Development of Glucose Regulated Protein 94-Selective Inhibitors Based on the BnIm and Radamide Scaffold

Article

pubs.acs.org/jmc

Development of Glucose Regulated Protein 94-Selective Inhibitors

Based on the BnIm and Radamide Scaﬀold

Vincent M. Crowley,^†Anuj Khandelwal,^†Sanket Mishra,^†Andrew R. Stothert,^‡Dustin J. E. Huard,^§

Jinbo Zhao,^†Aaron Muth,^†Adam S. Duerfeldt,^†James L. Kizziah,^§Raquel L. Lieberman,^§

Chad A. Dickey,^‡and Brian S. J. Blagg*^,†

^†Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott Hall 4070, Lawrence, Kansas

66045-7563, United States

^‡Department of Molecular Medicine and Byrd Alzheimer’s Research Institute, University of South Florida, Tampa, Florida 33613,

United States

^§School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 United States

S

* Supporting Information

ABSTRACT: Glucose regulated protein 94 (Grp94) is the

endoplasmic reticulum resident of the heat shock protein 90

kDa (Hsp90) family of molecular chaperones. Grp94

associates with many proteins involved in cell adhesion and

signaling, including integrins, Toll-like receptors, immunoglo-

bulins, and mutant myocilin. Grp94 has been implicated as a

target for several therapeutic areas including glaucoma, cancer

metastasis, and multiple myeloma. While 85% identical to

other Hsp90 isoforms, the N-terminal ATP-binding site of

Grp94 possesses a unique hydrophobic pocket that was used

to design isoform-selective inhibitors. Incorporation of a cis-

amide bioisostere into the radamide scaﬀold led to develop-

ment of the original Grp94-selective inhibitor, BnIm. Structure−activity relationship studies have now been performed on the

aryl side chain of BnIm, which resulted in improved analogues that exhibit better potency and selectivity for Grp94. These

analogues also manifest superior antimigratory activity in a metastasis model as well as enhanced mutant myocilin degradation in

a glaucoma model compared to BnIm.

The majority of Hsp90 research has focused on the

development of N-terminal inhibitors, which compete with

ATP within the binding site.¹⁰⁻¹²N-terminal inhibitors have

entered clinical studies for the treatment of various

cancers.¹³⁻¹⁵However, some concerns have been raised that

must be addressed before new Hsp90 inhibitors are developed.

A common factor among all clinical candidates is that they

exhibit pan-Hsp90 inhibition, meaning they manifest similar

aﬃnities against all four Hsp90 isoforms: Hsp90α and Hsp90β

are found in the cytosol, tumor necrosis factor receptor-

associated protein 1 (Trap1) is localized to the mitochondria,

and glucose regulated protein 94 (Grp94) resides in the

endoplasmic reticulum (ER).

Many clients of the Hsp90-dependent protein folding

process have been identiﬁed, however, clients with speciﬁc

dependency on each isoform remain underinvestigated

although some isoform-dependent substrates have been

determined. For instance, maturation of the hERG channel

and its traﬃcking to the cell surface was found to be solely

INTRODUCTION

■

Molecular chaperones are responsible for the conformational

maturation of nascent polypeptides into their bioactive

conformations.¹One family of chaperones is the 90 kDa heat

shock proteins (Hsp90), which has emerged as a promising

therapeutic target for the treatment of many diseases.²⁻⁷The

Hsp90 family is responsible for the maturation of more than

200 client proteins, many of which belong to signaling

pathways that are commonly hijacked in cancer. In fact,

Hsp90 clients are directly associated with all ten hallmarks of

cancer and thus represents a unique opportunity to

simultaneously target multiple oncogenic pathways.^8,9Hsp90

exists as a homodimer within cells and consists of three

domains: an N-terminal domain, a middle domain, and a C-

terminal dimerization domain. The N-terminal domain

contains an ATP-binding pocket in which ATP hydrolysis

provides the necessary energy for client protein maturation.

The middle domain is responsible for client protein binding

and interactions with cochaperones and partner proteins. The

C-terminal domain contains a dimerization motif and is

responsible for modulating client protein release.

Received: January 28, 2016

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dependent upon the Hsp90α isoform and suggests that

inhibition of Hsp90α may contribute to some of the

cardiotoxicity observed in clinical trials.¹⁶It is also likely that

other isoform-dependent client proteins contribute to other

toxicities, which highlights the need to develop new strategies

for Hsp90 inhibition. An alternative to pan-inhibition is the

development of isoform-selective inhibitors. Such molecules

would provide an opportunity to elucidate isoform-dependent

client proteins associated with each isoform while simulta-

neously reducing/derisking potential liabilities associated with

pan-inhibition. Unfortunately, the development of isoform-

selective inhibitors is challenging due to the fact that 85%

identity is shared within the N-terminal ATP-binding site of all

four Hsp90 isoforms. In fact, Hsp90α and Hsp90β are 95%

identical within their N-terminal ATP-binding pocket, while

Grp94 is least similar to only 85% identity.¹⁷⁻¹⁹

Grp94 is responsible for the maturation of proteins

associated with cell-to-cell signaling and cell adhesion. Client

proteins dependent upon Grp94 include many integrins (α-2,

α-4, α-L, and β-4), Toll-like receptors (TLR1, TLR2, TLR4,

and TLR9), insulin-like growth factor-I and -II, immunoglobu-

lins, and mutant myocilin.^5,20−22Grp94 is essential only during

embryonic development and therefore appears to represent a

nontoxic drug target. However, overexpression of Grp94 is

associated with tumor aggressiveness and poor clinical

prognosis for cancer patients.²³In fact, Grp94 is responsible

for the maturation and traﬃcking of proteins required for

metastasis and cell migration and thus represents a nontoxic

target for the development of antimetastatic agents. More

recently, mutant myocilin has also been identiﬁed as a Grp94-

dependent substrate and studies have shown that inhibition of

Grp94 leads to the disaggregation of mutant myocilin and

consequently represents a novel target for the treatment of

primary open-angle glaucoma (POAG).^4,21,24While Grp94 may

represent an ideal target for cell migration and glaucoma,

Grp94 has also been shown to be essential in multiple

myeloma, which appears to possess increased ER stress.

Inhibition of Grp94 has been shown to induce cell death in

multiple myeloma, as disruption of interactions between Grp94

and LRP6 results in reduced cell-traﬃcking of LRP6 to the cell

surface.^3,5,25LRP6 is a coreceptor of Frizzled in the Wnt

pathway and reduction of these interactions leads to caspase 9

activation and apoptosis. Grp94 has also been shown to interact

with Her2 and, consequently, disruption of these interactions

leads to Her2 degradation via the lysosome and may represent

an alternative mechanism to treat Her2 overexpressing breast

cancers.^26,27

Grp94 is least similar, in its N-terminal ATP-binding pocket,

to other Hsp90 isoforms due to a ﬁve amino acid insertion

(QEDGQ) into the primary sequence.²²This insertion results

in the creation of a hydrophobic, secondary binding region

within the N-terminal ATP-binding site of Grp94. As such, this

unique pocket provides an opportunity to develop Grp94-

selective inhibitors.^28,29Prior studies via a high throughput

screening method identiﬁed NECA (Figure 1) as a Grp94-

selective inhibitor that bound to this secondary pocket.^22,30

Recently, a high throughput screen identiﬁed a series of Grp94-

selective inhibitors based on the purine scaﬀold and subsequent

optimization led to the development of 2.²⁶Co-crystallization

of 3 (Figure 1) with Grp94 showed this inhibitor to induce a

conformational change within Grp94 that revealed an extended

binding pocket distinct from the secondary pocket observed

with NECA and RDA (4).^{26,27,30−32}Co-crystallization of the

Figure 1. Purine-based Grp94-selective inhibitors (1, 2, and 3).

Resorcinol-based pan-Hsp90 (4) and Grp94-selective (5) inhibitors.

resorcinol containing pan-Hsp90 inhibitor, radamide (RDA,

Figure 1) with canine Grp94 (cGrp94), revealed that the amide

bond existed in both the cis- and trans-amide conformations.³³

The cis-amide conformation projected the quinone moiety into

the secondary binding pocket, while the trans-amide did not.

On the basis of the RDA co-crystal structure, it was clear that a

cis-amide conformation was required for selective Grp94

inhibition. The incorporation of a cis-amide bioisostere (e.g.,

imidazole) into the RDA scaﬀold predisposes the aryl ring into

the unique binding pocket found in Grp94 and ultimately led to

the discovery of BnIm (Figure 1).²⁸BnIm demonstrates Grp94

selectivity in vitro as determined by inhibition of IGF-II

secretion and the traﬃcking of Toll-like receptors at

concentrations that did not result in cytosolic Hsp90-

dependent client protein degradation. BnIm provided a lead

compound for Grp94-selective inhibition, however, analysis of

the unique binding pocket of Grp94 revealed further

interactions could be realized improving the selectivity and

aﬃnity of this scaﬀold for Grp94, reported herein.

GRP94-SELECTIVE INHIBITOR DESIGN, SYNTHESIS,

AND BIOCHEMICAL SCREENING

■

The co-crystal structure of RDA with cGrp94 revealed the cis-

amide to project the aryl side chain into the secondary binding

pocket, which is surrounded by hydrophobic amino acids

(Val82, Ile166, Ala167, Phe195, Val197, Phe199, Tyr200, and

Trp223), suggesting that aﬃnity may be increased through

additional hydrophobic interactions (Figure 2a).³³In contrast,

Hsp90β contains the backbone carbonyl of Asn92 and the ε-

NH₂of Lys98, which blocks access to these hydrophobic

residues and can be used as a paradigm to predict Grp94-

selective inhibition (Figure 2b). Docking of BnIm into the RDA

co-crystal structure with Grp94 (PDB 2GFD) suggested that

substitutions on the aryl side chain would provide additional

interactions with the secondary pocket of Grp94, allowing for

optimization of the aryl side chain for Grp94-selective

inhibition. Nonpolar substitutions at the 4-position were

proposed to extend further into the hydrophobic pocket and

to produce increased aﬃnity and selectivity. However, larger

substitutions at this position would likely result in steric clash

with Val82 and exhibit decreased aﬃnity. Because of the close

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a

Scheme 1

a

Conditions: (a) LiAlH₄, THF, 0−25 °C, 12 h; (b) DBU, DPPA,

toluene, 0−25 °C, 12 h; (c) PPh₃, THF:H₂O (10:1), 25 °C, 12 h.

ammonium bicarbonate and glyoxal, followed by desilylation

with tetrabutylammonium ﬂuoride to provide the desired

analogues, 10−64 (Scheme 2).^28,35,36

Scheme 2

Figure 2. Co-crystal structures of RDA with Hsp90 isoforms. cGrp94

(A, PDB 2GFD) highlights the hydrophobic and π-rich nature of the

unique secondary binding pocket of Grp94. Only cis-RDA shown for

clarity. yHsp82 (B, PDB 2FXS) highlights the hydrogen bonding

network present and restricted access to the aromatic residues.

Residues are numbered as in their respective proteins. BnIm docked in

to Gpr94 (C) highlighting substitution positions on the aryl side chain.

Once in hand, the analogues were screened via a competitive

binding ﬂuorescence polarization assay using Grp94 or Hsp90α

and FITC-labeled geldanamycin (FITC-GDA). Geldanamcyin

is a potent, natural product N-terminal pan-Hsp90 inhibitor

that competes with Grp94 inhibitors for the N-terminal ATP-

binding site.¹⁰Analogues were originally screened at 25 μM to

determine the percent of FITC-GDA (tracer) bound compared

to vehicle control (0% tracer displaced, Table 1).

The 25 μM screen provided insight into the structural

requirements for binding of the aryl side chain to the Grp94

secondary pocket. Substitutions at the 4-position typically

maintained similar aﬃnity as BnIm, but provided increased

selectivity in several cases. As hypothesized, polar substitutions

(17 and 21) manifested lower aﬃnity due to the hydrophobic

nature of the secondary binding pocket (Figure 2).

Incorporation of a bicyclic system was not tolerated as the

naphthyl (23 and 24) and quinoline (25) derivatives

manifested decreased aﬃnity for both Grp94 and Hsp90α.

Substitutions at the 3-position were not beneﬁcial, as all of the

analogues resulted in decreased aﬃnity for both isoforms

compared to BnIm. Alternatively, substitutions at the 2-position

typically increased Grp94 aﬃnity and improved selectivity

versus Hsp90α.

proximity of Tyr200 to the 3-position of the aryl side chain,

substitutions at this position appeared detrimental to Grp94

aﬃnity. Substitutions at the 2-position were likely to increase

aﬃnity through hydrophobic interactions with the secondary

pocket. Furthermore, these substitutions have the potential to

produce the conformational shift observed with the purine-

based Grp94-selective inhibitor, 3 (see Figure 4), and project

into the extended binding pocket.²⁶On the basis of these

observations, the aryl side chain of BnIm was investigated to

probe the unique pocket present in Grp94 and to elucidate

structure−activity relationships for optimal aﬃnity and

selectivity

Because the cis-amide bioisostere, imidazole, could be

prepared via a multicomponent cyclization reaction with an

aromatic aldehyde, access to the desired analogues was achieved

by varying the substitutions on the aryl amine component,

which were either commercially available or readily accessible

(Scheme 1). 4-Ethynylbenzylamine (6a) was synthesized from

4-iodobenzylamine following literature precedent.³⁴Aromatic

nitriles were reduced using lithium aluminum hydride to

produce the requisite amines (6b−e). The substituted benzoic

acid was reduced to the corresponding benzyl alcohol, 8, using

lithium aluminum hydride, and then converted to the

corresponding azide, which was subsequently reduced to the

desired amine, 6f, via Staudinger reduction. The basic amines

were then cyclized with aldehyde 9 in the presence of

Analogues that displaced ≥70% of the tracer when incubated

with Grp94 at 25 μM were subsequently evaluated to

determine their apparent K_dagainst Grp94 and Hsp90α.

BnIm manifests an apparent K_dof 1.1 μM for Grp94 and 13.1

μM for Hsp90α. Substitutions at the 4-position were designed

to extend further into the hydrophobic pocket present in Grp94

and to increase selectivity over other isoforms. Modest

increases in selectivity were observed with these compounds

(11, 12, and 18), while increases in aﬃnity were observed with

C

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Table 1. Apparent K_dValues with Grp94 and Hsp90α

a

% Tracer bound determined when incubated with 25 μM of inhibitors. Data are the average of at least two experiments SEM. n.d. = not

determined.

analogues containing halogen substitutions. Incorporation of a

larger methyl group (14) resulted in a substantial increase in

selectivity for Grp94, as well as increased aﬃnity. Extended

alkyl chains (15 and 16) were too large for accommodation

into the secondary pocket of Grp94 and resulted in decreased

aﬃnity.

D

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Figure 3. (A) Tertiary structure of RDA bound to Grp94 (PDB 2GFD) showing the open conformation of the N-terminal domain of Grp94. (B)

Tertiary structure of 3 bound to Grp94 (PDB 3O2F) showing the conformational shift (blue, green, and orange helices) of the N-terminal domain

induced by the inhibitor binding to Grp94 revealing the extended binding pocket.

