Development of radamide analogs as Grp94 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.05.075

Hsp90 isoform-selective inhibition is highly desired as it can potentially avoid the toxic side-effects of pan-inhibition. The current study developed selective inhibitors of one such isoform, Grp94, predicated on the chimeric and pan-Hsp90 inhibitor, radamide (RDA). Replacement of the quinone moiety of RDA with a phenyl ring (2) was found to be better suited for Grp94 inhibition as it can fully interact with a unique hydrophobic pocket present in Grp94. An extensive SAR for this scaffold showed that substitutions at the 2- and 4-positions (8 and 27, respectively) manifested excellent Grp94 affinity and selectivity. Introduction of heteroatoms into the ring also proved beneficial, with a 2-pyridine derivative (38) exhibiting the highest Grp94 affinity (K _d = 820 nM). Subsequent cell-based assays showed that these Grp94 inhibitors inhibit migration of the metastatic breast cancer cell line, MDA-MB-231, as well as exhibit an anti-proliferative affect against the multiple myeloma cell line, RPMI 8226.

Full text of DOI:10.1016/j.bmc.2014.05.075

Bioorganic & Medicinal Chemistry 22 (2014) 4083–4098
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of radamide analogs as Grp94 inhibitors
⇑
Aaron Muth, Vincent Crowley, Anuj Khandelwal, Sanket Mishra, Jinbo Zhao, Jessica Hall, Brian S. J. Blagg
Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott 4070, Lawrence, KS 66045-7563, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Hsp90 isoform-selective inhibition is highly desired as it can potentially avoid the toxic side-effects of
pan-inhibition. The current study developed selective inhibitors of one such isoform, Grp94, predicated
on the chimeric and pan-Hsp90 inhibitor, radamide (RDA). Replacement of the quinone moiety of RDA
with a phenyl ring (2) was found to be better suited for Grp94 inhibition as it can fully interact with a
unique hydrophobic pocket present in Grp94. An extensive SAR for this scaffold showed that substitu-
tions at the 2- and 4-positions (8 and 27, respectively) manifested excellent Grp94 affinity and selectivity.
Introduction of heteroatoms into the ring also proved beneficial, with a 2-pyridine derivative (38) exhib-
iting the highest Grp94 affinity (Kd = 820 nM). Subsequent cell-based assays showed that these Grp94
inhibitors inhibit migration of the metastatic breast cancer cell line, MDA-MB-231, as well as exhibit
an anti-proliferative affect against the multiple myeloma cell line, RPMI 8226.
Received 10 April 2014
Revised 21 May 2014
Accepted 28 May 2014
Available online 12 June 2014
Keyword:
Hsp90
Grp94
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
detrimental activities that include cardiovascular, ocular, and/or
hepatotoxicities, amongst others have been observed for many of
Heat shock protein 90 (Hsp90) is a molecular chaperone that
plays an important role in cellular activity by maintaining the con-
formational maturation of approximately 200 client proteins. In
normal cells, Hsp90 is associated with proteins responsible for traf-
ficking, cell signaling, protein folding, as well as the maintenance
and degradation of client proteins.1,2 Hsp90 functions are modu-
lated through three domains; the N- and C-termini and the middle
domain. The N-terminus contains an ATP-binding site which upon
binding ATP closes to induce ATP hydrolysis and provides the chap-
erone machinery the energy required for protein folding. The mid-
these investigational new drugs.13–15 Unfortunately, the N-termi-
nal ATP-binding site is >85% identical across all four Hsp90 iso-
forms, which includes cytosolic Hsp90
a and b, the endoplasmic
reticulum-localized isoform, Grp94, and the mitochondrial chaper-
one, Trap1.16 Since pan-inhibition of all Hsp90 isoforms can result
in the degradation of more than 200 Hsp90-dependent client
proteins, it is likely that toxic side effects cannot be overcome by
traditional approaches that focus upon pan-Hsp90 inhibition.
Recently, it was shown that Hsp90a is the isoform responsible
for maturation of the hERG channel, and is therefore more likely
responsible for the clinically observed cardiotoxicity.17 Conse-
quently, new approaches toward Hsp90 inhibition are needed in
an effort to refine the number of Hsp90-dependent proteins as well
as to escape the deleterious consequences of pan-Hsp90 inhibition.
The development of isoform-selective Hsp90 inhibitors represents
an attractive strategy for the discovery of such attributes.
dle domain binds the
c-phosphate of ATP and is responsible for
interactions with client proteins. The C-terminus controls Hsp90
homodimerization and also contains a nucleotide-binding site that
allosterically regulates the N-terminal domain.3–9 In cancer cells,
Hsp90 is overexpressed and involved in numerous pathways
required for the rapid growth and proliferation of cancer cells. As
a consequence of its essential role in so many processes, Hsp90
is a highly sought after therapeutic target, as disruption can simul-
taneously affect multiple pathways required for cancer cell
growth.10–12,41–50
Hsp90 N-terminal inhibition has been shown to be an effective
therapeutic strategy as 17 inhibitors of the ATP-binding site have
advanced to clinical trials for the treatment of various cancers.
Despite the number of N-terminal inhibitors in clinical trials,
Compared to cytoplasmic Hsp90a and b, the biological role
manifested by glucose regulated protein-94 kDa (Grp94) is far less
explored.16 Preliminary studies on Grp94 indicate that many of its
client proteins are involved in intercellular communication and
adhesion (i.e., Toll-like receptors, myocilin, integrins, immunoglob-
ulins).18 These properties suggest that Grp94 inhibition may be
useful for the treatment of specific disease states. For example,
since Grp94 plays an integral role in the trafficking of integrins
to the cell membrane, it represents a potential target for the devel-
opment of anti-metastatic agents.19 This is further illustrated by
recent work showing that Grp94 inhibition (via siRNA knockdown)
⇑
Corresponding author. Tel.: +1 (785) 864 2288; fax: +1 (785) 864 5326.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
http://dx.doi.org/10.1016/j.bmc.2014.05.075
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

4084
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
led to inhibition of cell proliferation, migration and metastasis in
two aggressive breast cancer cell lines (MDA-MB-231 cells and
reactive oxygen species resistant (ROS) MCF-7 cells).20 Grp94 has
also been ascribed a role in glaucoma due to its involvement in
mutant myocilin clearance.21 In addition, Grp94 was found to be
overexpressed in multiple myeloma as a consequence of endoplas-
mic reticulum stress,22–24 in fact, Grp94 inhibition was shown to
decrease the proliferation rate of multiple myeloma cells.25
hydrophobic pocket (compounds 2–5) as suggested by Figure 1.
This is necessary to determine how deep these molecules can
project into the pocket and to provide a scaffold upon which sub-
stituents can be readily incorporated for elucidation of structure–
activity relationships. Substitutions around the aryl ring would
then probe for beneficial interactions with the peptide binding
pocket as outlined in Scheme 1. These observations led to the
design and synthesis of compounds that would provide an initial
set of data to direct subsequent optimization efforts. Since RDA
Despite the highly homologous nature of Hsp90 isoforms,
Grp94 possesses a five-amino acid insertion (QEDGQ) between res-
idues 182 and 186, which translates into a small pocket that can be
accessed by Grp94 inhibitors.29 This insertion does not exist in
other Hsp90 isoforms, and therefore creates a unique hydrophobic
environment within the ATP-binding domain that can lead to
selective inhibition.26–29 In 2009, a novel mode of binding was
identified for the radicicol/geldanamycin chimeric inhibitor, rada-
mide (RDA)30,31,37–40 bound to Grp94 (Fig. 1). Co-crystal structures
of RDA bound to both Hsp90 and Grp94 (Fig. 1)30 indicated two key
elements were needed to target the hydrophobic binding pocket
and to selectively bind Grp94. The first proposed modification
included replacement of the quinone ring with a phenyl substitu-
ent that interacts favorably with the hydrophobic pocket exclusive
to Grp94. The second requirement for binding to Grp94 was projec-
tion of the phenyl group into this hydrophobic environment. Such
observations led to the development of BnIm, which demonstrated
good Grp94 inhibitory activity in a number of assays.26 However,
little effort towards the development of RDA as a Grp94 inhibitor
has been pursued, until now. Therefore, the goal of this project
was to further evaluate RDA as a Grp94 inhibitor and to establish
structure–activity relationships for this class of inhibitors.
bound to Grp94 (Scheme 1) indicated an extended p-stacking envi-
ronment with Phe199 and Tyr200, naphthyl, indole, and indoline
scaffolds were also pursued to take advantage of potential interac-
tions. These aryl systems could then be modified to contain addi-
tional substitutions about the ring for further access into the A
and B pockets. Due to
p–p interactions with Phe199 and Tyr200,
probing the electronic nature of the aryl ring via the addition of
heteroatoms that could enhance interactions was also investigated
and led to the identification of additional compounds. Lastly, since
the cis-amide conformation was found important for binding
Grp94 (Fig. 1), tertiary amides were prepared to promote the cis-
conformation.
Projection of the phenyl ring into the hydrophobic binding
region (Scheme 1) required optimization of the linker length
between the amide and phenyl groups, leading to the synthesis
of 2–5 (Fig. 2). Molecular modeling studies indicated that phenyl
amide 2 would provide the ideal linker for binding Grp94. In an
effort to validate this model and to support this hypothesis, linkers
between the amide and the phenyl ring were synthesized and ana-
lyzed for inhibitory activity. In the event, TBS-protected acid 1, was
coupled with the corresponding aniline, benzylamine, phenethyl-
amine, or phenyl hydrazine, utilizing 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDCI) and pyridine in dichloromethane, to
generate the corresponding amides. These compounds were subse-
quently treated with tetrabutylammonium fluoride (TBAF) to
unmask the phenols, 2–5, in good yields. These compounds were
evaluated for their ability to bind Grp94 via a competitive binding
fluorescence polarization assay (Fig. 2).32,33 As suggested by
2. Results and discussion
Optimization of the RDA scaffold required several modifications
to access the unique hydrophobic pocket within Grp94. The first
investigation needed to determine the optimal linker length
between the amide and the aromatic ring that projects into the
A
O
OMe
O
O
O
N
HN
OMe
H
O
N
N
O
O
O
O
Cl
O
HO
OMe
O
OMe
O
O
Cl
Cl
O
O
HO
OH
O
HO
OH
HO
OH
H2N
Radamide (RDA)
BnIm
Radicicol (RDC)
Geldanamycin (GDA)
C
B
Gly121
Tyr200
Phe199
Gly196
Lys98
Phe124
trans-RDA
cisRDA
Asn107
trans-RDA
Lys44
Asn162
trans-RDA
Asn92
Asn91
Trp223
Glu88
Figure 1. (A) Structures of natural product Hsp90 inhibitors GDA and RDC, chimeric inhibitor RDA, and Grp94-selective inhibitor BnIm; (B) Co-crystal structure of RDA bound
to Hsp90 (PDB: 2FXS); (C) Co-crystal structure of RDA bound to Grp94 (PDB: 2GFD).

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4085
region. Molecular modeling indicated that the 2-position of the
phenyl ring is directed towards pocket A, whereas the 3- and 4-
positions point at B. Substitutions at these positions could also pro-
H
N
cis-RDA
O
O
B
O
OMe
vide information about the potential for
bonding interactions.
p-stacking and hydrogen
Cl
CO2Me
A
Functionalization of 2 at the 2-position on the phenyl ring was
pursued with the goal of projecting substituents into pocket A and/
HO
OH
trans-RDA
or altering the electronic nature for additional
p–p interactions.
Radamide
EDCI-mediated coupling with the corresponding anilines, 7a–15a,
followed by silyl-deprotection, generated amides 7–15 in good
yields. Evaluation of 7–15 by the fluorescence polarization assay
demonstrated enhanced binding affinities for these compounds
(620% tracer bound) as compared to the unsubstituted lead com-
pound. These substitutions illustrated that both electronic and ste-
ric effects influence the ability of these amides to bind Grp94, and
the 2-chloro exhibited the highest affinity (8, 0.1% tracer bound). A
trend is observed in Figure 3 which suggests that substituents lar-
ger than chlorine (for example, 2-bromo and 2-trifluoromethyl,
compounds 9 and 11, respectively) exhibit a size-dependent
decrease in binding affinity. The 2-fluoro derivative, 7, also exhib-
ited a decrease in binding affinity, which may result from its inabil-
ity to fill this hydrophobic environment. However, this could also
be a consequence of the electronic effects manifested by fluorine,
which prevents the amide bond from adopting the cis-conforma-
tion.34 Electron donating groups, hydrogen bond acceptors and
hydrogen bond donors all exhibit a decreased affinity for binding
Grp94. Ultimately, it was discovered that a chlorine atom at the
2-position (compound 8) proved optimal for binding to pocket A.
Additional structure–activity relationships were pursued by the
incorporation of substitutions at the 3-position of the phenyl ring.
As shown in Scheme 1, the 3-position of the phenyl ring provides
access to pocket B and therefore, substitutions were incorporated
to elucidate the size of this pocket, the electronic nature that can
R1
N
H
O
N
O
R2
n
Cl
CO2Me
Cl
CO2Me
OH
HO
OH
R
n=0,1,2, NHNH;
R1=alkyl, OH,
R2=cyclohexyl,
substituted aryl rings;
7-65
2-6
Scheme 1. Development of RDA-based Grp94 inhibitors.
computational studies, phenyl amide 2, was found to contain the
optimal linker as compared to benzyl amide, phenethyl amide,
and hydrazine amide (3, 4, and 5, respectively). As described ear-
lier, RDA analogs appear to manifest
p–p interactions with
Phe199 and Tyr200, therefore, compound 6, containing a cyclo-
hexyl ring in lieu of the phenyl ring, was synthesized and evalu-
ated. As expected, this compound demonstrated a decrease in
binding affinity (compound 2 Grp94 Kd, 20.5
l
M, compound 6
M), which was in accord with our predictive model
that supported p–p stacking interactions between Grp94 and com-
enhance p–p stacking interactions, and potentially, hydrogen
Grp94 Kd, 35.2
l
bonding interactions with Gly196. This led to the synthesis of 3-
substituted amides by an EDCI-mediated coupling of acid 1 with
the corresponding anilines, 16a–24a, followed by silyl-deprotec-
tion to give the desired amides, 16–24, in good overall yields. As
seen in Figure 4, these substitutions followed a similar trend for
Grp94 binding as observed for the 2-position, as chlorine at the
3-position (17) displayed the highest binding affinity for Grp94.
In comparison, larger (compounds 18–20) and smaller (compound
16) substituents resulted in diminished binding. In contrast to the
2-position, incorporation of a hydroxyl or amino group at the
pound 2. These studies validated compound 2 as our starting point
for further development and allowed further interrogation of this
scaffold.
Functionalization of phenyl amide 2 at the 2-, 3-, and 4-posi-
tions was pursued to explore the space surrounding the phenyl
ring and to identify key interactions by the inclusion of electron
donating and withdrawing groups. As seen in Scheme 1, there
are two main pockets (A and B) within the hydrophobic binding
R1
N
O
OH
O
R2
1.) EDCI, pyridine,
amine, CH2Cl2
Cl
CO2Me
OTBS
Cl
CO2Me
OH
2.) TBAF, THF
TBSO
HO
1
2-6
entry
R1
R2
H
H
H
H
H
% Tracer Bound
19.8 ( 1.2)
Ph
2
3
4
5
6
CH2Ph
CH2CH2Ph
NHNHPh
cyclohexyl
65.8 ( 3.5)
33.0 ( 2.1)
26.3 ( 1.5)
26.1 ( 1.4)
Figure 2. Optimization of Amide Linker and Hydrophobic Moiety.a–d aCompounds 2–6 (25
l
M) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before
fluorescence polarization values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested
at 25 l
M. dAll compounds were tested in triplicate.

