Narrow SAR in odorant sensing Orco receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2014.04.081

The systematic exploration of a series of triazole-based agonists of the cation channel insect odorant receptor is reported. The structure-activity relationships of independent sections of the molecules are examined. Very small changes to the compound structure were found to exert a large impact on compound activity. Optimal substitutions were combined using a 'mix-and-match' strategy to produce best-in-class compounds that are capable of potently agonizing odorant receptor activity and may form the basis for the identification of a new mode of insect behavior modification.

Full text of DOI:10.1016/j.bmcl.2014.04.081

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2613–2616
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Narrow SAR in odorant sensing Orco receptor agonists
Ian M. Romaine a, Robert W. Taylor d, , Samsudeen P. Saidu d, Kwangho Kim a,b, Gary A. Sulikowski a,b
,
Laurence J. Zwiebel a,c,d, Alex G. Waterson a,b,c,
⇑
a Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37322, United States
b Department of Chemistry, Vanderbilt University, Nashville, TN 37322, United States
c Department of Pharmacology, Vanderbilt University, Nashville, TN 37322, United States
d Department of Biological Sciences, Vanderbilt University, Nashville, TN 37322, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The systematic exploration of a series of triazole-based agonists of the cation channel insect odorant
receptor is reported. The structure–activity relationships of independent sections of the molecules are
examined. Very small changes to the compound structure were found to exert a large impact on com-
pound activity. Optimal substitutions were combined using a ‘mix-and-match’ strategy to produce
best-in-class compounds that are capable of potently agonizing odorant receptor activity and may form
the basis for the identification of a new mode of insect behavior modification.
Received 6 March 2014
Revised 18 April 2014
Accepted 21 April 2014
Available online 29 April 2014
Keywords:
Odorant receptor
VUAA1
Ó 2014 Elsevier Ltd. All rights reserved.
Structure activity relationship
Drosophila melanogaster
Insect chemosensory receptors from the odorant receptor (OR)
superfamily are responsible for a large portion of the peripheral
signal transduction that is required for the detection of a wide
array of volatile odorant compounds that exist in an insect’s envi-
ronment.1 In contrast to their mammalian counterparts, which are
G-protein coupled receptors, insect ORs act as heteromeric ligand
(odorant)-gated cation channels consisting of two 7-transmem-
brane domain protein subunits: the obligate OR co-receptor (Orco)
and an odorant-interacting ‘tuning’ OR (ORx).2–5 While Orco is
highly conserved across insect taxa, the tuning ORs tend to be
highly divergent and, for the most part, specific to individual insect
species.6 Recognition of an odorant ligand by the tuning OR is
thought to open the non-selective channel complex, which initi-
ates action potentials in odorant receptor neurons (ORNs), leading
to downstream neuronal activity that allows an insect to sample
and respond to its chemical environment.4,7 As many aspects of
insect behavior are directed on the basis of these olfactory pro-
cesses, adversely affecting or otherwise modulating the ability of
an insect to correctly sense and interpret environmental cues rep-
resents an established method of altering behavior to reduce the
impact of a wide range of economically and medically important
insect pests and disease vectors.
We have previously reported the identification and character-
ization of VUAA1 as an agonist of Orco action.8–10 Similar indepen-
dent efforts have identified additional active analogs in this
compound class.13–14 We have previously reported that more
potent analogs from the same chemical class are capable of affect-
ing the behavior of mosquito larvae in mobility assays at concen-
trations similar to, or lower than, those required for VUAA1
activity.11 Thus, we have continued to seek more potent analogs
of VUAA1 that would be capable of agonizing the ion channel at
more therapeutically useful doses. Here, we report the full details
of the structure–activity relationships (SAR) in the VUAA1 series
that led to the identification of more potent analogs. We have per-
formed a systematic evaluation of each section of the chemical
template and have noted that only an extremely narrow group of
substitutions leads to agonist activity. Following this initial survey,
a ‘mix and match’ strategy was undertaken to produce potent
compounds.
The VUAA1-based agonists were assembled using a straightfor-
ward and flexible synthetic route (Scheme 1). The route begins
with commercially available hydrazides (2), which were reacted
with isothiocyanates and then subsequently cyclized to generate
3-thio-1,2,4-triazoles (3). If necessary, carboxylic acid esters (1)
were first converted to the hydrazides. To construct the final ana-
logs, the thiols (3) were alkylated in a two-stage process beginning
with the condensation of anilines (4) with chloroacetyl chloride.
