Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor

Article abstract of DOI:10.1016/j.bmcl.2014.04.013

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.

Full text of DOI:10.1016/j.bmcl.2014.04.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2473–2476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon
receptor
Kaitlin J. Basham a, , Vasudev R. Bhonde b, , Collin Kieffer a,à, James B. C. Mack b, Matthew Hess b,
Bryan E. Welm a,c, Ryan E. Looper b,
⇑
a Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
b Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA
c Department of Surgery, University of Utah, Salt Lake City, UT 84112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological
activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morpho-
genesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary
differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity
relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are crit-
ical for AHR activation. We validated our results with a functional biological assay, using desmosome for-
mation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of
oxadiazole derivative therapeutics with reduced off-target toxicity profiles.
Received 31 January 2014
Revised 2 April 2014
Accepted 4 April 2014
Available online 13 April 2014
Keywords:
Oxadiazole
Aryl hydrocarbon receptor
Mammary gland
Branching morphogenesis
Dioxin
Ó 2014 Elsevier Ltd. All rights reserved.
Small molecule libraries are widely used as a tool in chemical
biology,1 both to probe biological pathways and to develop new
therapeutics. However, the success of chemical library screening
efforts is limited by library composition and size. One strategy to
produce a large number of drug-like compounds is to use scaffolds
that have previously generated biologically active chemicals.2 In
particular, the oxadiazole nucleus has been used extensively as a
scaffold in drug development3 due to the range of activities
reported for its derivatives, including antimicrobial, anticancer,
anti-inflammatory, and antiviral effects.4–7
As a heteroaromatic ring, oxadiazoles can be prepared as sev-
eral constitutional isomers. The 1,2,4-oxadiazole isomer has been
used in numerous pharmacologic drugs, including metabotropic
glutamate subtype 5 receptor antagonists,8 sphingosine-1-phos-
phate-1 receptor agonists,9 and anticancer apoptosis inducers.10
Additionally, we previously identified a derivative of this isoform
as a potent compound that blocks mammary branching morpho-
genesis.11 In our assay, 1,2,4-bis-aryloxadiazole 1 (referred as
1023 in our previous communication) was the lead compound
identified in a chemical genetic screen for molecules that block
mammary branching morphogenesis. Further analysis showed 1
had an EC50 of 1.2 0.050 M and blocked branching through acti-
vation of the aryl hydrocarbon receptor (AHR).
l
In addition to influencing mammary branching, AHR agonists
also block differentiation and lactation in the mammary gland12–
and exhibit a wide range of toxic effects in other tissues.15,16
14
Our previous observations that compound 1 potently activated
AHR suggested that other 1,2,4-oxadiazole derivatives may display
unwanted drug effects due to AHR stimulation. Given the structural
relationship of these derivatives to 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD), a known carcinogen and environmental toxin that
also activates AHR, we performed structure–activity relationship
(SAR) studies of 1 to identify key elements of the molecule that con-
tribute to AHR activation. A library of bis-aryloxadiazoles was pre-
pared by Lewis-acid mediated coupling of benzoyl chlorides with
benzamidoximes (Scheme 1). The activity of each analog was deter-
mined by measuring expression of the AHR target gene, Cyp1a1,17,18
in HC11 mammary epithelial cells (MECs) treated for 48 h with
10
l
M compound.
We initially made systematic modifications on the C-ring of 1
(Table 1). Based on a previous homology model,11 this ring was
predicted to form charge/polar interactions with amino acid
residues His-291 and Gln-383 in the AHR binding pocket. Our
results indicated that replacing the o-Cl substituent with an amino
group (compound 2) increased AHR activity ꢀ5-fold, as shown by
⇑
Corresponding author. Tel.: +1 (801) 585 0408.
E-mail address: r.looper@utah.edu (R.E. Looper).
These authors contributed equally.
Present address: Division of Biology, California Institute of Technology, Pasadena,
à
CA 91125, USA.
http://dx.doi.org/10.1016/j.bmcl.2014.04.013
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2474