4086
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
R
H
O
OH
O
N
R
1.) EDCI, pyridine,
amine, CH2Cl2
Cl
CO2Me
OTBS
Cl
CO2Me
OH
H2N
2.) TBAF, THF
TBSO
HO
1
7-15
7a-15a
entry
7
R
% Tracer Bound
F
16.3 ( 0.1)
0.1 ( 0.1)
6.1 ( 0.1)
7.9 ( 0.1)
13.4 ( 0.5)
18.0 ( 0.3)
16.1 ( 0.6)
20.5 ( 1.0)
3.4 ( 0.1)
Cl
Br
I
CF3
OH
OMe
NH2
CH3
8
9
10
11
12
13
14
15
Figure 3. Evaluation of 2-phenyl substitutions.a–d aCompounds 7–15 (25
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization
values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested at 25
were tested in triplicate.
l
M. dAll compounds
H
O
OH
O
N
R
1.) EDCI, pyridine,
amine, CH2Cl2
Cl
CO2Me
OTBS
Cl
CO2Me
OH
H2N
R
2.) TBAF, THF
TBSO
HO
1
16-24
16a-24a
entry
16
R
% Tracer Bound
11.0 ( 0.1)
0.1 ( 0.1)
F
Cl
Br
17
9.3 ( 0.3)
18
CF3
39.4 ( 3.0)
46.8 ( 4.6)
3.5 ( 0.1)
27.4 ( 0.9)
17.4 ( 0.8)
8.3 ( 0.1)
19
Ph
20
OH
21
OMe
NH2
22
23
NHAc
24
Figure 4. Evaluation of 3-phenyl substitutions.a–d aCompounds 16–24 (25
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization
values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested at 25
were tested in triplicate.
l
M. dAll compounds
3-position (compounds 21 and 22, respectively) led to increased
affinity for Grp94 (potentially through a gained hydrogen bonding
interaction with Gly196). Both amino and N-acetamides mani-
fested decreased binding affinity as compared to the phenol, as
they are known to form weaker hydrogen bonds. Ultimately, the
3-chloro derivative proved to bind Grp94 more efficiently than
the 3-phenol, indicating a preference for hydrophobic over hydro-
gen bonding interactions at this location.
The 4-position of the phenyl ring was also explored by the
introduction of electron donating and withdrawing groups. As
indicated in Scheme 1, the 4-position appears optimal for directing
substituents into pocket B, however, a potential hydrogen bonding
interaction with Gly196 could also be rationalized. Similar to 3-
substitutions, the 4-position was investigated for steric constraints
in pocket B, as well as elucidation of the electronic nature for p–p
interactions with Phe199 and Tyr200. Compounds 25–37 were
synthesized via EDCI-mediated coupling of TBS-acid 1 with the
corresponding anilines, 25a–37a, followed by silyl-deprotection
to provide amides 25–37 in good overall yields. When evaluated
for their ability to bind Grp94, the 4-position followed a very clear
SAR trend with particular sensitivity to sterics (as shown in Fig. 5).
In contrast to the 2- and 3-positions, the 4-position appears to
accommodate larger functionalities, as illustrated by the inclusion
of bromine at the 4-position (compound 27), which displayed
excellent affinity for Grp94. The enhanced affinity for 27 was antic-
ipated as the 4-bromine can project well into the hydrophobic
pocket of area B (Scheme 1). A decrease in binding affinity was
observed (as compared to 27) for both smaller substitutions

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4087
H
O
OH
O
N
R
1.) EDCI, pyridine,
amine, CH2Cl2
H2N
Cl
CO2Me
OTBS
Cl
CO2Me
OH
2.) TBAF, THF
R
TBSO
HO
1
25-37
25a-37a
entry
R
% Tracer Bound
10.9 ( 0.5)
15.7 ( 0.3)
2.1 ( 0.1)
29.8 ( 1.7)
34.5 ( 3.3)
85.0 ( 16.4)
18.7 ( 0.7)
6.5 ( 0.2)
19.8 ( 1.3)
15.7 ( 0.5)
5.4 ( 0.1)
12.7 ( 1.1)
59.0 ( 3.2)
F
25
26
27
28
29
30
31
32
33
34
35
36
37
Cl
Br
I
CF3
Ph
CH3
OH
OMe
NH2
CN
CCH
CH(CH3)2
Figure 5. Evaluation of 4-phenyl substitutions.a–d aCompounds 25–37 (25
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization
values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested at 25
were tested in triplicate.
l
M. dAll compounds
(4-fluoro and 4-chloro, compounds 25 and 26, respectively) and
larger substitutions (4-iodo, 4-trifluoromethyl, and 4-isopropyl,
compounds 28, 29, and 37, respectively). Similar to the trends
observed for the 2- and 3-positions, electron withdrawing groups
at the 4-position also displayed higher binding affinity than elec-
tron donating groups. A hydrogen bond donor at the 4-position
also displayed good binding affinity (4-hydroxyl, compound 32),
and this affinity could be altered by conversion to the methyl ether
(34), which manifested decreased affinity. Compound 35, contain-
ing a nitrile group at the 4-position, also displayed enhanced Grp94
binding, which may be attributed to its electron withdrawing abil-
ity as well as its ability to serve as a hydrogen bond acceptor. As
predicted by molecular modeling studies which clearly outlined
the A and B pockets as important for binding, both the 2- and 3-
substituted compounds (8 and 17, respectively) exhibited the
greatest influence on Grp94 binding as compared to the 4-substi-
tuted derivatives (27).
coupling of TBS-protected acid 1 with corresponding amines
38a–48a, followed by silyl-deprotection to afford the desired
amides, 38–48. Evaluation of the pyridine analogs showed that a
nitrogen atom at the 2-position (38) resulted in the highest binding
affinity, followed by the 3- (39) and 4-incorporation (40), respec-
tively (as shown in Fig. 6). Molecular modeling studies suggest this
trend was due at least in part by the ability of the nitrogen atom to
project towards the open channel, while simultaneously maintain-
ing optimal orientation for
p-stacking with Phe199 and Tyr200.
However, when the nitrogen was moved to the 3-position (40),
nitrogen is shifted towards the channel, resulting in less favorable
p-stacking interactions. The 4-pyridine derivative (40) demon-
strated the lowest binding affinity due to its inability to access
the open channel, and instead was forced into unfavorable interac-
tions with the hydrophobic binding pocket of B. Pyridazine 42 was
prepared to determine whether the incorporation of 2- and 3-con-
taining nitrogen atoms could simultaneously exist while providing
Preliminary molecular modeling studies (Scheme 1) indicated
that one explanation for the enhanced activity of the phenyl amide
p–p interactions. Unfortunately 42 did not improve binding
affinity, suggesting that the inclusion of a nitrogen atom at the
meta-position is not favored. Subsequent analogs that contained
the 2-pyridine nitrogen and other nitrogen locations (41–45,
Fig. 6) were explored, but unfortunately, did not improve affinity.
In contrast, thiazole 46 and thiadiazoles 47 and 48 displayed good
binding affinity and reinforced the importance of nitrogen
incorporation at the 2-position.
An understanding of this scaffold’s ability to selectively bind
Grp94 required binding affinity evaluation to another isoform,
Hsp90a. As shown in Figure 8, phenyl amide 2 did not demonstrate
scaffold was due to its potential to
p-stack with the electron-rich
rings of Phe199 and Tyr200, suggesting that
p-stacking with these
residues could be enhanced by alteration of the electron density on
the phenyl ring. Structure–activity relationships at the 2-, 3-, and
4-positions supported this hypothesis as decreased electron den-
sity (via electron withdrawing groups) led to enhanced binding.
Further validation of this hypothesis prompted the introduction
of heteroatoms into the aromatic ring to enhance these
interactions.
p-stacking
Investigation of the influence that electron withdrawing groups
exhibit led to a series heterocycles that exhibited decreased elec-
tron density within the aromatic ring. Sulfur containing 5-mem-
bered heterocycles (46–48) were synthesized to represent a ring
similar in size as the corresponding 6-membered analogs (38–
45), while manifesting some electron deficiency. As described
previously, these analogs were generated by an EDCI-mediated
selectivity for Grp94 (ꢀ1:1), illustrating the need to optimize this
scaffold for greater selectivity. However, substitution at the 2-, 3-
, and 4-positions on the phenyl ring (compounds 8, 17, and 27,
respectively) did enhance selectivity for Grp94. Specifically, com-
pound 27 showed the greatest selectivity (ꢀ27-fold at 25
lM),
indicating the importance of substituents at the 4-position for
selective inhibition, while 8 and 17 manifested 8- and 25-fold

4088
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
H2N
H2N
V
Y
W
X
H
N
H
N
O
OH
O
V
Y
O
W
Y
Z
W
X
X
Z
Z
38a-45a
1.) EDCI, pyridine,
amine, CH2Cl2
Cl
CO2Me
OTBS
Cl
CO2Me
Cl
CO2Me
W
X
Y
or
2.) TBAF, THF
Z
TBSO
HO
OH
HO
OH
1
38-45
46-48
46a-48a
%Tracer
entry
V
W
X
Y
Z
Bound
N
CH
N
CH
CH
N
CH
CH
CH
N
CH
N
CH
N
CH
CH
N
CH
CH
CH
CH
CH
CH
N
1.0 ( 0.1)
14.9 ( 1.0)
34.9 ( 2.3)
16.6 ( 0.7)
12.2 ( 1.0)
11.6 ( 0.7)
16.7 ( 1.2)
19.5 ( 1.2)
6.6 ( 0.2)
8.7 ( 0.6)
10.2 ( 0.6)
38
39
40
41
42
43
44
45
46
47
48
CH
CH
N
CH
CH
N
CH
CH
CH
CH
CH
CH
N
N
CH
N
N
CH
CH
N
N
N
S
N
N
S
CH
S
Figure 6. Evaluation of heterocyclic amides.a–d aCompounds 38–48 (25
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization
values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested at 25
were tested in triplicate.
l
M. dAll compounds
R
HN
R
N
1.) EDCI, pyridine,
O
OH
O
O
N
amine, CH2Cl2,
or
49a-51a
53a
(COCl)2, DMF,
CH2Cl2 then
DIPEA, amine,
Cl
CO2Me
OR1
Cl
CO2Me
OH
Cl
CO2Me
OH
HN
or
2.) TBAF, THF, or
H2, Pd/C, MeOH
R1O
HO
HO
1
53
: R1=TBS
1a: R1=Bn
49
50
51
: R=Me
: R=Et
: R=Pr
i
R
N
O
H
O
1.) nitrosobenzene,
DBU, catalyst,
CH2Cl2
BF4
Cl
CO2Me
OTBS
N
Cl
CO2Me
OH
N
C6F5
N
2.) TBAF, THF
TBSO
HO
1b
52
: R=OH
catalyst
entry
% Tracer Bound
16.7 ( 2.1)
18.6 ( 2.8)
30.2 ( 3.2)
2.5 ( 0.8)
49
50
51
52
53
23.0 ( 4.4)
Figure 7. Evaluation of amides 49–53.a–d aCompounds 49–53 (25
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization values
were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. cAll compounds tested at 25
tested in triplicate.
l
M. dAll compounds were
selectivity, respectively. It was also observed that heterocycles 38,
46 and 47 exhibited selectivity, albeit to a lesser extent than 8, 17,
and 27. In an effort to further improve Grp94-selective binding,
tertiary amides 49–53 were developed. These compounds were
designed to enhance isomerization to the cis-amide conformation,
by making the cis-conformation lower in energy, which appears

