Oxadiazole-isopropylamides as potent and noncovalent proteasome inhibitors

Article abstract of DOI:10.1021/jm400221d

Screening of the 50 000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited chymotrypsin-like (CT-L) activity (IC₅₀ = 0.60 μM) with little effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and postglutamyl-peptide-hydrolysis-like (PGPH-L). LC-MS/MS and dialysis show that 1 is a noncovalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC₅₀ = 27 nM). Detailed SAR studies indicate that the amide moiety and the two phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl in the para position (not ortho or meta) of the A-ring and a m-pyridyl group as B-ring, significantly improve activity. Compound 11ad (IC₅₀ = 0.37 μM) is more potent than 1 (IC₅₀ = 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further preclinical investigation of this class as noncovalent proteasome inhibitors.

Full text of DOI:10.1021/jm400221d

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Article
Oxadiazole-isopropylamides as potent
and non-covalent proteasome inhibitors.
Sevil Ozcan, Aslamuzzaman Kazi, Frank Marsilio, Bin Fang, Wayne C.
Guida, John Matthew Koomen, Harshani Lawrence, and Saïd M. Sebti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400221d • Publication Date (Web): 02 Apr 2013
Downloaded from http://pubs.acs.org on April 28, 2013
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Oxadiazoleꢀisopropylamides as Potent and Nonꢀcovalent Proteasome Inhibitors.
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†
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‡
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†,‡, ║, §
Sevil Ozcan, Aslamuzzaman Kazi, Frank Marsilio, Bin Fang, Wayne C. Guida,
¥,#,§
,†,‡,§
†, §
John Koomen,
Harshani R. Lawrence,*
Saïd M. Sebti
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Drug Discovery Department, Molecular Oncology Department, Chemical Biology Core, Proteomics
Core, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA.
§
║
Department of Oncologic Sciences, Department of Chemistry University of South Florida, Tampa, FL
3
3620, USA
ABSTRACT
Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazoleꢀ
isopropylamide 1 (PIꢀ1833) which inhibited CTꢀL activity (IC 0.60 ꢁM) with little effects on the other 2
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0
major proteasome proteolytic activities, TꢀL and PGPHꢀL. LC/MSꢀMS and dialysis show that 1 is a nonꢀ
covalent and rapidly reversible CTꢀL inhibitor. Focused library synthesis provided 11ad (PIꢀ1840) with CTꢀ
L activity (IC 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are
50
sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the paraꢀposition (not
ortho or meta) of the Aꢀring and a metaꢀpyridyl group as Bꢀring significantly improve activity. Compound
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1ad (IC 0.37 ꢂM) is more potent than 1 (IC 3.5 ꢂM) at inhibiting CTꢀL activity in intact MDAꢀMBꢀ468
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human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further preꢀclinical
investigation of this class as nonꢀcovalent proteasome inhibitors.
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INTRODUCTION
The ATPꢀdependent ubiquitinꢀproteasome pathway is responsible for the controlled degradation of
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proteins in eukaryotic cells.
The 26S proteasome is a multifunctional complex consisting of a 19S
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regulatory particle (RP) and a 20S core particle (CP). The three main catalytic activities of the proteasome;
peptidylglutamyl peptide hydrolyzingꢀlike (PGPHꢀL), trypsinꢀlike (TꢀL), and chymotrypsinꢀlike (CTꢀL) are
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mediated by three distinct catalytic βꢀ1, βꢀ2, and βꢀ5 subunits, respectively. For each of the catalytic βꢀ
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,10,11
subunits, the Nꢀterminal Thrꢀ1 serves as the nucleophile that initiate cleavage of the peptide bond.
Development of inhibitors of CTꢀL activity has been the subject of considerable interest in the treatment of
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,10,12
cancer due to its critical role in the degradation of apoptotic and tumor suppressor proteins.
The
proteasome inhibitors advanced to clinic or clinical trials are derived from 3 structural classes (Figure 1A): 1.
Boronic acid containing compounds such as bortezomib, a dipeptide boronic acid (clinically approved by
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3, 14
15ꢀ17
18ꢀ21
FDA),
MLN9708
and CEPꢀ18770;
(Figure 1A). 2. βꢀlactones such as salinosporamide A (NPIꢀ
22,23
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052)
which is a marine microbial natural product (Figure 1A) and 3. Epoxyketone containing
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tetrapeptide carfilzomib (Figure 1A), clinically approved by FDA which is related to the natural product
epoxomicin. Each inhibitor class reacts with the proteasome Nꢀterminal threonine (Thrꢀ1 at the active site)
by a distinct mechanism. Peptide boronic acids form a covalent and slowly reversible tetrahedral adducts
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1,25
with the OH group of the catalytic Thrꢀ1.
For the βꢀlactone salinosporamide A, attack of the lactone ring
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by catalytic Thrꢀ1 forms an ester bond (that undergoes intramolecular rearrangement) which makes this
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compound an irreversible inhibitor. The epoxyketone moiety of carfilzomib reacts with the OH and the αꢀ
amino group of Thrꢀ1 to form 2 covalent bonds, also making the inhibition irreversible.
Covalent proteasome inhibitors are classified as slow reversible or irreversible inhibitors according to
their chemical structure and mechanism of inhibition. Covalent irreversible or covalent slow reversible
inhibitors as described above possess a chemically reactive group that bind to the proteasome covalently. In
contrast, nonꢀcovalent inhibitors inhibit the proteasome through a network of interactions (hydrophobic,
hydrogen bonds, electrostatic and/or van der Waals). Although some proteasome inhibitors have been
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suggested to bind nonꢀcovalently
structural evidence to support this was provided for only three of these.
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35ꢀ37
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The Xꢀray structures of TMCꢀ95A,
hydroxyl urea (Figure 1B) and the peptide from Millenium
(Figure 1B) bound to proteasome demonstrate that the binding mode of these compounds are nonꢀcovalent.
Since nonꢀcovalent inhibitors do not have a reactive electrophilic moiety or ‘warhead’, which is often
associated with metabolic instability, poor specificity, and excessive reactivity, they have the advantage of
exerting fewer side effects over the covalent ones. It has been shown that the proteasome activity recovers at
the same rate with covalent irreversible inhibitors as with covalent slow reversible inhibitors, presumably via
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de novo proteasome synthesis. The clinical advantages/benefits of nonꢀcovalent proteasome inhibitors in
cancer treatment are not well understood. Figure 1B shows the structures of small molecules that have been
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identified as nonꢀcovalent proteasome inhibitors.
We have been actively engaged in the discovery of
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novel proteasome inhibitors.
We reported the discovery of the compound 1 as a proteasome inhibitor in a
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poster at the 2011 American Association for Cancer Research (AACR) meeting. Villoutreix et al also have
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reported oxadiazoleꢀisopropylamide containing compounds as proteasome modulators.
Although
Villoutreix et al and our group have independently identified similar scaffolds, each group focused on
different modifications of the hits that led to important findings that are complementary but not overlapping.