K. J. Basham et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2473–2476
Table 2
O
Cl
O
N
NH
BF3 . OEt2
dioxane, 100 o
B
SAR study of the A-ring of 1,2,4-bis-aryloxadiazole
OH
Cl
N
N
C
A
+
C
Cl
H
R1
O
R4
N
Cl
Cl
12h, 92%
B
1023
N
C
R2
A
X
R3
Scheme 1. General strategy for synthesis of bis-aryloxadiazoles.
Compound
X
R1
R2
R3
R4
Relative Cyp1a1
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CH o-Cl
CH o-Cl
CH o-F
CH o-F
CH o-Cl
CH o-Cl
CH o-Cl
CH o-Cl
p-Cl
p-Cl
H
H
H
H
H
H
H
H
H
H
H
H
p-Cl
H
H
H
m-CF3
m-CO2Me
m-CO2Me
m-CO2H
o-Cl
1.84 0.28
0.18 0.49
0.29 0.74
3.71 0.22
5.91 0.18
1.59 0.30
8.15 0.14
Table 1
SAR study of the C-ring of 1,2,4-bis-aryloxadiazole
R1
H
O
N
B
p-Cl
p-Cl
p-Cl
p-Cl
N
C
R2
A
m-Cl
p-Cl
R3
p-O-Propargyl 0.66 0.26
p-O-Propargyl 0.29 0.35
p-O-Allyl
Compound
R1
R2
R3
Relative Cyp1a1
CH o-NO2 p-Cl
CH o-NH2 p-Cl
CH o-NH2 p-Cl
0.62 0.16
1
2
3
4
5
6
7
8
9
o-Cl
p-Cl
p-Cl
H
H
H
H
H
H
H
H
128.53 0.17
638.80 0.14
0.36 0.15
0.42 0.25
0.25 0.14
0.39 0.16
2.43 0.19
122.64 0.12
58.63 0.15
25.79 0.17
p-O-Propargyl 0.38 0.42
o-NH2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NH2
o-Cl
N
N
N
N
o-Cl
o-Cl
o-NO2 p-Cl
m-Cl
p-Cl
H
H
H
H
0.53 0.28
0.37 0.13
8.25 0.13
0.35 0.41
p-CF3
p-OMe
p-OH
o-NO2 p-OAc
p-OCO2Me
p-O-Propargyl
p-F
Expression of an AHR response gene, Cyp1a1, was measured in HC11 MECs treated
with 10 M compound for 48 h.
l
p-CF3
H
m-Cl
10
p-Cl
Expression of an AHR response gene, Cyp1a1, was measured in HC11 MECs treated
with 10 M compound for 48 h.
l
increased Cyp1a1 gene expression with compound 2. In contrast,
placement of an electron-withdrawing group (NO2) at the ortho
position of the C-ring dramatically decreased AHR activity (com-
pounds 3–7). These results suggested that the C-ring of bis-arylox-
adiazole is compatible with an electroneutral or protic-polar
substitution that can be stabilized by hydrogen bonding with
His-291 (Fig. 1). This was confirmed by replacing the nitro group
at the ortho position of compound 3 with an amino group (com-
pound 9), which partially restored Cyp1a1 gene expression.
In addition to the ortho position, the para group of the C-ring
also influenced AHR activity. Specifically, p-CF3 (compound 9)
showed significantly lower Cyp1a1 gene expression compared to
p-Cl (compound 2). This result is likely due to the electron with-
drawing and sterically bulky nature of CF3. Surprisingly, p-F (com-
pound 8) offset the decreased activity of o-NO2 observed in other
analogs. This may be explained by the small size of p-F, which
would decrease van der Waals repulsion and contribute to aro-
matic stabilization through charge interaction. Together, modifica-
tions on the C-ring suggested bis-aryloxadiazole requires subtle
Figure 2. Homology model structure of human AHR (gray) and compound 11
(purple). Residues predicted to contribute to compound binding are shown in green.
Steric interactions of the A-ring with Phe324 and Phe287 lead to decreased AHR
activation.
electronic demand at the ortho and para positions and tight van
der Waals radii at the para position to elicit significant AHR
activation.
Next, we extended our SAR study to the A-ring of bis-arylox-
adiazole (Table 2). Previous modeling studies11,19 suggested this
portion of the molecule binds within a tight hydrophobic cavity
of AHR and is stabilized by aromatic
Phe-287. As a result, we hypothesized that functional groups on
the A-ring of bis-aryloxadiazole able to distort this -stacking
p-stacking of Phe-324 and
p
would also diminish AHR activation (Fig. 2). Supporting this
hypothesis, we previously showed that addition of m-CF3 to the
A-ring (compound 11) dramatically decreased AHR activity.11 Sim-
ilarly, polar carbomethoxy or carboxylate substituents at R4 (com-
pounds 12–14) showed low Cyp1a1 gene expression, irrespective
of identities at R1 and R2. Importantly, these substitution patterns
are seen in lead compounds for the treatment of nonsense muta-
tion disorders (e.g., Ataluren).20
In addition to meta substitutions, we altered other positions of
the A-ring and modified the A-ring itself. Substitution of CF3 with
Cl at different positions resulted in only subtle AHR activation,
with p-Cl showing the highest Cyp1a1 gene induction (compound
15–17). Similarly, larger para-substituents on the A-ring (com-
pounds 18–21) or substitution of the A-ring with a heterocycle
Figure 1. Homology model structure of human AHR (gray) and compound
2
(purple), with residues predicted to contribute to compound binding shown in
green. Hydrogen bonding between the amino group at the ortho position of the C-
ring and His-291 is predicted to stabilize binding.