The resulting crude intermediate was reacted with the thiol to gen-
erate the agonists (5).
⇑
Corresponding author. Tel.: +1 615 322 9971; fax: +1 615 875 3236.
E-mail address: alex.g.waterson@vanderbilt.edu (A.G. Waterson).
Current address: Lurie Children’s Hospital Research Center, Chicago, IL 60614,
United States.
http://dx.doi.org/10.1016/j.bmcl.2014.04.081
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2614
I. M. Romaine et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2613–2616
Table 2
Indoline-based agonists
a
Compd
R
n
EC50
(lM)
% VUAA1 efficacyb
6a (VUAA1)
N/A
H
Br
F
35.1
—
21.5
—
—
38.6
—
—
—
—
100%
No agonism
82%
No agonism
No agonism
128%
No agonism
No agonism
No agonism
No agonism
7a
7b
7c
7d
7e
7f
7g
7h
7i
1
1
1
1
1
1
1
1
1
Scheme 1. Synthetic route to VUAA1-based odorant agonists. aReagents and
conditions: (a) hydrazine, 150 °C,
wave, 15 min; (c) K2CO3, H2O, reflux, 16 h; (d) ClCH2COCl, triethyl amine, 2 h; and
(e) 3, CsCO3, acetonitrile, 16 h.
lwave, 15 min; (b) isothiocyanates, 150 °C,
Methyl
Ethyl
Methoxy
N,N-Dimethylamino
Isopropyl
tertButyl
l
All analogs were evaluated for Orco agonist activity using a
high-throughput calcium mobilization/imaging assay, as previ-
ously described.9,10,12 For this SAR study, we used HEK293 cells
stably expressing Orco derived from Drosophila melanogaster
(DmOrco) on its own or, in some instances, together with addi-
tional tuning OR subunits. Using this assay across a range of com-
pound concentrations allowed the determination of the half
maximal effective concentration (EC50) of the compound, as well
as the maximal agonist activity, which was expressed as a percent-
age of VUAA1 activity (Tables 3 and 4).
c
7j
47.6
—
71%
7k
Br
2
No agonism
a
b
c
Mean result of four experiments.
Maximum agonism of the compound, normalized to the activity of VUAA1.
Entire ring replaced.
these results suggest a very narrow steric constraint at this end of
the molecule. We next examined branching on the substituent at
the 4-position of the aniline. The isopropyl analog (6f) was superior
to VUAA1 in both EC50 and overall agonist activity. However, the
tert-butyl moiety (6g) displayed reduced agonism.
Along with the steric constraints at the 4-position of the phenyl
ring, we also explored changes to the nature of the substitution.
Although a methyl ketone (6h) was somewhat tolerated, other
changes (e.g., methoxy and halides) were not (6i,j). Unsaturated
carbon chains were partially effective, with a vinyl group (6k)
showing activity, although reduced in potency relative to VUAA1.
With the addition of a further degree of unsaturation using an
alkynyl group (6l), all activity was lost. In general, additional sub-
stitutions on the ethylaniline were not tolerated (e.g., 6m,n), and
non-phenyl analogs were ineffective as agonists (6o,p).
Initially, we examined the SAR of the aniline portion of the mol-
ecule. VUAA1 has a 4-ethyl aniline in this position. We observed an
extremely narrow constraint for active compounds—only struc-
tures very close to the parent maintained activity (Table 1). VUAA1
(6a) activates DmOrco with an EC50 of 35 lM. Simply moving the
ethyl substitution to the meta position (6b) completely abrogated
activity (other substitutions in the 2- and 3-position of the phenyl
ring also failed to demonstrate agonist activity, data not shown).
While removing one carbon sharply reduced activity (6c), we
found that the addition of one carbon to form the propyl analog
(6d) was tolerated, while a butyl substitution (6e) was inactive.