In our study, we have extensively explored SAR (Figure 2) on the oxadiazoleꢀisopropylamide containing
compounds as proteasome inhibitors by systematically synthesizing focused libraries around key features of
the pharmacophore. We present compound 1 and its most potent analogs as nonꢀpeptidic, nonꢀcovalent and
reversible proteasome inhibitors that have the potential to become clinical candidates.
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Figure 1: A. Structures of clinically advanced covalent proteasome inhibitors B. Structures of nonꢀcovalent
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small molecule proteasome inhibitors; hydroxyurea pharmacophore and peptidic pharmacophore from
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Millennium.
CHEMISTRY
The screening hit 1 was identified as a CTꢀL proteasome inhibitor with an IC value of 0.60 ± 0.18 ꢁM
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(inꢀvitro). Validation of the synthetic route to 1 was undertaken to confirm both the structure and the in vitro
CTꢀL inhibitory activity. Synthesis of 1 was achieved using the route shown in Scheme 1. The substituted
acetyl chloride building block library 5 (Scheme 1) was synthesized from readily available phenol derivatives
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via the ester 3 and acid 4 using reported protocols.
The oxadiazole portion of the compound 1 was
synthesized from readily available nitrile building blocks 6. The nitrile building blocks were reacted with
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hydroxylamine hydrochloride and sodium carbonate at 70 ºC in water to yield the hydroxyamidines
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(Scheme 1, condition g). The 5ꢀsubstituted pyrimidinehydroxyamidine 7h (see supporting information) was
synthesized starting from the commercially available methyl pyrimidineꢀ5ꢀcarboxylate via amide 24 and
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nitrile 25. The intermediate hydroxyamidine library 7 was reacted with chloroacetyl chloride (Scheme 1,
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condition h) to provide the library 8, which was cyclized in refluxing toluene to provide the oxadiazole
portion of the pharmacophore 9. The library 9 was subsequently reacted with appropriate alkyl amines
(
isopropylꢀ, isobutylꢀ, methylꢀ. ethylꢀ, cyclopropylꢀ and tertꢀbutyl amines) to obtain the amine building block
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library 10 (Scheme 1, condition j). For the synthesis of 10n (R = H), compound 9a (R = paraꢀtolyl, see
supporting information compound 26) was first reacted with phthalimide in the presence of potassium
carbonate in refluxing acetonitrile, followed by reaction with hydrazine to obtain the compound 10n in high
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yield. We were able to generate library 10 with a variety of substituted aryl and heteroꢀaryl R moieties and
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library 5 with substituted/unsubstituted aromatic R moieties (Scheme 1). Modifications around 1 for library
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synthesis are shown in Figure 2, and initially we focused our synthetic efforts to modify R , R and R
moieties. The two key building block libraries 10 and 5 were then reacted in the presence of triethylamine to
provide the compound 1, library 11 and 12 (Scheme 1, Tables 1 and 3) in good yields. The route described
in Scheme 1 was efficient and convenient for rapid synthesis and optimization of the substituted phenyl and
amide moieties. The final libraries 11, 12 and compound 1 were characterized using NMR, LCꢀMS, HRMS
and the purity was > 95% as determined by HPLC. The final compound library 11 and 12 (including
compound 1) showed formation of approximately 3:1 atropisomers (hindered rotation about the CꢀN amide
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bond) by H NMR and C NMR spectroscopy (see the experimental section and supporting information).
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Analysis of H NMR spectra of compound 1 at variable temperatures (20 °C to 50 °C) showed that the peaks
from the minor rotamer coalesced with the major rotamer as the temperature increased.
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Figure 2: Modifications and library synthesis around 1 for design of new proteasome inhibitors and SAR
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Scheme 1. Synthetic route to compound 1, libraries 11 and 12. Reagents and conditions: a. Ethyl
bromoacetate, K CO , Acetone, reflux, 14 h. b. tertꢀButyl bromoacetate , DMF, 80 ºC, 14 h. c. Ethyl
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bromoacetate, K CO DMF, r.t., 14 h. d. NaOH, THF, reflux, 2 h. (R= Ethyl). e. CF COOH, DCM, r.t., 2 h
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t
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R = Bu). f. SOCl , benzene, reflux, 3 h. g. NH OH.HCl, Na CO water, 70 ºC, 14 h. h. Chloroacetyl
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chloride, acetone, r.t., 30 min. i. toluene, reflux, 2 h. j. Alkylamine, K CO , CH CN, reflux, 30 min. k.
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Et N, THF, r.t., 15 min.
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Next, we focused our efforts to modify the chemical space between the amide moiety, A and B rings
(Figure 2) in compound 1. First, we introduced a urea moiety to assess the SAR. To install the urea moiety,
the intermediate 10d was reacted with commercially available isocyanate 13 in the presence of triethylamine
in refluxing benzene, and under these conditions urea 14 was obtained in good yield (Scheme 2, condition q).
Further modifications included replacement of the ether moiety (Hꢀbond acceptor) in 1 (Figure 2) with an
NH (Hꢀbond acceptor/donor) group. The amine 10d was first reacted with chloroacetyl chloride in the
presence of triethylamine in THF at room temperature to obtain intermediate 15 (Scheme 2) followed by
coupling 15 with paraꢀmethylaniline using sodium acetate in refluxing ethanol to obtain the final compound
16 (Scheme 2, conditions r and s respectively) also in good yield. The ether moiety in 1 (Figure 2) was also
replaced by a methylene unit using 3ꢀ(4ꢀ(trifluoromethyl)phenyl)propanoic acid building block (17a). The
acid starting material 17a (Scheme 2) was converted to the corresponding acid chloride 18a and coupled with
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0d to provide the oxadiazole 19a (Scheme 2). The final compound 19b with bulky Rꢀgroups was
synthesized following the route in Scheme 2 starting from benzofuranꢀ2ꢀcarboxylic acid (17b) via the
formation of acid chloride 18b and subsequent coupling with 10f. The intermediate 10d was chosen for
synthesis of compounds 14, 16, and 19a since our early SAR indicated unsubstituted B ring is desirable to
retain in vitro CTꢀL potency together with paraꢀCH group on the A ring and the isopropyl moiety in the
3
amide group. Extension of the linker between the oxadiazole moiety and amide group in 1 (Figure 2) by one
carbon atom was achieved by first reacting the intermediate 7d with 3ꢀchloropropanoyl chloride to generate
intermediate 20 which provided 21 upon refluxing in toluene. The intermediate 21 was next reacted with
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isopropylamine to obtain 22, which was further reacted with intermediate 5a (see supporting information) to
obtain the final compound 23 in good yield. The intermediates and final compounds were characterized by
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H NMR, C NMR, HRMS, LCꢀMS; the purity of final compounds was determined using HPLC.
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Scheme 2: Reagents and conditions: q. Et N, benzene, reflux, 14 h, 78%. r. Chloroacetyl chloride, Et N,
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THF, r.t., 15 min., 80%. s. paraꢀMethylaniline, NaOAc, ethanol, reflux, 15 h, 78%. f. SOCl , benzene,
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reflux, 3 h, 94%. i. toluene, reflux, 2 h, 81%. j. Isopropylamine, K CO , CH CN, reflux, 30 min., 86%. k.