As observed in the 25 μM screen, substitutions at the 3-

position did not manifest improved aﬃnity nor selectivity for

Grp94 compared to BnIm (26−32). Alternatively, substitutions

at the 2-position provided additional insights into the modes of

binding for these analogues. Small substitutions at the 2-

position were tolerated, however, such substitutions did not

increase selectivity or aﬃnity. Larger substitutions produced

increased selectivity for Grp94 versus other Hsp90 isoforms, as

38, 39, and 40 manifested >40-fold selectivity for Grp94 over

Hsp90α, which is likely due to induction of a conformational

shift in the tertiary structure of Grp94. Patel and colleagues

previously reported the co-crystallization of 3 with Grp94

(PDB 3O2F), in which a conformational shift was observed

that resulted in an extended binding region within the Grp94

ATP-binding site.^26,31This pocket is distinct from the

secondary pocket observed with NECA and RDA (Figure 3),

as this region opens due to the migration of Phe199, which

allows additional access to the binding pocket as observed with

3. Substitutions at the 2-position may also induce a similar

conformational change in Grp94, which allows access to this

region and increases selectivity for Grp94. In the case of 40

(Grp94 apparent K_d= 0.2 μM; 41-fold selective), the increased

selectivity was accompanied by a ∼6-fold increase in aﬃnity for

Grp94 compared to BnIm. Extension or branching of the alkyl

ether (41 and 42) improved aﬃnity for Grp94, although a loss

in selectivity was observed. Larger alkyl ethers were not

accommodated within the secondary binding pocket of Grp94

and likely push the benzyl side chain toward the solvent

exposed region to form hydrogen bonds with polar amino

acids. Such polar residues are present in both Grp94 and Hsp90

and provides an explanation for the loss of selectivity and an

increase in aﬃnity. 44 was synthesized in an attempt to

combine beneﬁcial substitutions observed with the 2- and 4-

positions, however, aﬃnity was compromised as compared to

the parent compounds (14 and 38), suggesting the eﬀects of

these substitutions are not additive (Table 1).

(Scheme 3). Chlorination of thiophen-2-ylmethanamine via

sulfuryl chloride provided 6m (Scheme 4). Radical bromination

a

Scheme 3. Synthesis of Amines from Aromatic Aldehydes

a

Conditions: (a) HNO₃, H₂SO₄, Ac₂O, 0 °C, 1 h; (b) 50% H₂SO₄

(aq), 110 °C, 5 min; (c) conc HCl, 40 °C, 12 h; (d) NH₂OH·HCl,

NaOAc, MeOH, 25 °C, 8 h; (e) LiAlH₄, THF, 0−25 °C, 12 h; (f)

potassium vinyltriﬂuoroborate, Pd(dppf)Cl₂, ⁱPr₂EtN, toluene, 110 °C,

14 h; (g) H₂, 10% Pd/C, EtOH, 25 °C, 6 h.

of 5-methylisoxazole followed by conversion to the azide and

subsequent reduction resulted in 6n. The aromatic carboxylic

acid 68 was reduced to the corresponding alcohol using lithium

aluminum hydride followed by conversion to the azide and

then Staudinger reduction to yield 6o. Deprotonation of 3-

chlorothiophene with n-butyllithium followed by the addition

of CO₂(g) provided a mixture of carboxylate regioisomers

which were separable via ﬂash chromatography after reduction

to the corresponding alcohols (69b and 69c) with lithium

aluminum hydride. These isomers were then converted to the

corresponding azides, followed by Staudinger reduction to

provide the requisite basic amines, 6p and 6q (Scheme 4). The

desired analogues were obtained through the multicomponent

reaction detailed in Scheme 2 to produce analogues 45−64.

As can be seen in Table 2, the incorporation of an electron

poor (compared to benzene) pyridine ring (61−64) resulted in

decreased selectivity and aﬃnity for Grp94. In contrast, the

incorporation of electron rich ﬁve-membered heterocycles

proved beneﬁcial for Grp94 aﬃnity. Converting the phenyl ring

of BnIm to the bioisosteric replacement, thiophene (50),

resulted in a signiﬁcant increase in Grp94 aﬃnity compared to

BnIm. The furan and thiophene (46 and 50, respectively)

analogues exhibited both increased aﬃnity and selectivity and

Because of the high density of aromatic amino acids within

the unique secondary binding pocket of Grp94, the

incorporation of heterocycles was proposed to improve both

π−π interactions and aﬃnity for Grp94.³³The requisite

heterocyclic amines (6g−l) were synthesized from the

corresponding aldehydes through conversion to the oximes

(66a−e) followed by reduction via lithium aluminum hydride

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a

Scheme 4

Table 2. Apparent K_dof Heterocyclic Analogues with Grp94

and Hsp90α

a

Conditions: (a) SO₂Cl₂, 15 °C, AcOH:Et₂O (9:1), 1 h; (b) NBS,

AIBN, CCl₄, 80 °C, 4 h; (c) NaN₃, MeOH:H₂O (10:1), 25 °C, 12 h;

(d) PPh₃, THF:H₂O (10:1), 25 °C, 12 h; (e) LiOH, THF:MeOH:-

H₂O (9:1:1), 25 °C, 10 h; (f) LDA, MeI, THF, −40 °C, 3 h; (g)

LiAlH₄, THF, 0−25 °C, 12 h; (h) DBU, DPPA, toluene, 25 °C, 12 h;

(i) n-BuLi, CO₂(g), THF, −78 °C, 2 h.

consequently, substitutions about these rings were explored.

The energy minimized structures of BnIm and 46 were overlaid

and revealed the 5-position of the heterocycle to align similar to

the 4-position of the BnIm benzyl side chain (Figure 4).

Therefore, substitutions at the 5-position of both the furan and

thiophene rings were sought to achieve increased selectivity and

aﬃnity.

53 was shown to manifest improved selectivity for Grp94

compared to the 3- and 4-chloro substituted thiophene

analogues (55 and 55), supporting the hypothesis that 5-

substituents bind similar to the 4-substituted phenyl ring.

Incorporation of a 5-chloro substitution on the smaller furan

ring (48) resulted in increased aﬃnity for Grp94 while

maintaining selectivity. However, incorporation of an ethyl

substitution at the 3-position of the thiophene to mimic the 2-

position of the benzene ring resulted in both reduced aﬃnity

and selectivity.

CO-CRYSTALLIZATION OF GRP94 WITH A

GRP94-SELECTIVE INHIBITOR

■

The co-crystal structure of the N-terminal domain of Grp94

lacking the acidic linker (NΔ41, see Experimental Section) in

complex with 48 (referred to as VC3 in the PBD ﬁle) was

solved to 2.6 Å resolution to support the proposed docking

interactions (Figure 5a, Supporting Information, Table 1). The

structure reveals 48 in the ATP-binding site is stabilized by

hydrogen bonding interactions with Asp149 (Figure 5b,c),

similar to other resorcinol-based pan-Hsp90 inhibitors. There

are few other direct interactions between 48 and the NΔ41

ATP-binding site, which are mediated through conserved water

molecules (Figure 5c). In contrast to RDA, 48 is bound to

Grp94 in a single orientation, illustrating the eﬀectiveness of the

cis-amide bioisostere in reducing binding heterogeneity.

The electron density for 48 was largely continuous for the

length of the molecule, with the notable exception of the

chlorinated furan moiety (Figure 5d), which apparently samples

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Table 2. continued

substituent is nestled in an ordered loop (residues 165−170) at

the entry to the ATP-binding site (arrow, Figure 5a,b). Notably,

this loop is disordered in the structure complexed with RDA.

The furan moiety also appears to be involved in a cation−π

interaction with Lys168 to stabilize this loop. In general, phenyl

rings form stronger cation−π interactions due to a larger

quadrapole moment compared to furan rings. However,

modeling studies suggest that the phenyl ring of BnIm cannot

orient in a manner that allows this interaction (data not shown)

and therefore accounts for the increased aﬃnity manifested by

the smaller heterocycles (45−60). Taken together, 48, and by

analogy other analogues described within this series, bind to the

ATP-binding site of Grp94 in a mode that manifests increased

selectivity over the other Hsp90 isoforms.

a

% Tracer bound determined when incubated with 25 μM of

inhibitors. Data are the average of at least two experiments SEM.

n.d. = not determined.

Figure 4. Overlay of the minimized structures of BnIm (cyan) and 46

(green) highlighting the overlapping 4- and 5-positions, respectively.

GRP94-SELECTIVE INHIBITION IN CANCER

■

Grp94 is responsible for the maturation and traﬃcking of

several proteins associated with cell signaling and adhesion.

One such client of Grp94 are the integrins, which are essential

for cell adhesion and migration through promoting interactions

between the intracellular actin cytoskeleton and the extrac-

ellular matrix.³⁷⁻³⁹Integrins are dependent upon Grp94 for not

only their maturation but also their transport to the cell surface.

Therefore, inhibition of Grp94 leads to decreased traﬃcking of

integrins to the cell surface and results in decreased integrin

expression at the cell surface. As a result, decreased cell

migration is observed and provides a new opportunity for the

development of antimetastatic agents.^29,40,41For example,

selective inhibition of Grp94 results in decreased migration of

MDA-MB-231 cells, an aggressive form of metastatic breast

cancer. In a wound-healing scratch assay, Grp94-selective

inhibitors, 40 and 48, produced decreased wound closing at 24

h compared to BnIm and vehicle control (70% and 73% closed

at 500 nM, respectively, Figure 6). In fact, these analogues

manifested superior antimigratory activity compared to BnIm at

10-fold lower concentrations. Furthermore, these analogues

were evaluated for antiproliferative activity against the same cell

line and were found to manifest no antiproliferative activity up

to 100 μM (78% and 107% viable at 100 μM, respectively,

Figure 6), conﬁrming that the antimigratory activity is not

linked to cell viability. These data are in agreement with the

nonessential nature of Grp94 and provides a large therapeutic

window for the development of Grp94-selective inhibitors as a

new class of antimetastatic agents.

Recently, integrin α2 was determined to be dependent upon

Grp94 for its maturation and traﬃcking to the cell surface

through proteomic and Western blot analyses of Grp94

knockdown cells.⁴²Similarly, selective inhibition of Grp94

resulted in the degradation of integrin α2 (Figure 7A,B).

Integrin α2 forms a heterodimer with integrin β1 on the cell

surface,³⁸which is responsible for binding to collagen in the

extracellular matrix to promote metastasis and invasion. 40 and

48 both induced the degradation of integrin α2, providing

evidence that the decrease in cell migration results from Grp94-

selective inhibition. Furthermore, 40 and 48 do not result in

the degradation of Akt (Figure 7C), a well-known client protein

that is dependent upon the cytosolic isoforms of Hsp90. It

should be noted that a slight induction of the pro-survival heat

shock response was observed with 40 and 48 (Figure 7D).

However, this occurred at concentrations 10−50-fold higher

than antimigratory activity was demonstrated.

Figure 5. Crystal structure of 48 bound to Grp94. (a) Surface

representation of ATP-binding pocket with 48 as ball and stick. Arrow

points to a well-ordered loop at the mouth of the active site. (b)

Cartoon representation of the structure as in (a) highlighting H-

bonding interactions (≤3.5 Å) and loop conﬁguration. (c) Zoomed

view of 48 in active site with H-bonding interactions as in (b). (d)

Final 2F_o− F_celectron density contoured at 1 σ (gray mesh)

superimposed with initial F_o− F_cdensity contoured at 3 σ (green)

after molecular replacement (see Experimental Section).

multiple conformations within the ATP-binding site. It is

possible that the chlorinated furan dwells in the extended

hydrophobic region to increase selectivity (as predicted through

modeling studies); however, the ﬁnal binding mode modeled,

based on optimal ﬁt to 2F_o− F_c(Figure 5d) and F_o− F_c

diﬀerence density (not shown), is one in which the chloride

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Figure 7. (A) Western blot analysis of MDA-MB-231 cells treated

with Grp94-selective inhibitors, geldanamycin (GDA, a pan-Hsp90

inhibitor), or vehicle control (DMSO, 0.25% ﬁnal concentration).

Ratio of Integrin α2 (B), Akt (C), and Hsp70 (D) normalized to actin

for each compound concentration.

aggregation and leads to TM cell death.^43,45Recently, it was

demonstrated that Grp94 associates with amyloid-like

aggregates of mutant myocilin but cannot triage these

aggregates for degradation through the ER-associated degrada-

tion (ERAD) pathway.⁴Our prior studies showed that

inhibition of Grp94 allows mutant myocilin degradation

through autophagy, which decreases intracellular levels of

myocilin and, ultimately, reduces toxicity.²¹Therefore, these

newly designed Gpr94-selective inhibitors were evaluated for

their ability to promote mutant myocilin clearance in an

inducible HEK model. Treatment with 48 resulted in a

substantial decrease of mutant myocilin levels at 1 μM (Figure

8A,C), however, treatment with 40 only resulted in a slight

decrease in myocilin levels. The diﬀerence in activity between

40 and 48 is not readily clear and is currently being investigated

in our laboratory. As expected, 40 and 48 did not induce the

degradation of the cytosolic Hsp90 isoform-dependent client,

Figure 6. Wound-healing scratch assay results after 24 h treatment

with BnIm, 40, 48, or vehicle. Antiproliferative activity given is relative

to vehicle control (n = 4). ND = not determined.

PRIMARY OPEN ANGLE GLAUCOMA AND

GRP94-SELECTIVE INHIBITION

■

Although POAG is characterized clinically by irreversible optic

nerve damage and retinal ganglion cell death that leads to vision

loss, the risk factor of elevated intraocular pressure typically

results from decreased outﬂow of the aqueous humor through

Schlemm’s canal of the trabecular meshwork (TM) extracellular

matrix in the anterior segment of the eye. A contributor to TM

dysfunction is TM cell death,^24,43which can be brought about

by the aggregation of mutant myocilin.⁴⁴Nonsynonymous

mutations in myocilin, localized to its olfactomedin domain,

result in non-native tertiary structures, which promote facile

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Figure 9. Summary of structure−activity relationships for the BnIm

series of Grp94-selective inhibitors.

Hsp90 isoforms. Replacing the phenyl ring of the side chain

with ﬁve-membered heterocycles also resulted in increased

aﬃnity for Grp94 as observed with 48. A crystal structure of the

N-terminal domain of Grp94 was solved revealing a cation−π

interaction between the furan ring and Lys168, which accounts

for the increased aﬃnity observed with the ﬁve-membered

heterocycles. Grp94-selective inhibition reduced cell migration

of aggressive breast cancer cells without manifesting toxicity

and thus provided a large therapeutic index. Additionally,

Grp94 inhibition resulted in the degradation of myocilin

aggregates and provides a nontoxic approach to the treatment

of POAG. These data presented herein provide a strong

foundation for the further development of rationally designed

Grp94-selective inhibitors for the treatment of metastasis and

POAG.

EXPERIMENTAL SECTION

■

Chemistry General. ¹H NMR were recorded at 400 (Bruker

AVIIIHD 400 MHz NMR with a broadband X-channel detect gradient

probe) or 500 MHz (Avance AVIII 500 MHz spectrometer with a dual

carbon/proton cryoprobe), and ¹³C were recorded at 125 MHz

(Bruker AVIII spectrometer equipped with a cryogenically cooled

carbon observe probe); chemical shifts are reported in δ (ppm)

relative to the internal standard (CDCl_3,7.26 ppm or MeOD, 3.31

ppm). HRMS spectra were recorded with a LCT Premier (Waters

Cor., Milford, MA). The purity of compounds was determined by

HPLC (Agilent 1100 series quaternary pump; 60% MeCN/40% water;

Agilent C-18 column, 4.6 mm × 150 mm, 5 μM) with UV detection.

All biologically tested compounds were determined to be >95% pure.

TLC analysis was performed on glass backed silica gel plates and

visualized by UV light. All solvents were reagent grade and used

without further puriﬁcation.

Figure 8. Western blot analysis of HEK cells overexpressing the I477N

myocilin mutant and treated with 1 μM of indicated Grp94-selective

inhibitors illustrating (A) myocilin degradation and (B) lack of AKT

degradation and Hsp70 induction. Ratio of myocilin (C), AKT (D),

and Hsp70 (E) normalized to actin for each compound. Vehicle =

DMSO and 17-AAG = 17-(allylamino)-17-demethoxygeldanamycin

(pan-Hsp90 N-terminal inhibitor).

