Optimization of a 1,3,4-oxadiazole series for inhibition of Ca2+/calmodulin-stimulated activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain

Article abstract of DOI:10.1016/j.ejmech.2018.11.036

Adenylyl cyclases type 1 (AC1) and 8 (AC8) are group 1 transmembrane adenylyl cyclases (AC) that are stimulated by Ca²⁺/calmodulin. Studies have shown that mice depleted of AC1 have attenuated inflammatory pain response, while AC1/AC8 double-knockout mice display both attenuated pain response and opioid dependence. Thus, AC1 has emerged as a promising new target for treating chronic pain and opioid abuse. We discovered that the 1,3,4-oxadiazole scaffold inhibits Ca²⁺/calmodulin-stimulated cyclic adenosine 3‘,5‘-monophosphate (cAMP) production in cells stably expressing either AC1 or AC8. We then carried out structure-activity relationship studies, in which we designed and synthesized 65 analogs, to modulate potency and selectivity versus each AC isoform in cells. Furthermore, molecular docking of the analogs into an AC1 homology model suggests the molecules may bind at the ATP binding site. Finally, a prioritized analog was tested in a mouse model of inflammatory pain and exhibited modest analgesic properties. In summary, our data indicate the 1,3,4-oxadiazoles represent a novel scaffold for the cellular inhibition of Ca²⁺/calmodulin-stimulated AC1- and AC8 cAMP and warrant further exploration as potential lead compounds for the treatment of chronic inflammatory pain.

Full text of DOI:10.1016/j.ejmech.2018.11.036

Accepted Manuscript
2+
Optimization of a 1,3,4-oxadiazole series for inhibition of Ca /calmodulin-stimulated
activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain
Jatinder Kaur, Monica Soto-Velasquez, Zhong Ding, Ahmadreza Ghanbarpour,
Markus A. Lill, Richard M. van Rijn, Val J. Watts, Daniel P. Flaherty
PII:
S0223-5234(18)30995-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.036
EJMECH 10893
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 August 2018
Revised Date: 12 November 2018
Accepted Date: 15 November 2018
Please cite this article as: J. Kaur, M. Soto-Velasquez, Z. Ding, A. Ghanbarpour, M.A. Lill, R.M. van Rijn,
2+
V.J. Watts, D.P. Flaherty, Optimization of a 1,3,4-oxadiazole series for inhibition of Ca /calmodulin-
stimulated activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.036.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Optimization of a 1,3,4-oxadiazole series for inhibition of Ca2+/calmodulin-
stimulated activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain
Jatinder Kaura, Monica Soto-Velasqueza, Zhong Dinga, Ahmadreza Ghanbarpoura, Markus A. Lilla,b,c
,
Richard M. van Rijna,b,c, Val J. Wattsa,b,c, and Daniel P. Flahertya,b,c,
*
a
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University,
575 Stadium Mall Dr., West Lafayette, IN 47907.
b Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907
c Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN 47907
* Corresponding author E-mail address: dflaher@purdue.edu (D.P. Flaherty)
Keywords
Adenylyl cyclase inhibitors; 1,3,4-oxadiazole; Chronic pain treatment, Molecular modeling
Abstract
Adenylyl cyclases type 1 (AC1) and 8 (AC8) are group 1 transmembrane adenylyl cyclases (AC) that
are stimulated by Ca2+/calmodulin. Studies have shown that mice depleted of AC1 have attenuated
inflammatory pain response, while AC1/AC8 double-knockout mice display both attenuated pain response and
opioid dependence. Thus, AC1 has emerged as a promising new target for treating chronic pain and opioid
abuse. We discovered that the 1,3,4-oxadiazole scaffold inhibits Ca2+/calmodulin-stimulated cyclic adenosine
3’,5’-monophosphate (cAMP) production in cells stably expressing either AC1 or AC8. We then carried out
structure-activity relationship studies, in which we designed and synthesized 65 analogs, to modulate potency
and selectivity versus each AC isoform in cells. Furthermore, molecular docking of the analogs into an AC1
homology model suggests the molecules may bind at the ATP binding site. Finally, a prioritized analog was
tested in a mouse model of inflammatory pain and exhibited modest analgesic properties. In summary, our
data indicate the 1,3,4-oxadiazoles represent a novel scaffold for the cellular inhibition of Ca2+/calmodulin-
stimulated AC1- and AC8 cAMP and warrant further exploration as potential lead compounds for the treatment
of chronic inflammatory pain.

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1. Introduction
Adenylyl cyclases (ACs) are key effectors of G protein-coupled receptor (GPCR) signaling pathways
that catalyze the production of the secondary messenger molecule cyclic adenosine 3’,5’-monophosphate
(cAMP) from adenosine triphosphate (ATP) [1]. Many cellular responses are mediated by changes in
intracellular cAMP, and ACs play crucial roles in various physiological processes including learning, memory,
chronic pain, and drug abuse [2, 3]. ACs serve as downstream effectors for various GPCRs including the µ-
opioid receptor (µOR), the preferred target for analgesic drugs [4]. Agonism of the µOR induces dissociation of
the inhibitory G-protein αi (Gαi) subunit from the heterotrimeric G-protein complex, which then inhibits AC
activity and reduces cAMP levels and propagating the desired analgesic effect [5]. Subsequently, µOR
activation also leads to the recruitment of β-arrestins, which mediate the undesired side-effects such as
tolerance, respiratory depression and constipation commonly associated with opioid pain management [4, 6-8].
Thus, there is ongoing research to design biased modulators of the µOR with the aim of increasing the desired
analgesic properties via the G-protein, while simultaneously reducing the unwanted side-effects induced by β-
arrestin signaling [9]; however, this has been notoriously difficult to achieve. Alternatively, we [10], and others
[11], have proposed inhibiting the activity of the downstream ACs – effectively bypassing the µOR – may
represent a promising strategy for the development of drugs to treat chronic pain while limiting the undesired
side-effects associated with opioid treatment.
There are nine membrane-bound AC isoforms categorized into four distinct groups [12]. All ACs are
activated by the stimulatory Gαs protein and can also be regulated by alternative mechanisms including Gβγ
subunits, calmodulin, protein kinases and cations, but these regulatory mechanisms differ between the four
distinct groups. Group 1 ACs include AC1, AC3, and AC8 and are characterized by regulation with
Ca2+/calmodulin; group 2 ACs comprising AC2, AC4, and AC7 are conditionally activated by Gβγ subunits;
group 3 ACs consisting of AC5 and AC6 and are inhibited by Ca2+; and the Group 4 AC, AC9, is the only AC
insensitive to forskolin activation. Although cells typically express multiple AC isoforms, specific tissue
expression patterns coupled with distinct regulatory properties of the AC isoforms have led to certain isoforms
being implicated with particular pathophysiology [12]. For example, AC1 and AC8 are highly expressed in the
brain, within the hippocampus (associated with learning and memory) [13, 14] and the anterior cingulate cortex