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Et N, THF, r.t., 15 min., 88% (19a), 82% (19b), 87%, (23). l. DCM, r.t., 14 h, 76%.
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RESULTS AND DISCUSSIONS
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Unlike lactacystin, 1 is a nonꢀcovalent and reversible CTꢀL inhibitor
To determine whether our hit compound 1 is a nonꢀcovalent CTꢀL inhibitor, we used two approaches, Liquid
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Chromatography Tandem Mass Spectrometry (LC/MSꢀMS) and dialysis. With both approaches we used
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lactacystin, a known covalent and irreversible CTꢀL inhibitor as a control.
For LC/MSꢀMS, 1 or
lactacystin were incubated with the 20S proteasome, digested with trypsin and the resulting peptides purified
by HPLC and analyzed by LC/MSꢀMS as described under Methods. Figure 3A shows that peptides purified
from the vehicleꢀtreated samples contained unmodified TTTLAFK peptide (observed as a protonated
molecule at m/z 781.4401) corresponding to the Nꢀterminal tryptic peptide of rabbit proteasome subunit β
typeꢀ5. Figure 3B shows that peptides purified from the compound 1 treated samples also contained the
unmodified Thrꢀ1 containing TTTLAFK peptide (Figure 3D). Both intact mass spectrum and tandem mass
spectrum indicate unmodified Thrꢀ1 containing peptide. Figure 3C shows that lactacystinꢀtreated samples
contained a lactacystinꢀmodified Threonine adduct corresponding to the doubly charged modified peptide at
m/z 497.7795 (structure shown in Figure 3E). The searches matching experimental data to peptides, from the
database of rabbit proteasome subunits produced only one modified peptide, which indicated that Thrꢀ1 on βꢀ
5
was modified by lactacystin. No other modifications to βꢀ5 subunit from samples treated with DMSO,
compound 1 or lactacystin were observed. In addition, no modifications were detected for other proteasome
subunits, such as βꢀ1 and βꢀ2 subunits included in the database (n =21). These results suggest that, unlike
lactacystin, 1 does not bind covalently to the proteasome.
To confirm these results, we incubated 1 and lactacystin with the 20S proteasome and determined the
reversibility of binding by dialysis as described under Methods. The Figure 4 shows that more than 70% of
the CTꢀL activity in the dialysis compartment from the sample that was treated with 1 was recovered within
the first 20 min., and recovered fully by 2 hours of dialysis. In contrast, very little CTꢀL activity was
recovered from the sample that was treated with lactacystin even after 18 hours of dialysis (Figure 4). Taken
together these results confirm the well established fact that lactacystin is a covalent and irreversible CTꢀL
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inhibitor and indicate that 1 is a nonꢀcovalent and reversible CTꢀL inhibitor. This is consistent with the
chemical structure of 1 that lacks any reactive groups, unlike clastoꢀlactacystin that contains the
reactive betaꢀlactone moiety. The βꢀlactone moiety of the clastoꢀlactacystin covalently modifies the βꢀ
subunits of the proteasome and is responsible for inhibiting 20S proteasome in an irreversible mode of
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Figure 3: A: LC/MSꢀMS analysis of tryptic digests from proteasome CTꢀL subunit after incubation with
vehicle. LC/MSꢀMS analysis of Thrꢀ1 containing peptide from the proteasome CTꢀL subunit β5 after tryptic
digestion is shown. Singly charged unmodified peptide was observed at m/z 781.4401, which represents a mass
error of 6.9 ppm. The tandem mass spectrum was matched to peptide TTTLAFK. The b ions (labeled in red),
contain the Nꢀterminus of the peptide; and y ions, (labeled in blue), contain the Cꢀterminus of the peptide. The
number associated with each ion indicates the number of amino acids in that fragment (for example, y4 contains
LAFK from Cꢀterminus of the peptide). B: LC/MSꢀMS analysis of tryptic digests from proteasome CTꢀL
subunit after incubation with compound 1. The Thrꢀ1 containing peptide didn’t show any modification. Both
intact mass spectrum and tandem mass spectrum indicate unmodified Thrꢀ1 containing peptide. C: LC/MSꢀMS
analysis of tryptic digests from proteasome CTꢀL subunit after incubation with lactacystin shows clastoꢀ
lactacystinꢀmodified Thrꢀ1 containing peptide. Doubly charged lactacystinꢀThr modified peptide was detected
at m/z 497.7795, which represents a mass error 6.1 ppm. The tandem mass spectrum confirms the modification
of the peptide by lactacystin. D: Structure of the unmodified TTTLAFK tryptic peptide. E: Structure of the
clastoꢀlactacystin modified peptide.
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Figure 4: Recovery of CTꢀL activity upon dialysis of the 20S proteasomeꢀcompound complexes after preꢀ
incubation with lead 1 and lactacystin.
Structure Activity Relationship studies and chemistry leading to compound 11ad.
Our screening efforts of the 50,000 inꢀhouse ChemBridge compound library led to the discovery of the
hit 1, an inhibitor of the CTꢀL activity of the proteasome. After confirming the structure and in vitro CTꢀL
activity of the inꢀhouse synthesized 1 (Scheme 1), we embarked on synthetic modifications to develop
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structure and activity relationship (SAR) data to identify novel, potent and selective CTꢀL proteasome
inhibitors that block the action of the proteasome in a nonꢀcovalent manner. Proteasome CTꢀL activity was
4
1
measured using a fluorogenic assay as previously described. Focused library synthesis was undertaken by
1
2
3
independently varying the R , R and R groups in compound 1 (Figure 2). Initially, we replaced the
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isopropyl R group in 1 with H, isobutyl, ethyl, methyl, tertꢀbutyl and cyclopropyl moieties (Table 1). The
loss of CTꢀL activities of these analogs indicated that the isopropyl group is essential and optimal for
proteasome inhibitory activity (see Table 1). The isobutyl amide 12a and ethyl amide 12b (Table 1, Entries
2
and 3) showed 4ꢀ to 10ꢀfold loss of activity with IC values of 2.37 and 6.02 ꢂM respectively compared to
50
3
1. Methyl, H, tertꢀbutyl and cyclopropyl as R groups were detrimental for CTꢀL activity (12c, 12d, 12e, 12f
1
13
in Table 1 showed weak or no proteasome inhibition at 10 ꢂM). The H and C NMR spectra of the
compound 1 with isopropyl amide group indicated the presence of a mixture of 3:1 atropisomers (isomers
that exist due to the hindered rotation about the carbonꢀnitrogen bond). We observed a similar ratio of
3
3
1
13
atropisomers with 12b (R = ethyl), and 12c (R = methyl) by analysis of H and C NMR spectra.
3
Compounds 12d with unsubstituted amide (Entry 5, R = H, Table 1), 12e with bulky tertꢀbutyl group (Entry
3
t
3
6
, R = Bu, Table 1) and 12f with cyclopropyl group (Entry 7, R = cyclopropyl) did not show any
1
13
atropisomers by H or C NMR spectra.