General Procedure for Multicomponent Cyclization Reac-

tion. Basic amines (e.g., 6a−6q, 0.26 mmol) were added to a stirred

solution of 9 (125 mg, 0.26 mmol) in wet MeOH (2 mL) and stirring

continued for 30 min at rt before the addition of NH₄HCO₃(0.26

mmol) and glyoxal (0.26 mmol). After stirring for 12 h,

tetrabutylammonium ﬂuoride (0.52 mL of 1 M solution in THF,

0.52 mmol) was added and then stirred for 30 min before the reaction

was quenched with saturated aqueous NH₄Cl (10 mL) and extracted

with EtOAc (3 × 10 mL). The organic layers were combined, dried

(Na₂SO₄), ﬁltered, and concentrated. The residue was puriﬁed via ﬂash

chromatography (SiO₂, 1:49 MeOH:DCM) to aﬀord the desired

product as amorphous solids.

Akt (Figure 8B,D), nor was there any measurable induction of

the pro-survival heat shock response, as monitored by Hsp70

induction (Figure 8E). This is in contrast to Western blot

analysis of the MDA-MB-231 cell line above (Figure 7) and is

due to the Hsp90 isoforms in cancer cells having a higher

aﬃnity for inhibitors (and ATP) compared to the Hsp90

isoforms in nontransformed cells.⁴⁶

Methyl 2-(2-(1-Benzyl-1H-imidazol-2-yl)ethyl)-3-chloro-4,6-dihy-

1

CONCLUSION

droxybenzoate (5). Yield 54 mg (54%), white amorphous solid. H

■

NMR (500 MHz, CDCl₃, MeOD) δ 7.28−7.23 (m, 3H), 7.01−6.97

(m, 2H), 6.95 (d, J = 1.5 Hz, 1H), 6.81 (d, J = 1.5 Hz, 1H), 6.40 (s,

1H), 5.02 (s, 2H), 3.79 (s, 3H), 3.48−3.41 (m, 2H), 2.89−2.83 (m,

2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 170.81, 162.06, 158.06,

147.65, 141.73, 136.17, 128.93, 127.99, 126.91, 126.53, 120.10, 114.76,

105.88, 102.46, 52.45, 49.38, 30.77, 26.08. HRMS (ESI) m/z [M −

H]⁻C₂₀H₁₉ClN₂O₄385.0955, found 385.0953. t_R= 5.062 min, 99.0%.

(4-Ethynylphenyl)methanamine (6a). 6a was synthesized from 4-

iodobenzylamine hydrochloride following procedures detailed in ref

Structure−activity relationship studies were performed on the

aryl side chain of BnIm, which interacts with the unique

secondary pocket of Grp94 (Figure 9). Substitutions at the 2-

and 4-positions were tolerated within this binding region.

Incorporation of an ethoxy group at the 2-position manifested a

signiﬁcant increase in aﬃnity, as well as selectivity. 40 manifests

nearly a 10-fold increase in aﬃnity for Grp94 compared to

BnIm and 40-fold selectivity for Grp94 over the cytosolic

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1

34. Yield 93 mg (78%) as a dark-yellow oil. H NMR (500 MHz,

MeOD) δ 7.54 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 4.13 (s,

2H), 3.60 (s, 1H). ¹³C NMR (126 MHz, MeOD) δ 133.56, 132.27

(2C), 128.71 (2C), 123.34, 82.10, 78.58, 42.51. HRMS (ESI) m/z [M

+ H]⁺for C₉H₁₀N 132.0813, found 132.0809.

2H). ¹³C NMR (126 MHz, CDCl₃) δ 156.24, 156.23, 135.05, 107.49,

106.62, 39.35. HRMS (ESI) m/z [M + H]⁺for C₅H₇ClNO 132.0216,

found 132.0221.

1

Thiazol-2-ylmethanamine (6h). Yield 42 mg (9%) as a red oil. H

NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 3.1 Hz, 1H), 7.26 (d, J = 3.2

Hz, 1H), 4.20 (s, 2H), 1.80 (br, s, 2H). ¹³C NMR (126 MHz, CDCl₃)

δ 174.02, 142.60, 118.56, 44.04. HRMS (ESI) m/z [M + H]⁺for

C₄H₇N₂S 115.0330, found 115.0331.

(5-Methylthiophen-2-yl)methanamine (6i). Yield 72 mg (16%) as

a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 6.70 (d, J = 3.4 Hz, 1H),

6.57 (dd, J = 3.3, 1.3 Hz, 1H), 3.96 (s, 2H), 2.72 (br s, 2H), 2.44 (s,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 143.88, 138.89, 124.79, 124.03,

41.20, 15.40. HRMS (ESI) m/z [M + H]⁺for C₆H₁₀NS 128.0534,

found 128.0532.

General Procedure for Reduction of Aromatic Nitriles.

Benzonitriles (e.g., 7a−7c, 0.7 M in THF, 1 equiv) were added

dropwise to a stirred solution of LiAlH₄(0.5 M in dry THF, 5 equiv)

at 0 °C. The reaction mixture warmed to rt and stirred for 12 h. H₂O

(1 mL/g of LiAlH₄) was added dropwise to quench the excess hydride,

followed by 4 M NaOH (1 mL/g of LiAlH₄) and EtOAc (3 mL/g of

LiAlH₄). The resulting suspension was ﬁltered through a pad of Celite

and the Celite washed with warm EtOAc (30 mL) and the eluent

concentrated. The residue was puriﬁed via ﬂash chromatography

(SiO₂, 1:49, MeOH:DCM to 1:20, MeOH:DCM) to aﬀord the

desired product as oils.

(2-Ethylphenyl)methanamine (6b). Yield 653 mg (79%) as a

colorless oil. H NMR (400 MHz, MeOD) δ 6.77−6.69 (m, 1H),

6.63−6.56 (m, 2H), 6.58−6.48 (m, 1H), 4.51 (s, 2H), 2.07 (q, J = 7.6

Hz, 2H), 0.60 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ

143.24, 129.80, 129.39, 129.10, 127.47, 126.97, 42.31, 26.37, 15.96.

HRSM (ESI) m/z [M + NH₄]⁺for C₉H₁₇N₂153.1392, found

153.1395.

(2-Ethoxyphenyl)methanamine (6c). Yield 215 mg (59%) as a

colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.25−7.16 (m, 2H), 6.90

(t, J = 7.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.07 (q, J = 7.6, 7.1 Hz,

2H), 3.82 (s, 2H), 1.73 (br s, 2H), 1.44 (t, J = 7.0 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 156.82, 131.81, 128.52, 128.05, 120.37, 111.07,

63.33, 42.88, 14.98. HRMS (ESI) m/z [M + H]⁺for C₉H₁₄NO

152.1075, found 152.1077.

(2-Isopropoxyphenyl)methanamine (6d). Yield 243 mg (59%) as a

colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.19 (t, J = 6.6 Hz, 2H),

6.92−6.83 (m, 2H), 4.59 (hept, J = 6.0 Hz, 1H), 3.79 (s, 2H), 1.74 (br

s, 2H), 1.36 (d, J = 6.1 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ

155.74, 132.68, 128.74, 127.95, 120.24, 112.53, 69.73, 43.07, 22.23

(2C). MS (EI) m/z [M]⁺for C₁₀H₁₅NO 165.1, found 165.2.

(2-Propoxyphenyl)methanamine (6e). Yield 265 mg (67%) as a

colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.24−7.16 (m, 2H), 6.90

(t, J = 7.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 3.96 (t, J = 6.4 Hz, 2H),

3.83 (s, 2H), 1.90−1.78 (m, 4H), 1.06 (t, J = 7.4 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 156.94, 131.72, 128.50, 128.08, 120.33, 111.02,

69.26, 42.85, 22.70, 10.78. MS (EI) m/z [M]⁺for C₁₀H₁₅NO 165.1,

found: 165.2.

(3-Vinylthiophen-2-yl)methanamine (6j). Yield 152 mg (42%) as a

1

yellow oil. H NMR (400 MHz, CDCl₃) δ 7.20−7.07 (m, 1H), 6.73

(dd, J = 17.5, 11.0 Hz, 1H), 5.54 (dd, J = 17.4, 1.3 Hz, 1H), 5.24 (dd, J

= 11.0, 1.3 Hz, 1H), 4.07 (s, 1H), 2.03 (s, 1H). ¹³C NMR (126 MHz,

CDCl₃) δ 142.46, 135.23, 128.76, 125.43, 123.24, 114.19, 38.92.

HRMS (ESI) m/z [M + H]⁺for C₇H₁₀NS 140.0534, found 140.0531.

(3-Vinylfuran-2-yl)methanamine (6k). Yield 57 mg (24%) as a

1

yellow oil. H NMR (400 MHz, CDCl₃) δ 7.29 (dd, J = 2.0, 0.7 Hz,

1H), 6.57 (dd, J = 17.3, 10.7 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 5.42

(dd, J = 17.4, 1.4 Hz, 1H), 5.14 (dd, J = 10.8, 1.4 Hz, 1H), 3.85 (s,

2H), 1.67 (br s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 152.77, 141.68,

126.24, 119.34, 113.23, 107.72, 37.05. HRMS (ESI) [M + H]⁺for

C₇H₁₀NO 124.0762, found 124.0768.

(3-Ethylthiophen-2-yl)methanamine (6l). Pd/C 10% (5 mol %)

was added to a stirred solution of (3-vinylthiophen-2-yl)methanamine

(6k, 50 mg, 0.36 mmol) in MeOH (5 mL) and stirred at rt under a

hydrogen atmosphere for 12 h. The reaction mixture was ﬁltered

through a pad of Celite, and the eluent was concentrated to produce

the title compound which was used without further puriﬁcation. Yield

45 mg (89%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.16 (d, J

= 5.1 Hz, 1H), 6.87 (d, J = 5.2 Hz, 1H), 4.56 (s, 2H), 4.05 (s, 2H),

2.61 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 141.48, 134.90, 128.62, 123.84, 37.77, 21.46, 15.29.

HRMS (ESI) m/z [M + H]⁺for C₇H₁₂NS 147.0690, found 147.0692.

(5-Chlorothiophen-2-yl)methanamine (6m). A solution of thio-

phene-2-ylmethanamine (1 mL, 9.7 mmol, 1 equiv) in AcOH:Et₂O

(9:1, 0.5 M) was cooled to 5 °C followed by the dropwise addition of

SO₂Cl₂(1.18 mL, 14.6 mmol, 1.5 equiv), maintaining the reaction

temperature under 20 °C. The reaction was stirred at rt for 1 h, at

which time Et₂O (20 mL) was added and stirred for an additional 30

min. The precipitate (hydrochloride salt of desired product) was

ﬁltered and the solid washed with Et₂O. The isolated solid was

dissolved in DCM (25 mL) and washed with sat’d NaHCO₃(2 × 30

mL). The organic layer was separated, dried (Na₂SO₄), and

concentrated. The residue was puriﬁed via ﬂash chromatography

(SiO₂, 1:49 MeOH:DCM to 1:20 MeOH:DCM) to provide the title

General Procedure for Staudinger Reduction. PPh₃(1.1

equiv) was added to a stirred solution of the benzyl azide (1 equiv) in

THF:H₂O (0.1 M, 10:1) and stirred at rt for 12 h. The reaction

mixture was concentrated, and the residue was puriﬁed via ﬂash

chromatography (SiO₂, 1:49, MeOH:DCM to 1:20, MeOH:DCM) to

aﬀord the desired product as oils.

(2-Methoxy-4-methylphenyl)methanamine (6f). Yield 99 mg

(73%) as a colorless oil. H NMR (500 MHz, CDCl₃) δ 7.08 (d, J

1

compound. Yield 658 mg (37%) as a yellow oil. H NMR (400 MHz,

= 7.4 Hz, 1H), 6.72 (d, J = 7.4 Hz, 1H), 6.69 (s, 1H), 3.83 (s, 3H),

3.77 (s, 2H), 2.34 (s, 3H), 1.87 (br s, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 157.32, 138.16, 128.47, 120.96, 111.24, 74.80, 55.12, 42.39,

21.55. HRMS (ESI) m/z [M + H]⁺for C₉H₁₄NO 152.1075, found

152.1076.

General Procedure for Oxime Reduction. Aryl oximes (e.g.,

66a−66e, 0.7 M in THF, 1 equiv) were added dropwise to a stirred

solution of LiAlH₄(0.5 M in dry THF, 5 equiv) at 0 °C. The reaction

mixture warmed to rt and stirred for 12 h. H₂O (1 mL/g of LiAlH₄)

was added dropwise to quench the excess hydride, followed by 4 M

NaOH (1 mL/g of LiAlH₄) and EtOAc (3 mL/g of LiAlH₄). The

resulting suspension was then ﬁltered through a pad of Celite and the

Celite washed with warm EtOAc (30 mL), and the eluent was

concentrated. The residue was puriﬁed via ﬂash chromatography

(SiO₂, 1:49, MeOH:DCM to 1:20, MeOH:DCM) to aﬀord the

desired products as oils.

MeOD) δ 5.51 (d, J = 3.0 Hz, 1H), 5.40 (d, J = 3.8 Hz, 1H), 2.69 (s,

2H), 1.73 (br s, 2H). ¹³C NMR (126 MHz, MeOD) δ 134.70, 132.71,

130.62, 127.93, 38.70. HRMS (ESI) m/z [M + H]⁺for C₅H₆ClNS

148.9990, found 148.9988.

Isoxazol-5-ylmethanamine (6n). Yield 159 mg (77%) as a

1

colorless oil. H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 6.13 (s,

1H), 4.00 (s, 2H), 1.53 (br, s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ

172.47, 150.28, 100.04, 37.62. MS (EI) m/z [M]⁺for C₄H₆N₂O 98.0,

found 98.1.

(3,5-Dimethylfuran-2-yl)methanamine (6o). Yield 98 mg (65%)

1

as a yellow oil. H NMR (400 MHz, MeOD) δ 5.78 (s, 1H), 3.70 (s,

2H), 2.17 (s, 3H), 1.92 (s, 3H). ¹³C NMR (126 MHz, MeOD) δ

152.01, 147.87, 118.48, 110.06, 36.39, 13.41, 9.78. HRMS (ESI) m/z

[M + H]⁺for C₇H₁₂NO 126.0919, found 126.0924.

(3-Chlorothiophen-2-yl)methanamine (6p). Yield 113 mg (59%)

1

as a yellow oil. H NMR (400 MHz, CDCl₃) δ 7.15 (dd, J = 5.2, 1.2

(5-Chlorofuran-2-yl)methanamine (6g). Yield 93 mg (47%),

yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 6.12 (d, J = 3.2 Hz,

1H), 6.06 (d, J = 3.2 Hz, 1H), 3.76 (d, J = 0.8 Hz, 2H), 1.58 (br s,

Hz, 1H), 6.86 (dd, J = 5.3, 1.1 Hz, 1H), 3.98 (s, 2H), 1.79 (br s, 2H).

¹³C NMR (126 MHz, CDCl₃) δ 148.24, 124.45, 123.90, 118.33, 41.51.

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HRMS (ESI) m/z [M + H]⁺for C₅H₇ClNS 147.9988, found

147.9990.

(4-Chlorothiophen-2-yl)methanamine (6q). Yield 79 mg (67%) as

a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 6.96 (s, 1H), 6.78 (s, 1H),

3.99 (s, 2H), 1.71 (br s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 148.24,

124.45, 123.90, 118.33, 41.51. HRMS (ESI) m/z [M + H]⁺for

C₅H₇ClNS 147.9988, found 147.9995.

General Procedure for Alkylation of 2-Hydroxybenzonitrile.

Alkyl iodides (3.7 mmol, 1.1 equiv) and K₂CO₃(930 mg, 6.7 mmol, 2

equiv) were added to a stirred solution of 2-hydroxybenzonitrile (400

mg, 3.4 mmol) in DMF (20 mL) at rt and stirred for 4 h. The reaction

was quenched with the addition of H₂O (20 mL) and EtOAc (40 mL).