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(ACC, associated with chronic pain perception) [2, 15]. Additionally, AC1 knockout (AC1-/-) and AC1/AC8
double knockout (DKO) mice have exhibited reduced or abolished behavioral responses to two inflammatory
pain stimuli (formalin and complete Freund’s adjuvant, CFA) [16, 17]. Even though, the DKO mice also
exhibited behaviors consistent with severe memory impairment, these memory deficits were attenuated or not
observed in the AC1 knockout mice [18]. Hence, these results and the distinct tissue specific expression of
AC1 in pain perception regions of the ACC, suggests AC1 represents an attractive target for therapeutic
development of pain management treatments.
The current state-of-the-art for AC1 inhibitors (Fig. 1) includes
adenosine-based inhibitors represented by SQ22536 [19] that are
proposed to bind in a non-competitive way at the catalytic site “P”-site.
NB001
SQ22536
ST034307
This class typically only exhibits modest (> 10 µM) AC potency in
cellular assays, and non-selectively inhibit the catalytic activity of AC
isoforms [20]. This weak activity was confirmed with our data for
SQ22536 against HEK-AC1 cells stimulated by forskolin (Fig. S1).
More recently, pharmacological data from the molecule NB001
supported genetic evidence that implicated AC1s role in pain
1
Figure 1. Representative structures of
the current state of AC1 inhibitors.
Molecule
screening campaign and the basis for
the structure-activity relationship
optimization described.
1
discovered in our
perception by attenuating chronic pain response (inflammatory and neuropathic) in both mice and rats treated
with the molecule [21, 22]. However, while effective in in vivo models for chronic pain, NB001 has moderate
activity against AC1 (IC50 = 10 µM) in the cell-based Ca2+/calmodulin-mediated cAMP accumulation assay [22].
Furthermore, while it is hypothesized to bind at the P-site recent data suggests that it does not directly inhibit
AC1 but rather it acts through an alternative mechanism [23]. Additionally, due to the adenine-like structures of
NB001 and SQ22536 concerns have been raised that adenosine-based molecules could interfere with other
cellular processes such as DNA synthesis [19, 20, 23] limiting the advancement of P-site inhibitors for further
development. Finally, our team has previously reported a chromone-based inhibitor, ST034307, with single-
digit micromolar potency versus AC1 (cellular IC50 value of 2.3 µM), no inhibition versus AC8, and efficacy
against allodynia in a phenotypic mouse model of inflammatory pain [24]. Unfortunately, the potency of the
chromone-based molecule was not able to be further optimized through medicinal chemistry efforts
(unpublished results). Additionally, the trichloromethyl moiety can act as an electrophilic center leading to

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undesired toxicity via non-specific covalent modification of off-target proteins. Attempts to remove this
functionality led to complete loss of activity versus AC1 and, therefore, the scaffold was not pursued further.
These representative AC1 inhibitors is not an exhaustive list and have significant drawbacks, however, the
preliminary in vivo data suggest AC1 is a viable target for the treatment of chronic pain. Therefore, our team
has embarked upon an AC1 inhibitor discovery program.
To this end, we carried out a phenotypic cell-based high-throughput screen in human embryonic kidney
(HEK) cells stably expressing AC1 to identify small molecules with the ability to reduce AC1-mediated
production of cAMP upon stimulation with the Ca2+ ionophore, A23187 (manuscript in preparation to describe
the screen and results). This screen identified the 1,3,4-oxadiazole containing molecules, represented by hit 1,
as a promising scaffold that reduced cAMP levels in cells stably expressing AC1 upon stimulation with
Ca2+/calmodulin by more than 90% at a single dose of 10 µM compared to DMSO-treated controls. Hit
compound 1 was synthesized by our team (described below) and validated as an inhibitor of AC1-mediated
cAMP production with a cellular IC50 value of 3.4 ± 1.0 µM in AC1 expressing cells. This molecule was also
observed to inhibit AC8 activity with an IC50 of 19 ± 5 µM and displayed approximately 5.5-fold selectivity for
AC1 over AC8 and data from the resynthesized molecule was comparable to the commercially available dry
powder. Our team then designed and synthesized novel analogs for this 1,3,4-oxadiazole series with the aim to
improve potency and/or selectivity in the HEK-AC1 and AC8 cell models. A prioritized analog was selected to
test for in vivo efficacy in an inflammatory pain model. The results of these studies are presented below.
2. Results
2.1.
Structure-activity relationship (SAR) strategy
The 1,3,4-oxadiazole series emerged as a hit scaffold from a cell-based cAMP accumulation assay in
HEK cells stably expressing AC1. The
screen identified 41 hits that contained the
1,3,4-oxadiazole core and displayed more
than 90% inhibition of Ca2+/calmodulin-
stimulation of AC1 activity when tested at a
single dose of 10 µM compared to DMSO
R1 =
A
B
C
D
E
Figure 2. R1 functional groups prioritized to be part of SAR optimization
design strategy of the 1,3,4-oxadiazole scaffold.