3
In the next generation of synthetic analogs, we retained the isopropyl R group (Figure 2) and modified
the phenyl rings in 1 [i.e. library 11, (Scheme 1 and Table 3)]. Initial SAR studies demonstrated that
1
2
substitutions on the paraꢀposition of the R and R with small hydrophobic groups are tolerable. For
1
2
example, changing the paraꢀmethyl in R and R to trifluoromethyl or chlorine as in compounds 11a, 11c,
1d, 11e, 11f, 11g, 11i and 11n retained the in vitro CTꢀL inhibitory activities (Entries 14, 16ꢀ20, 22, 27,
1
1
2
Table 3). Next we demonstrated that the R methyl is required whereas the R methyl is dispensable.
Indeed, compounds 11b, 11h and 11m (Entries 15, 21 and 26, Table 3) with an unsubstituted phenyl ring as
2
2
R showed slightly improved IC values around 0.3 to 0.5 ꢂM indicating paraꢀsubstitution of R phenyl in 1
5
0
is not required for inhibitory activity. However, compound 11j with both unsustituted phenyl rings resulted
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in 10 fold loss of CTꢀL activity (Entry 23, Table 3, IC = 6.22 ꢂM). Removal of the R paraꢀmethyl group
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as in compounds 11k and 11l (Entries 24 and 25, Table 3) also led to 14ꢀ and 18ꢀfold loss of activity with
IC values of 8.5 and 11 ꢂM, respectively. The loss of in vitro potency of compounds 11j, 11k and 11l
50
1
suggest that paraꢀsubstitution in R phenyl ring is critical to maintain CTꢀL proteasome activity. Changing
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the R paraꢀmethyl to metaꢀ or orthoꢀpositions as in compounds 11o and 11p (Entries 28 and 29, Table 3)
1
was detrimental for in vitro potency further suggesting that R paraꢀsubstitution is important for CTꢀL
1
2
3
proteasome activity. Overall our SAR indicated that the paraꢀmethyl group in R but not in R and the R
isopropyl amide are key features that are responsible for retaining the CTꢀL proteasome activity of
1
compound 1 and loss of activity was consistent with unsubstituted R aromatic rings in this class of
compounds.
1
2
We next modified the chemical space between the amide moiety and R /R in the parent compound 1
(Figure 2) using the synthetic routes and protocols described in Scheme 2 (also see supporting information).
First, replacement of the etherꢀoxygen by methylene showed significant loss of CTꢀL activity (19a, IC50
5
3.48 ꢂM, Entry 8, Table 2). Furthermore, substituting the amide group by urea as in 14 (Entry 9, IC > 10
50
ꢂM, Table 2) also led to loss of activity. Replacement of the ether (Hꢀbond acceptor) with NH (Hꢀbond
donor/acceptor) also decreased the in vitro CTꢀL activity (16, IC 5.67 ꢂM, Entry 10, Table 2). These
5
0
modifications confirmed that the ether moiety, most likely, as Hꢀbond acceptor, is critical for focused library
synthesis and improving the CTꢀL inhibitory activity. Extending the spacer between the amide and the
oxadiazole by one carbon as shown in 23 (Entry 11, IC > 10 ꢂM, Table 2) was detrimental for CTꢀL
5
0
inhibitory activity, probably due to the increased flexibility of the molecule. Compound 19b (IC 0.37 ꢂM,
5
0
1
2
Entry 12, Table 2), a bulky R substituent with a rigid ether moiety and metaꢀpyridyl as R showed improved
inhibitory activity compared to 1 (pyridyl moiety was chosen for the synthesis of 19b, based on the most
1
2
potent analogs described in Table 3). Overall, synthetic modifications of the linkers between R , R and the
amide moiety in 1 were not tolerated.
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To gain more insight in the interactions between R and the binding region, we modified the R via
several synthetic modifications. For example, synthesis of 11q and 11r that possessed large hydrophobic
1
groups such as phenyl and Brꢀnaphthyl as paraꢀR groups respectively further revealed that large
hydrophobic groups are not tolerated in the binding region (Entries 30, 31, IC50 > 10 ꢂM, Table 3).
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Replacement of the R methyl group in compound 1 by small hydrophobic fluorine as in 11s (Entry 32, Table
3) also displayed poor CTꢀL inhibitory activity. The 16ꢀfold loss of CTꢀL inhibitory activity in compound
1
1s with paraꢀfluorine compared to 11b with paraꢀCF (Entry 15, IC = 0.43 ꢂM) was not unexpected since
3 50
5
8
fluorine is isosteric to hydrogen and as described above we have already observed the detrimental effects of
1
unsubstituted R rings in compounds 11k, 11l and 11ac (Entries 24, 25 and 42, Table 3) toward the CTꢀL
t
1
inhibitory activity. The hydrophilic COOH, COO Bu and OH groups in the paraꢀposition of the R as in
compounds 11t, 11aq and 11v respectively also failed to maintain the CTꢀL inhibitory activity (Entries 33,
5
6 and 35, Table 3) indicating Hꢀbond acceptor/donor moieties are not desirable. In contrast compound 11u
1
2
(
Entry 34, Table 3) with paraꢀhydroxyphenyl as R and metaꢀpyridyl as R showed an IC value of 1 ꢂM,
50
and comparison of in vitro CTꢀL activities of 11u and 11v (Entries 34 and 35, Table 3) with paraꢀ
1
2
hydoxyphenyl as R highlights the significance of the R metaꢀpyridyl group toward CTꢀL activity in this
class of compounds.
We next made 2 major observations that led us to our most potent compounds. First, we demonstrated
that increasing the alkyl length from methyl in 1 to a propyl as in 11ap (IC = 0.27 ꢂM) increased potency
50
2
(
Entry 55, Table 3). Second, replacing the tolyl R in 1 by a metaꢀpyridyl (as in 11x, Entry 37, Table 3) or
paraꢀ pyridyl (as in 11y, Entry 38, Table 3) but not orthoꢀpyridyl (as in 11w, Entry 36, Table 3) improved
potency with the metaꢀpyridyl (IC 0.22 ꢁM) being slightly better than the paraꢀpyridyl (IC 0.37 ꢁM,
5
0
50
1
2
Table 3). Substituting both, the R methyl with a propyl and the R tolyl with a metaꢀpyridyl as in 11ad
(combined features of compounds 11ap and 11x) resulted in our most potent compound with an IC value of
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1
2
7 nM. Furthermore, the length of the alkyl group in paraꢀposition of the R ꢀring is critical with the propyl
or butyl groups being the optimal size. Indeed, decreasing the size from propyl (or butyl) to ethyl, methyl
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and hydrogen as in 11ab, 11x and 11ac, resulted in progressive loss of inhibitory activity from 27 nM to 99
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
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4
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4
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4
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5
5
5
5
5
5
5
5
5
6
1
nM, 220 nM and 2710 nM, respectively. Increasing the size of the R paraꢀhydrophobic moiety also resulted
in progressive loss of potency from propyl or butyl to pentyl, hexyl, isobutyl, isopropyl, cyclohexyl and tertꢀ
butyl as in 11af (IC 120 nM), 11ag (IC 430 nM), 11ak (IC 140 nM), 11ai (IC 440 nM), 11ah (IC
50
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0
50
50
50
0
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2
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1
356 nM) and 11aj (IC 46.49 ꢁM), respectively. Furthermore, substituting the pyridyl in 11ad with 3,5ꢀ
50
pyrazine as in 11al did not alter the potency (IC 32 nM) which further supports the importance of a metaꢀ
5
0
positioned nitrogen atom in the ring. In contrast, substituting the pyridyl in 11ad by 2,5ꢀpyrazine as in 11am
Entry 52, IC 105 nM, Table 3) with one metaꢀpositioned nitrogen or 2,6ꢀpyrimidine as in 11ao (Entry 54,
(
5
0
IC 1269 nM) which possess two orthoꢀpositioned nitrogen atoms, led to 3ꢀfold and 40ꢀfold loss of CTꢀL
5
0
activity respectively (Table 3) further highlighting the importance of the nitrogen in the metaꢀposition of the
ring. Compounds 11am and 11an that show CTꢀL inhibitory activities with IC values in the range of 100
50
1
nM also demonstrate propyl or butyl in the paraꢀposition of the R are equally tolerated in the binding
region. Interestingly, compound 11ar and 11as with hydrophobic, electron withdrawing fluorine and
1
2
chlorine in the paraꢀposition of the R and metaꢀpyridyl as R (Entries 57 and 58, Table 3) showed 3ꢀ5ꢀfold
improvement in inhibitory activities respectively comparing to its related analogs 11s (Entry 32, Table 3)
1
1h and (Entry 21, Table 3). Overall, our detailed SAR studies signify the importance of metaꢀpyridyl,
isopropylamide and paraꢀpropyl/ꢀbutyl phenyl groups in the oxadiazole pharmacophore shown in 1 for
inhibition of CTꢀL activity of the proteasome.