The organic layer was washed with water (5 × 40 mL), dried

(Na₂SO₄), ﬁltered, and concentrated. The residue was puriﬁed by

column chromatography (SiO₂, 4:1 Hex:EtOAc) to aﬀord the desired

alkyl ethers as colorless oils.

2-Ethoxybenzonitrile (7a). Yield 360 mg (71%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.63−7.45 (m, 2H), 7.04−6.89 (m,

2H), 4.15 (q, J = 7.1 Hz, 2H), 1.48 (t, J = 7.0 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 161.02, 134.65, 134.17, 120.91, 116.99, 112.49,

102.36, 64.99, 14.91. MS (EI) m/z [M]⁺for C₉H₉NO 147.1, found

147.1; [M + H]⁺for C₉H₁₀NO 148.1, found 148.1.

27.26. HRMS (ESI) m/z [M + H] for C₂₀H₁₉ClFN₂O₄405.1017,

found 405.1009.

Methyl 3-Chloro-2-(2-(1-(4-chlorobenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (11). Yield 28 mg, (25%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.24 (d, J

= 8.4 Hz, 2H), 6.98 (d, J = 1.4 Hz, 1H), 6.90 (d, J = 8.4 Hz, 2H), 6.78

(d, J = 1.4 Hz, 1H), 6.40 (s, 1H), 4.97 (s, 2H), 3.81 (s, 3H), 3.46−3.40

(m, 2H), 2.89−2.80 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ

170.63, 162.31, 157.66, 147.56, 141.56, 134.60, 134.02, 129.19 (2C),

127.92 (2C), 127.05, 120.00, 114.57, 106.08, 102.68, 52.59, 30.87,

29.69, 26.04 HRMS (ESI) m/z [M + H] for C₂₀H₁₉Cl₂N₂O₄421.0722,

found 421.0714.

Methyl 2-(2-(1-(4-Bromobenzyl)-1H-imidazol-2-yl)ethyl)-3-

chloro-4,6-dihydroxybenzoate (12). Yield 45 mg (38%), oﬀ-white

amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.36 (d, J =

8.4 Hz, 2H), 6.91 (d, J = 1.4 Hz, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.77

(d, J = 1.5 Hz, 1H), 6.35 (s, 1H), 4.93 (s, 2H), 3.78 (s, 3H), 3.42−3.34

(m, 2H), 2.84−2.77 (m, 2H). ¹³C NMR (101 MHz, CDCl₃, MeOD) δ

170.23, 161.42, 157.64, 147.25, 141.09, 134.83, 131.69 (2C), 127.85

(2C), 126.55, 121.59, 119.68, 114.27, 102.14, 99.99, 52.03, 30.38,

25.69. HRMS (ESI) m/z [M + H]⁺for C₂₀H₁₉BrClN₂O₄465.0217,

found 465.0237. t_R= 4.17 min, 95.3%.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(4-iodobenzyl)-1H-imida-

zol-2-yl)ethyl)benzoate (13). Yield 22 mg (35%), white amorphous

solid. ¹H NMR (400 MHz, CDCl₃, MeOD): δ 7.59−7.57 (dd, J = 11.6

Hz, 2H), 7.14−7.11 (dd, J = 10.0 Hz, 2H), 7.05 (s, 1H), 6.86 (s, 1H),

6.44 (s, 1H), 5.14 (s, 2H), 3.87 (s, 3H), 3.48−3.44 (t, J = 16.8, 2H),

2.91−2.87 (t, J = 18.4 Hz, 2H.). ¹³C NMR (125 MHz, CDCl₃,

MeOD): δ 170.6, 162.4, 157.6, 147.6, 141.5, 138.1, 128.4, 127.2, 120.0,

114.6, 106.1, 102.7, 93.6, 52.6, 52.2, 49.0, 30.9, 26.1, 25.2, 20.1. HRMS

(ESI) m/z [M + H]⁺for C₂₀H₁₈ClIN₂O₄513.0020, found 513.0070.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(4-methylbenzyl)-1H-imi-

dazol-2-yl)ethyl)benzoate (14). Yield 44 mg (43%), oﬀ-white

2-Isopropoxybenzonitrile (7b). Yield 405 mg (71%) as a colorless

oil. ¹H NMR (400 MHz, CDCl₃) δ 7.57 (dt, J = 7.7, 1.5 Hz, 1H), 7.52

(td, J = 7.9, 7.3, 1.5 Hz, 1H), 6.99 (dd, J = 8.2, 5.2 Hz, 2H), 4.68 (hept,

J = 6.1 Hz, 1H), 1.43 (d, J = 6.1 Hz, 6H). ¹³C NMR (126 MHz,

CDCl₃) δ 159.92, 134.11, 133.94, 120.45, 116.77, 113.64, 102.99,

71.78, 21.86 (2C). MS (EI) m/z [M]⁺for C₁₀H₁₁NO 161.1, found

161.1; [M + H]⁺for C₁₀H₁₂NO 162.1, found 162.1.

2-Propoxybenzonitrile (7c). Yield 385 mg (71%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J = 7.8 Hz, 1H), 7.50 (td, J =

8.1, 7.4, 1.5 Hz, 1H), 7.00−6.93 (m, 2H), 4.03 (t, J = 6.5 Hz, 2H), 1.88

(h, J = 7.0 Hz, 2H), 1.08 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz,

CDCl₃) δ 160.83, 134.27, 133.76, 120.52, 116.56, 112.19, 102.00,

70.45, 22.33, 10.46. MS (EI) m/z [M]⁺for C₁₀H₁₁NO 161.1, found

161.1; [M + H]⁺for C₁₀H₁₂NO 162.1, found 162.1.

General Procedure for the Reduction of Carboxylic Acids.

The aromatic carboxylic acids (e.g., 68, 0.7 M in THF, 1 equiv) were

added dropwise to a stirred solution of LiAlH₄(0.5 M in THF, 5

equiv) at 0 °C. The reaction warmed to rt and stirred for 12 h. H₂O (1

mL/g of LiAlH₄) was added, to quench the excess hydride, then 4 M

NaOH (1 mL/g of LiAlH₄) and EtOAc (3 mL/g of LiAlH₄). The

resulting suspension was ﬁltered through a pad of Celite then the

Celite was washed with warm EtOAc and the eluent concentrated. The

residue was puriﬁed via ﬂash chromatography (SiO₂, 3:10 EtOAc:hex-

anes to 3:5 EtOAc:hexanes) to provide the desired alcohol as colorless

oils:

(2-Methoxy-4-methylphenyl)methanol (8). Yield 187 mg (50%),

colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 7.5 Hz, 1H),

6.78 (dd, J = 7.5, 0.8 Hz, 1H), 6.75 (s, 1H), 4.32 (s, 2H), 3.86 (s, 3H),

2.38 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 157.58, 140.13, 130.11,

121.13, 120.80, 111.52, 55.33, 50.01, 21.71. HRSM (ESI) m/z [M]⁺

for C₉H₁₂O₂152.0837, found 152.0846.

Methyl 4,6-Bis((tert-butyldimethylsilyl)oxy)-3-chloro-2-(3-

oxopropyl)benzoate (9). 9 was synthesized following procedures

detailed in refs 28 and 35. Yield 1.8 g (68%), white amorphous solid.

¹H NMR (500 MHz, chloroform-d) δ 9.80 (s, 1H), 6.31 (s, 1H), 3.84

(s, 3H), 2.96−2.92 (m, 2H), 2.80−2.74 (m, 2H), 1.02 (s, 9H), 0.95 (s,

9H), 0.23 (s, 6H), 0.21 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ

201.47, 168.36, 153.48, 151.94, 138.10, 121.44, 118.81, 110.15, 109.97,

52.69, 43.83, 26.00 (3C), 25.83 (3C), 18.74, 18.43, −3.96 (2C), −4.00

(2C). HRMS (ESI) m/z [M + H]⁺for C₂₃H₄₀ClO₅Si₂487.2103,

found 487.2120.

Methyl 3-Chloro-2-(2-(1-(4-ﬂuorobenzyl)-1H-imidazol-2-yl)ethyl)-

4,6-dihydroxybenzoate (10). Yield 22 mg (21%), white amorphous

solid. ¹H NMR (500 MHz, MeOD) δ 7.07−6.94 (m, 5H), 6.86 (d, J =

1.4 Hz, 1H), 6.29 (s, 1H), 5.00 (s, 2H), 3.69 (s, 3H), 3.22−3.19 (m,

2H), 2.84 (m, 2H). ¹³C NMR (126 MHz, MeOD) δ 171.22, 164.76,

162.81, 160.69, 158.81, 148.82, 141.37, 134.18, 130.01, 129.94, 127.28,

121.71, 116.75, 116.58, 112.63 (d, J = 558.4 Hz), 103.34, 52.85, 31.45,

1

amorphous solid. H NMR (500 MHz, MeOD) δ 7.15 (d, J = 7.9

Hz, 2H), 7.03 (d, J = 1.4 Hz, 1H), 7.00−6.96 (m, 2H), 6.94 (d, J = 1.5

Hz, 1H), 6.39 (s, 1H), 5.05 (s, 2H), 3.77 (s, 3H), 3.37−3.30 (m, 2H),

2.97−2.89 (m, 2H), 2.31 (s, 3H). ¹³C NMR(125 MHz, MeOD) δ

171.3, 160.9, 159.0, 148.8, 141.5, 138.9, 135.1, 130.5 (2C), 127.9 (2C),

127.1, 121.8, 115.0, 110.1, 103.3, 52.8, 50.1, 31.5, 27.3, 21.1. HRMS

(ESI) m/z [M + H]⁺for C₂₁H₂₂ClN₂O₄401.1268, found 401.1266.

Methyl 3-Chloro-2-(2-(1-(4-ethylbenzyl)-1H-imidazol-2-yl)ethyl)-

4,6-dihydroxybenzoate (15). Yield 37 mg (39%), oﬀ white

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.14 (d, J

= 8.1 Hz, 2H), 7.11−7.08 (m, 1H), 6.98−6.91 (m, 2H), 6.85 (d, J =

1.5 Hz, 1H), 6.44 (s, 1H), 4.96 (s, 3H), 3.54−3.45 (m, 2H), 3.04 (t, J

= 7.9 Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.17 (td, J = 7.6, 1.0 Hz, 3H).

¹³C NMR (125 MHz, CDCl₃, MeOD) δ 170.6, 162.1, 158.2, 147.2,

145.0, 140.9, 132.1, 128.8 (2C), 127.2 (2C), 124.2, 120.6, 114.9, 106.2,

103.0, 50.0, 49.9, 30.7, 28.6, 25.6, 15.6. HRMS (ESI) m/z [M + H]⁺

for C₂₂H₂₄ClN₂O₄415.1425, found 415.1432.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(4-isopropylbenzyl)-1H-

imidazol-2-yl)ethyl)benzoate (16). Yield 25 mg (22%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃) δ 7.13 (d, J = 8.1

Hz, 2H), 7.03 (s, 1H), 6.90 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 1.5 Hz,

1H), 6.49 (s, 1H), 4.95 (s, 2H), 3.79 (s, 3H), 3.50 (t, J = 7.9 Hz, 2H),

3.00−2.92 (m, 2H), 2.82 (hept, J = 6.9 Hz, 1H), 1.16 (d, J = 7.0 Hz,

6H). ¹³C NMR (126 MHz, CDCl₃) δ 170.63, 162.91, 157.32, 149.25,

147.18, 127.20 (2C), 126.81 (2C), 120.19, 114.50, 106.24, 103.00,

99.98, 52.76, 49.56, 33.81, 30.77, 29.72, 23.93 (2C). HRMS (ESI) m/z

[M + H]⁺for C₂₃H₂₆ClN₂O₄429.1581, found 429.1588.

Methyl 3-Chloro-2-(2-(1-(4-cyanobenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (17). Yield 37 mg (39%), oﬀ white

amorphous solid. ¹H NMR (500 MHz, CDCl₃) δ 7.79 (q, J = 7.8, 6.0

Hz, 2H), 7.61−7.36 (m, 2H), 7.20−7.12 (m, 1H), 7.05 (s, 1H), 6.57

(s, 1H), 5.44−5.25 (m, 2H), 4.00 (s, 3H), 3.57 (q, J = 7.7 Hz, 2H),

3.02 (t, J = 7.9 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): 170.4, 161.5,

158.0, 147.8, 142.0, 141.2, 132.8, (2C), 127.7, 127.4, 127.0 (2C),

120.2, 118.2, 114.5, 111.6, 102.4, 52.4, 52.3, 30.8, 26.0. HRMS (ESI)

m/z [M + H]⁺for C₂₁H₁₉ClN₃O₄412.1064, found 412.1049.

Methyl 3-Chloro-2-(2-(1-(4-ethynylbenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (18). Yield 37 mg (35%), tan

K

DOI: 10.1021/acs.jmedchem.6b00085

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

amorphous solid. ¹H NMR (500 MHz, CDCl₃) δ 7.46 (d, J = 8.3 Hz,

1H), 7.06 (d, J = 1.4 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 1.4

Hz, 1H), 6.48 (s, 1H), 5.12 (s, 1H), 3.84 (s, 2H), 3.50 (dd, J = 9.3, 6.7

Hz, 1H), 3.09 (s, 1H), 2.94 (dd, J = 9.0, 7.0 Hz, 1H). ¹³C NMR (126

MHz, CDCl₃) δ 170.71, 162.73, 158.41, 147.66, 141.27, 136.69,

132.78, 129.10, 126.79, 126.61, 126.52, 122.09, 120.19, 120.15, 114.98,

105.65, 102.81, 82.85, 52.61, 49.28, 30.86, 26.01. HRMS (ESI) m/z

[M + H]⁺for C₂₂H₂₀ClN₂O₄411.1112, found 411.1121.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(4-(triﬂuoromethyl)-

benzyl)-1H-imidazol-2-yl)ethyl)benzoate (19). Yield 24 mg (36%),

white amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD): δ

7.56−7.55 (dd, J = 9.7 Hz, 2H), 7.15 (s, 1H), 7.10−7.08 (dd, J = 11.3

Hz, 2H), 6.86 (s, 1H), 6.44 (s, 1H), 5.06 (s, 2H), 3.86 (s, 3H), 3.47−

3.43 (t, J = 16.3 Hz, 2H), 3.04−3.00 (t, J = 16.3 Hz, 2H). ¹³C NMR

(125 MHz, CDCl₃, MeOD) δ 170.2, 162.1, 157.8, 147.3, 140.3, 138.7,

131.1, 130.8, 127.1, 126.3, 126.2, 124.7, 122.6, 120.5, 114.6, 111.4,

106.1, 103.1, 52.9, 30.6, 25.3. HRMS (ESI) m/z [M + H⁺] for

C₂₁H₁₈ClF₃N₂O₄455.0937, found 455.0972.

110.78, 101.68, 51.80, 48.38, 29.92, 26.18. HRMS (ESI) m/z [M +

H]⁺for C₂₄H₂₁ClN₂O₄437.1268, found 437.1280.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(quinolin-5-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (25). Yield 29 mg (25%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 8.87 (dd,

J = 4.3, 1.6 Hz, 1H), 8.20−8.16 (m, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.60

(dd, J = 8.5, 7.2 Hz, 1H), 7.49−7.44 (m, 1H), 6.99 (s, 1H), 6.86−6.83

(m, 1H), 6.76 (s, 1H), 6.36 (s, 1H), 5.48 (s, 2H), 3.83 (s, 3H), 3.48−

3.44 (m, 2H), 2.95−2.89 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ

170.55, 161.86, 157.87, 150.21, 147.86, 141.56, 132.32, 131.06, 129.45,

129.37, 127.44, 125.58, 124.79, 121.64, 120.13, 114.59, 106.08, 102.48,

99.99, 52.49, 46.63, 30.93, 26.20. HRMS (ESI) m/z [M + H]⁺for

C₂₃H₂₁ClN₃O₄438.1221, found 438.1207.