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4089
80
70
60
50
40
30
20
10
0
affinities (Figs. 7 and 8). In contrast, compound 52, which contains
an N-hydroxyl, demonstrated a significant increase in selectivity,
however, at the consequence of lower affinity (Figs. 7 and 8).
Further optimization of this scaffold was pursued to develop
more potent and selective Grp94 inhibitors by combining the fea-
tures identified above to produce 54–65, which contain the phenyl
amide, an ortho-pyridine, a thiazole, or a thiadiazole ring along
with substitution patterns based on earlier studies (Fig. 9). These
compounds were also synthesized by the EDCI-mediated coupling
of acid 1 with anilines 54a–65a. Subsequent silyl-deprotection of
the resulting amides afforded 54–65. Unfortunately, 54–58, 60,
61, and 63–65 were unable to exhibit Grp94 binding affinity simi-
lar to 8, 17, 27 or 38. Some of the compounds (60, 61, and 63)
exhibited a slight enhancement for Grp94 selectivity, but also
demonstrated lesser affinity. However, 58, 59 and 62 demon-
strated a high Grp94 binding affinity (61% tracer bound), while
Grp94
Hsp90α
GDA RDA
2
3
4
8
17 21 27 38 46 47 49 50 51 52 53 58 59 62
Figure 8. Inhibition of Grp94 and Hsp90
a
at 25
l
M Illustrating Grp94-Selectivity.a–
aCompounds (25
lM) were incubated with cGrp94 or Hsp90a and FITC-GDA
e
(tracer) for 24 h before fluorescence polarization values were determined. bDMSO
(1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive
control. cAll compounds tested at 25 M. dAll compounds were tested in triplicate.
l
also manifesting good selectivity (4–18% tracer bound for Hsp90a).
Despite these characteristics, compounds 54–65 did not manifest
greater binding affinity or selectivity for Grp94 than previously
prepared analogs.
Compounds 8, 17, 27, 38, 46 and 47 were evaluated for determi-
nation of their respective Kd values for Grp94 binding. Kd’s were
preferred for Grp94-inhibition (Fig. 1). Compounds 49, 50 and 53
were prepared by an EDCI-mediated coupling of TBS-protected
acid 1 with anilines 49a, 50a, and 53a, followed by silyl-deprotec-
tion to afford the desired amides, 49, 50, and 53. 51 and 52
required an alternative method for their preparation. Compound
51 was generated by conversion of the benzyl-protected acid 1a
to the corresponding acid chloride, followed by coupling with ani-
line 51a and subsequent debenzylation to afford desired amide 51
(Fig. 7). Compound 52 was prepared by catalyst-mediated coupling
of aldehyde 1b with nitrosobenzene, followed by silyl-deprotec-
tion to afford amide 52 (Fig. 7).35 Unfortunately, compounds 49–
51 and 53 exhibited little to no improvement in Grp94-selectivity
(Fig. 8), while simultaneously manifesting decreased binding
determined in a manner similar to the 25 lM fluorescence polari-
zation screen, wherein compounds were incubated with Grp94 and
FITC-GDA for 24 h at increasing concentrations. From the data, the
Kd values for 8, 17, 27, 38, 46, and 47 were determined as shown in
Table 1. In accord with earlier studies, substitutions at the 2- and
4-positions (8 and 27, respectively) manifested better activity than
the 3-position (17). The importance of a nitrogen atom at the 2-
position was also confirmed as 38 manifested the highest affinity
(0.82
lM), while both 46 and 47 also displayed less affinity
R2
R2
R1
R3
R4
R1
X
R1
R3
R4
Y
Z
X
X
NH2
O
OH
O
NH
O
NH
54a-62a
R1
Y
1.) EDCI, pyridine,
amine, CH2Cl2
Cl
CO2Me
OTBS
Cl
CO2Me
OH
Cl
CO2Me
OH
X
Z
or
2.) TBAF, THF
NH2
TBSO
HO
HO
1
54-62
63-65
63a-65a
%
Tracer
entry
R1
R2
R3
R4
X
Y
Z
Bound
H
Br
H
Br
H
H
Br
H
H
H
H
Cl
H
Cl
H
H
Cl
H
H
Br
H
H
Br
H
H
H
Cl
Br
CH
50.7 ( 3.3)
15.6 ( 0.4)
71.4 ( 13.2)
24.9 ( 1.0)
1.4 ( 0.1)
0.1 ( 0.1)
7.7 ( 0.3)
9.8 ( 0.4)
0.2 ( 0.1)
22.4 ( 1.6)
27.7 ( 1.8)
5.1 ( 0.3)
54
55
56
57
58
59
60
61
62
63
64
65
Cl
Cl
H
CH
CH
CH
N
Cl
H
N
H
N
H
N
H
N
Cl
Br
Br
S
CH
CH
N
N
N
N
S
S
Figure 9. Combinations of key features and their Grp94 inhibition.a–d aCompounds 54–65 (25
fluorescence polarization values were determined. bDMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control. ND = not determined.
cAll compounds tested at 25 M. dAll compounds were tested in triplicate.
lM) were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before
l

4090
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
Table 1
Kd’s of 8, 17, 27, 38, and 46 for Grp94a–d
entry
8
Kd (µM)
1.85 ( 0.22)
6.42 ( 0.92)
1.50 ( 0.18)
0.82 ( 0.09)
1.08 ( 0.13)
1.54 ( 0.26)
17
27
38
46
47
a
b
c
Compounds 8, 17, 27, 38, 46 and 47 were incubated with cGrp94 and FITC-GDA (tracer) for 24 h before fluorescence polarization values were determined.
DMSO (1%) served as a negative control (vehicle), and GDA (50 nM) served as the positive control.
All compounds were tested in triplicate.
d
Millipolarization (mP) units were used to determine % tracer bound.
(1.08
l
M and 1.54
l
M, respectively). Compounds 38 and 46 man-
which cells can migrate and heal the wound. Migration was
recorded at 0 h, 16 h, and 24 h using a digital camera mounted
ifested the highest affinity for Grp94, and therefore were used in
subsequent cell-based assays.
microscope. In addition, each compound was tested at 2.5
lM
(Fig. 10) up to 25 M (Supporting information, Figs. S1–S2) where
l
2.1. Inhibition of migration and proliferation of MDA-MB-231
cells
they demonstrated no cytotoxicity. The results at 2.5
shown in Figure 10.
lM are
Compounds 38 and 46 were evaluated in this assay as they
manifested the most potent (Kd values, Table 1) Grp94 inhibition
while also manifesting good selectivity for Grp94 binding (Fig. 8).
As shown in Figure 10, they inhibited cell migration consistent
with Grp94 inhibition, which was also in accord with prior studies
by Verrax et al.20, wherein they showed that Grp94 knockdown led
to migratory inhibition of MDA-MB-231 cells. The ability of these
compounds to inhibit migration mirrored the trend observed in
the fluorescence polarization assay, where compound 38 mani-
fested a greater anti-migratory effect than compound 46. The
anti-migratory effect of these compounds was determined to be
dose-dependent (Supporting information, Figs. S1 and S2). Ulti-
mately, the wound-healing assay illustrates the potential for
Inhibition of Grp94 has been linked to decreased metastasis,
migration and proliferation in the highly metastatic breast cancer
cell lines, MDA-MB-231 and ROS-resistant MCF-7.20 Specifically,
silencing Grp94 expression caused almost complete inhibition of
migration as well as a significant decrease in proliferation of the
highly aggressive metastatic breast cancer cell line, MDA-MB-
231.20 While compounds 38 and 46 manifested modest anti-prolif-
erative activities against MDA-MB-231 cells (38: IC50 = 12
lM, 46:
IC50 = 16 M), they did demonstrate potent anti-migratory proper-
l
ties against the same cell line. This was determined by employing a
wound healing assay where a monolayer of cells was grown in a
12-well plate format. In each well, two wounds were created in
Figure 10. a,bInhibition of MDA-MB-231 Cell Migration by Compounds 38 and 46a,b aMDA-MB-231 cells were incubated for 24 h prior to compound addition. bCells were
incubated for 16 h and 24 h at 37 °C with the respective conjugate indicated above (2.5 M).
l

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4091
Grp94-inhibitors to be used in the treatment of highly metastatic
breast cancers, while manifesting minimal toxicity.
Compounds 38 and 46 demonstrated the ability to bind Grp94
in MDA-MB-231 cells. The anti-Grp94 (9G10) antibody recognizes
the open conformation of Grp94, and when an inhibitor is bound to
this region, Grp94 switches to the closed conformation. Therefore,
the 9G10 antibody does not recognize Grp94 bound to an inhibi-
tor.26,37 Compounds 38 and 46 demonstrated a dose-dependent
effect by inducing a conformational change in Grp94, and thus,
prevented the 9G10 antibody from recognizing and immunopre-
cipitating Grp94 from MDA-MB-231 cells (as shown in Fig. 11).
These findings correspond well with the observed anti-prolifera-
tive and anti-migratory concentrations, indicating these effects
parallel Grp94-inhibition.
Figure 13. Western blot analyses of Hsp90-dependent client proteins from MDA-
MB-231 breast cancer cell lysate upon treatment with amides 38 and 46. H (high)
represents a concentration equal to 5-fold the anti-proliferative IC50 value. L (low)
represents
Radicicol (RDC, 15
a
concentration equal to 0.5-fold the anti-proliferative IC50 value.
M) and dimethylsulfoxide (DMSO, 100%) were employed as
l
positive and negative controls, respectively.
Previous studies have shown that Grp94-inhibition decreases
insulin-like growth factor-II (IGF-II) secretion, which also plays
an important role in the proliferation and migration of certain can-
cers.26,51,52 Compounds 38 and 46 decreased secretion of the
Grp94-dependent client protein, IGF-II, as shown in Fig. 12. The
dramatic decrease in IGF-II secretion was observed at concentra-
tions that parallel the concentrations needed to exhibit anti-migra-
tory activity.
Compounds 38 and 46 also demonstrated selective Grp94-inhi-
bition by Western blot analysis of Hsp90 client proteins Akt and
Cyclin D1 from MDA-MB-231 cell lystates (Fig. 13). Degradation
of Hsp90-dependent cytosolic clients, Akt and Cyclin D1, was not
2.2. Anti-proliferative effect against RPMI 8226 cells
Recently, elevated levels of Grp94 were linked to the acceler-
ated proliferation of multiple myeloma cells,22–24 identifying
Grp94 as a potential therapeutic target for the treatment of
myeloma.25 The RPMI 8226 multiple myeloma cell line was shown
to be particularly sensitive to Grp94 inhibition, which prompted
investigation of compounds 38 and 46 for their anti-proliferative
activity against this cell line.
As anticipated, the two most potent compounds for binding
Grp94 (Table 1) also exhibited potent anti-proliferative properties
observed at a high concentration of compound 38 (60
ing other Hsp90 isoforms were not affected at the concentration
needed to exhibit anti-migratory activity (2.5–25 M). Compound
46 demonstrated modest client-protein degradation at 80 M,
lM), indicat-
against the RPMI 8226 cell line (IC50 <10
ingly, 38 displayed slightly lower anti-proliferative effect
(9.12 M) than 46 despite its superior binding affinity. These com-
lM, Table 2). Interest-
a
l
l
l
pounds were then evaluated for their ability to selectively inhibit
Grp94 in RPMI 8226 cells in a similar fashion seen for MDA-MB-
231 cells.
Compounds 38 and 46 did demonstrate an ability to bind Grp94
in RPMI 8226 cells in a dose-dependent manner. This was first
determined by use of the 9G10 antibody to recognize Grp94 in
immunoprecipitated RPMI8226 cell lysates treated with com-
pounds 38 and 46 (Fig. 14). These data indicate that compounds
38 and 46 bind Grp94 at concentrations needed to inhibit the pro-
liferation of RPMI 8226 cells.
however, this effect was observed at concentrations significantly
higher than the concentration needed for anti-migratory activity.
This finding along with the observed effects of utilizing the 9G10
antibody (Fig. 11) and decreased IGF-II secretion (Fig. 12) indicate
these compounds inhibit Grp94 at concentrations relevant to the
observed anti-migratory activity.
Compounds 38 and 46 also decreased secretion of the Grp94-
dependent client protein, IGF-II; at concentrations similar to the
observed anti-proliferative activity manifested by both compounds
Table 2
a–cAnti-proliferative evaluation of 38 and 46 in RPMI 8226 cells
Entry
IC50 (lM)
Figure 11. Effect of compounds 38 and 46 on Grp94 conformation. MDA-MB-231
cells were treated with the indicated concentrations of 38 and 46 (concentrations
listed are lM) or radicicol (R 15 lM) overnight, and cell lysates were immunopre-
38
46
9.12 ( 0.4)
7.83 ( 2.2)
cipitated with the conformational-specific antibody 9G10 and subsequently were
immunoblotted for Grp94. Lower panel, immunoblot of whole cell lysates with
9G10.
a
b
c
RPMI 8226 cells were incubated for 24 h prior to drug addition.
Cells were incubated for 72 h at 37 °C in 5% CO2 with the respective conjugate.
All experiments were performed in triplicate.
Figure 14. Effect of compounds 38 and 46 on Grp94 conformation. RPMI 8226 cells
were treated with the indicated concentrations of 38 and 46 (concentrations listed
are lM) or radicicol (R 15 lM) overnight, and cell lysates were immunoprecipitated
with the conformational-specific antibody 9G10 and subsequently were immuno-
blotted for Grp94. Lower panel, immunoblot of whole cell lysates with 9G10.
Figure 12. Western blot analysis of IGF-II secretion into MDA-MB-231 breast
cancer cell media upon treatment with amides 38 and 46. H (high) represents a
concentration equal to 10
Radicicol (RDC, 15 M) and dimethylsulfoxide (DMSO, 100%) were employed as
positive and negative controls, respectively.
lM. L (low) represents a concentration equal to 1 lM.
l

4092
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
synthesized, and evaluated for their ability to inhibit Grp94. A
comprehensive SAR study of this scaffold indicated several key
features necessary for selective inhibition of Grp94 (Fig. 17). Ini-
tial optimization of the linker led to the identification of phenyl
amide, 2, manifesting the highest Grp94 affinity (blue, Fig. 17).
This finding demonstrated both the importance of a short linker,
as well as the presence of an aromatic ring for p-stacking interac-
tions. Substituents at the 2- and 4-positions (pink and green,
Fig. 17) of the phenyl amide demonstrated increased Grp94 bind-
ing affinity and selectivity, with chlorine and bromine substitu-
Figure 15. Western blot analysis of IGF-II secretion into RPMI 8226 cell media upon
treatment with amides 38 and 46. H (high) represents a concentration equal to 2-
fold the anti-proliferative IC50 value. L (low) represents a concentration equal to
ents identified as optimal (Table 1, compounds
8 and 27,
0.25-fold the anti-proliferative IC50 value. Radicicol (RDC, 15 lM) and dimethyl-
sulfoxide (DMSO, 100%) were employed as positive and negative controls,
respectively.
respectively). Substitution at the 3-position (orange, Fig. 17) also
proved important; however, both potency and selectivity were
decreased relative to substitutions at the 2- and 4-positions
(Table 1, compound 17). In contrast, modifications to the 4-posi-
tion resulted in the greatest effect on selectivity, as most substit-
uents at this position manifested enhanced selectivity. Decreasing
the electron density of the aromatic ring via the introduction of
heteroatoms (38, 46, and 47) not only increased binding affinity,
but also increased selectivity. However, this finding was specific
to the incorporation of
a nitrogen atom at the 2-position
(Fig. 6). The 2-pyridine ring was successfully incorporated with
the 4-bromine or 3-chlorine derivative to provide potent and
selective Grp94-inhibitors. Subsequent in vitro analysis of 38
and 46 further validated the therapeutic potential for Grp94
inhibitors as well as validated selective Grp94-inhibition. The
ability of these compounds to inhibit Grp94 was correlative with
their anti-migratory activity against the highly metastatic breast
cancer cell line, MDA-MB-231, as well as anti-proliferative activ-
ity against the multiple myeloma cell line, RPMI 8226.
Figure 16. Western blot analyses of Hsp90-dependent client proteins from RPMI
8226 multiple myeloma cell lysate upon treatment with amides 38 and 46. H (high)
represents a concentration equal to 5-fold the anti-proliferative IC50 value. L (low)
represents
Radicicol (RDC, 15
a
concentration equal to 0.5-fold the anti-proliferative IC50 value.
M) and dimethylsulfoxide (DMSO, 100%) were employed as
l
positive and negative controls, respectively.
(Fig. 15). Along with the use of the 9G10 antibody (Fig. 14), this
data also suggests that the observed anti-proliferative activity of
compounds 38 and 46 against RPMI 8226 cells is attributed to
the inhibition of Grp94 in RPMI 8226 cells.
4. General experimental methods
4.1. Fluorescence polarization
Compounds 38 and 46 also demonstrated that their anti-prolif-
erative activities are likely due to selective Grp94 inhibition. This
was illustrated by the inability of compound 38 to degrade
Hsp90 client-proteins Akt and cyclin D1 (Fig. 16) at concentrations
above and below its respective anti-proliferative IC50 value. Com-
pound 46 demonstrated modest client-protein degradation at con-
centrations five-fold higher than its IC50, indicating a slightly lower
selectivity for Grp94. However, this data does indicate that com-
pounds 38 and 46 inhibit Grp94 in RPMI 8226 cells, which results
in their anti-proliferative activity. The ability of this scaffold to
selectively inhibit Grp94 and manifest anti-proliferative activity
against the RPMI 8226 multiple myeloma cell line demonstrates
the therapeutic potential of Grp94 inhibition for the treatment of
these cancers.
Assay buffer (25 lL, 20 mM HEPES pH 7.3, 50 mM KCl, 5 mM
MgCl2, 1 mM DTT, 20 mM Na2MoO4, 0.01% NP-40, and 0.5 mg/mL
BGG) containing desired compound or GDA were plated in 96-well
plates (black well, black bottom) at the indicated final concentra-
tions in DMSO (1% DMSO final concentration).32 Recombinant
cGrp94 and FITC-GDA were then added (10 and 6 nM, respec-
tively). Plates were incubated with rocking for 24 h at 4 °C. Fluores-
cence was read using excitation and emission filters of 485 and
528 nm, respectively. Percent FITC-GDA bound was determined
by using the DMSO millipolarization unit (mP) as the 100% bound
value and the mP value of free FITC-GDA as the 0% bound value. Kd
values were calculated from separate experiments performed in
triplicate using GraphPad Prism.
3. Conclusion
Using the previously reported co-crystal structure of RDA
4.2. Anti-proliferation assays
bound to Grp94,
a
series of RDA analogs were designed,
RPMI 8226 and MDA-MB-231 cells were maintained in
RPMI1640 (Cellgro) and DMEM (Cellgro), respectively, supple-
Cl > Br > F
Br > F > Cl
mented with nonessential amino acids,
tomycin (500 g/mL), penicillin (100 units/mL) and 10% FBS. Cells
were grown to confluence in a humidified atmosphere (37 °C, 5%
CO2), seeded (2000/well, 100 L) in 96-well plates, and allowed
L-glutamine (2 mM), strep-
l
Cl > Br > F
l
H
2-pyridine > phenyl > cyclohexyl
to attach overnight. Compound at varying concentrations in DMSO
(1% DMSO final concentration) was added, and cells were returned
to the incubator for 72 h. After 72 h, the number of viable cells was
determined using an MTS/PMS cell proliferation kit (Promega) per
the manufacturer’s instructions. Cells incubated in 1% DMSO were
use as 100% proliferation, and values were adjusted accordingly.
IC50 values were calculated from separate experiments performed
in triplicate using GraphPad Prism.
O
N
n
n = 0 > 1 > 2
Cl
CO2Me
OH
HO
Figure 17. Summary of SAR for Grp94 binding of RDA based derivatives.