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controls. The scaffold was not flagged in cheminformatics filters that identify problematic PAINS [25, 26] or
aggregators [27]. Therefore, dry powders were reordered of twelve hit compounds, and dose-response curves
were generated to determine the potency and maximal efficacy of the compounds against both AC1 and AC8
(IC50 values for the reordered dry powders reported in Supplementary Table S1). All re-tested hit compounds
inhibited Ca2+/calmodulin-mediated cAMP production in both AC1 and AC8 containing cells in a dose-
dependent manner, with IC50 values ranging from 2 µM to 10 µM against AC1 and 5 µM to 21 µM against AC8.
The goals of the SAR optimization campaign were then prioritized in the following order: 1) improve potency
toward AC1-mediated cAMP production, 2) improve selectivity over AC8-mediated cAMP production, 3)
maintain low cellular toxicity, and 4) improve physicochemical properties. Due to convenience of synthesis and
diversification of R2 substituents at the final synthetic step (Scheme 1), our medicinal chemistry approach
focused on only five R1 group substituents identified in the original hit set with varying levels of cellular potency
against AC1 and/or selectivity over AC8 (Figure 2). Initial rounds of SAR combined common R2 substituents
with the prioritized R1 moieties to create nearest-neighbor analogs for comparison of activity data. Then SAR
was continued with further diversification to R2 and cellular AC1 and AC8 cAMP accumulation data was used
to assess analogs for futher iterative design cycles. In total 65 novel analogs were designed and synthesized
using the protocol described below.
2.2.
Chemistry
All 1,3,4-oxadiazole derivatives were synthesized using the same 3-step condensation and amide bond
formation protocol (Scheme 1). First, either substituted aryl or cycloalkyl aldehydes (2) were reacted with
semicarbazide hydrochloride salt and sodium acetate in methanol/water solution (1:1) at room temperature
(RT) for up to 60 minutes at which point a white precipitate was formed to create a suspension. This precipitate
was filtered out by vacuum filtration to produce the desired hydrazine carboxamide intermediate (3) typically as
a white solid and in good yield. Intermediate 3 was subjected to halogen-mediated oxidative cyclization
adapted from a published protocol [28] using bromine and sodium acetate in acetic acid while heating to 60 °C
for 2 hours to produce the desired 2-amino-1,3,4-oxadiazole intermediate 4 in good yields. Intermediate 4 was
then used as the nucleophile in an amide bond formation reaction with various substituted benzoic acids or
acyl chlorides (5) to form the final products represented by 6 - 70. All analogs were fully characterized for
identity by proton and carbon NMR and mass spectrometry. All analogs were analyzed for purity using high-

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performance liquid chromatography (HPLC) and were required to meet or exceed 95% purity before any
biological testing of the molecules.
2A-E
3A-E
4A-E
5
1, 6 - 70
Scheme 1. Representative synthetic protocol for 1,3,4-oxadiazole analogs Reagents and conditions: a)
semicarbazide HCl (1.0 eq), sodium acetate (2.0 eq), MeOH:water (1:1), RT, 30 – 60 min, 86 – 98%; b) Br2 (1.1
eq), sodium acetate (2.0 eq), acetic acid, 60 °C, 2 hr, 70 – 95%; c) if X = OH: oxalyl chloride (1.1 eq), DMF (1
drop), DCM, 0 °C – rt, 1 hr, crude product carried into next step; d) if X = Cl; pyridine, rt, 16 – 24 hr, 2 – 77%. R1
(A-E) groups depicted in Figure 2.
2.3.
AC1 and AC8 activity
The ability to inhibit Ca2+/calmodulin-stimulated production of cAMP mediated by both AC1 and AC8
was evaluated for all analogs by measuring cAMP accumulation in HEK cells stably expressing each
respective AC. Dose-response curves were generated for each analog starting at 30 µM concentration, and
IC50 values were calculated for molecules that showed > 50% inhibition at the 30 µM dose. All the other
analogs that exhibited < 50% inhibition in the respective cell lines were labeled with IC50 values as not
determined (ND) in data tables with the observed % inhibition at 30 µM reported in parentheses.
Table 1. SAR data for A-series
a
b,c
b,c
Compd
R1
R2
AC1 IC50
AC8 IC50
Fold selectivityd
1
6
7
8
9
10
11
12
13
14
15
16
A
A
A
A
A
A
A
A
A
A
A
A
H
3-CF3
4-Br
3.4 ± 1.0
1.6 ± 0.2
12 ± 3
19 ± 5
5.6
4.9
2.23
1.2
5.5
8.1
7.4
> 4.7
3.7
4.2
2.4
7.9 ± 3.0
27 ± 0.2
12 ± 6
9.4 ± 2.0
25 ± 4
25 ± 6
ND (34%)
22 ± 4
4-OCH(CH3)2
3-SCH3
3-OCH3
4-CH3
4-OCH3
4-F
3-F
4-Cl
4-CH2CH3
10 ± 7
1.7 ± 0.7
3.1 ± 0.4
3.4 ± 1.0
6.4 ± 1.0
5.9 ± 1.0
1.0 ± 0.3
1.5 ± 1.0
1.2 ± 0.2
4.2 ± 0.2
3.6 ± 0.4
2.0 ± 0.4
1.7
a R1 modifications corresponding to each letter shown in Fig. 2. b IC50 values (µM) represent the mean and
SEM for three independent experiments. c If analog exhibits > 50% inhibition at 30 µM concentration, the
IC50 value was reported. If molecules exhibit < 50% inhibition then the data is listed as not determined
(ND) with the percentage inhibition shown in parentheses, ND (XX%). d Fold selectivity = AC8 IC50 / AC1
IC50. If the AC8 IC50 value of the compound is ND, then 30 µM used for the AC8 IC50 value to calculate the
fold selectivity that is shown with a > symbol.
Beginning with hit 1 our design strategy was to maintain the 5,6,7,8-tetrahydronaphthalene R1
substituent (A-series) while modifying the phenyl functional group at R2. Most modifications made to the R2
component resulted in low-to-mid single digit micromolar potencies (Table 1) with selectivity over AC8 ranging