Methyl 3-Chloro-2-(2-(1-(3-ﬂuorobenzyl)-1H-imidazol-2-yl)ethyl)-

4,6-dihydroxybenzoate (26). Yield 26 mg (25%), pale-yellow

amorphous solid. ¹H NMR (500 MHz, CDCl₃) δ 7.29−7.20 (m,

1H), 7.01 (d, J = 1.4 Hz, 1H), 6.92 (m, 1H), 6.82 (d, J = 1.4 Hz, 1H),

6.75 (m, 1H), 6.70−6.58 (m, 1H), 6.46 (s, 1H), 5.01 (s, 2H), 3.80 (s,

3H), 3.52−3.40 (m, 2H), 2.88−2.74 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 157.05, 147.61, 141.78, 130.85, 127.79, 122.12, 120.22 (2C),

115.28, 115.12, 113.69, 113.51, 106.66, 102.98 (2C), 100.13, 53.58,

48.96, 31.01, 26.26. HRMS (ESI) m/z [M + H] for C₂₀H₁₉ClFN₂O₄

405.1017, found 405.1009.

Methyl 2-(2-(1-([1,1′-Biphenyl]-4-ylmethyl)-1H-imidazol-2-yl)-

ethyl)-3-chloro-4,6-dihydroxybenzoate (20). Yield 35 mg (29%),

1

tan amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.54−

7.48 (m, 4H), 7.39 (ddd, J = 8.0, 7.0, 1.7 Hz, 2H), 7.31 (td, J = 7.2, 1.4

Hz, 1H), 7.07 (dd, J = 8.3, 1.6 Hz, 2H), 6.98 (s, 1H), 6.86 (s, 1H),

6.41 (s, 1H), 5.07 (s, 2H), 3.83 (s, 3H), 3.52−3.45 (m, 2H), 2.92−

2.87 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.80, 162.08, 157.84,

147.65, 141.83, 140.99, 140.24, 135.21, 128.81 (2C), 127.62 (2C),

127.52, 127.16, 127.00 (2C), 126.98 (2C), 120.08, 114.67, 106.06,

102.49, 52.50, 49.64, 30.89, 26.17. HRMS (ESI) m/z [M + H]⁺for

C₂₆H₂₃ClN₂O₄463.1425, found 463.1447.

Methyl 3-Chloro-2-(2-(1-(3-chlorobenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (27). Yield 47 mg (44%), pale-yellow

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.25−7.20

(m, 2H), 7.03 (d, J = 1.6 Hz, 1H), 6.99- 6.96 (m, 1H), 6.91−6.84 (m,

2H), 6.40 (s, 1H), 5.00 (s, 2H), 3.82 (s, 3H), 3.49−3.33 (m, 2H),

3.01−2.83 (m, 2H). ¹³C NMR (125 MHz, CDCl₃, MeOD) δ 170.6,

161.9, 158.2, 147.5, 141.1, 137.7, 135.1, 130.5, 128.5, 126.8, 125.9,

124.9, 120.5, 114.8, 106.2, 102.7, 52.6 (2C), 30.6, 25.8. HRMS (ESI)

m/z [M + H]⁺for C₂₀H₁₉Cl₂N₂O₄421.0722, found 421.0728.

Methyl 2-(2-(1-(3-Bromobenzyl)-1H-imidazol-2-yl)ethyl)-3-

chloro-4,6-dihydroxybenzoate (28). Yield 23 mg (19%), white

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(4-methoxybenzyl)-1H-

imidazol-2-yl)ethyl)benzoate (21). Yield 25 mg (23%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 6.86−6.84

(m, 1H), 6.82 (d, J = 1.4 Hz, 1H), 6.74−6.69 (m, 4H), 6.31 (s, 1H),

4.85 (s, 2H), 3.72 (s, 3H), 3.65 (s, 3H), 3.38−3.32 (m, 2H), 2.82−

2.76 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 174.64, 165.64,

163.22, 162.05, 151.40, 145.46, 135.37, 132.06 (2C), 132.02, 130.37,

123.92, 118.20 (2C), 110.16, 106.36, 99.99, 59.12, 56.29, 34.61, 30.03.

HRMS (ESI) m/z [M−H]⁻for C₂₁H₂₁ClN₂O₅415.1061, found

415.1061.

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.38 (m,

1H), 7.19−7.09 (m, 2H), 6.99 (d, J = 1.5 Hz, 1H), 6.91 (m, 1H), 6.84

(d, J = 1.5 Hz, 1H), 6.40 (s, 1H), 5.00 (s, 2H), 3.83 (s, 3H), 3.46−3.38

(m, 2H), 2.92−2.79 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ

168.02, 159.25, 155.49, 145.00, 138.77, 135.70, 128.71, 128.01, 126.95,

124.05, 122.60, 120.52, 117.64, 112.15, 103.58, 100.01, 49.95, 46.22,

28.09, 23.35. HRMS (ESI) m/z [M + H] for C₂₀H₁₉BrClN₂O₄

465.0217, found 465.0225.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(3-iodobenzyl)-1H-imida-

zol-2-yl)ethyl)benzoate (29). Yield 4 mg (11%), white amorphous

solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.67−7.64 (dd, J = 7.05 Hz,

1H), 7.43 (s, 1H), 7.18−7.14 (m, 2H), 7.08−7.06 (dd, J = 9.24 Hz,

1H), 6.86 (s, 1H), 6.47 (s, 1H), 5.14 (s, 2H), 3.68 (s, 3H), 2.92−2.88

(t, J = 16.3 Hz, 2H), 2.73−2.67 (t, J = 18.2 Hz, 2H). ¹³C NMR (125

MHz, DMSO-d₆, MeOD): δ 167.9, 155.0, 154.5, 146.4, 140.2, 137.7,

136.2, 135.1, 130.9, 126.0, 120.6, 113.8, 110.8, 101.8, 99.5, 95.2, 52.0,

47.4, 29.8, 26.1. HRMS (ESI) m/z [M + H⁺] for C₂₀H₁₈ClIN₂O₄

513.0023, found 513.0065.

Methyl 3-Chloro-2-(2-(1-(3,4-dichlorobenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (22). Yield 27 mg (23%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.30 (d, J

= 8.3 Hz, 1H), 7.05 (d, J = 1.9 Hz, 2H), 6.92 (d, J = 1.7 Hz, 1H), 6.81

(dd, J = 8.3, 2.2 Hz, 1H), 6.32 (s, 1H), 4.94 (s, 2H), 3.76 (s, 3H),

3.36−3.29 (m, 2H), 2.95 (t, J = 7.8 Hz, 2H). ¹³C NMR (126 MHz,

CDCl₃, MeOD) δ 173.55, 164.94, 161.93, 141.26, 137.43, 135.20,

133.07, 130.90, 130.47, 125.31 (2C), 118.43, 116.40, 110.96, 107.00

(2C), 56.54, 53.05, 33.98, 28.86. HRMS (ESI) m/z [M + H]⁺for

C₂₀H₁₈Cl₃N₂O₄455.0332, found 455.0354.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(naphthalen-1-ylmethyl)-

1H-imidazol-2-yl)ethyl)benzoate (23). Yield 32 mg (28%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.79 (dd,

Methyl 2-(2-(1-(3-Methoxybenzyl)-1H-imidazol-2-yl)ethyl)-3-

J = 6.8, 2.5 Hz, 1H), 7.71 (dd, J = 8.0, 3.9 Hz, 2H), 7.48−7.40 (m,

2H), 7.28 (d, J = 1.2 Hz, 1H), 6.93 (d, J = 1.4 Hz, 1H), 6.72 (d, J = 1.5

Hz, 1H), 6.71−6.67 (m, 1H), 6.32 (s, 1H), 5.43 (s, 2H), 3.73 (s, 3H),

3.45−3.35 (m, 2H), 2.94−2.84 (m, 2H). ¹³C NMR (126 MHz, CDCl₃,

MeOD) δ 170.58, 161.56, 158.02, 147.88, 141.47, 133.57, 131.62,

130.34, 128.92, 128.68, 126.80 (2C), 126.15, 125.35, 123.93, 121.99,

120.36, 114.62, 106.32, 102.38, 52.29, 47.31, 30.74, 26.17. HRMS

(ESI) m/z [M + H]⁺for C₂₄H₂₁ClN₂O₄437.1268, found 437.1250.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(naphthalen-2-ylmethyl)-

1H-imidazol-2-yl)ethyl)benzoate (24). Yield 28 mg (25%), tan

chloro-4,6-dihydroxybenzoate (30). Yield 32 mg (31%), white

1

amorphous solid. H NMR (400 MHz, CDCl₃) δ 7.06 (s, 1H), 6.88

(s, 1H), 6.82 (d, J = 9.0 Hz, 1H), 6.62 (d, J = 7.7 Hz, 1H), 6.55 (s,

1H), 6.53 (s, 1H), 5.04 (s, 2H), 3.85 (s, 3H), 3.76 (s, 3H), 3.59−3.51

(m, 2H), 2.95−2.89 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.68,

162.88, 160.13, 157.00, 147.45, 141.62, 137.81, 130.13, 127.12, 120.17,

118.69, 114.33, 113.03, 112.49, 106.43, 102.82, 55.26, 52.64, 49.34,

30.90, 26.09. HRMS (ESI) m/z [M + H]⁺for C₂₁H₂₂ClN₂O₅

417.1217, found 417.1221.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(3-methylbenzyl)-1H-imi-

dazol-2-yl)ethyl)benzoate (31). Yield 18 mg (17%), tan amorphous

solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 7.06 (t, J = 7.6 Hz, 1H),

6.95 (d, J = 7.7 Hz, 1H), 6.85 (d, J = 1.5 Hz, 1H), 6.75 (d, J = 1.5 Hz,

1H), 6.72−6.66 (m, 2H), 6.30 (s, 1H), 4.87 (s, 2H), 3.69 (s, 3H),

3.36−3.30 (m, 2H), 2.81 (dd, J = 9.2, 6.9 Hz, 2H), 2.16 (s, 3H). ¹³C

NMR (126 MHz, CDCl₃, MeOD) δ 174.49, 165.52, 161.99, 151.39,

145.16, 142.73, 139.62, 132.79 (2C), 131.28, 129.76, 127.67, 124.32,

1

amorphous solid. H NMR (400 MHz, DMSO-d₆) δ 7.88 (dd, J =

9.1, 4.0 Hz, 2H), 7.83−7.78 (m, 1H), 7.51−7.46 (m, 1H), 7.25 (dd, J

= 8.4, 1.9 Hz, 1H), 7.18 (d, J = 1.3 Hz, 1H), 6.85 (d, J = 1.3 Hz, 1H),

6.42 (s, 1H), 5.29 (s, 2H), 3.57 (s, 3H), 2.92−2.86 (m, 2H), 2.77−

2.71 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 167.86, 155.00,

154.47, 146.42, 137.73, 135.17, 132.79, 132.21, 128.35, 127.60 (2C),

127.54, 126.80, 126.43 (2C), 126.04, 124.94, 124.84, 120.56, 113.74,

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DOI: 10.1021/acs.jmedchem.6b00085

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Journal of Medicinal Chemistry

Article

118.57, 110.31, 106.43, 56.27, 53.48, 34.48, 29.85, 25.07. HRMS (ESI)

m/z [M + H]⁺for C₂₁H₂₂ClN₂O₄401.1268, found 401.1255.

Methyl 2-(2-(1-([1,1′-Biphenyl]-3-ylmethyl)-1H-imidazol-2-yl)-

ethyl)-3-chloro-4,6-dihydroxybenzoate (32). Yield 46 mg (39%),

pale-yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ

7.53−7.47 (m, 3H), 7.41 (dd, J = 9.1, 7.3 Hz, 3H), 7.38−7.33 (m,

1H), 7.23 (s, 1H), 7.03 (d, J = 1.3 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H),

6.89 (d, J = 1.4 Hz, 1H), 6.46 (s, 1H), 5.12 (s, 2H), 3.82 (s, 3H),

3.56−3.46 (m, 2H), 2.97−2.89 (m, 2H). ¹³C NMR δ (100 MHz,

CDCl₃): 170.8, 163.0, 157.9, 147.7, 142.4, 141.5, 140.5, 136.6, 129.8

(3C), 129.1(2C), 128.0, 127.3, 127.3, 125.7, 125.6, 120.5, 114.9, 106.3,

103.2, 52.8, 49.9, 31.1, 26.2. HRMS (ESI) m/z [M − H]⁻for

C₂₆H₂₂ClN₂O₄461.1268, found 461.1259.

129.33, 127.59, 126.69, 124.66, 120.72, 120.13, 114.71, 110.27, 105.90,

102.51, 55.27, 52.50, 44.85, 30.85, 26.04. HRMS (ESI) m/z [M + H]⁺

for C₂₁H₂₂ClN₂O₅417.1217, found 417.1211.

Methyl 3-Chloro-2-(2-(1-(2-ethylbenzyl)-1H-imidazol-2-yl)ethyl)-

4,6-dihydroxybenzoate (39). Yield 33 mg (31%), tan amorphous

solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.43 (d, J = 2.1 Hz, 1H),

7.40 (d, J = 3.0 Hz, 1H), 7.32−7.28 (m, 1H), 7.26 (d, J = 7.7 Hz, 1H),

6.83 (d, J = 7.7 Hz, 1H), 6.79 (s, 1H), 6.55 (s, 1H), 4.98 (s, 2H), 4.02

(s, 3H), 3.68 (t, J = 7.3 Hz, 2H), 3.45 (t, J = 7.1 Hz, 3H), 2.52 (q, J =

7.5 Hz, 3H), 1.19 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃,

MeOD) δ 169.85, 161.95, 158.20, 146.18, 145.33, 142.11, 138.91,

129.92, 129.32, 128.17, 127.04, 120.75, 119.05, 114.84, 106.28, 103.49,

53.17, 30.86, 30.04, 25.20, 24.25, 14.49. HRMS (ESI) m/z [M + H]⁺

for C₂₂H₂₄ClN₂O₄415.1425, found 415.1419. t_R= 4.27 min, 95.3%.

Methyl 3-Chloro-2-(2-(1-(2-ethoxybenzyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (40). Yield 44 mg (39%), tan

Methyl 3-Chloro-2-(2-(1-(2-ﬂuorobenzyl)-1H-imidazol-2-yl)ethyl)-

4,6-dihydroxybenzoate (33). Yield 23 mg (22%), white amorphous

1

solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.27−7.23 (m, 1H),

1

7.06−6.98 (m, 2H), 6.94 (d, J = 1.4 Hz, 1H), 6.81 (d, J = 1.4 Hz, 1H),

6.78 (dd, J = 7.5, 1.7 Hz, 1H), 6.40 (s, 1H), 5.05 (s, 2H), 3.82 (s, 3H),

3.64−3.38 (m, 2H), 3.05−2.68 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 170.80, 162.21, 157.79, 147.67, 141.77, 130.04, 128.42,

127.27, 124.63, 123.62, 123.50, 119.93, 115.60 (d, J = 21.0 Hz),

114.66, 106.02, 102.55, 52.51, 43.46, 30.83, 26.04. HRMS (ESI) m/z

[M + H] for C₂₀H₁₈ClFN₂O₄405.1029, found 405.1017.