The parent compound 1 and 11ad were further evaluated for TꢀL and PGPHꢀL activities of the
proteasome as shown in the Table 4. The in vitro data (IC ) indicated that this class of compounds shows
5
0
excellent selectivity for CTꢀL inhibitory activity over both TꢀL and PGPHꢀL activities. This level of CTꢀL
selectivity is impressive for a nonꢀpeptidic, small molecule as compared to the covalent proteasome
inhibitors reported to date. Only a small number of compound classes have been reported with this level of
11, 38
activity.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
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4
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Table 1: Synthetic analogs of Library 12 and SAR.
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7
8
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2
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6
7
8
9
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2
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4
5
6
7
8
9
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Compound
ID
3
a
Entry
R
IC50 (ꢁM) CTꢀL
1
1
isopropyl
isobutyl
ethyl
0.60 ± 0.18 (n=27)
2.37 ± 0.40 (n=2)
2
3
4
5
6
12a
12b
12c
12d
12e
6.04 ± 1.34 (n=3)
methyl
H
29.90 ± 3.9 (n=2)
No inhibition @ 10 ꢀM
tertꢀbutyl
No inhibition @ 10 ꢀM
No inhibition @ 10 ꢀM
7
12f
cyclopropyl
a
IC values are given as the average of 2 or more determinations; n = number of determinations.
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Table 2. Modifications of the spacer between the amide, R and R groups and SAR.
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
N
R1
N
N
R2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
a
Entry #
IC50 (ꢁM) CTꢀL
Structure and ID
8
53.48 ± 10.89 (n=3)
9
No inhibition @ 10 ꢀM
1
1
0
1
5.67 ± 0.96 (n=4)
No inhibition @ 10 ꢀM
O
O
N
N
N
O
1
2
N
0.379 ± 0.06 (n=3)
1
9b
a
IC values are given as the average of 2 or more determinations; n = number of determinations.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
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6
Table 3: Compound 1, synthetic analogs of library 11 and SAR.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
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9
0
Compound
1
2
a
Entry
R
R
IC50 (ꢁM) CTꢀL
ID
13
1
0.60 ± 0.18 (n=27)
1
1
4
5
11a
11b
1.08 ± 0.33 (n=4)
0.43 ± 0.12 (n=4)
F
3
C
F
3
C
1
1
6
7
11c
11d
11e
11f
2.53 ± 0.95 (n=5)
0.94 ± 0.26 (n=8)
1.12 ± 0.33 (n=5)
1.41 ± 0.19 (n=2)
1.07 ± 0.05 (n=4)
0.51 ± 0.16 (n=6)
CF
3
F
3
C
C
F
3
Cl
18
19
Cl
Cl
Cl
Cl
CF
3
2
2
0
1
11g
11h
Cl
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11i
11j
0.45 ± 0.15 (n=4)
6.20 ± 1.1 (n=2)
8.50 ± 0.60 (n=2)
11.20 ± 2.1 (n=3)
0.31 ± 0.08 (n=4)
0.97 ± 0.15 (n=2)
Cl
11k
11l
10
11
12
13
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15
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CF
3
11m
11n
27
CF
3
2
2
8
9
11o
11p
No inhibition @ 10 ꢀM
No inhibition @ 10 ꢀM
30
11q
No inhibition @ 10 ꢀM
3
3
1
2
11r
11s
No inhibition @ 10 ꢀM
Br
10.09 ± 1.63(n=3)
F
3
3
11t
No inhibition @ 10 ꢀM
HO
O
N
34
11u
0.98 ± 0.50 (n=3)
HO
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5
11v
10.12 ± 4.5 (n=3)
HO
N
36
37
11w
11x
3.75 ± 1.47 (n=6)
0.22 ± 0.084 (n=6)
0
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9
0
N
38
11y
0.37 ± 0.05 (n=4)
N
N
39
11z
4.0 ± 0.90 (n=3)
0.26 ± 0.05 (n=6)
4
4
0
1
11aa
N
N
N
11ab
11ac
11ad
0
.099 ± 0.032 (n=14)
4
4
2
3
2.71 ± 0.56 (n=5)
0
.027 ± 0.014 (n=20)
N
4
4
4
5
11ae
11af
0.039 ± 0.01 (n=7)
N
0.120 ± 0.04 (n=3)
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N
4
4
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7
11ag
11ah
0.43 ± 0.04 (n=3)
1.36 ± 0.18 (n=3)
N
N
10
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11ai
11aj
11ak
0.44 ± 0.07 (n=3)
46.49 ± 1.23(n=3)
0.140 ± 0.05 (n=3)
N
N
4
9
50
51
N
N
11al
0.032 ± 0.003 (n=3)
0.105 ± 0.031 (n=3)
N
5
5
2
3
11am
N
N
11an
N
0.107 ± 0.013 (n=3)
1.27 ± 0.22 (n=3)
N
N
5
5
4
5
11ao
11ap
0.273 ± 0.10 (n=3)
5
5
6
7
11aq
11ar
No inhibition @ 10 ꢀM
N
1.93 ± 0.61 (n=4)
F
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N
5
8
11as
0.14 ± 0.064 (n=4)
Cl
a
IC values are given as the average of 2 or more determinations; n = number of determinations.
5
0
Table 4: IC values of 1 and 11ad for CTꢀL, TꢀL and PGPHꢀL activities of the proteasome.