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from 1.2 – 8.1-fold. In particular, the 3-CF3 (6, AC1 IC50 = 1.6 ± 0.2 µM), 3-SCH3 (9, 1.7 ± 0.7 µM), 3-F (14, 1.0
± 0.3 µM), 4-Cl (15, 1.5 ± 1.0 µM), and 4-CH2CH3 (16, 1.2 ± 0.2 µM) were slightly more potent against AC1-
mediated cAMP production compared to 1 (3.4 ± 1.0 µM). The 3-substituted analogs (6, 9, and 14) displayed
moderate selectivity over AC8, with values of 4.9, 5.5, and 4.2, respectively. However, selectivity was reduced
by at least half in the 4-substituted analogs to 2.4 for the 4-Cl (15) and 1.7 for the 4-CH2CH3 (16) analogs.
Compared to the hit, analogs 10 and 11 were equipotent versus AC1 (IC50s = 3.1 ± 0.4 µM, and 3.4 ± 1.0 µM)
and more selective over AC8 (8.1 and 7.4-fold selectivity, respectively). Rounding out the SAR for the A-series
are four 4-position modifications that displayed at least 3.5-fold reduced activity against AC1-mediated cAMP
production and reduced selectivity over AC8 compared to the hit. Two pairs of analogs in this series illustrate
the apparent preference for 3-substituted over 4-subsituted derivatives. The pair represented by 10 and 12 (3-
OCH3 and 4-OCH3) displayed preference for the meta over para position in AC1 potency. Selectivity was more
difficult to assess given that for 12 the IC50 value against AC8 activity was not determined; however, when
using % inhibition as a guideline, the selectivity is within the same value as 10. The second pair represented by
analogs 14 and 13 (3-F and 4-F) also clearly indicated a preference for the 3-position substitution in AC1
potency (3-F was 5.9 times more potent than 4-F) with slightly improved selectivity over AC8. Based on the A-
series analogs it appears that R2 modifications at the meta-position were generally beneficial for either AC1
potency and/or selectivity over AC8.
Table 2. SAR data for B- and C-series
a
b,c
b,c
Compd
R1
R2
AC1 IC50
AC8 IC50
Fold selectivityd
17
18
19
20
21
22
23
24
25
26
27
28
29
B
B
B
B
B
B
B
B
C
C
C
C
C
H
3-SCH3
3-CF3
31 ± 3
ND (24%)
ND (37%)
32 ± 12
ND (39%)
23 ± 2
ND (18%)
ND (25%)
14 ± 13
ND (18%)
ND (41%)
ND (34%)
ND (35%)
ND (10%)
>1
> 3.2
6.3
> 4
6.8
> 3.1
> 2.5
2.6
> 1.1
> 3.9
> 2.7
> 2.7
> 2
9.5 ± 1.0
5.1 ± 0.7
7.5 ± 0.7
3.4 ± 0.6
9.6 ± 3.0
12 ± 4
5.3 ± 0.4
28 ± 3
7.7 ± 1.0
4-OCH(CH3)2
4-Br
3-OCH3
4-CH3
3,4-di-CH3
H
4-Br
3-SCH3
4-OCH(CH3)2
4-OCH3
11 ± 2
11 ± 2
15 ± 2
a R1 modifications corresponding to each letter shown in Fig. 2. b IC50 values (µM) represent the mean and
SEM for three independent experiments. c If analog exhibits > 50% inhibition at 30 µM concentration, the
IC50 value was reported. If molecules exhibit < 50% inhibition then the data is listed as not determined
(ND) with the percentage inhibition shown in parentheses, ND (XX%). d Fold selectivity = AC8 IC50 / AC1
IC50. If the AC8 IC50 value of the compound is ND, then 30 µM used for the AC8 IC50 value to calculate the
fold selectivity that is shown with a > symbol.

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B-series analogs maintained the 2,3-dihydro-1,4-dioxin-6-yl moiety as the R1 while altering the R2
(Table 2). In total only eight analogs were synthesized containing a subset of the same R2 modifications
utilized in the A-series. When comparing nearest neighbor analogs between the A- and the B-series the
5,6,7,8-tetrahydronaphthalene generally outperformed the 2,3-dihydro-1,4-dioxin-6-yl substituent versus AC1
with the exceptions of the 4-Br (21 compared to 7) and the 4-OCH(CH3)2 (20 compared to 8), in which the B-
series derivatives were slightly more potent against AC1 activity. The H, 3-CF3, 3-SCH3, 3-OCH3, and 4-CH3 at
the R2 position in the B-series were less potent than their A-series counterparts. The B-series analogs,
however, displayed reduced potency against AC8-mediated cAMP production, but it is difficult to conclude if
this reduction arose from a gain in selectivity or just the overall reduced AC potency in general.
C-series analogs containing a 2-thiophene at the R1 position (Table 2) also proved to be inferior to the
A-series analogs. Five analogs were synthesized and tested, and all displayed modest activity on both AC1
and AC8. Therefore, this series was deprioritized from further SAR optimization.
Table 3. SAR data for D-series
a
b,c
b,c
Compd
R1
R2
AC1 IC50
AC8 IC50
Fold selectivityd
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
H
4-Br
3-CF3
29 ± 10
10 ± 1
5.0 ± 0.8
7.3 ± 2.0
6.5 ± 1.0
15 ± 4
13 ± 3
16 ± 4
11 ± 3
17 ± 2
ND (12%)
8.2 ± 2.0
8.9 ± 1.0
2.8 ± 0.6
2.3 ± 0.4
ND (8%)
ND (33%)
29 ± 1
> 1
> 3
5.8
4-CH3
ND (31%)
ND (42%)
ND (23%)
ND (20%)
ND (38%)
ND (35%)
ND (14%)
ND (13%)
ND (32%)
ND (11%)
7.5 ± 1.0
7.0 ± 1.0
> 3.6
> 4.6
> 2
> 2.3
> 1.9
> 2.7
> 1.8
-
3,4-di-CH3
3-OCH3
4-OCH3
3-SO2-piperidine
3-F
4-F
2-F
4-CH2CH3
4-Cl
4-phenyl
3-phenyl
> 3.6
> 3.4
2.7
3.0
a R1 modifications corresponding to each letter shown in Fig. 2. b IC50 values (µM) represent the mean and
SEM for three independent experiments. c If analog exhibits > 50% inhibition at 30 µM concentration, the
IC50 value was reported. If molecules exhibit < 50% inhibition then the data is listed as not determined
(ND) with the percentage inhibition shown in parentheses, ND (XX%). d Fold selectivity = AC8 IC50 / AC1
IC50. If the AC8 IC50 value of the compound is ND, then 30 µM used for the AC8 IC50 value to calculate the
fold selectivity that is shown with a > symbol.
The D-series consists of 15 analogs carrying a 4-F-phenyl substituent at the R1 position (Table 3). In
general, the D-series analogs still underperformed compared to their A-series counterparts. There were three