Although the binding site for these allosteric agonists is unknown,
We surveyed a small set of indoline-based amides (Table 2)
with substitution at the 5-position, which corresponds to the ani-
line 4-position that generated positive results. Similar to the ani-
lines, a very narrow SAR was observed, with only the bromo (7b)
Table 1
Structure–activity relationships on the aniline ring
Table 3
a
Compd
R
X
EC50
(lM)
% VUAA1 efficacyb
Examination of the pyridyl ring SAR
6a (VUAA1)
4-Ethyl
3-Ethyl
4-Methyl
4-Propyl
4-Butyl
4-Isopropyl
4-tertButyl
4-Acetyl
4-Methoxy
4-Bromo
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
N
35.1
—
—
94.1
—
11.7
LAd
84.7
—
—
102
—
—
—
100%
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
No agonism
No agonism
57%
No agonism
127%
a
Compd
Ar
EC50
(l
M)
% VUAA1 efficacyb
42%e
96%
6f
3-Pyridyl
H
Br
2-Pyridyl
4-Pyridyl
2-F, 4-pyridyl
3-Pyrrolyl
3-Furyl
3-Thiophenyl
1-Methyl,3-pyrazolyl
4-Pyrazolyl
1-Methyl,4-pyrazolyl
5-Thiazolyl
11.7
—
—
127%
No agonism
No agonism
57%
No agonism
No agonism
No agonism
No agonism
No agonism
8a
8b
8c
8d
8e
8f
8g
8h
8i
No agonism
No agonism
No agonism
150%
4-Vinyl
—
4-Ethynyl
2-Methyl-4-ethyl
2-Bromo-4-ethyl
Cyclohexylc
4-Ethyl
6.8
60.1
—
—
—
—
—
107
10.7
148%
No agonism
No agonism
No agonism
No agonism
No agonism
34%
—
—
a
b
c
Mean result of four experiments.
Maximum agonism of the compound, normalized to the activity of VUAA1.
Entire ring replaced.
8j
8k
8l
36%
d
LA = low agonism. Compound shows agonism only at the highest concentration
a
tested, but no EC50 could be calculated.
Mean result of four experiments.
Maximum agonism of the compound, normalized to the activity of VUAA1.
e
b
Maximum observed agonism, but from an incomplete curve.

I. M. Romaine et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2613–2616
2615
Table 4
Examination of substitution on the triazole nitrogen
a
Compd
R
EC50
(lM)
% VUAA1 efficacyb
8d
9a
9b
9c
9d
9e
9f
9g
9h
9i
Ethyl
Methyl
6.8
—
—
—
84.0
—
35.9
3.9
LAc
—
150%
No agonism
No agonism
No agonism
28%
No agonism
111%
162%
13%
No agonism
No agonism
i-Propyl
t-Butyl
n-Propyl
n-Butyl
Allyl
Cyclopropyl
Cyclopentyl
Cyclohexyl
Phenyl
Figure 1. VUAA analogs as agonists of DmOrco. Concentration response curves of
DmOrco expressing HEK cells in the presence of a series of increasing concentra-
tions of test compound (LogM) versus the % Max fluorescence using VUAA1.
9j
—
a
b
c
Mean result of four experiments.
Maximum agonism of the compound, normalized to the activity of VUAA1.
LA = low agonism. Compound shows agonism only at the highest concentration
position. Analogs with various heterocyclic isosteres (8f–l) met
with minimal success, although both a methylated pyrazole (8k)
and a thiazole (8l) were noted as weak agonists. Interestingly, 8l
displayed good potency although the overall agonist activity was
weak.
tested, but no EC50 could be calculated.
As the final portion of the systematic evaluation of the SAR of
the compound class, we examined the impact of the nitrogen sub-
stitution of the triazole. For this study, we employed the 4-pyridyl
moiety and the 4-isopropyl aniline of 8d. Notably, an oxadiazole
derivative with no substitution at this position was not active
(not shown), nor was a methyl substitution (9a). Substitution on
the ethyl moiety was not tolerated (9b,c), and lengthening the car-
bon chain was only marginally successful (9d,e). Interestingly,
unsaturation was tolerated (9f), although it led to reduced activity.
Although larger carbocycles were poorly tolerated (9h–9j), a cyclo-
propyl analog (9g) produced one of the most potent and efficacious
analogs in the study.
Having thoroughly explored the contributions of the individual
segments of the molecule, we engaged in an organized ‘mix and
match’ strategy to combine the best substituents at each position
with each other to seek possible complementarity in the SAR.
Selected successful examples are shown in Table 5. As expected,
the combination of the 3-pyridyl and 4-pyridyl aryl groups with
ethyl and cyclopropyl triazole substitutions produced consistently
good results compared with VUAA1. In particular, the cyclopropyl
and ethyl (7e) derivatives maintaining agonist activity. In addition,
we examined tetrahydroquinoline analogs, exemplified by 7k,
which were also inactive. Surprisingly, however, the indole (7j)
was noted as a moderate agonist, with activity inferior to VUAA1.