5
0
0
1
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0
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0
a
a
Compound
CTꢀL (ꢂM)
TꢀL (ꢂM)
PGPHꢀL (ꢂM)
1
0.60 ± 0.18 ( n= 27)
0.027 ± 0.014 (n =
>100
>100
1
1ad
>100
>100
2
0)
a
The values given are the means of 3 experiments; n = number of determinations.
Lead compound 11ad is more potent than the parent compound 1 at inhibiting CTꢀL activity and
survival/proliferation of intact human breast cancer cells
Our in vitro SAR studies demonstrated that substituting the methyl of ring A with a propyl and replacing
ring B tolyl with metaꢀpyridine in 1 (IC 600 nM) led to 22 fold more potent 11ad (IC 27 nM). To
5
0
50
determine whether these CTꢀL proteasome inhibitors are cell permeable and inhibit CTꢀL activity in intact
cells, human breast cancer MDAꢀMBꢀ468 cells were treated with increasing concentrations of 1 or 11ad for
4
1
2
hours and the cells were processed for CTꢀL activity determination as described under Methods.
Figure 5A shows that 1 inhibited CTꢀL activity in a dose dependent manner with an IC value of 3.5 ꢂM.
5
0
Figure 5A also shows that consistent with the in vitro results, 11ad was more potent than the parent
compound 1 and inhibited the CTꢀL activity with an IC value of 0.37 ꢂM. We next determined whether
5
0
compounds 1 and 11ad are capable of inhibiting tumor cell survival/proliferation. To this end, MDAꢀMBꢀ
468 cells were treated with parent compound 1 and 11ad and processed for MTT assay as described under
Methods. Figure 5B shows that treatment with both compounds inhibited survival/proliferation but that 11ad
was more potent than the parent compound 1.
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Figure 5. A: Lead 11ad is more potent at inhibiting proteasomal CTꢀL activity in intact human MDAꢀMBꢀ
4
68 cancer cells compared to the parent compound 1. B: Lead 11ad is more potent at inhibiting
proliferation/survival of human MDAꢀMBꢀ468 cells compared to the parent hit 1.
CONCLUSIONS: In this study we have extensively explored the SAR of oxadiazoleꢀisopropylamide
containing analogs as nonꢀcovalent CTꢀL proteasome inhibitors. The structure of the hit, 1 (IC50 = 0.60 ±
0
.18 ꢂM) served as a starting point for synthetic modifications and design and synthesis of novel, highly
potent and nonꢀcovalent proteasome inhibitors. The LC/MSꢀMS and dialysis experiments confirmed that 1
binds to CTꢀL βꢀ5 subunit of the proteasome in a nonꢀcovalent and rapidly reversible binding mode.
Compound 11ad with an IC value of 27 nM was identified as one of the most potent and cell permeable
5
0
1
proteasome inhibitors. Our detailed SAR analysis indicated that extending the R ꢀmethyl in 1 to an optimal
2
size with propyl or butyl in combination with metaꢀpyridyl as R significantly improves the CTꢀL
proteasome inhibitory activity (11ad and 11ae, IC 27 and 39 nM respectively). The isopropyl amide and
50
the ether moieties in 1 are critical for inhibitory activity and modification of these moieties substantially
reduced the CTꢀL inhibitory activity. The SAR demonstrated the importance of the length and composition
1
2
of the linking chain between the amide moiety, R and R groups. The potent lead 11ad demonstrated
efficacy in cellular assays and showed antiꢀproliferative activity at low micromolar concentrations. Our in
vitro data also highlighted that this class of compounds shows high level of selectivity for CTꢀL proteasome
inhibition over TꢀL and PGPHꢀL activities. Moreover, activities observed for some of the other analogs
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presented herein (such as 11al [IC 32 nM], 11am [IC 105 nM] and 11an [IC 107 nM]) could also be
5
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50
optimized in the development of novel nonꢀcovalent CTꢀL proteasome inhibitors.
MATERIALS AND METHODS
0
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CTꢀL, TꢀL, PGPHꢀL proteolytic activity assays. In the highꢀthroughput screen, we used fluorogenic peptides
as substrates to assay (at 10 ꢂM) the 50,000 compounds library from ChemBridge for inhibitory activity against
the CTꢀL proteolytic activity of the purified rabbit 20S proteasome, resulting in the identification of hit 1. To
test for selectivity for CTꢀL over TꢀL and PGPHꢀL we used the corresponding fluorogenic peptides. Briefly, 1
nM of purified 20S rabbit proteasome was incubated with 20 ꢂM SucꢀLeuꢀLeuꢀValꢀTyrꢀAMC for the CTꢀL
activity, BzꢀValꢀGlyꢀArgꢀAMC for the TꢀL activity, and benzyloxycarbonyl ZꢀLeuꢀLeuꢀGluꢀAMC for the
PGPHꢀL activity for 1 h at 37 ºC in 100 ꢂl of assay buffer (50 mM TrisꢀHCl, pH 7.6) with or without
compound. After incubation, production of hydrolyzed 7ꢀamidoꢀ4ꢀmethylꢀcoumarin (AMC) groups was
2
measured using a WALLAC Victor 1420 Multilabel Counter with an excitation filter of 355 nm and an
emission filter of 460 nm (Perkin Elmer Life Sciences, Turku, Finland). The amount of AMC released is within
the linear range. Bortezomib was used as a positive control for IC determinations (IC of Bortezomib was
5
0
50
typically around 10ꢀ30 nM). We used the following equation to calculate the in vitro IC values, A=1/1+
5
0
n
(
[I]/IC ) , where A is the fraction of activity remaining, [I] is the concentration of inhibitor, and n is the Hill
50
Slope Coefficient. To determine proteasome activity in whole cell, extracts (5 ꢂg) from cultured cell lysate was
used instead of 20S rabbit proteasome, and followed the same assay mentioned above except using the
following equation; Y = M + (L – M)/ (1+10^ ((XꢀlogIC )) from GraphPad software, where Y = % inhibition,
5
0
X = inhibitor concentration, M= maximum % inhibition, L = lowest % inhibition.
Cell culture and cell lysate preparation. Human MDAꢀMBꢀ468 breast cancer cells were cultured in
DMEM medium containing 10% fetal bovine serum (FBS) supplemented with 100 units/ml of penicillin and
1
00 ꢂg/ml of streptomycin. Cells were maintained at 37 ºC in a humidified incubator in an atmosphere of
% CO2.
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Whole cell lysates were prepared as follows. Cells were harvested, washed with PBS twice, and
homogenized in a lysis buffer (50 mM TrisꢀHCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NPꢀ40) for 30
min at 4 ºC. Cell lysates were centrifuged at 12,000 g for 15 min, and the supernatants were collected as
whole cell lysates.
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1
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1
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Dialysis using purified rabbit 20S proteasome. To measure the effect of dialysis on CTꢀL activity,
compounds 1 (10 ꢂM), lactacystin (2.5 ꢂM) or vehicle (DMSO) were added to rabbit 20S proteasome at a
final concentration of 1 nM in proteasome assay buffer (50 mM TrisꢀHCl, pH 7.6) and incubated at room
temperature for 30 min. After 30 min of incubation, proteasomeꢀcompound mixtures were added to 3500
MWCO Thermo Scientific SlideꢀAꢀLyzer Mini Dialysis Units (Rockford, IL) and dialyzed against
proteasome assay buffer. Immediately (t = 0) and 0.25, 1, 2, 4, and 18 hour of dialysis at 4 ºC, samples were
removed from the dialysis cassette and the CTꢀL activity of 20S proteasome was determined as described
above under “CTꢀL, TꢀL, and PGPHꢀL proteolytic activity assays” section. Proteasome activity was
normalized against proteasome activity of DMSO control.