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4093
4.3. Wound-healing assay
necessary, were purified and dried by standard methods. Concen-
tration of solutions after reactions and extractions involved the
use of a rotary evaporator operating at reduced pressure.
Cells were grown to confluence in a humidified atmosphere
(37 °C, 5% CO2), seeded (200,000/well, 1 mL) in 12-well plates,
and allowed to grow to a confluent monolayer (24 h) at 37 °C
4.8. General amide formation for compounds 2–50 and 53–65
and 5% CO2. Each well was then scratched with a 200
pipet tip, photographed with an Olympus IX-71 microscope (60ꢁ
objective, time = 0 h), the respective compound solutions (2.5 L,
final DMSO concentration is 0.25%) were added, and the plates
were placed back in the incubator. Cell migration was recorded
after 16 and 24 h of incubation with compound via photographs
taken by the Olympus IX-71 microscope. All experiments were
run in quadruplicate on two different days.
lL sterile
To a solution of acid 1 (0.05 mmol), EDCIꢂHCl (0.12 mmol), and
pyridine (0.13 mmol) in CH2Cl2 (1 mL) was added the correspond-
ing aniline (0.1 mmol) and stirred at room temperature under Ar
overnight. Upon complete consumption of acid 1, the solvent was
removed in vacuo and redissolved in THF (1 mL). The reaction mix-
ture was then treated with TBAF (0.2 mmol) and stirred for 30 min,
and upon completion saturated aqueous NH4Cl was added and
extracted 3ꢁ with EtOAc. The combined organic layers were then
dried over Na2SO4, filtered, and concentrated in vacuo to give a
crude oil. The residue was purified via flash chromatography
(SiO2, 49:1, CH2Cl2/MeOH) to afford the desired amide.
l
4.4. Western blotting
MDA-MB-231 and RPMI 8226 cells were plated in 6-well plates
and treated with high and low concentrations of compounds 38
and 46, as well as 15 lM RDC in DMSO (0.25% DMSO final concen-
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(phenylamino)propyl)
benzoate (2). 15 mg, 86% yield, white solid. 1H NMR (400 MHz,
tration), or DMSO only for 24 h. Cells were harvested in cold PBS
and lysed in mammalian protein extraction reagent (MPER, Pierce)
and protease inhibitors (Roche) on ice for 15 min. Lysates were
clarified at 15,000 rpm for 15 min at 4 °C. Protein concentrations
were determined with the Pierce BCA assay kit per the manufac-
CDCl3): d 11.23 (s, 1H), 7.53 (d, J = 7.91 Hz, 2H), 7.34 (t,
J = 7.66 Hz, 2H), 7.12 (t, J = 7.36 Hz, 1H), 6.59 (s, 1H), 6.33 (s, 1H),
3.97 (s, 3H), 3.50 (dd, J = 5.76, 10.15 Hz, 2H), 2.66–2.59 (m, 2H).
13C NMR (125 MHz, CDCl3): d = 170.43, 169.99, 162.91, 156.28,
141.66, 137.73, 129.10 (2C), 124.40, 119.72 (2C), 113.92,
106.81, 102.94, 52.75, 36.73, 28.81. HRMS (FAB) m/z [M+Na+] for
turer’s instructions. Equal amounts of protein (20
l
g) were electro-
PVDF
phoresed under reducing conditions, transferred to
a
C17H16ClNO5Na: 372.0609, found 372.0609.
membrane, and immunoblotted with the corresponding specific
antibodies. The corresponding amount of media necessary to main-
tain equal actin levels was also electrophoresed under reducing
conditions, transferred to a PVDF membrane, and immunoblotted
with the IGF-II antibody. Membranes were incubated with an
appropriate horseradish peroxidase-labeled secondary antibody,
developed with chemiluminescent substrate, and visualized.
Methyl 2-(3-(benzylamino)-3-oxopropyl)-3-chloro-4,6-dihydrox-
ybenzoate (3). 30 mg, 83% yield, white solid. 1H NMR (500 MHz,
CDCl3): d 7.32–7.21 (m, 5H), 6.44 (s, 1H), 4.40 (d, J = 4.7 Hz, 2H),
3.86 (s, 3H), 3.44–3.29 (m, 2H), 2.50–2.33 (m, 2H). 13C NMR
(125 MHz, CDCl3)
d 171.98, 170.68, 162.51, 157.03, 141.87,
138.12, 127.81, 127.60, 114.22, 106.34, 102.63, 52.53, 49.78,
43.73, 35.65, 28.92. HRMS (FAB) m/z [M+Na+] for C18H18ClNO5Na:
386.0874; found, 386.0877.
4.5. Grp94 immunoprecipitation
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(phenethylamino)pro-
pyl)benzoate (4). 17 mg, 90% yield, white solid. 1H NMR
(400 MHz, CDCl3): d 11.29 (s, 1H), 7.32 (dd, J = 6.63, 8.10 Hz, 2H),
7.26–7.23 (m, 1H), 7.22–7.17 (m, 2H), 6.57 (s, 1H), 5.48 (t,
J = 5.95 Hz, 1H), 3.91 (s, 3H), 3.56 (q, J = 6.58 Hz, 2H), 3.40–3.34
(m, 2H), 2.84 (t, J = 6.84 Hz, 2H), 2.41–2.35 (m, 2H). 13C NMR
Detergent lysates of the indicated cells were immunoprecipi-
tated with 9G10 monoclonal anti-Grp94 (Enzo Life Sciences)
followed by protein G-MagBeads (GenScript) as previously
described.36
(125 MHz, CDCl3):
d 171.92, 170.64, 162.90, 156.43, 141.74,
4.6. Molecular modeling
138.72, 128.77, 128.71, 126.63, 113.90, 106.64, 102.80, 52.58,
40.63, 35.81, 35.61, 29.05. HRMS (FAB) m/z [M+Na+] for
Surflex-Dock in Sybyl v8.0 was used for molecular modeling
and docking studies. The co-crystal structure of RDA bound to
Grp94 was utilized for all docking experiments.30 The docked mol-
ecules were locked into the cis-amide conformation and utilized
with 10 different starting conformations while rotation of the
rotatable bonds was unrestricted. Visual interpretation and figure
preparation were then carried out in Pymol (Scheme 1).
C
19H20ClNO5Na: 400.0922, found 400.0931.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(2-phenylhydrazinyl)
propyl)benzoate (5). 17 mg, 94% yield, orange solid. 1H NMR
(400 MHz, CD3OD, major conformer): d 7.23–7.13 (m, 2H), 6.83–
6.74 (m, 3H), 6.41 (s, 1H), 3.92 (s, 3H), 3.36–3.28 (m, 2H), 2.59–
2.53 (m, 2H); 13C NMR (125 MHz, CD3OD, major conformer): d
174.8, 171.6, 161.5, 159.0, 150.0, 142.1, 130.2, 130.0, 121.0,
115.0, 114.1, 113.3, 109.5, 103.2, 52.9, 34.3, 29.1. HRMS (FAB)
m/z [M+Na+] for C17H17ClN2O5Na: 387.0724; found, 387.0723.
Methyl 3-chloro-2-(3-(cyclohexylamino)-3-oxopropyl)-4,6-dihyd-
roxybenzoate (6). 33 mg, 88% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 6.41 (s, 1H), 3.89 (s, 3H), 3.68 (s, 1H), 3.40–
3.24 (m, 2H), 2.40–2.26 (m, 2H), 1.91–1.76 (m, 2H), 1.64 (dt,
J = 13.8, 3.8 Hz, 2H), 1.61–1.52 (m, 1H), 1.39–1.25 (m, 2H), 1.07
(ddd, J = 19.2, 9.8, 3.9 Hz, 3H). 13C NMR (125 MHz, CDCl3) d
171.44, 170.78, 162.49, 157.48, 142.07, 114.43, 106.04, 102.51,
52.46, 49.40, 48.22, 35.90, 33.07, 28.92, 25.46, 24.83. HRMS (FAB)
m/z [M+Na+] for C17H22ClNO5Na: 378.1187; found, 378.1167.
Methyl 3-fluoro-2-(3-((2-chlorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (7). 15 mg, 81% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 11.19 (s, 1H), 8.49–8.00 (m, 1H), 7.34 (s, 1H),
7.12–6.97 (m, 3H), 6.53 (s, 1H), 6.05 (s, 1H), 3.91 (s, 3H), 3.49–
3.37 (m, 2H), 2.67–2.57 (m, 2H).13C NMR (125 MHz, CDCl3) d
4.7. Chemistry general
1H NMR were recorded at 400 or 500 MHz (Bruker DRX-400
Bruker with a H/C/P/F QNP gradient probe) spectrometer and 13C
NMR spectra were recorded at 125 MHz (Bruker DRX 500 with
broadband, inverse triple resonance, and high resolution magic
angle spinning, HR-MA probe spectrometer); chemical shifts are
reported in d (ppm) relative to the internal chloroform-d (CDCl3,
7.27 ppm). FAB (HRMS) spectra were recorded with a LCT Premier
(Waters Corp., Milford, MA). The purity of all compounds was
determined to be >95% purity as determined by 1H NMR and 13C
NMR spectra, unless otherwise noted. The most active five com-
pounds were verified for >95% purity by HPLC analyses. TLC was
performed on glass backed silica gel plates (Uniplate) with spots
visualized by UV light. All solvents were reagent grade and, when

