without
affecting activity. As shown in Table 2, the activity of the
compounds 1, 7−10 against the purified proteasome
corresponded very well with the reported cellular inhibition
of NF-κB transcription. Incorporation of an amide rendered a
decrease of activity. Therefore, subsequent optimization studies
were focused around the ethyl ester (III) as the leading
scaffold. After having established the strong correlation between
the inhibition of cellular NF-κB activity and the inhibition of
proteasome activity in the purified protein assays, we evaluated
modification of the respective R1−R3 domains (Scheme 1) in
a
b
n = 1. Data are averages of a minimum of two independent
experiments, each as a technical triplicate. Individual values and
standard error of the means are listed in Supporting Information (SI)
Table S1.
B
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with the methylene-2-furan (15) moiety also resulted in loss of
activity compared to the parent compound (1). Neither
electron donating groups (16) nor withdrawing groups (17)
in the para-position of the aryl group affected potency
significantly. The incorporation of halogens (compounds 18−
19) did increase potency following a trend of favoring
lipophilicity. Unfortunately, the increase in lipophilicity came
with a cost of decreased solubility as indicted for 18 (<0.5 mg/
mL) and 19 (<0.5 mg/mL) compared to 1 (1.91 mg/mL) in
5% dextrose in water. Thus, considering the already lipophilic
nature of the imidazoline scaffolds, additional lipophilic
moieties were considered unfavorable. Aryl and alkyl groups
were previously found to lack cellular activity toward the
inhibition of NF-κB,13a thus these were not pursued in our
optimization studies.
Table 4. Inhibition of the Chymotryptic-Like Activity of
Purified Human 20S Proteasome by Compounds 37−49
a
Considering the lack of major positive changes in activity
compared to the original benzyl moiety, we next focused our
attention on the R2 group. Incorporation of a pyridine moiety
in the R2 position rendered the imidazoline (20) inactive,
whereas the more electron rich 2-furan heterocycle (21)
regained its low micromolar potency. Following this trend,
electron withdrawing groups (22, 24) resulted in a decrease in
activity. Electron donating groups (23, 27) and lipophilic
substitutions (25) retained activity but did not increase the
overall potency significantly. However, benzylation of the
aniline 23 yielded compound 29, which exhibited a strong
increase in activity over parent compound 1 (IC50 0.5 μM).
This represents the first small molecule noncompetitive
proteasome inhibitor with IC50 values below the 1 μM level
in purified protein assays.
Prior to following up on this new lead, we evaluated several
functionalities as R3 moieties. The goal was to combine all the
improved features in order to gain the highest efficacy. The R3
substituents can be incorporated by benzoylation of phenyl-
glycine with different acyl moieties (Scheme 1). Cyclic
dehydration of the benzoyl protected amino acid with
trifluoroacetic anhydride provided the oxazolone I. A 1,3-
dipolar cycloaddition reaction with the oxazolone and N-
benzyl-1-(phenyl)methanimine was invoked using TMSCl,
which generated the imidazoline II as a single diastereomer.
Esterification of the carboxylic acid was accomplished using
ethanol and EDCI with DMAP to provide compound 30−33.
The incorporation of an electron withdrawing group resulted in
a loss of activity as indicated by compounds 32 and 33.
Although the bromophenyl (30) did not significantly increased
activity, the incorporation of an electron donating methoxy
group resulted in an increase in activity, with the methox-
yphenyl (31) being more than twice as active as the parent
molecule 1.
a
Data are averages of a minimum of two independent experiments,
each as a technical triplicate. Individual values and standard error of
the means are listed in SI Table S2.