MTT (3ꢀ(4,5ꢀdimethythiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) proliferation/survival assay. Cells
were plated in 96ꢀwell plates in 100 ꢂl medium and allowed to attach overnight. Cells were then incubated
for 120 h with varying concentrations of inhibitors. Media was aspirated and replaced with 100 ꢂl complete
media containing 1 mg/ml MTT and incubated for three hours at 37 °C in 5% CO humidified incubator.
2
Media was then aspirated and DMSO was added. Cells were incubated for 10 min at room temperature
while shaking, and the absorbance was determined at 540 nm using a ꢂQuant spectrophotometric plate reader
(
BioꢀTEK, Winooski, VT). The IC values were determined using equation under CTꢀL, TꢀL, PGPHꢀL
50
proteolytic activity assays.
Protein Digestion and Peptide Purification.
Rabbit 20S Proteasome (1 nM), inhibitors and buffer (50 mM TrisꢀHcl, pH 7.6) were incubated (450 ꢂl total
reaction volume) for 30 min at room temperature. After incubation, 112.5 ꢂl of acetonitrile was added and
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trypsin was added to quench the reaction and denature the protein. Trypsin was added with an enzymeꢀtoꢀ
substrate ratio of 1:50. The digestion was carried out for 4 hours at 37 ºC. The digest was concentrated by
vacuum centrifugation (ISS110, Speedvac, Thermo), and the peptides were extracted with C18 reversed
phase pipette tip columns (Ziptip, Millipore). An aliquot (25%) of the total digest was injected into mass
spectrometer. To assess LC/MSꢀMS performance, tryptic peptides from horse apomyoglobin (25 fmol) were
spiked in each LC/MSꢀMS analysis.
0
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LC/MSꢀMS Analysis:
Liquid chromatographyꢀtandem mass spectrometry (LC/MSꢀMS) peptide sequencing experiments were
performed using a nanoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA) interfaced with an
electrospray ion trap mass spectrometer (LTQꢀOrbitrap, Thermo, San Jose, CA) in order to detect and
localize modified peptides from the proteasome. The sample was first loaded onto a trap column (5mm x
3
00 ꢂm ID packed with C18 reversedꢀphase resin, 5ꢂm particle size, 100Å pore size) and washed for 8
minutes with aqueous 2% acetonitrile and 0.04% trifluoroacetic acid. The trapped peptides were eluted onto
the analytical column, (C18 Pepmap 100, 75 ꢂm ID x 15 cm, Dionex, Sunnyvale, CA). The 120ꢀminute
gradient was programmed as: 95% solvent A (2% acetonitrile + 0.1% formic acid) for 8 minutes, solvent B
(90% acetonitrile + 0.1% formic acid) from 5% to 50% in 90 minutes, then solvent B from 50% to 90% B in
7
minutes and held at 90% for 5 minutes, followed by solvent B from 90% to 5% in 1 minute and reꢀ
equilibration for 10 minutes. The flow rate on analytical column was 300 nl/min. Five tandem mass spectra
were collected in a dataꢀdependent manner following each survey scan. The MS survey scans were
performed in Orbitrap to obtain accurate peptide mass measurement and the MS/MS scans were performed in
linear ion trap using 60 second exclusion for previously sampled peptide peaks.
Database Searching and Data Analysis
5
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Sequest searches were performed against a database containing all rabbit proteasome protein sequences (n
= 22) extracted from the UniProt (http://www.uniprot.org). Cleavage was set to fully tryptic, allowing up to
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two missed cleavages; the precursor mass tolerance was 1.08 Da and MS/MS mass tolerance was 0.8 Da.
Dynamic modifications included oxidation (Met + 15.99492), and potential modifications on threonine (Thr
2
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8
9
1
1
1
1
1
1
1
1
1
1
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+
379.18957 for compound 1, Thr +394.20047 for compound 11ad and +213.10009 for βꢀlactone
6
0
modification by lactacystin ). The search results were summarized in Scaffold 3.0.
www.proteomesoftware.com). The integrated peak areas for peptide quantification were calculated from
0
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(
extracted ion chromatograms (EIC) using QuanBrowser from Xcalibur 2.0. These values were restricted by
m/z (+/ꢀ 0.02) and retention time (120 seconds). The accuracy of the m/z values and the fragmentation
patterns of the target peptides were manually inspected to insure proper sequence assignment.
EXPERIMENTAL
General. All reagents were purchased from commercial suppliers and used without further purification.
Melting points were determined using a Barnstead international melting point apparatus or Optimelt
automated melting point system (Stanford Research Systems) and remain uncorrected. Proton NMR spectra
were recorded on an AgilentꢀVarian Mercury 400 MHz spectrometer with CDCl or DMSOꢀd as the
3
6
1
3
solvent. Carbon ( C) NMR spectra are recorded at 100 MHz. All coupling constants are measured in Hertz
(Hz) and the chemical shifts (δ and δ ) are quoted in parts per million (ppm) relative to TMS (δ 0), which
H
C
was used as the internal standard. Formula guided High resolution mass spectroscopy (HRMS) was carried
out on an Agilent 6210 LCꢀMS (ESIꢀTOF). HPLC analysis was performed using a JASCO HPLC system
equipped with a PUꢀ2089 Plus quaternary gradient pump and a UVꢀ2075 Plus UVꢀVIS detector, using an
Alltech Kromasil Cꢀ18 column (150 × 4.6 mm, 5 ꢁm) and Agilent Eclipse XDBꢀC18 (150 × 4.6 mm, 5 ꢁm).
Thin layer chromatography was performed using silica gel 60 F254 plates (Fisher), with observation under
UV when necessary. Anhydrous solvents (acetonitrile, dimethylformamide, ethanol, isopropanol, methanol
and tetrahydrofuran) were used as purchased from Aldrich. Burdick and Jackson HPLC grade solvents
(methanol, acetonitrile and water) were purchased from VWR for HPLC and high resolution mass analysis.
HPLC grade TFA was purchased from Fisher.