4094
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
170.45, 169.98, 163.02, 156.23, 141.37, 124.71, 124.68, 124.37,
124.31, 121.60, 114.87, 114.72, 113.86, 106.81, 102.99, 52.73,
36.80, 28.83. HRMS (FAB) m/z [M+Na+] for C17H15ClFNO5Na:
390.0521; found, 390.0547.
Methyl 3-chloro-2-(3-((2-chlorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (8). 14 mg, 73% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 11.22 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.61 (s,
1H), 7.31 (dd, J = 8.0, 1.5 Hz, 1H), 7.26–7.21 (m, 1H), 7.02–6.97
(m, 1H), 6.53 (s, 1H), 3.91 (s, 4H), 3.54–3.32 (m, 2H), 2.76–2.47
(m, 3H).13C NMR (125 MHz, CDCl3) d 170.49, 163.08, 156.23,
141.30, 134.49, 129.04, 127.85, 124.68, 121.47, 113.86, 106.83,
103.00, 52.75, 37.03, 28.90. HRMS (FAB) m/z [M+Na+] for C17H15Cl2
NO5Na: 406.0225; found, 406.0246.
Methyl 2-(3-((2-bromophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (9). 16.3 mg, 76% yield, pale yellow solid. 1H
NMR (500 MHz, CDCl3) d 11.25 (s, 1H), 8.49–7.19 (m, 7H), 6.93
(td, J = 7.7, 1.4 Hz, 1H), 6.53 (s, 1H), 6.13 (s, 1H), 3.91 (s, 3H),
3.53–3.36 (m, 2H), 2.64 (t, J = 8.3 Hz, 2H).13C NMR (125 MHz,
CDCl3) d 170.54, 170.04, 163.07, 156.29, 141.26, 135.54, 132.27,
128.50, 125.26, 121.80, 113.89, 113.18, 106.80, 103.01, 52.79,
37.05, 27.08. HRMS (FAB) m/z [M+Na+] for C17H15BrClNO5Na:
449.9720; found, 449.9767.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((2-iodophenyl)amino)-3-
oxopropyl)benzoate (10). 18.2 mg, 77% yield, pale yellow solid. 1H
NMR (500 MHz, CDCl3) d 11.27 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H),
7.77–7.23 (m, 3H), 6.82–6.78 (m, 1H), 6.53 (s, 1H), 6.03 (s, 1H),
3.92 (s, 3H), 3.51–3.37 (m, 3H), 2.72–2.52 (m, 3H). 13C NMR
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(o-tolylamino)propyl)
benzoate (15). 10 mg, 25% yield, white powder. 1H NMR
(400 MHz, CDCl3): d 11.19 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.28–
7.19 (m, 2H), 7.19–7.07 (m, 2H), 6.67 (bs, 1H), 6.60 (s, 1H), 4.00
(s, 3H), 3.60–3.48 (m, 2H), 2.78–2.62 (m, 2H), 2.29 (s, 3H); 13C
NMR (125 MHz, CDCl3 and CD3OD): d 170.8, 170.5, 161.9, 157.8,
141.9, 135.3, 130.4, 129.5, 126.6, 125.3, 123.4, 114.6, 106.0,
102.4, 52.4, 36.3, 28.9, 17.6. HRMS (FAB) m/z [M+Na+] for
C18H18ClNO5Na: 386.0771; found, 386.0769.
Methyl 3-chloro-2-(3-((3-fluorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (16). 27 mg, 74% yield, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 9.38 (br s, 1H), 7.86–7.64 (m,
1H), 7.37–7.22 (m, 2H), 6.81 (ddt, J = 9.1, 6.3, 2.6 Hz, 1H), 6.51 (s,
1H), 3.95 (s, 3H), 3.57–3.20 (m, 2H), 2.84–2.52 (m, 2H); 13C
NMR(125 MHz, (CD3)2CO): d 171.3, 164.7, 162.8, 162.3, 158.6,
143.0, 142.1, 131.0, 130.9, 115.5, 110.4, 108.2, 106.9, 103.2, 52.9,
36.8, 28.8; HRMS (FAB) m/z [M+H+] for C17H15ClFNO5: 368.0701;
found, 368.0701.
Methyl 3-chloro-2-(3-((3-chlorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (17). 30 mg, 78% yield, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 9.33 (s, 1H), 8.06–7.85 (m, 1H),
7.49 (ddd, J = 8.2, 2.1, 1.0 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.08
(ddd, J = 7.9, 2.1, 0.9 Hz, 1H), 6.51 (s, 1H), 3.95 (s, 3H), 3.69–3.33
(m, 2H), 2.85–2.53 (m, 2H); 13C NMR(125 MHz. (CD3)2CO): d
171.3, 171.3, 162.3, 158.6, 143.0, 141.8, 134.7, 131.0, 123.8,
119.8, 118.1, 114.8, 108.2, 103.2, 52.9, 36.8, 28.8; HRMS (FAB) m/
z [M–Hꢃ] for C17H14Cl2NO5: 382.0249; found, 382.0237.
(125 MHz, CDCl3)
d
170.58, 163.13, 156.24, 141.26, 138.84,
Methyl 2-(3-((3-bromophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (18). 27 mg, 74% yield, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 9.32 (s, 1H), 8.10 (q, J = 1.6 Hz,
1H), 7.66–7.46 (m, 1H), 7.38–7.15 (m, 2H), 6.51 (s, 1H), 3.95 (s,
3H), 3.60–3.33 (m, 2H), 2.73–2.61 (m, 2H). 13C NMR (125 MHz,
138.04, 129.38, 126.07, 121.88, 113.88, 106.85, 103.01, 52.84,
37.05, 28.97. HRMS (FAB) m/z [M+Na+] for C17H15ClINO5Na:
497.9581; found, 497.9593.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-((2-(trifluoromethyl)
phenyl)amino)propyl)benzoate (11). 33 mg, 79% yield, white amor-
phous solid: 1H NMR (400 MHz, CDCl3–MeOD) d 6.87 (d, J = 1.4 Hz,
1H), 6.84 (d, J = 1.4 Hz, 1H), 6.41 (s, 1H), 6.08 (d, J = 3.1 Hz, 1H),
5.84 (dd, J = 3.0, 1.3 Hz, 1H), 4.88 (s, 2H), 3.84 (s, 3H), 3.49–3.41
(m, 2H), 3.02–2.92 (m, 2H); 13C NMR (CDCl3+CH3OH, 125 MHz): d
171.0, 162.2, 158.3, 153.1, 147.3, 147.1, 141.9, 126.7, 119.7, 114.9,
109.7, 106.5, 102.6, 52.6, 42.7, 30.9, 26.2. (FAB) m/z [M+H+] for
(CD3)2CO)
d 171.32, 171.26, 158.63, 142.99, 141.93, 131.31,
126.77, 122.77, 122.70, 122.62, 118.63, 118.54, 114.81, 108.21,
103.18, 52.93, 36.81, 36.76, 28.84; HRMS (FAB) m/z [M+Na+] for
C17H15ClBrNO5Na: 449.9720; found, 449.9720.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-((3-(trifluoromethyl)
phenyl)amino)propyl)benzoate (19). 34 mg, 81% yield, white amor-
phous solid: 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 7.77–7.65
(m, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.32–7.28 (m, 1H), 6.41 (d,
J = 0.7 Hz, 1H), 5.27 (d, J = 0.7 Hz, 1H), 3.91 (d, J = 0.7 Hz, 3H),
3.50–3.39 (m, 2H), 2.67–2.45 (m, 2H); 13C NMR (125 MHz, CDCl3):
d 170.7, 161.8, 158.0, 147.6, 141.6, 136.8, 136.5, 129.4, 126.4,
126.0, 125.1, 124.5, 120.1, 106.0, 102.4, 52.3, 30.7, 26.1; HRMS
(FAB) m/z [M+H+] for C18H16ClF3NO5: 418.0669; found, 418.0661.
Methyl 2-(3-([1,10-biphenyl]-3-ylamino)-3-oxopropyl)-3-chloro-
4,6-dihydroxybenzoate (20). 35 mg, 72% yield, white amorphous
solid: 1H NMR (500 MHz, (CD3)2CO) d 8.10–8.01 (m, 1H), 7.66
(tdd, J = 6.3, 2.1, 1.0 Hz, 3H), 7.52–7.44 (m, 2H), 7.44–7.33 (m,
3H), 6.52 (s, 1H), 3.97 (s, 3H), 3.63–3.36 (m, 2H), 2.73–2.56 (m,
2H); 13C NMR(125 MHz, (CD3)2CO): d 143.2, 130.1, 129.7, 128.3,
127.7, 122.6, 118.9, 118.5, 103.1, 52.9, 36.8, 29.0;. HRMS (FAB)
m/z [M+Na+] for C23H20ClNO5Na: 448.0928; found, 448.0927.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((3-hydroxyphenyl)amino)-
3-oxopropyl)benzoate (21). 16 mg, 88% yield, off-white solid. 1H
NMR (400 MHz, CD3OD): d 7.16 (t, J = 2.27 Hz, 1H), 7.06 (td,
J = 2.36, 8.01 Hz, 1H), 6.92 (dd, J = 2.70, 7.62 Hz, 1H), 6.50 (dd,
J = 2.47, 8.35 Hz, 1H), 6.37 (s, 1H), 3.86 (s, 3H), 3.91 (s, 3H), 3.32–
3.25 (m, 2H), 2.60 (ddt, J = 3.13, 7.38 Hz, 10.18, 2H). 13C NMR
(125 MHz, CD3OD): d 173.26, 171.66, 161.48, 158.88, 142.34,
140.93, 130.50, 114.95, 114.93, 112.36, 112.09, 109.54, 108.40,
103.14, 52.87, 37.06, 29.09. HRMS (FAB) m/z [M+Na+] for
C18H16ClF3NO5: 418.0669; found, 418.0658.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((2-hydroxyphenyl)amino)-
3-oxopropyl)benzoate (12). 23 mg, 63% yield, white amorphous
solid: 1H NMR (500 MHz, (CD3)2CO) d 9.21 (br s, 1H), 7.49 (dd,
J = 8.1, 1.6 Hz, 1H), 7.03 (td, J = 7.8, 1.6 Hz, 1H), 6.91 (dd, J = 8.1,
1.5 Hz, 1H), 6.82 (td, J = 7.7, 1.5 Hz, 1H), 6.52 (s, 1H), 3.96 (s, 3H),
3.54–3.37 (m, 2H), 2.87–2.74 (m, 2H); 13C NMR (125 MHz,
(CD3)2CO): d 172.9, 171.3, 162.4, 158.7, 149.1, 142.8, 127.6,
126.5, 122.7, 120.6, 118.7, 114.9, 108.1, 103.2, 53.0, 36.3, 29.4;
(FAB) m/z [M+H+] for C17H16ClNO6: 388.0563; found, 388.0563.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((2-methoxyphenyl)amino)-
3-oxopropyl)benzoate (13). 14.5 mg, 77% yield, white solid. 1H
NMR (500 MHz, CDCl3) d 11.40 (s, 1H), 8.41 (dd, J = 7.9, 1.7 Hz,
1H), 7.77 (s, 1H), 7.10–6.85 (m, 3H), 6.59 (s, 1H), 3.96 (s, 3H),
3.87 (s, 3H), 3.56–3.46 (m, 2H), 2.71–2.62 (m, 2H).13C NMR
(125 MHz, CD3OD) d 173.39, 171.67, 161.61, 158.99, 151.43,
142.28, 128.21, 126.10, 123.37, 121.46, 114.98, 111.74, 109.46,
103.16, 56.19, 52.83, 37.23, 29.40. HRMS (FAB) m/z [M+H] for
C18H18ClNO6: 380.0901; found, 380.0920.
Methyl 2-(3-((2-aminophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (14). 7.5 mg, 41% yield, pale yellow solid. 1H
NMR (400 MHz, CD3OD) d 7.05–6.92 (m, 2H), 6.79–6.61 (m, 1H),
6.32 (d, J = 4.7 Hz, 1H), 3.84 (d, J = 5.2 Hz, 3H), 3.31–3.25 (m, 2H),
2.66–2.60 (m, 2H). 13C NMR (125 MHz, CD3OD) d 173.77, 171.72,
161.58, 159.34, 143.21, 142.29, 128.24, 127.13, 125.21, 119.62,
118.61, 115.21, 109.28, 103.28, 52.91, 36.45, 29.33. HRMS (FAB)
m/z [M+Na+] for C17H17ClN2O5Na: 387.0724; found, 387.0744.
C17H16ClNO6Na: 388.0558, found 388.0555.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((3-methoxyphenyl)amino)-
3-oxopropyl)benzoate (22). 14.6 mg, 76% yield, white solid. 1H
NMR (500 MHz, CDCl3)
d 11.15 (s, 1H), 7.30–7.11 (m, 3H),