a
Scheme 2
By combining the structural features with the best relative
potencies, a range of derivatives were prepared that varied in
the R4 position of imidazoline 37 (Table 4). Imidazoline 37 was
prepared via the exposure of oxazolone 34 to TMSCl in the
presence of N-benzyl-1-(4-nitrophenyl) methanimine (Scheme
2). This generated the HCl salt of the imidazoline 35 as a single
diastereomer. Esterification of the sterically hindered carboxylic
acid was accomplished using ethanol and EDCI with DMAP in
86% yield. Reduction of the nitro-moiety of imidazoline 36
with zinc proceeded well to produce the aniline 37 in 93%
yield. Aniline 37 was subsequently acylated with the
appropriate acylating agent in the presence of pyridine to
yield compounds 38 and 39. Treatment of the aniline with
a
(a) Na2CO3, H2O/1,4-dioxane, p-MeO-PhCO2Cl, rt, 85%; (b)
TFAA, DCM, rt, 88%; (c) (Z)-N-benzyl-1-(4-nitrophenyl)-
methanimine, TMSCl, DCM, reflux, 34%; (d) EDCI.HCl, DMAP,
EtOH, DCM, rt, 86%; (e) Zn, AcOH, rt, 93%; (f) Ac2O, pyr, DCM, rt,
46%; (g) BzCl, DMAP, DCE, rt, 31%; (h) N,N′-bis(t-
butoxycarbonyl)thiourea, DIPEA, EDCI·HCl, DCE, rt, 46%; (i) 1:1
TFA:DCM, rt, 68%; (j) sulfonation, RSO2Cl, pyr, DCM, 0 °C at rt,
47−94%; (k) reductive amination, NaBH(OAc)3, RCHO, DCE, 15−
87%.
(di-isopropylethylamine, DIPEA) provided the guanidine
derivative 40 after deprotection of the Boc-group with
trifluoroacetic acid (TFA). Sulfonation of the aniline using
N,N-bis(t-butoxycarbonyl)thiourea, EDCI, and Hunigs base
̈
C
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the respective sulfonyl chlorides and pyridine provided
analogues 41−45 in good yields. The aniline was also alkylated
using reductive alkylation. Treatment of the aniline with the
desired aldehyde formed the imine in situ, which was reduced
with NaBH(OAc)3 to yield compounds 46−49. Yields for the
formation of compounds 47 and 48 were relatively low (16%
and 28%, respectively), likely due to the poor electrophilic
nature of those aldehydes coupled with the poor nucleophilicity
of the aniline.
Acylation (to yield compound 38), benzoylation (to yield
compound 39), sulfonation (to yield compound 41),
benzylation (to yield compounds 46−48), and alkylation (to
yield compound 49) of compound 37 increased the potency of
the starting scaffold in all cases. Even the guanidine-based
imidazoline 40 (IC50 = 0.63 μM) exhibited excellent potency.
Sulfonation clearly provided several compounds with sub-
micromolar activity, as indicated by imidazoline 41 (IC50 = 0.58
μM) and 42 (IC50 = 0.53 μM). However, the alkylation and
benzylation (46−49) provided the first nanomolar non-
competitive, noncovalent proteasome inhibitors described in
the literature, with imidazoline 49 being the most potent at an
IC50 of 130 nM. Consistent with the biological mechanism of
the parent compound 1 (TCH-013), compound 46 indicates
similar kinetics for inhibition of the proteasome’s chymotryptic-
like activity (Supporting Information (SI) Figure S1).
The improved analogues were screened for cytotoxicity in
cell culture in human myelomonocytic THP-1 cells and a
bortezomib-resistant THP-1 cell line (BTZ500).4b,17 Consid-
ering that the mechanism of acquired resistance for bortezomib
in this cell line was previously determined to be due to the
overexpression of a mutated β5 catalytic subunit of the
proteasome,4b,18 compounds that inhibit the proteasome via a
non-competitive mechanism should be able to overcome this
resistance.8,10 The imidazolines 37, 46−49 were screened at 10
μM (Table 5) and 5 μM (SI Figure S2). Bortezomib was used
Thus, similar to the parent agent TCH-013, these agents were
able to overcome resistance to competitive inhibitors in the
bortezomib resistant cell line BTZ500 (Table 5), further
confirming their unique mechanism of proteasome inhibition.