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.18 (t, J

= 8.0 Hz, 1H), 6.88 (d, J = 1.9 Hz, 1H), 6.83−6.76 (m, 3H), 6.67 (d, J

= 7.6 Hz, 1H), 6.38 (s, 1H), 4.99 (s, 2H), 3.97 (q, J = 7.7, 6.8 Hz, 2H),

3.80 (s, 3H), 3.45−3.39 (m, 2H), 2.88 (t, J = 8.1 Hz, 2H), 1.32 (t, J =

6.8 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 170.97, 162.15,

157.97, 155.99, 147.73, 141.95, 129.25, 127.68, 126.65, 124.63, 120.46,

120.13, 114.73, 111.01, 105.90, 102.43, 63.51, 52.45, 44.95, 30.86,

26.12, 14.69. HRMS (ESI) m/z [M + H]⁺for C₂₂H₂₄ClN₂O₅

431.1374, found 431.1378. t_R= 2.71 min, 98.3%.

Methyl 2-(2-(1-(2-Chlorobenzyl)-1H-imidazol-2-yl)ethyl)-3-

chloro-4,6-dihydroxybenzoate (34). Yield 23 mg (21%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 7.27 (s,

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-isopropoxybenzyl)-1H-

1H), 7.24−7.16 (m, 2H), 7.01 (d, J = 1.6 Hz, 1H), 6.94 (dd, J = 1.9,

1.0 Hz, 1H), 6.84 (m, 2H), 6.38 (s, 1H), 4.98 (s, 2H), 3.80 (s, 3H),

3.45−3.35 (m, 2H), 2.94−2.85 (m, 2H). ¹³C NMR (126 MHz, CDCl₃,

MeOD) δ 170.58, 162.43, 157.43, 147.63, 141.17, 133.48, 132.58,

129.83, 129.62, 127.72, 127.52, 126.18, 120.16, 114.51, 106.20, 102.85,

52.73, 47.48, 30.89, 25.78. HRMS (ESI) m/z [M + H]⁺for

C₂₀H₁₉Cl₂N₂O₄421.0722, found 421.0714.

imidazol-2-yl)ethyl)benzoate (41). Yield 18 mg (16%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.21 (t, J

= 8.0 Hz, 1H), 6.95 (s, 1H), 6.86−6.78 (m, 3H), 6.72 (d, J = 7.5 Hz,

1H), 6.44 (s, 1H), 5.00 (s, 2H), 4.56 (hept, J = 10.8, 5.3 Hz, 1H), 3.85

(s, 3H), 3.52−3.45 (m, 2H), 2.97−2.87 (m, 2H), 1.27 (d, J = 6.0 Hz,

6H). ¹³C NMR (126 MHz, CDCl₃) δ 170.97, 162.15, 157.97, 156.00,

147.73, 141.95, 129.25, 127.68, 126.65, 124.63, 120.46, 120.13, 114.73,

111.01, 105.90, 102.43, 99.99, 63.51, 52.45, 44.95, 30.86, 26.12, 14.69.

HRMS (ESI) m/z [M + H]⁺for C₂₃H₂₆ClN₂O₅445.1530, found

445.1521. t_R= 2.70 min, 97.7%.

Methyl 2-(2-(1-(2-Bromobenzyl)-1H-imidazol-2-yl)ethyl)-3-

chloro-4,6-dihydroxybenzoate (35). Yield 26 mg (22%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃) δ 7.54 (dd, J = 8.0,

1.3 Hz, 1H), 7.24−7.20 (m, 1H), 7.15 (td, J = 7.7, 1.7 Hz, 1H), 7.10

(d, J = 1.6 Hz, 1H), 6.78 (d, J = 1.6 Hz, 1H), 6.67−6.57 (m, 1H), 6.48

(s, 1H), 5.06 (s, 2H), 3.85 (s, 3H), 3.48−3.33 (m, 2H), 3.06−2.89 (m,

2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.47, 162.64, 157.77, 147.55,

140.60, 133.23 (2C), 130.08, 128.23 (2C), 122.57, 120.27 (2C),

114.71, 105.94, 103.08, 52.85, 50.15, 30.78, 25.58. HRMS (ESI) m/z

[M + H] for C₂₀H₁₉BrClN₂O₄465.0217, found 465.0231.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-methylbenzyl)-1H-imi-

dazol-2-yl)ethyl)benzoate (36). Yield 32 mg (31%), tan amorphous

solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 7.25 (s, 1H), 7.08−7.02

(m, 2H), 6.88−6.83 (m, 1H), 6.66−6.58 (m, 1H), 6.47 (t, J = 6.7 Hz,

1H), 6.29 (s, 1H), 4.87 (s, 2H), 3.72 (s, 3H), 3.35−3.25 (m, 2H), 2.81

(dd, J = 10.9, 5.1 Hz, 2H), 2.10 (s, 3H). ¹³C NMR (126 MHz, CDCl₃,

MeOD) δ 174.43, 165.45, 161.97, 151.53, 145.12, 139.32, 137.71,

134.49, 132.09, 130.45, 130.31, 129.87, 124.09, 118.52, 110.38, 106.41,

56.28, 51.65, 34.50, 29.86, 22.59. HRMS (ESI) m/z [M + H]⁺for

C₂₁H₂₂ClN₂O₄401.1268, found 401.1261.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-propoxybenzyl)-1H-

imidazol-2-yl)ethyl)benzoate (42). Yield 34 mg (29%), tan

amorphous solid. ¹H NMR (500 MHz, CDCl₃) δ 7.29−7.26 (m,

1H), 7.02 (d, J = 1.4 Hz, 1H), 6.90−6.85 (m, 3H), 6.76−6.73 (m,

1H), 6.49 (s, 1H), 5.10 (s, 2H), 3.95 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H),

3.56−3.50 (m, 2H), 3.03−2.96 (m, 2H), 1.80 (qt, J = 7.4, 6.5 Hz, 2H),

1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.91,

162.91, 157.83, 156.11, 147.58, 141.58, 129.36, 127.76, 126.49, 124.55,

120.56, 120.15, 114.73, 111.04, 105.90, 102.72, 69.53, 52.55, 45.00,

30.85, 26.04, 22.57, 10.61. HRMS (ESI) m/z [M + H]⁺for

C₂₃H₂₆ClN₂O₅445.1530, found 445.1539. t_R= 2.73 min, 98.0%.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-(triﬂuoromethyl)-

benzyl)-1H-imidazol-2-yl)ethyl)benzoate (43). Yield 25 mg (21%),

1

white amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.65

(d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H),

7.02 (s, 1H), 6.79 (s, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.39 (s, 1H), 5.25

(s, 2H), 3.85 (s, 3H), 3.50−3.41 (m, 2H), 2.93−2.79 (m, 2H). ¹³C

NMR (126 MHz, CDCl₃, MeOD) δ 170.72, 162.08, 157.95, 148.07,

141.52, 134.95, 132.62, 127.97, 127.61, 126.91, 126.26 (q, J = 5.6 Hz),

125.20, 123.03, 120.21, 114.70, 105.88, 102.50, 52.46, 45.88, 30.87,

25.83. HRMS (ESI) m/z [M + H]⁺for C₂₁H₁₉ClF₃N₂O₄455.0985,

found 455.0979.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-methoxy-4-methylben-

zyl)-1H-imidazol-2-yl)ethyl)benzoate (44). Yield 45 mg (40%), tan

amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.28 (d, J =

2.1 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.76

(d, J = 7.3 Hz, 1H), 6.69 (s, 1H), 6.56 (s, 1H), 4.81 (s, 2H), 3.99 (s,

3H), 3.74 (s, 3H), 3.63 (t, J = 7.4 Hz, 2H), 3.44 (t, J = 7.4 Hz, 2H),

2.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.87, 161.98, 157.11,

151.39, 145.68, 142.01, 140.11, 129.96, 129.53, 121.73, 121.63, 120.51,

118.55, 112.14, 111.93, 55.44, 54.21, 46.62, 30.48, 29.73, 23.96, 21.75.

HRMS (ESI) m/z [M + H]⁺for C₂₂H₂₄ClN₂O₅431.1374, found

431.1370.

Methyl 2-(2-(1-(2-Aminobenzyl)-1H-imidazol-2-yl)ethyl)-3-

chloro-4,6-dihydroxybenzoate (37). Yield 20 mg (19%), white

1

amorphous solid. H NMR (500 MHz, MeOD) δ 7.07 (td, J = 7.8,

1.6 Hz, 1H), 6.98 (dd, J = 7.8, 1.4 Hz, 2H), 6.78 (dd, J = 8.0, 1.2 Hz,

1H), 6.65 (td, J = 7.5, 1.2 Hz, 1H), 6.52 (dd, J = 7.6, 1.5 Hz, 1H), 6.39

(s, 1H), 5.03 (s, 2H), 3.79 (s, 3H), 3.37−3.33 (m, 2H), 3.01−2.96 (m,

2H). ¹³C NMR (126 MHz, MeOD) δ 169.90, 159.44, 157.47, 147.55,

145.00, 140.02, 128.56, 127.00, 125.07, 120.41, 120.32, 117.92, 116.00,

113.55, 108.77, 101.92, 51.46, 45.78, 29.89, 25.71. HRMS (ESI) m/z

[M + H] for C₂₀H₂₀ClN₃O₄402.1221, found 402.1230.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(2-methoxybenzyl)-1H-

imidazol-2-yl)ethyl)benzoate (38). Yield 46 mg (42%), tan

amorphous solid. ¹H NMR (500 MHz, CDCl₃) δ 7.26−7.23 (m,

1H), 6.97 (d, J = 1.4 Hz, 1H), 6.89−6.84 (m, 2H), 6.83 (d, J = 1.4 Hz,

1H), 6.70 (dd, J = 7.8, 1.7 Hz, 1H), 6.44 (s, 1H), 5.03 (s, 2H), 3.84 (s,

3H), 3.81 (s, 3H), 3.49−3.44 (m, 2H), 2.94−2.90 (m, 2H). ¹³C NMR

(126 MHz, CDCl₃) δ 170.96, 162.35, 157.84, 156.56, 147.74, 141.91,

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DOI: 10.1021/acs.jmedchem.6b00085

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Journal of Medicinal Chemistry

Article

Methyl 3-Chloro-2-(2-(1-(furan-3-ylmethyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (45). Yield 40 mg (41%), oﬀ-white

amorphous solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 7.30 (d, J =

1.6 Hz, 1H), 7.20 (s, 1H), 6.84 (dd, J = 2.7, 1.4 Hz, 1H), 6.78 (d, J =

1.4 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 6.15 (d, J = 1.9 Hz, 1H), 4.80 (s,

2H), 3.78 (d, J = 1.9 Hz, 3H), 3.39 (tt, J = 8.9, 2.5 Hz, 2H), 2.95−2.80

(m, 2H). ¹³C NMR (125 MHz, CDCl₃, MeOD): δ 170.8, 161.8, 158.1,

147.3, 144.1, 141.6, 141.6, 140.1, 126.7, 121.0, 119.6, 114.7, 109.4,

106.2, 102.5, 52.4, 41.0, 30.8, 26.1. HRMS (ESI) m/z [M + H]⁺for

C₁₈H₁₈ClN₂O₅377.0904, found 377.0905.

Methyl 3-Chloro-2-(2-(1-(furan-2-ylmethyl)-1H-imidazol-2-yl)-

ethyl)-4,6-dihydroxybenzoate (46). Yield 44 mg (45%), oﬀ-white

amorphous solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 7.31 (dt, J =

2.4, 1.2 Hz, 1H), 6.87 (dd, J = 2.4, 1.4 Hz, 1H), 6.83 (d, J = 1.5 Hz,

1H), 6.40 (d, J = 1.8 Hz, 1H), 6.26 (dt, J = 3.5, 1.7 Hz, 1H), 6.18 (d, J

= 3.3 Hz, 1H), 4.94 (d, J = 1.6 Hz, 2H), 3.82 (d, J = 1.9 Hz, 3H), 3.46

(ddd, J = 10.2, 6.1, 2.0 Hz, 2H), 2.95 (ddd, J = 9.8, 5.9, 1.5 Hz, 2H).

¹³C NMR (125 MHz, CDCl₃, MeOD) δ 170.9, 162.0, 158.2, 149.1,

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-((5-methylthiophen-2-yl)-

methyl)-1H-imidazol-2-yl)ethyl)benzoate (52). Yield 23 mg (22%),

1

oﬀ-white amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ

7.34 (d, J = 1.8 Hz, 1H), 7.02−6.96 (m, 1H), 6.79 (d, J = 4.1 Hz, 1H),

6.60 (d, J = 2.7 Hz, 1H), 6.48 (s, 1H), 5.03 (s, 2H), 3.95 (s, 3H), 3.58

(t, J = 7.2 Hz, 2H), 3.39 (t, J = 7.2 Hz, 2H), 2.41 (s, 3H). ¹³C NMR

(126 MHz, CDCl₃, MeOD) δ 169.83, 161.87, 158.19, 145.63, 138.77,

131.10, 129.14 (2C), 125.60 (2C), 120.60, 119.05, 106.34, 103.54,

53.15, 46.03, 30.05, 24.21, 15.28. HRMS (ESI) m/z [M + H]⁺for

C₁₉H₂₀ClN₂O₄S 407.0832, found 407.0839.

Methyl 3-Chloro-2-(2-(1-((5-chlorothiophen-2-yl)methyl)-1H-imi-

dazol-2-yl)ethyl)-4,6-dihydroxybenzoate (53). Yield 20 mg (18%),

yellow amorphous solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 6.95

(s, 1H), 6.87 (s, 1H), 6.68 (d, J = 3.5 Hz, 1H), 6.63 (d, J = 3.5 Hz,

1H), 6.37 (s, 1H), 5.03 (s, 2H), 3.81 (s, 3H), 3.47−3.37 (m, 2H),

3.02−2.92 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 174.29,

165.57, 162.00, 150.88, 144.73, 140.23, 134.89, 130.21, 130.09, 129.35,

123.74, 118.59, 110.29, 106.65, 56.47, 48.84, 34.48, 29.63. HRMS

(ESI) m/z [M + H]⁺for C₁₈H₁₇Cl₂N₂O₄S 427.0286, found 427.0292.

Methyl 3-Chloro-2-(2-(1-((4-chlorothiophen-2-yl)methyl)-1H-imi-

dazol-2-yl)ethyl)-4,6-dihydroxybenzoate (54). Yield 23 mg (21%),

tan amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 6.97 (s,

1H), 6.90 (s, 1H), 6.83 (s, 1H), 6.67 (s, 1H), 6.38 (s, 1H), 5.07 (s,

2H), 3.81 (s, 3H), 3.45−3.39 (m, 2H), 2.88 (t, J = 8.2 Hz, 2H). ¹³C

NMR (126 MHz, CDCl₃, MeOD) δ 170.61, 161.82, 158.12, 147.29,

141.48, 139.55, 127.34, 126.18, 125.05, 120.14, 119.50, 114.71, 106.03,

102.51, 52.44, 44.42, 30.76, 26.05. HRMS (ESI) m/z [M + H]⁺for

C₁₈H₁₇Cl₂N₂O₄S 427.0286, found 427.0290.

147.4, 143.2, 141.8, 126.9, 119.7, 114.8, 110.6, 108.8, 106.2, 102.6,

52.6, 42.6, 30.8, 26.1. HRMS (ESI) m/z [M + H]⁺for C₁₈H₁₈ClN₂O₅

377.0904, found 377.0911.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-((5-methylfuran-2-yl)-

methyl)-1H-imidazol-2-yl)ethyl)benzoate (47). Yield 46 mg (46%),

1

oﬀ white amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ

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, 1.2 Hz, 1H), 4.88 (s, 2H), 3.84

(s, 3H), 3.49−3.42 (m, 2H), 3.08−2.93 (m, 2H), 2.18 (d, J = 1.0 Hz,

3H). ¹³C NMR (125 MHz, CDCl₃, MeOD) δ 171.0, 162.2, 158.3,

153.1, 147.3, 147.1, 141.9, 126.7, 119.7, 114.9, 109.7, 106.5, 106.1,

102.6, 52.6, 42.7, 30.9, 26.2, 13.5. HRMS (ESI) m/z [M + H]⁺for

C₁₉H₂₀ClN₂O₅391.1061, found 391.1066. t_R= 3.05 min, 95.1%.