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4095
6.92–6.58 (m, 2H), 6.52 (s, 1H), 6.25 (s, 1H), 3.90 (s, 3H), 3.75 (s,
3H), 3.46–3.37 (m, 2H), 2.58–2.51 (m, 2H).13C NMR (126 MHz,
CDCl3) d 170.82, 170.71, 162.10, 160.03, 157.67, 142.01, 139.24,
129.61, 114.59, 111.83, 109.92, 106.09, 105.39, 102.47, 55.27,
52.54, 36.41, 28.58. HRMS (FAB) m/z [M+Na+] for C18H18ClNO6:
402.0720; found, 402.0722.
Methyl 2-(3-((3-aminophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (23). 9 mg, 49% yield, pale yellow solid. 1H
NMR (400 MHz, CDCl3) d 7.12–7.05 (m, 1H), 7.01 (t, J = 8.0 Hz,
1H), 6.68 (dd, J = 7.8, 2.1 Hz, 1H), 6.44–6.26 (m, 2H), 3.87 (s, 3H),
3.38 (t, J = 8.4 Hz, 2H), 2.61–2.47 (m, 2H). 13C NMR (125 MHz,
CDCl3) d 170.50, 162.27, 157.40, 146.36, 141.98, 129.72, 114.46,
111.35, 110.11, 106.84, 106.20, 102.55, 52.57, 36.57, 29.69, 28.69.
HRMS (FAB) m/z [M+Na+] for C17H17ClN2O5Na: 387.0724; found,
387.0736.
Methyl 2-(3-((3-acetamidophenyl)amino)-3-oxopropyl)-3-chloro-
4,6-dihydroxybenzoate (24). 18 mg, 89% yield white solid. 1H NMR
(400 MHz, CD3OD): d 7.86 (t, J = 1.91 Hz, 1H), 7.34–7.21 (m, 3H),
6.40 (s, 1H), 3.91 (s, 3H), 3.36–3.32 (m, 2H), 2.69–2.62 (m, 2H),
2.12 (d, J = 1.77 Hz, 3H). 13C NMR (125 MHz, CD3OD): d 173.33,
171.69, 171.65, 161.43, 159.00, 142.26, 140.37, 140.33, 130.02,
116.94, 116.83, 114.97, 113.06, 109.58, 103.15, 52.84, 37.01,
29.04, 23.81. HRMS (FAB) m/z [M+Na+] for C19H19ClN2O6Na:
428.0829, found 429.0824.
Methyl 3-chloro-2-(3-((4-fluorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (25). 14.5 mg, 80% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 11.28 (s, 1H), 7.89 (s, 1H), 7.50 (ddd, J = 9.4,
4.8, 2.4 Hz, 2H), 7.02 (t, J = 8.7 Hz, 2H), 6.50 (s, 1H), 3.96 (s, 3H),
3.52–3.44 (m, 2H), 2.65–2.56 (m, 2H). 13C NMR (125 MHz, CDCl3)
d 170.34, 169.85, 162.91, 160.34, 156.21, 141.58, 121.57, 121.50,
115.82, 115.64, 113.88, 106.86, 102.98, 52.73, 36.57, 28.79.HRMS
(FAB) m/z [M+Na+] for C17H15ClFNO5Na: 390.0521; found,
390.0553.
Methyl 2-(3-([1,10-biphenyl]-4-ylamino)-3-oxopropyl)-3-chloro-
4,6-dihydroxybenzoate (30). 12.5 mg, 60% yield, white solid. 1H
NMR (400 MHz, CDCl3) d 7.57–7.48 (m, 6H), 7.41–7.23 (m, 3H),
7.15 (d, J = 5.6 Hz, 1H), 6.54 (s, 1H), 6.02 (s, 1H), 3.92 (s, 3H),
3.49–3.41 (m, 2H), 2.62–2.55 (m, 2H). 13C NMR (125 MHz, CDCl3)
d 170.41, 169.88, 162.96, 156.19, 141.64, 140.41, 137.25, 128.81,
127.70, 127.17, 126.84, 119.96, 113.88, 106.87, 102.96, 52.75,
36.75, 28.82. HRMS (FAB) m/z [M+Na+] for C23H20ClNO5Na:
448.0928; found, 448.0946.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(p-tolylamino)propyl)
benzoate (31). 14 mg, 76% yield, white solid. 1H NMR (500 MHz,
CD3OD) d 7.32 (dd, J = 8.5, 2.0 Hz, 2H), 7.08–7.02 (m, 2H), 6.47 (s,
1H), 3.88 (s, 3H), 3.45–3.37 (m, 2H), 2.56–2.49 (m, 2H), 2.24 (s,
3H). 13C NMR (125 MHz, CD3OD)
d 168.00, 167.60, 167.51,
154.20, 139.34, 132.64, 131.47, 131.44, 127.00, 117.33, 117.21,
111.59, 104.03, 100.23, 50.14, 34.05, 26.29, 18.34.HRMS (FAB)
m/z [M+Na+] for C18H18ClNO5Na: 386.0771; found, 386.1007.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((4-hydroxyphenyl)amino)-
3-oxopropyl)benzoate (32). 8.8 mg, 48% yield, white solid. 1H NMR
(500 MHz, CD3OD) d 7.36–7.27 (m, 2H), 6.74–6.63 (m, 2H), 6.38
(s, 1H), 3.88 (s, 3H), 3.30–3.28 (m, 2H), 2.64–2.55 (m, 2H). 13C
NMR (125 MHz, CD3OD) d 172.98, 171.64, 161.37, 158.85, 155.34,
142.31, 131.72, 123.38, 116.18, 114.89, 109.69, 103.11, 52.84,
36.88, 29.19.HRMS (FAB) m/z [M+Na+] for C17H16ClNO6Na:
388.0564; found, 388.0556.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((4-methoxyphenyl)amino)-
3-oxopropyl)benzoate (33). 32 mg, 73% yield, white powder. 1H
NMR (500 MHz, CD3OD): d 7.42–7.37 (m, 2H), 6.84–6.79 (m, 2H),
6.41 (s, 1H), 3.90 (s, 3H), 3.75 (s, 3H), 3.45–3.39 (m, 2H), 2.58–
2.52 (m, 2H). 13C NMR (125 MHz, CD3OD): d 170.74, 170.72,
161.9, 157.8, 156.1, 142.0, 131.1, 121.6, 114.6, 114.0, 106.0,
102.3, 55.4, 52.4, 36.1, 28.6. HRMS (FAB) m/z [M+Na+] for
C18H18ClNO6Na: 402.0720; found, 402.0725.
Methyl 3-chloro-2-(3-((4-chlorophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (26). 13.8 mg, 72% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 11.16 (s, 1H), 7.52–7.45 (m, 2H), 7.32–7.28
(m, 2H), 6.60 (s, 1H), 6.14 (s, 1H), 3.97 (s, 3H), 3.55–3.45 (m, 2H),
2.65–2.59 (m, 2H). 13C NMR (125 MHz, CDCl3) d 170.45, 170.35,
162.39, 157.07, 141.80, 136.48, 129.09, 128.99, 120.94, 120.84,
114.29, 106.38, 102.70, 52.62, 36.48, 28.64.HRMS (FAB) m/z
[M+Na+] for C17H15Cl2NO5Na: 406.0225; found, 406.0234.
Methyl 2-(3-((4-bromophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (27). 15 mg, 70% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 7.38 (s, 4H), 6.49 (s, 1H), 3.90 (s, 3H),
3.45–3.39 (m, 2H), 2.58–2.52 (m, 2H). 13C NMR (125 MHz,
Methyl 2-(3-((4-aminophenyl)amino)-3-oxopropyl)-3-chloro-4,6-
dihydroxybenzoate (34). 8 mg, 33% yield, dark brown solid. 1H
NMR (400 MHz, CD3OD) d 7.45–7.27 (m, 2H), 6.90–6.74 (m, 2H),
6.50 (s, 1H), 4.00 (s, 3H), 3.41 (dt, J = 3.2, 1.7 Hz, 13H), 2.77–2.65
(m, 2H). 13C NMR (125 MHz, CDCl3 and CD3OD) d 170.59, 169.91,
162.49, 156.88, 143.10, 141.99, 129.23, 121.91, 115.51, 106.47,
102.65, 52.63, 49.80, 36.43, 29.70, 28.90. HRMS (FAB) m/z
[M+Na+] for C17H17ClN2O5Na: 387.0826; found, 387.0836.
Methyl 3-chloro-2-(3-((4-cyanophenyl)amino)-3-oxopropyl)-4,6-
dihydroxybenzoate (35). 13.6 mg, 72% yield, white solid. 1H NMR
(500 MHz, CDCl3) d 7.71–7.60 (m, 4H), 6.56 (s, 1H), 3.98 (s, 3H),
3.53–3.46 (m, 2H), 2.70–2.64 (m, 2H). 13C NMR (125 MHz, CDCl3)
d 170.97, 170.41, 162.19, 157.38, 142.28, 141.72, 133.26, 119.32,
118.94, 114.44, 106.70, 106.26, 102.66, 52.59, 36.46, 28.41.HRMS
(FAB) m/z [M+H] for C18H15ClN2O5: 375.0748; found, 375.2276.
Methyl 3-chloro-2-(3-((4-ethynylphenyl)amino)-3-oxopropyl)-4,
6-dihydroxybenzoate (36). 18 mg, 95% yield, off-white solid. 1H
NMR (400 MHz, CDCl3): d 7.51–7.46 (m, 2H), 7.39–7.34 (m, 2H),
6.38 (s, 1H), 3.87 (s, 3H), 3.48–3.32 (m, 5 H), 3.01 (s, 1H), 2.60–
2.53 (m, 2H); 13C NMR (125 MHz, CDCl3 and CD3OD): d 171.2,
170.7, 161.8, 157.8, 141.9, 138.7, 132.7, 119.2, 117.1, 114.6,
106.0, 102.3, 83.3, 76.5, 52.3, 36.0, 28.3. HRMS (FAB) m/z
[M+Na+] for C19H16ClNO5Na: 396.0615; found, 396.0608.
CDCl3)
d 170.27, 169.88, 162.91, 156.18, 141.50, 136.83,
132.05, 121.18, 116.89, 113.864, 106.87, 103.01, 52.74, 36.71,
28.71.HRMS (FAB) m/z [M+Na+] for C17H15BrClNO5Na: 449.9720;
found, 449.9740.
Methyl 3-chloro-4,6-dihydroxy-2-(3-((4-iodophenyl)amino)-3-
oxopropyl)benzoate (28). 22 mg, 76% yield, white solid. 1H NMR
(500 MHz, CDCl3 and CD3OD) d 7.55 (dd, 2H), 7.28 (dd, 2H), 6.43
(s, 1H), 3.89 (s, 3H), 3.41–3.39 (m, 2H), 2.56–2.53 (m, 2H). 13C
NMR (125 MHz, CDCl3 and CD3OD) d 169.93, 162.88, 156.22,
149.99, 141.51, 137.99, 136.22, 129.72, 128.84, 126.65, 126.34,
121.46, 103.01, 52.74, 36.74, 28.69. HRMS (FAB) m/z [M+Na+] for
C
17H15ClINO5Na: 497.9683; found, 497.9656.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-((4-(trifluoromethyl)
Methyl 3-chloro-4,6-dihydroxy-2-(3-((4-isopropylphenyl)amino)-
3-oxopropyl)benzoate (37). 26 mg, 87% yield, white solid. 1H NMR
(500 MHz, (CD3)2CO) d 9.13 (s, 1H), 7.74–7.52 (m, 2H), 7.27–7.13
(m, 2H), 6.54 (t, J = 1.5 Hz, 1H), 4.08–3.89 (m, 3H), 3.54–3.36 (m,
2H), 2.90 (td, J = 7.0, 2.1 Hz, 1H), 2.69 (ddt, J = 8.1, 6.5, 1.6 Hz,
2H), 2.15–2.11 (m, 1H), 1.25 (dt, J = 6.9, 1.4 Hz, 7H). 13C NMR
(125 MHz, (CD3)2CO) d 171.36, 170.68, 162.29, 158.56, 144.45,
143.28, 138.23, 138.15, 127.23, 120.07, 114.76, 108.22, 103.09,
52.91, 36.73, 34.28, 29.03, 24.41. HRMS (FAB) m/z [M+Na+] for
phenyl)amino)propyl)benzoate (29). 31 mg, 74% yield, white amor-
phous solid: 1H NMR (500 MHz, (CD3)2CO) d 8.13–7.78 (m, 2H),
7.65 (dd, J = 8.8, 2.5 Hz, 2H), 6.51 (t, J = 1.7 Hz, 1H), 3.95 (t,
J = 1.8 Hz, 3H), 3.44 (ddt, J = 10.2, 6.1, 1.8 Hz, 2H), 2.72 (ddt,
J = 10.2, 6.3, 1.8 Hz, 2H); 13C NMR (125 MHz, (CD3)2CO) d (171.5,
171.4, 171.3, 162.2, 158.6, 142.9, 126.9, 126.9, 126.8, 126.8,
119.8, 119.8, 114.8, 108.2, 103.2, 52.9, 36.8, 28.8. HRMS (FAB)
m/z [M+H+] for C18H16ClF3NO5: 418.0669; found, 418.0669.
C20H22ClNO5Na: 414.1187; found, 414.1165.

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A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
Methyl
3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyridin-2-ylamino)
1H NMR (500 MHz, (CD3)2CO) d 7.40 (d, J = 3.6 Hz, 1H), 7.11 (d,
J = 3.6 Hz, 1H), 6.52 (s, 1H), 3.94 (s, 3H), 3.57–3.34 (m, 2H), 2.98–
2.78 (m, 2H); 13C NMR ((CD3)2CO, 125 MHz) d 171.3, 170.7,
162.4, 158.9, 158.7, 142.7, 138.5, 114.9, 113.9, 108.2, 103.3, 53.0,
35.4, 28.6. (FAB) m/z [M+H+] calcd for C14H14ClN2O5S: 357.0312;
found, 357.0323.
propyl)benzoate (38). 1H NMR (500 MHz, (CD3)2CO) d 8.39–8.07
(m, 2H), 7.80 (dd, J = 8.7, 7.4, 1.8 Hz, 1H), 7.10 (dd, J = 7.3, 4.8,
1.1 Hz, 1H), 6.55 (s, 1H), 3.99 (s, 3H), 3.57–3.42 (m, 2H), 2.98–
2.77 (m, 2H). 13C NMR (125 MHz, (CD3)2CO) d 171.60, 162.34,
158.67, 153.19, 148.85, 143.11, 138.72, 120.02, 114.86, 114.29,
108.11, 103.12, 52.92, 36.62, 28.77. HRMS (FAB) m/z [M+Na+] for
Methyl
loro-4,6-dihydroxybenzoate (47). 28 mg, 78% yield, white amor-
phous solid: 1H NMR (500 MHz, CDCl3–MeOD,)
8.79 (d,
2-(3-((1,3,4-thiadiazol-2-yl)amino)-3-oxopropyl)-3-ch-
C
16H15ClN2O5Na: 373.0669; found, 373.0630.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyridin-3-ylamino)
d
propyl)benzoate (39). 23 mg, 65% yield, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 8.80 (dd, J = 4.7, 1.5 Hz, 1H), 8.35
(dd, J = 9.0, 1.5 Hz, 1H), 7.51 (dd, J = 9.0, 4.7 Hz, 1H), 6.40 (s, 1H),
3.82 (s, 3H), 3.48–3.21 (m, 2H), 2.80–2.75 (m, 2H); 13C
NMR(125 MHz, (CD3)2CO): d 158.6, 145.2, 143.0, 141.9, 126.7,
124.2, 103.1, 52.9, 36.7, 36.6, 28.8; HRMS (FAB) m/z [M+H+] for
J = 0.9 Hz, 1H), 6.44 (d, J = 0.9 Hz, 1H), 3.89 (d, J = 0.9 Hz, 3H),
3.62–3.30 (m, 2H), 2.93–2.40 (m, 4H); 13C NMR(CDCl3–MeOD,
125 MHz): 171.0, 170.9, 162.3, 159.5, 158.1, 148.0, 141.3, 114.9,
106.1, 102.8, 52.6, 34.9, 28.0. (FAB) m/z [M+Na+] for C13H12ClN3O5
SNa: 380.0084; found, 380.0090.
Methyl 2-(3-((1,2,4-thiadiazol-5-yl)amino)-3-oxopropyl)-3-chl-
oro-4,6-dihydroxybenzoate (48). 19 mg, 53% yield, white amor-
phous solid: 1H NMR (400 MHz, CDCl3–MeOD) d 8.19 (s, 1H),
7.72 (s, 1H), 6.36 (s, 1H), 3.83 (s, 3H), 3.50–3.32 (m, 2H), 2.91–
2.69 (m, 2H); 13C NMR (CDCl3–MeOD, 125 MHz): d 179.3, 176.3,
174.5, 163.0, 162.2.161.6, 144.8, 118.6, 110.2, 106.4, 56.3, 38.1,
33.5. (FAB) m/z [M+Na+] for C13H12ClN3O5SNa: 380.0084; found,
380.0078.
C16H6ClN2O5: 351.0748; found, 351.0755.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyridin-4-ylamino)
propyl)benzoate (40). 10 mg, 51% yield, white solid. 1H NMR
(500 MHz, (CD3)2CO and CD3OD) d 9.55 (s, 1H), 8.53–8.40 (m,
2H), 7.73–7.61 (m, 2H), 6.52 (s, 1H), 3.95 (s, 3H), 3.52–3.40 (m,
2H), 2.75–2.71 (m, 2H). 13C NMR (125 MHz, (CD3)2CO) d 172.20,
171.54, 165.56, 162.82, 151.33 (2C), 146.82, 142.44, 113.98 (2C),
103.33, 54.98, 52.68, 36.96, 28.73. HRMS (FAB) m/z [M+Na+] for
Methyl
3-chloro-4,6-dihydroxy-2-(3-(methyl(phenyl)amino)-3-
C
16H15ClN2O5Na: 373.0669; found, 373.0695.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyrazin-2-ylamino)
oxopropyl)benzoate (49). 28 mg, 77% yield, white amorphous solid:
1H NMR (400 MHz, CDCl3–MeOD) d 7.34 (dd, J = 8.4, 6.8 Hz, 2H),
7.31–7.23 (m, 2H), 7.15–7.08 (m, 2H), 6.33 (s, 1H), 3.87 (s, 3H),
3.25 (d, J = 7.5 Hz, 5H), 2.35–2.27 (m, 2H); 13C NMR (CDCl3–CH3OH,
125 MHz) d 172.9, 171.0, 162.1, 157.9, 143.7, 142.3, 130.0 (2),
128.1, 127.2 (2), 114.8, 106.0, 102.3, 52.4, 37.6, 33.4, 28.9; (FAB)
m/z [M+H+]for C18H19ClNO5: 364.0952; found, 364.0943.
Methyl 3-chloro-2-(3-(ethyl(phenyl)amino)-3-oxopropyl)-4,6-dih-
ydroxybenzoate (50). 30 mg, 79% yield, white amorphous solid: 1H
NMR (500 MHz, CDCl3–MeOD) d 7.34 (t, J = 7.7 Hz, 2H), 7.31–7.24
(m, 2H), 7.06 (dd, J = 7.3, 1.7 Hz, 2H), 6.33 (d, J = 4.0 Hz, 1H), 3.87
(d, J = 3.9 Hz, 3H), 3.70 (q, J = 7.1 Hz, 3H), 3.27–3.19 (m, 2H),
2.34–2.16 (m, 2H), 1.07 (td, J = 7.2, 3.4 Hz, 3H); 13C NMR (CDCl3–
MeOD, 125 MHz) d 172.3, 171.0, 162.0, 157.9, 142.3, 141.9, 129.9
(2), 128.3 (2), 128.2, 114.7, 106.1, 102.2, 52.4, 44.3, 33.7, 28.8,
13.0; (FAB) m/z [M+H+] for C19H21ClNO5: 378.1108; found,
378.1108.
propyl)benzoate (41). 9 mg, 26% yield, white powder. 1H NMR
(400 MHz, CDCl3 and CD3OD): d 11.22 (s, 1H), 9.58 (s, 1H), 8.38–
8.35 (m, 1H), 8.27–8.23 (m, 1H), 8.02 (br s, 1H), 6.58 (s, 1H), 3.98
(s, 3H), 3.55–3.48 (m, 2H), 2.76–2.69 (m, 2H); 13C NMR
(125 MHz, CDCl3 and CD3OD): d 171.5, 170.6, 161.7, 158.0, 148.5,
142.2, 141.3, 139.3, 136.7, 114.6, 105.9, 102.3, 52.3, 35.7, 27.9.
HRMS (FAB) m/z [M+Na+] for C15H14ClN3O5Na: 374.0520; found,
374.0524.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyridazin-3-ylamino)
propyl)benzoate (42). 26 mg, yield 74%, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 10.04 (s, 1H), 8.92 (dd, J = 4.7,
1.5 Hz, 1H), 8.47 (dd, J = 9.0, 1.5 Hz, 1H), 7.63 (dd, J = 9.0, 4.7 Hz,
1H), 6.52 (s, 1H), 3.94 (s, 3H), 3.54–3.38 (m, 2H), 2.99–2.84 (m,
2H); 13C NMR (125 MHz, (CD3)2CO) d 158.78, 149.10, 142.85,
128.79, 118.60, 118.54, 103.20, 52.93, 36.60, 28.60; HRMS (FAB)
m/z [M+Na+] for C15H14ClN3O5Na: 374.0520; found, 374.0534.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyrimidin-5-ylamino)
propyl)benzoate (43). 23 mg, 66% yield, white amorphous solid:
1H NMR (400 MHz, CDCl3–MeOD) d 9.01 (s, 2H), 8.84 (s, 1H), 6.42
(s, 1H), 3.91 (s, 3H), 3.59–3.36 (m, 2H), 2.70–2.51 (m, 2H); 13C
NMR (CDCl3, 125 MHz): d 172.1, 170.9, 162.0, 158.1, 152.9, 147.6
(2), 141.8, 114.8 (2), 106.2, 102.6, 52.6, 35.7, 28.2; (FAB) m/z
[M+H+] for C15H15ClN3O5: 352.0712; found, 352.0712.
Methyl 3-chloro-4,6-dihydroxy-2-(3-(isopropyl(phenyl)amino)-3-
oxopropyl)benzoate (51). A solution of benzyl protected acid 1a
(100 mg, 0.22 mmol) in dry CH2Cl2 was treated with oxalyl chlo-
ride (23 lL, 0.33 mmol) under Ar atmosphere and then stirred at
room temperature for 2 h. The solvent was then removed in vacuo
and the residue was placed under high vacuum for 30 min to
remove any unreacted oxalyl chloride. The residue was then redis-
solved in dry CH2Cl2 (3 mL), cooled to 0 °C, and was treated
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(pyrimidin-2-ylamino)
propyl)benzoate (44). 21 mg, yield 60%, white amorphous solid:
1H NMR (500 MHz, (CD3)2CO) d 9.04 (d, J = 1.3 Hz, 2H), 8.83 (s,
1H), 6.51 (s, 1H), 3.94 (s, 3H), 3.57–3.31 (m, 2H), 2.82–2.69 (m,
2H); 13C NMR(125 MHz, (CD3)2CO): d 171.7, 171.2, 158.6, 154.2,
148.0, 147.9, 142.7, 135.6, 135.5, 114.8, 108.3, 103.2, 52.9, 36.4,
28.7; HRMS (FAB) m/z [M+Na+] for C15H14ClN3O5Na: 374.0520;
found, 374.0527.
Methyl 2-(3-((1,2,4-triazin-5-yl)amino)-3-oxopropyl)-3-chloro-4,
6-dihydroxybenzoate (45). 8.7 mg, 12% yield, yellow powder. 1H
NMR (500 MHz, CDCl3 and CD3OD): d 8.89 (d, J = 2.5 Hz, 1H), 8.47
(d, J = 2.5 Hz, 1H), 6.37 (s, 1H), 3.80 (s, 3H), 3.45–3.38 (m, 2H),
2.89–2.80 (m, 2H). 13C NMR (125 MHz, CDCl3 and CD3OD): d
171.8, 170.7, 161.8, 158.8, 157.9, 149.7, 145.1, 141.5, 114.6,
105.9, 102.3, 52.3, 36.2, 27.6. HRMS (FAB) m/z [M+Na+] for
sequentially with diisopropylethyl amine (57
lL, 0.33 mmol) and
N-isopropylaniline (72 L, 0.33 mmol). The resulting mixture was
l
stirred at room temperature overnight under Ar. The solvent was
removed in vacuo and the residue was passed through a short
pad of silica (1:1, EtOAc/Hexanes) and was concentrated to afford
the amide crude product, which was dissolved in MeOH (5 mL)
and treated with Pd/C (10 mol %, 23 mg). The resulting suspension
was charged with a hydrogen balloon and stirred under H2 at room
temperature. When debenzylation was found to be complete, the
mixture was filtered through celite, concentrated, and purified on
silica gel chromatography (1:1, EtOAc/Hexanes) to afford com-
pound 51 (30 mg, 35% yield over 2 steps) as a white solid. 1H
NMR (400 MHz, CDCl3): d 7.45–7.27 (m, 13H), 7.06–7.12 (m, 2H),
6.41 (s, 1H), 5.07 (hept, J = 6.8 Hz, 1H), 5.04 (s, 2H), 4.98 (s, 2H),
3.88 (s, 3H), 3.03–2.93 (m, 2H), 2.30–2.21 (m, 2H), 1.08 (d,
J = 6.8 Hz, 6 H). 13C NMR (125 MHz, CDCl3): d 171.1, 167.5, 155.4,
154.5, 138.6, 138.3, 136.2, 135.9, 130.3, 129.2, 128.6, 128.5,
128.1, 127.9, 126.9, 126.8, 118.4, 115.5, 98.2, 70.9, 70.8, 52.4,
C
14H13ClN4O5Na: 375.0472; found, 375.0468.
Methyl 3-chloro-4,6-dihydroxy-2-(3-oxo-3-(thiazol-2-ylamino)
propyl)benzoate (46). 30 mg, 84% yield, white amorphous solid:

A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
4097
45.8, 34.4, 27.9, 21.0. HRMS (FAB) m/z [M+Na+] for C20H22ClNO5Na:
414.1084; found, 414.1076.
36.8, 28.7; (FAB) m/z [M+Na+] for C17H14BrCl2NO5Na: 483.9330;
found, 483.9327.
Methyl 3-chloro-4,6-dihydroxy-2-(3-(hydroxy(phenyl)amino)-3-
oxopropyl)benzoate (52). A round bottom flask was charged with
aldehyde 1b (100 mg, 0.2 mmol), nitrosobenzene (22 mg,
0.2 mmol), and 6,7-dihydro-2-pentafluorophenyl-5H-pyrrolo[2,1-
c]-1,2,4-trazolium tetrafluoroborate (catalyst, 7 mg, 0.02 mmol)
and purged with argon. The mixture was then dissolved in CH2Cl2
Methyl
3-chloro-2-(3-((6-chloropyridin-2-yl)amino)-3-oxopro-
pyl)-4,6-dihydroxybenzoate (58). 27 mg, 70% yield, white amor-
phous solid: 1H NMR (500 MHz, (CD3)2CO) d 10.88 (1H, br s),
9.48 (1H, br s)8.09 (dd, J = 8.2, 1.8 Hz, 1H), 7.80–7.58 (m, 1H),
7.00 (dd, J = 7.9, 1.7 Hz, 1H), 6.63–6.10 (m, 1H), 3.93 (s, 3H),
3.46–3.10 (m, 2H), 2.91–2.55 (m, 2H); 13C NMR (125 MHz,
(CD3)2CO) d 172.1, 171.5, 158.8, 153.3, 149.6, 143.1, 142.2, 119.9
(2), 115.0, 112.8, 108.3, 103.3, 53.1, 36.7, 28.8; (FAB) m/z
[M+Na+] for C16H14Cl2N2O5Na: 407.0174; found, 407.0177.
(1.5 mL) followed by addition of DBU (3 lL, 0.02 mmol) and stirred
at room temperature for 30 min. Upon completion, the solvent
removed in vacuo. The resulting residue was dissolved in THF
(2 mL) and treated with TBAF (0.5 mL of 1 M solution). After com-
plete silyl-deprotection, saturated aqueous NH4Cl was added and
extracted 3ꢁ with EtOAc (15 mL). The crude material was purified
via flash chromatography (SiO2, 2:3 EtOAc/Hexanes) to yield 52 as
an off white solid (16 mg, 94%). 1H NMR (400 MHz, CDCl3): d 11.30
(s, 1H), 7.45–7.32 (m, 5 H), 6.52 (s, 1H), 3.88 (s, 3H), 3.42–3.36 (m,
2H), 2.73–2.48 (m, 2H). 13C NMR (125 MHz, CDCl3): d 170.74,
163.10, 156.32 (2C), 141.18, 137.80, 129.57, 126.54, 119.88,
114.00, 106.92, 103.03 (2C), 52.71, 31.51, 28.84, 28.82. HRMS
(FAB) m/z [M+Li+] for C17H16ClNO6Li: 372.0826, found 372.0823.
Methyl 3-chloro-4,6-dihydroxy-2-(3-(indolin-1-yl)-3-oxopropyl)
benzoate (53). 15 mg, 25% yield, white solid. 1H NMR (400 MHz,
CDCl3): d 1H NMR (400 MHz, CDCl3 and CD3OD) d 8.19 (d,
J = 8.0 Hz, 1H), 7.24–7.10 (m, 2H), 6.99 (d, J = 1.2 Hz, 1H), 6.42 (d,
J = 1.5 Hz, 1H), 4.00 (dd, J = 9.1, 7.8 Hz, 2H), 3.88 (d, J = 2.8 Hz,
3H), 3.52–3.40 (m, 2H), 3.16 (t, J = 8.4 Hz, 2H), 2.75–2.60 (m, 2H).
13C NMR (125 MHz, CDCl3 and CD3OD) d 170.88, 170.71, 162.06,
157.89, 142.74, 142.21, 131.21, 127.50, 124.61, 123.81, 116.90,
114.64, 106.04, 102.42, 102.35, 52.51, 50.07, 49.72, 49.55, 49.38,
49.21, 49.03, 48.86, 48.69, 47.97, 35.08, 27.95, 27.84. HRMS (FAB)
m/z [M+Na+] for C19H18ClNO5Na: 398.0771; found, 398.0773.
Methyl 3-chloro-2-(3-((2,4-dibromophenyl)amino)-3-oxopropyl)-
4,6-dihydroxybenzoate (54). 21 mg, 83%, white solid. 1H NMR
(500 MHz, CDCl3 and CD3OD) d 8.22 (d, J = 8.9 Hz, 1H), 7.63 (dd,
J = 2.2, 1.2 Hz, 1H), 7.49–7.33 (m, 1H), 6.44 (d, J = 1.0 Hz, 1H),
3.90 (d, J = 1.2 Hz, 3H), 3.47–3.39 (m, 2H), 2.72–2.54 (m, 2H). 13C
NMR (125 MHz, CDCl3 and CD3OD) d 170.52, 162.40, 157.48,
141.32, 134.74, 134.42, 131.43, 122.78, 116.73, 114.47, 113.56,
106.14, 102.72, 52.64, 36.99, 28.77. HRMS (FAB) m/z [M+Na+] for
C17H14Br2ClNO5Na: 527.8927, found 527.8935.
Methyl 3-chloro-2-(3-((3,5-dichlorophenyl)amino)-3-oxopropyl)-
4,6-dihydroxybenzoate (55). 19 mg, 91% yield, white solid. 1H
NMR (400 MHz, CDCl3): d 11.07 (s, 1H), 7.51 (d, J = 1.88 Hz, 2H),
7.29 (s, 1H), 7.11 (t, J = 1.7 Hz, 1H), 6.60 (s, 1H), 6.13 (s, 1H), 3.98
(s, 1H), 3.52–3.43 (m, 2H), 2.75–2.49 (m, 2H). 13C NMR
(125 MHz, CDCl3 and CD3OD): d 170.73, 170.50, 162.48, 157.22,
141.80, 139.98, 135.30, 124.08, 117.85, 114.43, 106.49, 102.87,
52.77, 36.52, 28.62. HRMS (FAB) m/z [M+Na+] for C17H14Cl3NO5Na:
439.9830, found 439.9840.
Methyl 2-(3-((4-bromo-3-chlorophenyl)amino)-3-oxopropyl)-3-
chloro-4,6-dihydroxybenzoate (56). 24 mg, 89% yield, white solid.
1H NMR (500 MHz, (CD3)2CO) d 8.12 (d, J = 2.5 Hz, 1H), 7.64 (d,
J = 8.8 Hz, 1H), 7.48 (dd, J = 8.8, 2.5 Hz, 1H), 6.52 (s, 1H), 3.96 (s,
3H), 3.50–3.37 (m, 2H), 2.80–2.63 (m, 2H). 13C NMR (125 MHz,
Methyl
2-(3-((5-bromopyridin-2-yl)amino)-3-oxopropyl)-3-
chloro-4,6-dihydroxybenzoate (59). 29 mg, 68% yield, white amor-
phous solid: 1H NMR (500 MHz, CDCl3–MeOD) d 8.31–8.19 (m,
1H), 8.19–8.05 (m, 1H), 7.84–7.71 (m, 1H), 6.54–6.28 (m, 1H),
3.94 (s, 3H), 3.42–3.35 (m, 2H), 2.63 (dq, J = 12.7, 3.8, 2.1 Hz, 2H);
13C NMR (CDCl3–MeOD, 125 MHz): d 171.5, 170.9, 162.1, 158.2,
150.2, 148.6, 141.6, 141.0, 115.5, 114.8, 114.5, 106.1, 102.6, 52.6,
36.4, 28.3. (FAB) m/z [M+Na+] for C16H14BrClN2O5Na: 450.9672;
found, 450.9681.
Methyl
3-chloro-2-(3-((4-chloropyridin-2-yl)amino)-3-oxopro-
pyl)-4,6-dihydroxybenzoate (60). 16 mg, 64% yield, white solid. 1H
NMR (500 MHz, (CD3)2CO) d 8.41 (dd, J = 2.0, 0.6 Hz, 1H), 8.28
(dd, J = 5.3, 0.6 Hz, 1H), 7.19 (dd, J = 5.3, 1.9 Hz, 1H), 6.55 (s, 1H),
3.99 (s, 3H), 3.56–3.42 (m, 2H), 2.98–2.81 (m, 2H). 13C NMR
(125 MHz, (CD3)2CO) d 172.16, 171.34, 162.41, 158.69, 154.30,
150.12, 145.43, 142.93, 120.15, 113.98, 108.12, 103.20, 52.93,
36.62, 28.62. HRMS (FAB) m/z [M+Na+] for C16H14Cl2N2O5Na:
407.0280; found, 407.0766.
Methyl
3-chloro-2-(3-((3-chloropyridin-2-yl)amino)-3-oxopro-
pyl)-4,6-dihydroxybenzoate (61). 21 mg, 55% yield, white amor-
phous solid: 1H NMR (500 MHz, (CD3)2CO) d 8.36 (dd, J = 4.7,
1.6 Hz, 1H), 7.92 (dd, J = 8.0, 1.6 Hz, 1H), 7.27 (ddd, J = 8.0, 4.7,
0.6 Hz, 1H), 6.52 (s, 1H), 3.97 (s, 3H), 3.55–3.39 (m, 2H), 3.00–
2.76 (m, 2H). 13C NMR (125 MHz, (CD3)2CO) d 171.4, 171.28,
158.6, 149.3, 147.5, 143.2 (2), 139.3, 122.9 (2), 114.8, 108.14,
103.1, 52.9, 35.9, 28.8; (FAB) m/z [M+Na+] for C16H14Cl2N2O5Na:
407.0174; found 407.0174.
Methyl
2-(3-((3-bromopyridin-2-yl)amino)-3-oxopropyl)-3-
chloro-4,6-dihydroxybenzoate (62). 31 mg, 71% yield, white amor-
phous solid: 1H NMR (400 MHz, CDCl3–MeOD) d 8.47 (dd, J = 4.7,
1.6 Hz, 1H), 7.98 (dd, J = 8.0, 1.6 Hz, 1H), 7.27–7.22(m, 1H), 6.34
(s, 1H), 3.85 (s, 3H), 3.35–3.29 (m, 2H), 2.91–2.65 (m, 2H). 13C
NMR (125 MHz, CDCl3,): d 172.8, 170.0, 161.2, 156.9, 149.7,
147.8, 141.7, 140.6, 124.9, 120.3, 113.8, 105.0, 101.4, 51.6, 36.3,
26.6. (FAB) m/z [M+Na+] for C16H14BrClN2O5Na: 450.9672; found,
450.9677.
Methyl
3-chloro-2-(3-((5-chlorothiazol-2-yl)amino)-3-oxopro-
pyl)-4,6-dihydroxybenzoate (63). 17 mg, 44.7% yield, pale yellow
amorphous solid: 1H NMR (400 MHz, (CD3)2CO) d 7.34 (s, 1H),
6.52 (s, 1H), 5.63 (s, 1H), 3.94 (s, 3H), 3.55–3.31 (m, 2H), 2.93–
2.71 (m, 2H). 13C NMR (125 MHz, CDCl3–MeOD): d 170.8 (2),
162.0, 158.1, 147.8, 141.8, 136.9, 129.5, 126.6, 125.3, 124.6,
120.9, 114.8, 106.2, 52.5, 30.8, 26.2;. HRMS (FAB) m/z [M+H+] for
C
14H13Cl2N2O5S: 390.9922; found, 390.9913.
(CD3)2CO)
d 170.45, 170.32, 161.23, 157.73, 141.96, 140.00,
Methyl 2-(3-((5-bromothiazol-2-yl)amino)-3-oxopropyl)-3-
133.72, 120.46, 119.04, 114.46, 113.91, 107.27, 102.20, 52.01,
35.87, 27.84. HRMS (FAB) m/z [M+Na+] for C17H14BrCl2NO5Na:
483.9432; found, 398.9333.
chloro-4,6-dihydroxybenzoate (64). 36 mg, 83% yield, gray amor-
phous solid: 1H NMR (500 MHz, (CD3)2CO) d 7.33 (s, 1H), 6.52 (s,
1H), 3.94 (s, 2H), 3.60–3.28 (m, 2H), 2.93–2.78 (m, 2H). 13C
NMR(125 MHz, (CD3)2CO): d 170.9, 170.9, 162.2, 159.6, 158.1,
141.4, 137.7, 114.9, 106.1, 102.7 (2), 52.7, 34.7, 28.1; HRMS (FAB)
m/z [M+Na+] for C14H12BrClN2O5SNa: 456.9237; found, 456.9238.
Methyl 2-(3-((2-bromo-3-chlorophenyl)amino)-3-oxopropyl)-3-
chloro-4,6-dihydroxybenzoate (57). 23 mg, 72% yield, white amor-
phous solid: 1H NMR (500 MHz, CDCl3–MeOD)
d 8.14 (d,
J = 7.9 Hz, 1H), 7.25–7.15 (m, 2H), 6.40 (d, J = 1.2 Hz, 1H), 3.90 (d,
J = 1.2 Hz, 3H), 3.46–3.37 (m, 3H), 2.72–2.62 (m, 2H); 13C NMR
(CDCl3–MeOD, 125 MHz): d 170.9, 170.6, 162.0, 158.1, 141.3,
137.3, 134.8, 128.5 (2), 125.6, 120.2, 114.7, 105.9, 102.5, 52.5,
Methyl
2-(3-((5-bromo-1,3,4-thiadiazol-2-yl)amino)-3-oxopro-
pyl)-3-chloro-4,6-dihydroxybenzoate (65). 12 mg, 28% yield, gray
amorphous solid: 1H NMR (500 MHz, (CD3)2CO) d 6.53 (s, 1H),
3.94 (s, 4H), 3.59–3.43 (m, 2H), 3.05–2.84 (m, 2H). 13C NMR