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new R2 functional groups introduced into these analogs that were not tested in the A-series. Analog 40
contained the first ortho-substitution (2-F) of all the series discussed, and it displayed a significant decrease in
potency against AC1 activity. Additionally, 4-phenyl (43) and 3-phenyl (44) derivative performed the best in the
D-series (AC1 IC50 values of 2.8 ± 0.6 µM and 2.3 ± 0.4 µM, respectively). However, while these molecules
exhibited the best potency toward AC1 it was coupled by a reduction in selectivity over AC8.
Finally, 13 analogs make up the E-series molecules containing a cyclohexyl functional group at the R1
position (Table 4). The change from aromatic to cycloalkyl functionality provided molecules that were generally
equipotent to the B-, C-, and D-series and still lagged behind the A-series. However, the E-series appeared to
outperform other series when comparing the AC8 potencies. Four analogs from this series (45, 51, 52, and 54)
displayed single-digit % inhibition values against AC8 when tested at 30 µM, compared to only one such
analog in the previous four series (30). Additionally, direct comparison of the IC50 values of the 3-F containing
analogs (14, 38, and 55), indicates the E-series was the least potent against AC8 activity. This trend did not
correlate with overall AC1 potency as analogs 38 and 55 were equipotent against this isoform but 55 was
significantly less potent against AC8. This overall trend is also observed when comparing analogs containing
4-OCH3 and 4-CH3 at the R2 position. However, this trend does not hold when comparing the AC8 potency
amongst the 3-SCH3 analogs. Notably, the 3-phenyl derivative was the most potent E-series molecule against
both AC1 and AC8 (IC50s = 1.4 ± 0.2 and 1.4 ± 0.4 µM, respectively).
Table 4. SAR data for E-series
a
b,c
b,c
Compd
R1
R2
AC1 IC50
AC8 IC50
Fold selectivityd
45
46
47
48
49
50
51
52
53
54
55
56
57
E
E
E
E
E
E
E
E
E
E
E
E
E
H
4-Br
3-SCH3
3-CF3
25 ± 2
ND (2%)
25 ± 8
ND (43%)
33 ± 7
> 1.2
2.9
> 4.5
> 3.6
5.4
8.6 ± 3.0
6.6 ± 3.0
6.0 ± 0.6
8.1 ± 2.0
10 ± 1
22 ± 3
16 ± 2
7.3 ± 3.0
13 ± 1
3,4-di-CH3
4-OCH(CH3)2
4-OCH3
4-CH3
44 ± 6
ND (35%)
ND (6%)
ND (1%)
ND (44%)
ND (7%)
ND (11%)
ND (10%)
1.4 ± 0.3
> 3
> 1.4
> 1.9
> 4.1
> 2.3
> 2.7
> 2.1
1
4-CH2CH3
4-F
3-F
4-Cl
3-phenyl
11 ± 2
14 ± 2
1.4 ± 0.2
a R1 modifications corresponding to each letter shown in Fig. 2. b IC50 values (µM) represent the mean and
SEM for three independent experiments. c If analog exhibits > 50% inhibition at 30 µM concentration, the
IC50 value was reported. If molecules exhibit < 50% inhibition then the data is listed as not determined
(ND) with the percentage inhibition shown in parentheses, ND (XX%). d Fold selectivity = AC8 IC50 / AC1
IC50. If the AC8 IC50 value of the compound is ND, then 30 µM used for the AC8 IC50 value to calculate the
fold selectivity that is shown with a > symbol.