Methyl 3-Chloro-2-(2-(1-((5-chlorofuran-2-yl)methyl)-1H-imida-

zol-2-yl)ethyl)-4,6-dihydroxybenzoate (48). Yield 50 mg (47%),

white amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ

6.91 (s, 2H), 6.31 (s, 1H), 6.23 (d, J = 3.4 Hz, 1H), 5.98 (d, J = 3.3

Hz, 1H), 4.87 (s, 2H), 3.73 (s, 3H), 3.37−3.27 (m, 2H), 2.99 (t, J =

7.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 173.88, 165.00,

161.93, 151.08, 150.68, 143.82, 141.62, 124.59, 118.45, 116.26, 111.28

(2C), 106.80 (2C), 56.40, 46.86, 34.09, 29.14. HRMS (ESI) m/z [M +

H]⁺for C₁₈H₁₇Cl₂N₂O₅411.0515, found 411.0527. t_R= 4.15 min,

95.5%.

Methyl 3-Chloro-2-(2-(1-((3,5-dimethylfuran-2-yl)methyl)-1H-

imidazol-2-yl)ethyl)-4,6-dihydroxybenzoate (49). Yield 36 mg

(25%), oﬀ-white amorphous solid. ¹H NMR (400 MHz, CDCl₃,

MeOD) δ 7.28 (d, J = 2.0 Hz, 1H), 6.92 (s, 1H), 6.44 (s, 1H), 5.77 (s,

1H), 4.80 (s, 2H), 3.89 (s, 3H), 3.59−3.48 (m, 2H), 3.37 (t, J = 7.1

Hz, 2H), 2.11 (s, 3H), 1.92 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

169.87, 161.98, 157.11, 151.39, 145.68, 140.11, 129.96, 121.63, 120.51,

118.85, 118.55, 112.14, 111.93, 55.44, 54.21, 46.62, 30.48, 29.73,

23.96, 21.75. HRMS (ESI) m/z [M + H]⁺for C₂₀H₂₂ClN₂O₅

405.1217, found 405.1210. t_R= 3.24 min, 95.5%.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(thiophen-2-ylmethyl)-

1H-imidazol-2-yl)ethyl)benzoate (50). Yield 37 mg (37%), yellow

amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.23 (dd, J =

5.1, 1.3 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 6.92 (dd, J = 5.1, 3.5 Hz,

1H), 6.87 (dd, J = 3.5, 1.9 Hz, 2H), 6.43 (s, 1H), 5.17 (d, J = 0.9 Hz,

2H), 3.84 (s, 3H), 3.56−3.44 (m, 2H), 3.01−2.92 (m, 2H). ¹³C NMR

(125 MHz, CDCl₃, MeOD) δ 170.9, 162.1, 158.2, 147.3, 141.7, 138.5,

127.2, 126.9, 126.4, 126.2, 119.7, 114.9, 106.2, 102.7, 52.6, 44.7, 30.9,

26.2. HRMS (ESI) m/z [M + H]⁺for C₁₈H₁₈ClN₂O₄S 393.0676,

found 393.0674.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(thiophen-3-ylmethyl)-

1H-imidazol-2-yl)ethyl)benzoate (51). Yield 28 mg (27%), yellow

amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.25 (d, J =

4.8 Hz, 1H), 6.98−6.92 (m, 2H), 6.83 (d, J = 1.5 Hz, 1H), 6.81−6.78

(m, 1H), 6.37 (s, 1H), 4.97 (s, 2H), 3.79 (s, 3H), 3.43−3.37 (m, 2H),

2.95−2.88 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 174.48,

165.67, 162.02, 151.06, 145.03, 140.31, 131.26, 130.04, 129.39, 126.69,

124.00, 118.61, 110.15, 106.53, 56.40, 49.27, 34.49, 29.68. HRMS

(ESI) m/z [M + H]⁺for C₁₈H₁₈ClN₂O₄S 393.0676, found 393.0693.

Methyl 3-Chloro-2-(2-(1-((3-chlorothiophen-2-yl)methyl)-1H-imi-

dazol-2-yl)ethyl)-4,6-dihydroxybenzoate (55). Yield 17 mg (15%),

1

tan amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.21

(dd, J = 5.9, 2.3 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.86 (dt, J = 5.2, 1.8

Hz, 2H), 6.41 (s, 1H), 5.09 (s, 2H), 3.84 (s, 3H), 3.54−3.45 (m, 2H),

2.96 (dd, J = 11.9, 5.4 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD)

δ 170.79, 162.08, 158.04, 147.22, 141.70, 131.26, 127.92, 127.32,

125.17, 124.20, 119.32, 114.80, 105.96, 102.55, 52.52, 42.02, 30.79,

26.15. HRMS (ESI) m/z [M + H]⁺for C₁₈H₁₆Cl₂N₂O₄S 427.0286,

found 427.0278.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(isoxazol-5-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (56). Yield 15 mg (15%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 8.17 (s,

1H), 6.96 (s, 1H), 6.89 (s, 1H), 6.42 (s, 1H), 6.04 (s, 1H), 5.17 (s,

2H), 3.88 (s, 3H), 3.50−3.41 (m, 2H), 2.97−2.89 (m, 2H). ¹³C NMR

(126 MHz, CDCl₃, MeOD) δ 170.55, 166.32, 157.95, 150.41, 147.60,

141.46, 127.82, 119.70, 114.64, 106.11, 102.64, 102.47, 102.14, 52.57,

41.22, 30.85, 25.99. HRMS (ESI) [M + H]⁺for C₁₇H₁₇ClN₃O₅

378.0857, found 378.0866.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(thiazol-2-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (57). Yield 41 mg (40%), tan

1

amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ 7.72 (d, J

= 3.2 Hz, 1H), 7.30 (d, J = 3.3 Hz, 1H), 6.99 (s, 1H), 6.95 (s, 1H),

6.43 (s, 1H), 5.35 (s, 2H), 3.87 (s, 3H), 3.51−3.46 (m, 2H), 2.98−

2.91 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 170.72, 165.84,

162.13, 157.91, 147.63, 143.06, 141.59, 127.84, 120.27, 119.78, 114.71,

106.01, 102.63, 52.57, 46.84, 30.83, 26.06. HRMS (ESI) m/z [M +

H]⁺for C₁₇H₁₇ClN₃O₄S 394.0628, found 394.0630.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-((3-vinylthiophen-2-yl)-

methyl)-1H-imidazol-2-yl)ethyl)benzoate (58). Yield 35 mg (32%),

tan amorphous solid. ¹H NMR (400 MHz, CDCl₃, MeOD) δ 7.19 (d,

J = 5.3 Hz, 1H), 7.15 (d, J = 5.3 Hz, 1H), 7.01 (s, 1H), 6.77 (s, 1H),

6.58 (dd, J = 17.3, 10.9 Hz, 1H), 6.45 (s, 1H), 5.56 (d, J = 17.3 Hz,

1H), 5.31 (d, J = 11.0 Hz, 1H), 5.11 (s, 2H), 3.87 (s, 3H), 3.58−3.49

(m, 2H), 3.07 (t, J = 7.8 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ

170.94, 163.17, 156.14, 142.75, 140.72, 139.77, 138.46, 130.54, 128.69,

128.46, 126.69, 126.54, 126.04, 113.85, 106.68, 102.61, 52.59, 38.44,

33.98, 32.45. HRMS (ESI) m/z [M + H]⁺for C₂₀H₂₀ClN₂O₄S

419.0832, found 419.0844.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-((3-vinylfuran-2-yl)-

methyl)-1H-imidazol-2-yl)ethyl)benzoate (59). Yield 35 mg (33%),

1

oﬀ-white amorphous solid. H NMR (400 MHz, CDCl₃, MeOD) δ

7.30 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 6.51 (d, J = 2.0 Hz,

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1H), 6.49 (s, 1H), 6.48−6.41 (m, 1H), 5.50 (d, J = 17.3 Hz, 1H), 5.26

(d, J = 11.0 Hz, 1H), 4.94 (s, 2H), 3.91 (s, 3H), 3.59 (t, J = 7.6 Hz,

2H), 3.22 (t, J = 7.7 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.35,

162.14, 157.96, 146.62, 143.64 (2C), 124.53 (2C), 123.65, 119.92

(2C), 116.57, 114.78, 108.33 (2C), 108.24, 106.19, 103.08, 52.84,

41.07, 30.43, 25.17. HRMS (ESI) m/z [M + H]⁺for C₂₀H₂₀ClN₂O₅

403.1061, found 403.1069.

rt. The reaction was quenched with the addition of H₂O (20 mL) and

EtOAc (20 mL). The organic layer was washed with H₂O (2 × 20

mL), separated, dried (Na₂SO₄), and concentrated. The residue was

puriﬁed via ﬂash chromatography (1:10 EtOAc:hexanes to 1:3

EtOAc:hexanes) to provide the desired products as amorphous solids:

5-Chlorofuran-2-carbaldehyde Oxime (66a). Yield 225 mg (75%)

1

as a white amorphous solid, 1:1 mixture of isomers. H NMR (400

MHz, CDCl₃) δ 7.91 (s, 1H), 7.46 (s, 1H), 7.35 (d, J = 3.5 Hz, 1H),

6.61 (d, J = 3.4 Hz, 1H), 6.36 (d, J = 3.5 Hz, 1H), 6.26 (d, J = 3.4 Hz,

1H). ¹³C NMR (126 MHz, CDCl₃) δ 146.42, 144.40, 139.54, 139.11,

135.93, 121.19, 114.68, 109.78, 109.37, 108.45. HRMS (ESI) m/z [M

+ H]⁺for C₅H₅ClNO₂146.0009, found 146.0003.

Methyl 3-Chloro-2-(2-(1-((3-ethylthiophen-2-yl)methyl)-1H-imi-

dazol-2-yl)ethyl)-4,6-dihydroxybenzoate (60). Yield 43 mg (39%),

tan amorphous solid. ¹H NMR (500 MHz, CDCl₃, MeOD) δ 7.16 (d,

J = 5.2 Hz, 1H), 6.91 (d, J = 1.4 Hz, 1H), 6.86 (d, J = 5.2 Hz, 1H),

6.73 (d, J = 1.5 Hz, 1H), 6.45 (s, 1H), 5.04 (s, 2H), 3.86 (s, 3H),

3.56−3.50 (m, 2H), 3.00−2.95 (m, 2H), 2.53 (q, J = 7.6 Hz, 2H), 1.12

(t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.80, 162.20,

157.88, 147.06, 142.21, 141.84, 130.82, 128.69, 126.96, 124.60, 119.11,

114.70, 106.03, 102.57, 52.57, 42.34, 30.78, 26.25, 21.49, 14.92. HRMS

(ESI) m/z [M + H]⁺for C₂₀H₂₂ClN₂O₄S 421.0989, found 421.0995.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(pyridin-2-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (61). Yield 15 mg (15%), tan

Thiazole-2-carbaldehyde Oxime (66b). Yield 510 mg (91%), white

1

amorphous solid. H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 7.86

(d, J = 3.2 Hz, 1H), 7.33 (dd, J = 3.3, 0.9 Hz, 1H). ¹³C NMR (126

MHz, CDCl₃) δ 144.69, 143.24, 142.28, 140.70, 123.45, 119.96.

HRMS (ESI) m/z [M + H]⁺for C₄H₅N₂OS 129.0123, found

129.0124.

5-Methylthiophene-2-carbaldehyde Oxime (66c). Yield 491 mg

1

amorphous solid. H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 5.0

(76%), white amorphous solid. H NMR (500 MHz, CDCl₃) δ 7.62

Hz, 1H), 7.68 (td, J = 7.7, 1.9 Hz, 1H), 7.13 (d, J = 1.5 Hz, 1H), 6.99

(d, J = 1.6 Hz, 1H), 6.89 (d, J = 7.9 Hz, 1H), 6.51 (s, 1H), 5.23 (s,

2H), 3.89 (s, 3H), 3.52 (t, J = 7.9 Hz, 2H), 3.05 (t, J = 8.0 Hz, 2H).

¹³C NMR (126 MHz, CDCl₃) δ 170.50, 162.80, 157.65, 149.98 (2C),

(s, 1H), 7.21 (d, J = 3.7 Hz, 1H), 6.77 (dd, J = 3.6, 1.1 Hz, 1H), 2.53

(d, J = 1.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 146.84, 141.47,

132.33, 128.93, 124.83, 15.32. HRMS (ESI) m/z [M + H]⁺for

C₆H₈NOS 142.0327, found 142.0330.

3-Vinylthiophene-2-carbaldehyde oxime (66d). Yield 407 mg

(95%), white amorphous solid, 2:1 mixture of isomers. ¹H NMR (400

MHz, CDCl₃) δ 8.45 (s, 1H), 8.36 (s, 2H), 7.92 (s, 2H), 7.87 (s, 1H),

7.30 (d, J = 5.3 Hz, 2H), 7.25 (d, J = 5.4 Hz, 2H), 7.20 (d, J = 5.3 Hz,

1H), 7.01 (d, J = 5.4 Hz, 2H), 6.84 (dd, J = 17.4, 10.9 Hz, 1H), 5.65

(d, J = 17.3 Hz, 1H), 5.38 (d, J = 11.0 Hz, 1H). ¹³C NMR (126 MHz,

CDCl₃) δ 144.35, 143.69, 140.75, 130.66, 130.27, 130.16, 128.09,

127.53, 127.10, 125.61, 116.88, 113.85. HRMS (ESI) m/z [M + H]⁺

for C₇H₈NOS 154.0327, found 154.0331.

147.44, 137.50 (3C), 123.42, 121.11, 120.70, 114.63, 106.01, 103.24,

52.94, 51.53, 30.62, 25.40. HRMS (ESI) m/z [M−H]⁻for

C₁₉H₁₈ClN₃O₄386.0908, found 386.0899.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(pyridin-3-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (62). Yield 25 mg (25%), yellow

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 8.44 (d, J

= 4.3 Hz, 1H), 8.30 (s, 1H), 7.32 (d, J = 6.0 Hz, 1H), 7.31−7.24 (m,

1H), 6.98 (s, 1H), 6.83 (s, 1H), 6.38 (s, 1H), 5.06 (s, 2H), 3.83 (s,

3H), 3.48−3.37 (m, 2H), 2.90−2.84 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃, MeOD) δ 174.42, 165.67, 162.04, 152.92, 152.83, 151.54,

145.18, 138.88, 136.30, 131.13, 128.14, 123.84, 118.57, 110.07, 106.52,

56.40, 50.83, 34.70, 29.95. HRMS (ESI) m/z [M − H]⁻

C₁₉H₁₈ClN₃O₄386.0908, found 386.0903.

3-Vinylfuran-2-carboxaldehyde Oxime (66e). Yield 105 mg

1

(48%), white amorphous solid. H NMR (400 MHz, CDCl₃) δ 8.14

(s, 1H), 7.40 (d, J = 1.6 Hz, 1H), 6.76 (dd, J = 17.4, 10.9 Hz, 1H), 6.62

(d, J = 1.9 Hz, 1H), 5.59 (dd, J = 17.5, 1.2 Hz, 1H), 5.32 (dd, J = 10.8,

1.2 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 144.28, 143.04, 139.55,

126.52, 125.37, 116.55, 108.93. HRMS (ESI) m/z [M + H]⁺for

C₇H₈NO₂138.0555, found 138.0549.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(pyridin-4-ylmethyl)-1H-

imidazol-2-yl)ethyl)benzoate (63). Yield 32 mg (30%), yellow

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 8.44−8.29

(m, 2H), 6.95 (d, J = 1.4 Hz, 1H), 6.88−6.82 (m, 2H), 6.81 (d, J = 1.5

Hz, 1H), 6.30 (s, 1H), 5.03 (s, 2H), 3.78 (s, 3H), 3.40−3.28 (m, 2H),

2.78−2.71 (m, 2H). ¹³C NMR (126 MHz, CDCl₃, MeOD) δ 170.39,

161.48, 157.93, 149.56 (2C), 147.76, 146.40, 141.07, 127.39, 121.31

(2C), 120.18, 114.46, 106.24, 102.43, 52.34, 30.70, 25.91, 25.11, 19.88.