4098
A. Muth et al. / Bioorg. Med. Chem. 22 (2014) 4083–4098
22. Gullo, C. A.; Teoh, G. Immunol. Lett. 2004, 94, 1.
23. Obeng, E. A.; Carlson, L. M.; Gutman, D. M.; Harrington, W. J.; Lee, K. P.; Boise, L.
H. Blood 2006, 107, 4907.
24. Davenport, E. L.; Moore, H. E.; Dunlop, A. S.; Sharp, S. Y.; Workman, P.; Morgan,
G. J.; Davies, F. E. Blood 2007, 110, 2641.
(125 MHz, (CD3)2CO): d 171.7, 161.6, 158.7, 142.2, 134.8, 130.6,
114.9, 108.1, 103.3, 79.3, 53.0, 35.2, 21.1. HRMS (FAB) m/z
[M+H+] for C13H12BrClN3O5S: 435.9370; found, 435.9377.
25. Hua, Y.; White-Gilbertson, S.; Kellner, J.; Rachidi, S.; Usmani, S. Z.; Chiosis, G.;
DePinho, R.; Li, Z.; Liu, B. Clin. Cancer Res. 2013, 19, 6242.
26. Duerfeldt, A. S.; Peterson, L. B.; Maynard, J. C.; Ng, C. L.; Eletto, D.; Ostrovsky, O.;
Shinogle, H. E.; Moore, D. S.; Argon, Y.; Nicchitta, C. V.; Blagg, B. S. J. J. Am. Chem.
Soc. 2012, 134, 9796.
27. Rosser, M. F.; Nicchitta, C. V. J. Biol. Chem. 2000, 275, 22798.
28. Patel, P. D.; Yan, P.; Seidler, P. M.; Patel, H. J.; Sun, W.; Yang, C.; Que, N. S.;
Taldone, T.; Finotti, P.; Stephani, R. A.; Gewirth, D. T.; Chiosis, G. Nat. Chem. Biol.
2013, 9, 677.
Acknowledgment
The authors gratefully acknowledge the support of this Project
by NIH (CA109265).
Supplementary data
29. Soldano, K. L.; Jivan, A.; Nicchitta, C. V.; Gewirth, D. T. J. Biol. Chem. 2003, 278,
48330.
30. Immormino, R. M.; Metzger, L. E.; Reardon, P. N.; Dollins, D. E.; Blagg, B. S.;
Gewirth, D. T. J. Mol. Biol. 2009, 388, 1033.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.05.075.
31. Clevenger, R. C.; Blagg, B. S. J. Org. Lett. 2004, 6, 4459.
32. Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.; Chiosis, G. J. Biomol.
Screen. 2004, 9, 375.
References and notes
1. Hartl, F. U. Nature 1996, 381, 571.
2. Hartl, F. U. Nature 2011, 475, 324.
33. Taldone, T.; Patel, P. D.; Patel, M.; Patel, H. J.; Evans, C. E.; Rodina, A.; Ochiana,
S.; Shah, S. K.; Uddin, M.; Gewirth, D.; Chiosis, G. J. Med. Chem. 2013, 56, 6803.
34. Hunter, L. Beilstein J. Org. Chem. 2010, 6.
35. Wong, F. T.; Patra, P. K.; Seayad, J.; Zhang, Y.; Ying, J. Y. Org. Lett. 2008, 10, 2333.
36. Melnick, J.; Dul, J. L.; Argon, Y. Nature 1994, 370, 373.
37. Hadden, K. M.; Blagg, B. S. J. J. Org. Chem. 2009, 74, 4697.
38. Duerfeldt, A. S.; Brandt, G. E. L.; Blagg, B. S. J. Org. Lett. 2009, 11, 2353.
39. Shen, G.; Wang, M.; Welch, T. R.; Blagg, B. S. J. J. Org. Chem. 2006, 71, 7618.
40. Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157.
3. Pearl, L. H.; Prodromou, C. Curr. Opin. Struct. Biol. 2000, 10, 46.
4. Prodromou, C.; Pearl, L. H. Curr. Cancer Drug Targets 2003, 3, 301.
5. Pearl, L. H.; Prodromou, C. Adv. Protein Chem. 2001, 59, 157.
6. Meyer, P.; Prodromou, C.; Hu, B.; Vaughan, C.; Roe, S. M.; Panaretou, B.; Piper, P.
W.; Pearl, L. H. Mol. Cell 2003, 11, 647.
7. Panaretou, B.; Siligardi, G.; Meyer, P.; Maloney, A.; Sullivan, J. K.; Singh, S.;
Millson, S. H.; Clarke, P. A.; Naaby-Hansen, S.; Stein, R.; Cramer, R.; Mollapour,
M.; Workman, P.; Piper, P. W.; Pearl, L. H.; Prodromou, C. Mol. Cell 2002, 10,
1307.
41. Grad, I.; Cederroth, C. R.; Walicki, J.; Grey, C.; Barluenga, S.; Winssinger, N.; De
Massy, B.; Nef, S.; Picard, D. PLoS One 2010, 5, e15770.
8. Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, M.; Neckers, L. M. J. Biol. Chem.
2000, 275, 37181.
42. Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S.
J. J. Am. Chem. Soc. 2005, 127, 12778.
9. Soti, C.; Racz, A.; Csermely, P. J. Biol. Chem. 2002, 277, 7066.
10. Whitesell, L.; Bagatell, R.; Falsey, R. Curr. Cancer Drug Targets 2003, 3, 349.
11. Whitesell, L.; Lindquist, S. L. Nat. Rev. Cancer 2005, 5, 761.
12. Zhang, H.; Burrows, F. J. Mol. Med. 2004, 82, 488.
43. Burlison, J. A.; Blagg, B. S. J. Org. Lett. 2006, 8, 4855.
44. Burlison, J. A.; Neckers, L.; Smith, A. B.; Maxwell, A.; Blagg, B. S. J. JACS 2006,
128, 15529; Burlison, J. A.; Avila, C.; Vielhauer, G.; Lubbers, D. J.; Holzbeierlein,
J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 2130.
13. Kim, Y. S.; Alarcon, S. V.; Lee, S.; Lee, M. J.; Giaccone, G.; Neckers, L.; Trepel, J. B.
Curr. Top. Med. Chem. 2009, 9, 1479.
14. Biamonte, M. A.; Van de Water, R.; Arndt, J. W.; Scannevin, R. H.; Perret, D.; Lee,
W. J. Med. Chem. 2010, 53, 3.
15. Holzbeierlein, J.; Windsperger, A.; Vielhauer, G. Curr. Oncol. Rep. 2010, 12, 95.
16. Sreedhar, A. S.; Kalmar, E.; Csermely, P.; Shen, Y. F. FEBS Lett. 2004, 562, 11.
17. Peterson, L. B.; Esker, J. D.; Vielhauer, G. A.; Blagg, B. S. Mol. Pharm. 2012, 9,
1841.
45. Donnelly, A. C.; Mays, J. R.; Burlison, J. A.; Nelson, J. T.; Vielhauer, G.;
Holzbeierlein, J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 8901.
46. Zhao, H.; Kusuma, B. R.; Blagg, B. S. J. ACS Med. Chem. Lett. 2010, 1, 311.
47. Matts, R. L.; Dixit, A.; Peterson, L. B.; Sun, L.; Voruganti, S.; Kalyanaraman, P.;
Hartson, S. D.; Verkhivker, G. M.; Blagg, B. S. J. ACS Chem. Biol. 2011, 6, 800.
48. Zhao, H.; Donnelly, A. C.; Kusuma, B. R.; Brandt, G. E. L.; Brown, D.; Rajewski, R.
A.; Vielhauer, G.; Holzbeierlein, J.; Cohen, M. S.; Blagg, B. S. J. J. Med. Chem.
2011, 54, 3839.
18. McLaughlin, M.; Vandenbroeck, K. Br. J. Pharmacol. 2011, 162, 328.
19. Goodman, S. L.; Picard, M. Trends Pharmacol. Sci. 2012, 33, 405.
20. Dejeans, N.; Glorieux, C.; Guenin, S.; Beck, R.; Sid, B.; Rousseau, R.; Bisig, B.;
Delvenne, P.; Buc Calderon, P.; Verrax, J. Free Radic. Biol. Med. 2012, 52, 993.
21. Suntharalingam, A.; Abisambra, J. F.; O’Leary, J. C. I. I. I.; Koren, J. I. I. I.; Zhang,
B.; Joe, M. K.; Blair, L. J.; Hill, S. E.; Jinwal, U. K.; Cockman, M.; Duerfeldt, A. S.;
Tomarev, S.; Blagg, B. S.; Lieberman, R. L.; Dickey, C. A. J. Biol. Chem. 2012, 287,
40661.
49. Kusuma, B. R.; Zhang, L.; Sundstrom, T.; Peterson, L. B.; Dobrowsky, R. T.; Blagg,
B. S. J. J. Med. Chem. 2012, 55, 5797.
50. Khandelwal, A.; Hall, J. A.; Blagg, B. S. J. J. Org. Chem. 2013, 78, 7859.
51. Ostrovsky, O.; Ahmed, N. T.; Argon, Y. Mol. Biol. Cell 2009, 20, 1855.
52. Wang, J. S.; Wang, F. B.; Zhang, Q. G.; Shen, Z. Z.; Shao, Z. M. Mol. Cancer Res.
2008, 6, 372.