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After design and synthesis of 57 analogs our team chose three R1 substituents (A, D, and E) to pair
with combined modifications to the R2 side of the molecule (Table 5) in an attempt to merge AC1 potency
driving functional groups with AC8 selectivity driving functional groups (58 – 66). We chose to incorporate a
meta-substituted CF3 in all combined R2 side modifications as this functional group was consistently among the
top potent molecules for each series. We then paired the CF3 with one other modification either meta-
substituted methyl or methoxy or para-substituted methyl. These functional groups were chosen because they
consistently provided reduced AC8 potency. The resulting analogs displayed no improvement to AC1 potency
or selectivity over AC8. However, one molecule (61) containing the 4-fluorophenyl R1 coupled with 3-CH3-5-
CF3 had an AC1 IC50 value of 1.4 ± 0.2 µM with 2.9-fold selectivity over AC8.
Four additional analogs that did not fit within any of the five series described for R1 modification are
reported in the supplemental information (Table S2). Two analogs contain a R1 = phenyl with R2 as either 4-
isopropxy (67) or 3-CF3 (68) while the other two analogs have a 2-furyl moiety at the R1 and either H (69) or 4-
isopropoxy (70). All analogs had reduced activity versus both AC1 and AC8 and were not expanded on further
as full series for SAR exploration.
Table 5. SAR for combined modifications
a
b,c
b,c
Compd
R1
R2
AC1 IC50
AC8 IC50
Fold selectivityd
58
59
60
61
62
63
64
65
66
A
A
A
D
D
D
E
E
E
3-CH3-5-CF3
3-CF3-4-CH3
3-OCH3-5-CF3
3-CH3-5-CF3
3-CF3-4-CH3
3-OCH3-5-CF3
3-CH3-5-CF3
3-CF3-4-CH3
3-OCH3-5-CF3
9.1 ± 3.0
26 ± 11
2.4 ± 0.1
13 ± 2
10 ± 0.4
4.1 ± 0.4
9.0 ± 5.0
29 ± 1
15 ± 2
7.0 ± 1.0
9.0 ± 1.0
0.26
0.5
1.1
2.9
1.9
5.8
2.9
1.9
1.9
9.4 ± 1.0
1.4 ± 0.2
4.6 ± 2.0
5.0 ± 0.8
9.6 ± 1
3.7 ± 1.0
4.8 ± 1.0
a R1 modifications corresponding to each letter shown in Fig. 2. b IC50 values (µM) represent the mean and
SEM for three independent experiments. c If analog exhibits > 50% inhibition at 30 µM concentration, the
IC50 value was reported. If molecules exhibit < 50% inhibition then the data is listed as not determined
(ND) with the percentage inhibition shown in parentheses, ND (XX%). d Fold selectivity = AC8 IC50 / AC1
IC50. If the AC8 IC50 value of the compound is ND, then 30 µM used for the AC8 IC50 value to calculate the
fold selectivity that is shown with a > symbol.
Selectivity versus two other AC isoforms was evaluated for a subset of analogs. AC1 and AC8
represent group 1 ACs that are stimulated by Ca2+/calmodulin binding. We explored activity of the oxadiazole
analogs against a group 2 AC (AC2, stimulated by protein kinase C) and a group 3 AC (robustly stimulated by
forskolin) to assess the scaffolds effects on other AC group subtypes. Analogs 6, 7, 11, 21, 23, 33, and 37
were tested at single-dose (30 µM, n = 3) and compared to DMSO-controls (normalized to 100% activity). The

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seven analogs tested exhibited roughly 2 – 4-fold increases in cAMP levels from AC2 and 1 – 2-fold increase
in cAMP levels from AC5 (Table S5). This data suggests that the oxadiazole series potentiates AC2-mediated
cAMP production and to a lesser degree AC5-mediated cAMP production.
We also compared the activity of analog 6 with SQ22536 in a novel cell model [29]. HEK-AC∆3/6 cells
transiently transfected with AC1 [29] were incubated with test compounds in the presence of three
concentrations of forskolin (3, 10 and 30 µM). Compound 6 displayed increased inhibitory activity with
increasing concentrations of forskolin (Fig. S1) similar to the P-site inhibitor, SQ22536. However, further
pharmacological testing is necessary to validate this hypothesis and will be carried out in future work.
To assess how the molecules may interact with the cellular target we tracked the efficiency metric
Lipophilic Ligand Efficiency (LLE = pIC50 – Log P) [30], which provides an indication of the degree of entropic
driven (hydrophobic) binding [31, 32]. Our analogs are not at “lead” status, but rather at the beginning stages of
hit optimization, and possess LLE values ranging from -1.14 up to 2.90 (Table S3) indicating the molecules
likely bind predominantly via hydrophobic interactions. To determine LLE for each molecule the Log P values
for the oxadiazole analogs were calculated using MarvinSketch (version 15.9.14, ChemAxon,
http://www.chemaxon.com).
2.4.
Docking of 1,3,4-Oxadiazole Series into an AC1 Homology Model
The structure of transmembrane ACs consist of a N-terminus, two membrane domains of six-spanning
helices and two cytosolic domains (C1 and C2 domains) [33]. The ATP and forskolin binding sites are found at
the interface of the C1 and C2 domains of the AC [34]. Due to the lack of a human transmembrane AC crystal
structure we constructed a homology model of the C1 and C2 domains of human AC1 based on the X-ray
structures of the cytosolic
B.
domains of other two other
mammalian ACs (PDB ID:
1AZS and 3C16) – similar to a
previous AC homology model
Figure 3. Predicted binding mode of ligand 16 in ATP-
developed by our team [35]. We
then utilized this model to
binding site of AC1. A. 3D representation of binding
mode. Hydrogen-bonding residues GLY 314 (C1
domain) and SER 1007 (C2 domain) are shown in
stick mode together with ligand. The surface of the
binding site is colored-coded according to hydrophobic
scale (white: hydrophilic, red: hydrophobic). B. 2D
interaction scheme representing interacting residues
with ligand.

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perform docking experiments with the analogs into both the forskolin and ATP binding sites.
Analyses of the docking results revealed significant variability among the top poses for the 60 ligands.
Furthermore, for most ligands, similar scores for different binding poses were identified, which is not surprising
considering the potency of the ligands is in the µM-affinity range and the significant size of the extended
binding pocket. Thus, we performed clustering over all poses for all the analogs to identify the most prevalent
binding mode represented by the largest cluster of poses.
The largest cluster in the forskolin binding site contained 244 poses from 32 ligands. For the ATP-
binding site, two large clusters were identified with 285 poses (47 ligands) and 265 poses (46 ligands),
respectively. As the average docking score for the largest cluster was also the most favorable among all three
large clusters, the poses within this ATP-binding site cluster were considered to represent the most likely mode
of interaction of the oxadiazole series with AC1 if the molecules were to engage the target in cells. Figure 3
highlights the dominant interactions of a representative compound 16 with AC1 in the predicted binding mode.
The 3-dimensional representation indicates the binding site is mostly hydrophobic (Fig. 3A). The R2
substituents (depicted by an ethyl group in the figure) fits into a shallow hydrophobic pocket. The binding pose
also suggests that only one meta-position can accommodate a substitution without sacrificing binding. If the
molecules engage AC1 in cells, and this binding mode is accurate, this may explain why the 3,5-substituted
analogs did not provide improved potencies. The 2-dimensional interaction map (Fig. 3B) suggests the central
hydrophilic core interacts via hydrogen bonds with the backbone of GLY 314 in the C1 domain and with the
hydroxyl group of SER 1007 of the C2 domain. The terminal hydrophobic ligand moieties are enclosed by
hydrophobic regions including residues ILE 309, VAL 310, GLY 311, PHE 312, VAL 397, ALA 431, ALA 432
(all in the C1 domain) for the R2 substituents and LEU 315, GLY 351 (both in the C1 domain), ILE 922 and
MET 927 (both in the C2 domain) for the R1 substituents. The docking data supports the hypothesis that these
molecules bind to their targets via predominantly hydrophobic interactions. Additionally, the data indicates that
there is a higher probability the 1,3,4-oxadiazole series would bind within the ATP-binding site of AC1 rather
than the forskolin binding site.
2.5.
Cell viability