Several changes to the sulfide-containing linker region were
examined, including the addition of a methylene to the linker
length, as well as substitution on the linker and replacement of
the sulfur with a oxygen atom or a sulfonyl moiety. Although these
were explored within the context of the optimal aniline of 6f, no
activity was noted with any of these analogs (not shown). Even
simple methylation of the amide nitrogen produced inactive ana-
logs (not shown), reinforcing a preference for the unmodified
linker.
We also examined the contribution of the aromatic ring at the
3-position of the triazole, using the 4-isopropyl aniline found for
compound 6f. Once again, a narrow SAR space for active com-
pounds was observed. Compounds without an aromatic ring (e.g.,
8a and 8b) were inactive. While a 2-pyridyl was inactive, a 4-pyr-
idyl analog showed improved potency and agonist efficacy relative
to both VUAA1 and 6f, both of which contain a 3-pyridyl in this
Table 5
Selected results from the combination strategy
a
Compd
Ar
R
R0
EC50
(l
M)
% VUAA1 efficacyb
6a (VUAA1)
10a
10b
10c
10d
10e
10f
10g
10h
3-Pyridyl
3-Pyridyl
3-Pyridyl
3-Pyridyl
Ethyl
4-Ethylaniline
4-Ethylaniline
4-Isopropyl aniline
4-Vinylaniline
4-Ethylaniline
35.1
11.9
3.64
34.4
37.0
LAc
10.7
22.0
15.0
16.7
17.6
17.9
100%
171%
178%
126%
150%
122%d
175%
130%
113%
98%
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Ethyl
Cyclopropyl
Ethyl
Cyclopropyl
Cyclopropyl
n-Propyl
Allyl
4-Pyridyl
1-Methyl,4-pyrazolyl
1-Methyl,4-pyrazolyl
4-Pyridyl
4-Pyridyl
3-Pyridyl
4-Ethylaniline
4-Isopropyl aniline
5-Ethylindoline
5-Ethylindoline
5-Bromoindoline
5-Ethylindoline
5-Ethylindoline
10i
10j
10k
3-Pyridyl
4-Pyridyl
99%
76%
a
b
c
Mean result of four experiments.
Maximum agonism of the compound, normalized to the activity of VUAA1.
LA = low agonism. Compound shows agonism only at the highest concentration tested, but no EC50 could be calculated.
Maximun observed agonism, but from an incomplete curve.
d

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I. M. Romaine et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2613–2616
triazole substitution allowed a range of anilines to have activity
(10a–10d). Notably, some of these generated an EC50 below 5
manipulate of the destructive insect behaviors and/or alter the
behavior of economically and medically important insects. Further
work to define the binding interactions of these compounds and to
explore their use in the context of insect behavior modification is
in progress and will be reported in due course.
lM
and showed efficacy superior to VUAA1 (e.g., 10b). Surprisingly,
however, the 4-pyridyl moiety did not appear to provide as large
a boost in this context (compare 10d with 10a). Interesting results
were also obtained with the methyl pyrazole substitution (10e,f).
Strikingly, some of the compounds showed massive increases in
agonist efficacy, but without corresponding potency improve-
ments (e.g., 10e). The mix and match combination strategy
revealed a surprising tolerance for the indoline-based compounds,
with several examples possessing EC50 values superior to VUAA1,
along with comparable efficacy (10g–k). Interestingly, a wider than
anticipated range of triazole substitutions were allowed (10j and
10k) with some substitution patterns. Representative dose
response curves (Fig. 1) demonstrate the range of agonist activities
observed with selected compounds from this combination
strategy.
In summary, we have thoroughly explored the SAR around a tri-
azole-based series of DmOrco agonists. Significant activity was
obtained within only a very narrow set of compound structures.
The most successful analogs presented a five- or six-membered
heterocycle at the 3-position of a 1,2,4-triazole, and a small alkyl
substituent on the nitrogen. A range of aniline-based end pieces
were tolerated, but all active analogs possessed a small 4-position
substitution (or the equivalent substitution on an indoline ring).
This narrow, but definable, SAR may be indicative of a specific
binding location with tight steric and electronic tolerances. How-
ever, the binding site and the precise kinetics of these allosteric
ORco agonists remain unknown. Compounds in this series have
been shown to affect the behavior of insect larvae11 as well as
adults (L.J.Z., unpublished observations). Thus, these compounds
may have potential as excito-repellents that may be able to
Acknowledgments
We would like to thank Paul Reid for his invaluable insight and
contributions to this project. This work was supported by a grant
from the Foundation for the National Institutes of Health through
the Grand Challenges in Global Health Initiative (VCTR121 to L.J.Z.).
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