HRMS (ESI) m/z [M−H]⁻C₁₉H₁₈ClN₃O₄386.0908, found

386.0912.

5-(Bromomethyl)isoxazole (67). N-Bromosuccinimide (2.14 g, 12

mmol, 1 equiv) and benzoyl peroxide (290 mg, 1.2 mmol, 0.1 equiv)

were added to a stirred solution of 5-methylisoxazole (1 g, 12 mmol)

in CCl₄(50 mL). The reaction was heated to 80 °C for 5 h then

concentrated. The resulting residue was redissolved in EtOAc (30 mL)

and washed with H₂O (3 × 20 mL). The organic layer was separated,

dried (Na₂SO₄), and concentrated. The residue was puriﬁed via ﬂash

chromatography (SiO₂, 1:10 EtOAc:hexanes to 3:10 EtOAc:hexanes)

to provide the title compound. Yield 725 mg (38%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 1.6 Hz, 1H), 6.33 (d, J =

1.7 Hz, 1H), 4.49 (s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 166.90,

150.56, 103.07, 18.34. MS (EI) m/z [M]⁺for C₄H₄BrNO 160.9, found

161.0; [M − Br]⁺for C₄H₄NO 82.0, found 82.0.

Methyl 3-Chloro-4,6-dihydroxy-2-(2-(1-(pyrimidin-5-ylmethyl)-

1H-imidazol-2-yl)ethyl)benzoate (64). Yield 45 mg (45%), white

1

amorphous solid. H NMR (500 MHz, CDCl₃, MeOD) δ 9.09 (s,

1H), 8.46 (s, 2H), 7.05 (s, 1H), 6.92 (s, 1H), 6.40 (s, 1H), 5.12 (s,

2H), 3.86 (s, 3H), 3.52−3.36 (m, 2H), 3.00−2.79 (m, 2H). ¹³C NMR

(126 MHz, CDCl₃, MeOD) δ 174.09, 165.38, 161.94, 159.48, 159.42,

159.38, 151.44, 144.71, 134.07, 130.84, 123.84, 118.47, 110.37, 106.66,

56.46, 48.81, 34.66, 29.80. HRMS (ESI) m/z [M − H]⁻

C₁₈H₁₇ClN₄O₄387.0860, found 387.0861.

3,5-Dimethylfuran-2-carboxylic Acid (68). A solution of 3-

methylfuran-2-carboxylic acid (1 g, 8 mmol, 1 equiv) in THF (3

mL) was added to a stirred solution of LDA (1 M in THF, 16 mmol,

2.1 equiv) at −78 °C and stirred at this temperature for 15 min

followed by the addition of iodomethane (1 mL, 16 mmol, 2 equiv).

The reaction was stirred for an additional 15 min at −78 °C before

removal of the ice bath and slowly warming to room temperature. The

reaction was quenched with the addition of saturated NH₄Cl (50 mL).

The organic layer was separated, and the aqueous layer was extracted

with EtOAc (2 × 40 mL). The organic layers were combined, dried

(Na₂SO₄), and concentrated. The residue was puriﬁed via ﬂash

chromatography (SiO₂, 3:10 EtOAc:hexanes to 3:5 EtOAc:hexanes) to

provide the title compound. Yield 774 mg (69%) as a white

5-Chlorofuran-2-carbaldehyde (65). 5-Nitrofuran-2-carbaldehyde

(730 mg, 5.1 mmol) was stirred in conc HCl (40 mL) at 40 °C for 15

h. The reaction was poured into H₂O (80 mL) and extracted with

EtOAc (3 × 50 mL). The organic layers were combined, dried

(Na₂SO₄), and concentrated. The residue was puriﬁed via ﬂash

chromatography (SiO₂, 1:4 EtOAc:hexanes) to provide the title

1

compound. Yield 266 mg (40%), yellow oil. H NMR (400 MHz,

CDCl₃) δ 9.53 (s, 1H), 7.23 (d, J = 3.6 Hz, 1H), 6.42 (d, J = 3.6 Hz,

1H). ¹³C NMR (126 MHz, CDCl₃) δ 176.37, 152.03, 144.15, 122.67,

109.77. HRMS (ESI) m/z [M + H]⁺for C₅H₄ClO₂130.9900, found

130.9900.

General Procedure for Oxime Formation. NH₂OH·HCl (3

equiv) and NaOAc (3 equiv) were added to a stirred solution of

aromatic aldehydes (1 equiv) in MeOH (0.2 M) and stirred for 8 h at

1

amorphous solid. H NMR (400 MHz, CDCl₃) δ 12.35 (br s, 1H),

6.00 (s, 1H), 2.31 (s, 3H), 2.30 (s, 3H). ¹³C NMR (126 MHz, CDCl₃)

δ 164.38, 157.04, 138.03, 135.34, 112.39, 13.97, 11.83. HRMS (ESI)

m/z [M + H]⁺for C₇H₉O₃141.0552, found 141.0551.

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(3,5-Dimethylfuran-2-yl)methanol (69a). Yield 575 mg (82%),

soaked for ∼30 min in a solution of mother liquor containing 10 mM

compound 48 (diluted from a 100 mM stock prepared in DMSO).

After soaking, 100% glycerol was added to the drop containing the

crystals (to a total concentration of 25%) and the crystals were

cryocooled in liquid nitrogen. Diﬀraction data were collected at the

Advanced Photon Source, Argonne National Laboratories Beamline

Southeast Region Collaborateve Access Team (SER-CAT) 22-BM.

Data were processed using XDS/XSCALE.⁴⁹The initial model was

obtained by molecular replacement using the polypeptide chain of the

Grp94-RDA structure (PDB 2GFD) the search model in Phaser.⁵⁰

The model was iteratively built and reﬁned suing Coot and Phenix

reﬁne.⁵¹PEG 400 and glycerol ligand models were prepared by

inputting the corresponding SMILES string, and additional restraints

were generated with eLBOW in Phenix reﬁne.⁵⁰The model of 48 was

prepared using the PRODRG2 server, with additional restraints

generated using eLBOW.⁵²The structure has been deposited in the

Protein Data Bank with the PDB accession code 5IN9.

1

colorless oil. H NMR (400 MHz, CDCl₃) δ 5.80 (s, 1H), 4.52 (s,

2H), 2.24 (s, 3H), 1.99 (s, 3H), 1.62 (br s, 1H). ¹³C NMR (126 MHz,

CDCl₃) δ 145.18, 124.22, 115.31, 108.96, 61.40, 13.55, 9.68. MS (EI)

m/z [M + H]⁺for C₇H₁₁O₂127.1, found 127.1.

(3-Chlorothiophen-2-yl)methanol (69b). Yield 240 mg (36%),

1

yellow oil. H NMR (400 MHz, CDCl₃) δ 7.25 (dd, J = 5.4, 1.1 Hz,

1H), 6.90 (dd, J = 5.4, 1.1 Hz, 1H), 4.81 (dd, J = 6.1, 1.3 Hz, 2H), 2.04

(br s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 135.98, 127.80, 124.60,

123.00, 57.60. MS (EI) m/z [M]⁺for C₅H₅ClOS 148.0, found 148.0.

(4-Chlorothiophen-2-yl)methanol (69c). Yield 165 mg (25%),

yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.05 (s, 1H), 6.88 (s, 1H),

4.77 (d, J = 5.8 Hz, 2H), 1.92 (br s, 1H). ¹³C NMR (126 MHz,

CDCl₃) δ 144.73, 125.54, 124.79, 119.88, 60.09. MS (EI) m/z [M]⁺

for C₅H₅ClOS 148.0, found 148.0; [M + H]⁺for C₅H₆ClOS 149.0,

found 149.0.

Fluorescence Polarization. Assay was performed in 96-well

format in black, ﬂat bottom plates (Santa Cruz Biotechnology) with a

ﬁnal volume of 100 μL. 25 μL of assay buﬀer (20 mM HEPES, pH 7.3,

50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄, 2 mM DTT, 0.1 mg/

mL BGG, and 0.01% NP-40) containing 6 nM FITC-GDA

(ﬂuorescent tracer, stock in DMSO and diluted in assay buﬀer) and

50 μL of assay buﬀer containing 10 nM of either Grp94 or Hsp90α

were added to each well. Compounds were tested in triplicate wells

(1% DMSO ﬁnal concentration). For each plate, wells containing

buﬀer only (background), tracer in buﬀer only (low polarization

control), and protein and tracer in buﬀer with 1% DMSO (high

polarization control) were included. Plates were incubated at 4 °C with

rocking for 24 h. Polarization values (in mP units) was measured at 37

°C with an excitation ﬁlter at 485 nm and an emission ﬁlter at 528 nm.

Polarization values were correlated to % tracer bound and compound

concentrations. The concentration at which the tracer was 50%

displaced by the inhibitor was determined using Graphpad Prism.

X-ray Crystallography. The gene for the truncated N-terminal

domain of cGrp94, NΔ41 (amino acid residues 69−337, with residues

287−327 substituted with GGGG, as in Dollins et al.⁴⁷) was

synthesized and codon-optimized by DNA2.0 (Menlo Park, CA).

The construct was designed with an N-terminal maltose binding

protein (MBP) fusion, which is cleavable by tobacco etch virus (TEV)

protease. Fidelity of the ﬁnalized MBR-TEV-NΔ41 plasmid was

conﬁrmed by Euroﬁns MWG Operon. The plasmid for MBP-TEV-

NΔ41 was transformed in Escherichia coli BL21(DE3). Cells were

grown in Luria−Bertani broth supplemented with 100 mg/L ampicillin

(Amresco) at 37 °C with shaking at 225 rpm. Once cells reached an

OD₆₀₀= 0.6−0.8, they were induced with 0.5 mM isopropyl-β-D-1-

thiogalactopyranoside (Calbiochem) and were harvested by centrifu-

gation after an additional 4 h of growth with shaking. Cells were

solubilized in phosphate buﬀer (PBS, 10 mM Na₂HPO₄, 10 mM

KH₂PO₄, 0.2 M NaCl, pH 7.2) containing protease inhibitor cocktail

(Complete EDTA-free tablets, Roche), lysed with passage through a

French Press, and the resulting lysate was clariﬁed by ultra-

centrifugation. The MBP-TEV-NΔ41 fusion protein was puriﬁed

using a 20 mL column packed with amylose resin (New England

BioLabs) equilibrated with PBS. Protein was eluted with a buﬀer

containing PBS supplemented with 1 mM EDTA and 10 mM maltose.

MBP-TEV-NΔ41 was further fractionated with a prep grade Superdex

75 column (GE Healthcare) equilibrated with PBS. MBP-TEV-NΔ41

was cleaved by TEV protease (prepared in house using pRK793

plasmid⁴⁸), and the protease was removed by passing the mixture over

a 5 mL HisTrap HP column (GE Healthcare) equilibrated with 50

mM Tris buﬀer with 0.5 M NaCl at pH 8.0. The mixture of MBP and

NΔ41 were separated by amylose aﬃnity puriﬁcation, followed by a

Superdex 75 polishing step, as above. Any fractions of NΔ41

containing trace amounts of MBP were subjected to a ﬁnal round of

amylose aﬃnity puriﬁcation. The NΔ41 protein purity was assessed by

SDS-PAGE concentrated to 30 mg/mL in a 100 mM Tris buﬀer at pH

7.5, as quantiﬁed by the Bradford assay (reagents from Amresco) using

BSA as a standard. Crystals of NΔ41 were grown by the handing drop

method by equilibration against a reservoir solution containing 35%

PEG400, 100 mM Tris at pH 7.5, and 120 mM MgCl₂. Crystals were

Wound-Healing Scratch Assay. MDA-MB-231 cells (ATCC)

were grown to conﬂuence and seeded at 200,000 cells/well/mL in a

12-well plate and grown to conﬂuence. Each well was scratched twice

with a 20−200 μL pipet tip. Compounds were then added (0.25%

DMSO ﬁnal concentration) and 0 h pictures were taken with an

Olympus IX-71 microscope (10× objective) and the plates were

returned to the incubator until 24 h pictures were taken. Images were

processed and % wound closure was determined using ImageJ. All

experiments were performed in quadruplicate.

Anti-Proliferative Assay. MDA-MB-231 cells were grown to

conﬂuence and seeded at 2000 cells/well/0.1 mL in a 96-well plate

and placed back in the incubator for 24 h. Compounds or vehicle were

administered at the desired concentrations (1% DMSO ﬁnal

concentration) and incubated for 72 h. The % viable cells was

determined using the MTS/PMS cell proliferation kit (Promega) per

the manufacturer’s instructions. Cells treated with vehicle were

normalized to 100% proliferation and values adjusted accordingly.

Western Blot Analysis. MDA-MB-231 cells were grown to

conﬂuence and seeded at 200,000 cells/mL in a 10 cm dish and placed

back in the incubator for 24 h. Compounds or vehicle were

administered at the desired concentrations (0.25% DMSO ﬁnal

concentration) and incubated for 24 h. Cells were then harvested in

cold PBS and lysed using MPER (Thermo Scientiﬁc) supplemented

with protease inhibitors (Roche) according to the manufacturer’s

instructions. Cell lysates were obtained by centrifugation at 14000g for

15 min at 4 °C. Protein concentrations for each sample were

determined using the Pierce BCA assay kit following the

manufacturer’s directions. Equal amounts of proteins (20 μg) were

separated using gel electrophoresis under reducing conditions (10%

acrylamide gels) then transferred to PVDF membranes and

immunoblotted with the corresponding primary antibodies. Mem-

branes were then incubated with the correct HRP-labeled secondary

antibody, developed with a chemiluminescent substrate, and visualized.

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00085.

¹H and ¹³C NMR spectra for all new compounds and

HPLC traces for most active compounds (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 785-864-2288. Fax: 785-864-5326. E-mail: bblagg@ku.

edu.

Notes

The authors declare no competing ﬁnancial interest.

P

DOI: 10.1021/acs.jmedchem.6b00085

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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ACKNOWLEDGMENTS

■

This work was supported by grants from The National

Institutes of Health to CA109265 (B.S.J.B.), EY024232

(B.S.J.B. and C.A.D.), and EY021205 (R.L.L.). Support for

the NMR instrumentation was provided by NIH Shared

Instrumentation Grants (S10OD016360, S10RR024664) and

NSF Major Research Instrumentation Grant (0320648).

ABBREVIATIONS USED

■

Grp94, glucose-regulated protein 94; Hsp90 and Hsp70, heat

shock protein 90 and 70; ER, endoplasmic reticulum; TLR, toll-

like receptor; IGF, insulin-like growth factor; NECA, N-

ethylcarboxamideadenosine; RDA, radamide; GDA, geldana-

mycin; POAG, primary open angle glaucoma; ERAD,

endoplasmic reticulum associated degradation; TM, trabecular

meshwork

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N.; Crowley, V. M.; Mishra, S.; Blagg, B. S.; Lieberman, R. L.; Dickey,

C. A. Exploiting the interaction between Grp94 and aggregated

myocilin to treat glaucoma. Hum. Mol. Genet. 2014, 23, 6470−6480.

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Structure of the N-terminal domain of GRP94. Basis for ligand

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Development of Glucose Regulated Protein 94-Selective Inhibitors Based on the BnIm and Radamide Scaffold

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