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NꢀIsopropylꢀNꢀ((3ꢀpꢀtolylꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl)methyl)ꢀ2ꢀ(pꢀtolyloxy)acetamide (1): To a solution of 10a
187 mg, 0.81 mmol) and triethylamine (164 mg, 1.62 mmol) in THF (15 ml) at r.t., was added 5a (179 mg,
.97 mmol) in THF (3 mL) dropwise (1ꢀ2 min). Upon addition of the acetyl chloride 5a, a precipitate was
(
0
formed and the reaction was completed in 15 min (monitored by tlc, R = 0.75, TLC, EtOAc/Hexane [1:1]).
f
0
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The THF was evaporated and the residue was dissolved in EtOAc (20 ml), washed with HCl (4M, 2 x15 ml)
and water (15 ml). The organic phase was dried (MgSO ), evaporated and the crude product obtained was
4
purified by SiO chromatography (EtOAc/Hexane gradient elution) to obtain 1 (270 mg, 88%) as a white
2
º
1
solid. mp 142.1ꢀ143.4 C. HPLC 100% (t = 11.8 min, 60% CH CN in 0.1% TFA water 30 min); The H
R
3
1
NMR showed 3:1 ratio of atropisomers: H NMR (400 MHz, CDCl ) δ 7.91 (d, J = 8.0 Hz, 2H, [7.93 minor
3
isomer]), 7.30ꢀ7.25 (m, 2H), 7.10 (d, J = 8.3 Hz, 2H, [7.05 minor isomer]), 6.87 (d, J = 8.5 Hz, 2H, [6.82
minor isomer]), 4.78 (s, 2H, [4.84 minor isomer]), 4.70 (s, 2H, [4.83 minor isomer]), 4.45ꢀ4.39 (m, 1H), 2.40
(s, 3H, [2.42 minor isomer]), 2.28 (s, 3H, [2.25 minor isomer]), 1.29 (d, J = 6.6 Hz, 6H, [1.15 minor
1
isomer]); H NMR (400 MHz, d ꢀDMSO) δ 7.84 (d, J = 8.2 Hz, 2H, [7.87 minor isomer]), 7.36 (d, J = 7.9
6
Hz, 2H, [7.37 minor]), 7.01 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H, [6.75 minor isomer]), 4.88 (s, 2H,
[
(
4.98 minor isomer]), 4.71 (s, 2H, [4.82 minor isomer]), 4.31ꢀ4.21 (m, 1H, [4.62ꢀ4.52 minor isomer]), 2.37
1
3
s, 3H), 2.18 (s, 3H), 1.26 (d, J = 6.6 Hz, 6H, [1.06 minor isomer]); C NMR (100 MHz, CDCl ) δ 176.40
3
[176.54 minor isomer], 168.51, 168.56, 156.01 [155.81 minor isomer], 141.68, 131.23 [131.25 minor isomer
shown], 130.29 [130.24 minor isomer], 129.68 [129.84 minor isomer shown], 127.65, 124.08, 114.68
[
114.64 minor isomer], 67.99 [68.74 minor isomer], 48.96 [46.96 minor isomer], 37.20 [38.40 minor isomer],
21.49 [19.97 minor isomer], 20.81, 20.73; Anal. Calcd for C H N O : C, 69.64; H, 6.64; N, 11.07. Found:
2
2
25
3
3
+
C, 69.51; H, 6.74; N, 11.13; LCꢀMS (ESI+) m/z 380.20 (M+H) ; HRMS (ESI+ve) m/z calculated for
+
C H N O (M+H) 380.1969, found 380.1975.
22 26
3
3
NꢀIsopropylꢀNꢀ((3ꢀpꢀtolylꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl)methyl)ꢀ2ꢀ(4ꢀ(trifluoromethyl)phenoxy)acetamide (11a):
This compound was prepared from 5b (149 mg, 0.62 mmol) and 10a (120 mg, 0.52 mmol) using
triethylamine (105 mg, 1.04 mmol) in a similar manner as described for compound 1. The crude product was
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Journal of Medicinal Chemistry
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purified using SiO chromatography (EtOAc/Hexane gradient elution) to afford pure 11a (196 mg, 87%) as a
2
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
º
white solid. mp 76.6ꢀ78.5 C. HPLC 99.5% (t = 14.8 min, 60% CH CN in 0.1% TFA water 30 min); The
R
3
1
1
H NMR showed 3:1 ratio of atropisomers: H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 8.1 Hz, 2H), 7.57ꢀ
3
7
.49 (m, 2H), 7.25 (d, J = 7.9 Hz, 2H, [7.29 minor isomer]),7.04 (d, J = 8.5 Hz, 2H, [6.99 minor isomer]),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
.88 (s, 2H, [4.94 minor isomer]), 4.71 (s, 2H, [4.76 minor isomer]), 4.38ꢀ4.32 (m, 1H), 2.41 (s, 3H, [2.43
1
3
minor isomer]), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]);
C NMR (100MHz, CDCl ) δ 176.16
3
[
[
176.23 minor isomer], 168.57 [167.84 minor isomer], 167.80 [167.87 minor isomer], 160.48, 141.86
142.43 minor isomer], 129.73, 127.58, 127.29 (q, J = 3.67 Hz), 124.47 (q, J = 270 Hz), 124.11 (q, J = 32.5
Hz), 123.88 [123.86 minor isomer], 114.96 [114.82 minor isomer], 67.48 [67.90 minor isomer], 49.00 [47.05
19
minor isomer], 37.23 [38.35 minor isomer], 21.81, 21.45 [19.96 minor isomer];
F NMR (376 MHz,
+
CDCl ) δ ꢀ62.02 [ꢀ62.06 minor isomer]; LCꢀMS (ESI+) m/z 434.18 (M+H) ; HRMS (ESI+ve) m/z
3
+
calculated for C H F N O (M+H) 434.1686, found 434.1711.
2
2
23
3
3
3
NꢀIsopropylꢀNꢀ((3ꢀphenylꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl)methyl)ꢀ2ꢀ(4ꢀ(trifluoromethyl)phenoxy)acetamide (11b):
This compound was prepared from 5b (131 mg, 0.55 mmol) and 10d (100 mg, 0.46 mmol) using
triethylamine (93 mg, 0.92 mmol) in a similar manner as described for compound 1. The crude product was
purified using SiO chromatography (EtOAc/Hexane gradient elution) to afford pure 11b (172 mg, 89%) as a
2
º
white solid. mp 97.9ꢀ99.3 C. HPLC 100% (t = 10.4 min, 60% CH CN in 0.1% TFA water 30 min); The
R
3
1
1
H NMR showed 3:1 ratio of atropisomers: H NMR (400 MHz, CDCl ) δ 8.02ꢀ7.98 (m, 2H), 7.55ꢀ7.43 (m,
3
5
H), 7.04 (d, J = 8.8 Hz, 2H, [7.01 minor isomer]), 4.88 (s, 2H, [4.94 minor isomer]), 4.72 (s, 2H, [4.79
1
3
minor isomer]), 4.39ꢀ4.35 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); C NMR (100MHz,
CDCl ) δ 176.36 [176.47 minor isomer], 168.58 [168.54 minor isomer shown], 167.66 [166.71 minor
3
isomer], 160.50 [160.47 minor isomer], 131.47 [131.92 minor isomer], 129.00 [129.20 minor isomer],
127.65, 127.29 (q, J = 3.74 Hz), 126.78 [126.17minor isomer], 124.46 (q, J = 270 Hz), 124.14 (q, J = 32.7
Hz), 114.97 [114.90 minor isomer], 67.56 [68.07 minor isomer], 48.96 [47.07 minor isomer], 37.21 [38.41
19
minor isomer], 21.48 [19.98 minor isomer]; F NMR (376 MHz, CDCl ) δ ꢀ62.03 [ꢀ62.07 minor isomer];
3
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