K. J. Basham et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2473–2476
2475
expression (compounds 2, 8, 9, and 10). We next validated these
compounds as AHR agonists in a functional biological assay.
Previously, we showed 1 potently blocked mammary branching
morphogenesis of primary MECs.11 Using this same assay, we
observed that compounds 2, 8, and
9 recapitulated the
unbranched, cyst phenotype (Fig. 3a) and displayed an EC50 similar
to compound 1 (Fig. 3b). In contrast, compound 10, which induced
the lowest level of Cyp1a1 gene expression compared to the other
active analogs, did not inhibit branching and displayed a relatively
high EC50 (Fig. 3).
Finally, we assessed the level of desmosomal adhesion in pri-
mary MECs treated with compounds 2, 8, 9, and 10. We previously
identified desmosomes as a novel target of activated AHR, which
functionally disrupts mammary branching morphogenesis.11 Using
desmoglein 3 (DSG3) protein levels as an indicator of desmosome
complexes,21 we observed high levels of desmosomal proteins in
primary MECs treated with 10 nM TCDD, which is a known AHR
agonist, and 10 lM of compounds 1, 2, 8, and 9 (Fig. 4a and b).
Interestingly, compounds 10 and 11 showed the lowest levels of
DSG3, which correlated with their low Cyp1a1 gene expression lev-
els and poor EC50 values in our branching assay (Fig. 4b and c). The
shared patterns of Cyp1a1 gene expression and DSG3 protein levels
in these biological assays suggest DSG3 is a functional indicator of
AHR activity.
In summary, we performed SAR studies of 1,2,4-bis-arylox-
adiazole to identify components of the molecule critical for AHR
activation. Our results indicated the C-ring is agonistic when
substituted with electronically neutral or protic-polar moieties,
particularly when tight van der Waals radii at the para position
are maintained. In contrast, modification of the A-ring dramatically
reduced AHR activity in all cases, suggesting this portion of the
molecule significantly contributes to AHR binding and activation.
These findings indicate that chemical substitutions of the A-ring
that minimize AHR activation, but do not significantly alter thera-
peutic activity, should be considered for bis-aryloxadiazole com-
pounds. We validated our findings by assessing the biological
effects of these compounds on mammary branching morphogene-
sis. In agreement with our previous observations, there was a
Figure 3. Characterization of mammary branching morphogenesis in the presence
of 1,2,4-bis-aryloxadiazole analogs. (a) Representative images and (b) dose–
response analysis for branching in primary MECs. Scale bar = 40
EC50 values: compound 1 (1.2 0.4 M), compound 2 (1.4 0.1 M), compound 8
(2.2 0.1 M), compound 9 (3.2 0.1 M), compound 10 (>10 M).
lm. Calculated
l
l
l
l
l
(pyridine) nearly abolished AHR activity (compounds 22–25).
Taken together, these results show that any modification of the
A-ring of bis-aryloxadiazole has a profound effect on AHR binding
and activation.
From our SAR study, we identified
4 analogs of 1,2,
4-bis-aryloxadiazole that retained significant Cyp1a1 gene
Figure 4. Effect of analog compounds on desmosomal adhesion and AHR readout genes in MECs. (a) Western blot analysis and (b) quantification of desmoglein 3 (DSG3) in
primary MECs. (c) Relative Cyp1a1 gene expression in HC11 MECs.

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Acknowledgments
We thank Prof. David J. Bearss and Dr. Hariprasad Vankayalapati
at the Center for Investigational Therapeutics, Huntsman Cancer
Institute, for the modeling studies of compound 1 with human
AHR. National Institutes of Health (R01-GM090082, R01-
CA143815, R01-CA140296) and the Department of Defense Breast
Cancer Research Program (W81XWH-09-1-04310) supported this
work. K.J.B. is supported by National Institutes of Health Develop-
mental Biology Training Grant 5T32 HD07491.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.013.
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