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In parallel to the assessment of the effects of the compounds on cAMP accumulation in the HEK cell
lines stably expressing AC1 and AC8, the toxicity of the molecules was also examined. Upon a single dose
(30 µM), the viability of the cells was determined after incubation for 90 minutes with the analogs using a
luminescent reporter for the metabolic marker ATP, and % cell viability determined compared to DMSO (100%
viable) and Triton-X (0% viable) treated controls (Table S4). The results indicated little to no potential toxic
liability for the 1,3,4-oxadiazole series as the overwhelming majority of molecules exhibited cell viability greater
than 80%. Only three of the reported analogs displayed lower cell viability below 80% (44 – 71%; 68 – 78%; 69
– 73%).
2.6.
inflammatory pain.
Based on the pharmacological
In vivo efficacy in a mouse model of
profile combining low micromolar potency,
moderate selectivity over AC8, and mid-
range LLE compared to similarly potent
analogs we next assessed the ability of (61)
to provide analgesia in an inflammatory
pain model in C57BL/6 mice. Intraplantar
injection of CFA (Complete Freund’s
adjuvant) induced robust mechanical
hypersensitivity the following day in the
Figure 4. Efficacy of 61 in mouse inflammatory pain model. A. Time
course of induction and treatment of inflammatory mechanical
hypersensitivity: Male C57BL/6 mice (n=7) were injected with CFA in the
ipsilateral paw (Ips, closed symbols) and paw sensitivity was measured in
the ipsilateral and contralateral paw (Con, open symbols). On day 2 mice
received an i.p. injection of vehicle (veh, triangles) or 5 mg/kg 61
(squares). B. Statistical analysis of drug/vehicle treatment (filled bars) on
CFA induced mechanical hypersensitivity (open bars) of contralateral and
ipsilateral paws. **P < 0.01 (Two-way repeated measure ANOVA with
Bonferroni test). The data shown represent the average and SEM of each
group of measurements.
ipsilateral paw (Fig. 4A, CFA day2, filled symbols) as evidenced by mice withdrawing their paw upon tactile
stimulation with von Frey filaments of lower diameter and force than when stimulating the contralateral paw
(Fig. 4A, day2, open symbols). Following the measurement of CFA induced hypersensitivity on day 2, mice
received a systemic intraperitoneal (i.p.) injection of 5 mg/kg of compound 61 or vehicle and mechanical
sensitivity was recorded 30 minutes post-injection. Two-way repeated measure ANOVA revealed a significant
effect of treatment (F1,36 = 113.3, p < 0.0001) and interaction (F3,36 = 4.272, p = 0.011). Post-hoc Bonferroni
analysis revealed that analog 61 (t = 3.17, p < 0.05) but not vehicle (t = 0.244, p > 0.05) significantly
attenuated, the hypersensitivity in the ipsilateral paw without significantly altering response in the contralateral

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paw (Fig. 4B). The effect of 61 was short-lived and mice regained the hypersensitivity in their ipsilateral paw
the following day (data not shown).
3. Discussion
With the recent genetic and pharmacologic evidence suggesting inhibition of AC1 activity may be a
viable strategy for treatment of chronic inflammatory and neuropathic pain, our team set out to discover novel
small molecules that may be used to validate this strategy for pain therapy. A high-throughput screen in
search of small molecules that reduce Ca2+/calmodulin-mediated cAMP accumulation in HEK cells stably
expressing AC1 was carried out. The 1,3,4-oxadiazole series was identified as a hit scaffold (represented by 1)
and its activity was validated in further cellular cAMP accumulation assays against AC1 and AC8. We then
embarked upon a SAR optimization campaign to improve potency toward AC1 and/or selectivity over AC8. In
total, 65 new 1,3,4-oxadiazole containing analogs were designed and synthesized incorporating five common
R1 elements observed in the HTS hits. During optimization there were modest gains in potency versus AC1
and selectivity over AC8, albeit not to the desired sub-micromolar level for AC1 potency or the > 10-fold
selectivity over AC8. Nonetheless, there were some SAR trends that were observed from this structural class.
In particular, SAR at the R2 position revealed that substitution with the same functional group at the meta-
position was favored over the para-position for AC1 potency as illustrated by comparison of 10 (3-OCH3) vs 12
(4-OCH3), 14 (3-F) vs 13 (4-F), and 38 (3-F) vs 39 (4-F). This trend could be observed in the combined
modifications as well in which 58 (3-CH3-5-CF3) outperformed 59 (3-CF3-4-CH3) and 61 (3-CH3-5-CF3)
outperformed 62 (3-CF3-4-CH3). However, in the combined modification analogs there was an exception to this
observation in which the 3,4-di-substitution was modestly more potent than the 3,5 when combined with the
cyclohexyl moiety at the R1 (65 and 64, respectively), suggesting that the favored meta- substitution is best
when combined with an appropriate R1 substitution. The preference of functional group at on the R2 side of the
molecule is less clear. There appears to be a slight preference for halogenated moieties especially fluorine
containing substituents. However, two series (D and E) had the introduction of an aromatic phenyl substituent
at R2, and these typically boosted AC1 potency compared to the rest of the analogs within those series.
There were clear preferences in the R1 functional groups that could be observed during our SAR
analysis. Analogs containing either 2,3-dihydro-1,4-dioxin-6-yl (B-series) or 2-thiophene (C-series) consistently
underperformed in terms of AC1 potency when compared to their counterparts from the A, D, and E-series.