Taken together, we have evaluated the structural features of
the R1−R3 groups of the groups surrounding the imidazoline
scaffold. The in vitro data obtained for inhibition of the
chymotryptic activity of the 20S proteasome is consistent with
the earlier findings and follows the same trend as the inhibition
of NF-κB mediated gene expression in cell culture. Our new
data presented herein indicates that the R4 domain is critical in
achieving submicromolar IC50 values for proteasome inhibition.
From this series, compound 49 (IC50 = 130 nM) represents the
first nanomolar, noncompetitive proteasome inhibitor reported.
Importantly, this data demonstrates the potential and/or
viability of developing new classes of proteasome inhibitors
that regulate proteasome activity via a mechanism distinct from
all current leading drugs or second-generation drugs under
clinical evaluation.
CONCLUSION
■
All current therapeutics targeting the proteasome are
competitive inhibitors that compete for substrate binding via
the formation of a covalent and often irreversible bond with the
N-terminal threonine of the proteasome’s catalytic site. Reports
in the literature of small molecule, noncompetitive inhibitors
are scarce and are typically only effective at micromolar or
nonphysiologically relevant concentrations. Here we present
the first report of the optimization of a noncompetitive small
molecule proteasome inhibitor. The imidazoline-based micro-
molar inhibitor TCH-013 (1) was optimized for potency
toward the 20S proteasome in vitro to render nanomolar
noncompetitive proteasome inhibitors, with imidazoline 49
being the most potent agent (IC50 = 130 nM). The R1−R3
domains surrounding the imidazoline scaffold were found to be
critical for activity, yet optimization of those domains resulted
in little gain in terms of overall potency. However, the R4
domain was critical in obtaining submicromolar activities.
These studies illustrate the feasibility of the development of
noncompetitive proteasome inhibitors as possible additives
and/or alternatives to classical competitive agents.
Table 5. Viability Data of THP-1 Cells in Human
Myelomonocytic THP-1 Cells and Bortezomib-Resistant
THP-1 Cell Line (BTZ500) Exposed to Bortezomib and
a
Imidazolines for 72 h
ASSOCIATED CONTENT
* Supporting Information
■
S
IC50 values, kinetic analysis, and cell viability values of each
independent experiment and their error analysis; detailed
protocols of the biological experiments, solubility evaluation,
and synthesis and compound characterization of all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
a
Viability data are means of three independent experiments.
Bortezomib was used at 0.5 μM. Individual values and standard errors
of the means are listed in SI Table S2 and Figure S2.
AUTHOR INFORMATION
Corresponding Author
*Phone: 517-355-9715. Fax: 517-353-1793. E-mail: tepe@
chemistry.msu.edu.
■
at 0.5 μM concentration (∼100× its reported IC50 of 5−8 nM
for proteasome inhibition).1−3 As anticipated, the potent MM
drug, bortezomib, exhibits <5% cell viability in the wild-type
THP-1 cell line but is unable to induce significant cytotoxicity
in the resistant cells (Table 5) at a concentration of 100× its
IC50 value. Consistent with its moderate inhibition of the
proteasome, analogue 37 (proteasome IC50 1.97 μM) exhibits
weak cytotoxic activity in both cell lines. However, the more
potent proteasome inhibitors 46−49 exhibited excellent
cytotoxicity in the THP-1 wild-type and mutant cell lines.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support in part from the
Multiple Myeloma Research Foundation (MMRF) and the
National Institutes of Health (CA-142644-01). In addition,
D
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Journal of Medicinal Chemistry
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Kay, L. E. TROSY-based NMR evidence for a novel class of 20S
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L.M.A. and J.R.L. greatly appreciate financial support from the
Michigan State University College of Osteopathic Medicine
DO/Ph.D. program and the College of Natural Science,
respectively. A great deal of appreciation is given to Drs. J.
Cloos, G. Jansen, and J. Meerloo for the kind gift of the
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ABBREVIATIONS USED
■
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