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Thus, analogs for the B and C series were deprioritized in our design. Direct comparison of R2 moieties
between series, R1 = 5,6,7,8-tetrahydronaphthalene (A-series) consistently outperformed the 4-F-phenyl (D-
series) and cyclohexyl (E-series) in terms of AC1 potency. The A-series also produced the most selective
molecules when comparing fold-selectivity over AC8. Given that we only determined IC50 values for molecules
that displayed ≥ 50% inhibition at 30 µM we were not able to determine a large number of IC50 values against
AC8, thus comparison of selectivity is partially incomplete without full IC50 values against both isoforms. When
comparing % inhibition of AC8 across each series the cyclohexyl R1 may potentially display similar selectivity
toward to AC1, however, this is only an estimation and appears to come at the detriment of AC1 potency.
The 5,6,7,8-tetrahydronaphthalene proved to be the best when comparing analogs between series,
however, to our surprise the selectivity of this R1 moiety was not retained when paired with the combined R2
modifications suggesting the combination of R1 with specific R2 moieties are necessary to maintain or improve
AC1 selectivity over AC8. In fact, the most potent and selective analogs in the combined sets belong to the R1
= 4-F-phenyl (D-series), however, most analogs exhibited similar AC1 and AC8 potency. Thus, it is difficult to
deduce any SAR trends from these combined modifications without further investigation.
Even though there are some trends to be observed the overall SAR is relatively “flat” as almost all
analogs remained in the 1 to 30 µM range for AC1 potency. To this point we have not tested any analogs that
exhibited significant improvement in AC1 potency or selectivity attributable to a particular substituent.
Alternatively, only two analogs exhibited a reduction of potency toward AC1 beyond 30 µM. This flat SAR may
indicate that the molecules may bind to the cellular target predominantly via non-specific hydrophobic
interactions. The relatively high LogP for this scaffold series would support this assertion as increased LogP
tends to result in an increased interaction with hydrophobic protein surfaces [36]. In fact, the A-series of
molecules, containing the 5,6,7,8-tetrahydronaphthalene at the R1 position, were on average the most potent
toward AC1 (average AC1 IC50 value of 4.3 µM) and possessed the highest average LogP (average LogP =
5.65, calculated using ChemDraw v17). As described above, tracking the efficiency metric Lipophilic Ligand
Efficiency (LLE = pIC50 – Log P) [30], provides an indication of the degree of entropic driven (hydrophobic)
binding [31, 32]. Acceptable LLE values for lead candidates for clinical evaluation is > 5, with a positive
correlation to increased enthalpic (specific, polar interactions) binding energy [30]. Our analogs are not yet to
lead status, but rather at the beginning stages of hit optimization, and possess LLE values ranging from -1.14

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up to 2.90. These values indicate the molecules most likely bind predominantly via hydrophobic interactions.
While this is not an attribute that disqualifies these molecules from further development it does indicate that
more emphasis should be placed on incorporating hydrogen-bond donors and acceptors capable of forming
polar interactions within the active site. Nonetheless, the 1,3,4-oxadiazoles still represent an expansion of the
known chemical space for AC1 inhibitors and there are clear areas that can be targeted for improvement on
this scaffold in the next phase of the project.
After evaluation of all analogs in the cellular models we chose to test one analog from our study in an in
vivo mouse model to evaluate for its efficacy to attenuate inflammatory pain. While it would be desirable to
have a highly selective AC1 molecule, the AC1 inhibitor NB001 – that has moderate selectivity for AC1 over
AC8 (10-fold) – has displayed analgesic effects in various models of neuropathic and inflammatory pain [22,
37, 38]. Therefore, we selected analog 61 due to its low-µM potency against cellular AC1 activity (IC50 value of
1.4 µM), moderate selectivity over AC8 (2.9-fold), and mid-range LLE compared to the entire analog set (1.29).
The molecule dosed at 5 mg/kg i.p. provided a modest, but significant analgesic effect in the CFA model as
mice exhibited a significantly reduced allodynia compared to the vehicle control. A higher dose may provide a
greater analgesic effect, however, due to the limited solubility of the molecule, we were not able to increase
dosage. The scaffold also potentiates cAMP production from AC2 and AC5, however, these AC isoforms are
not directly involved in signaling pathways associated with chronic pain. Therefore, we propose that the in vivo
efficacy observed in this experiment is a result of AC1 inhibition and not related to the effects at AC2 or AC5.
Regardless, the preliminary results for this scaffold in the treatment of chronic pain are promising and warrant
further investigation.
Because the Ca2+/calmodulin-mediated AC1 and AC8 cAMP production is the output for evaluation of
our molecules we should stress that this is a cellular assay and does not necessarily indicate the molecules
are engaging AC1 or AC8 in the cells. The possibility remains that these molecules may be leading to
decreased cAMP production via an alternative pathway, similar to what has been suggested for NB001 [23].
Additionally, simply expressing and purifying recombinant human AC isoforms for in vitro binding analysis
remains extremely difficult. Even though direct target engagement has not been completely verified in our
system at this point we chose to make an educated suggestion as to the potential AC1 binding site if
engagement is indeed taking place in the cells. The AC1 homology model suggests the analogs would

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preferentially bind into a hydrophobic region of the ATP-binding site, which aligns with the observation that
increased hydrophobicity provides the greatest AC1 potency. However, further experiments are necessary to
validate these proposed binding interactions. Nonetheless, the desired goal is to reduce cAMP levels mediated
through AC1 activity in the cellular context, and these HEK cell lines express the AC1 or AC8 isoforms at much
higher levels relative to other AC isoforms. Therefore, the reduction in cAMP levels can be attributed mostly to
inhibition of the respective isoform, whether it be a result of direct engagement with AC1 or through an
alternative mechanism. Further studies will be done to pharmacologically evaluate this series, validate the
intracellular target and assess activity against other AC isoforms as part of future research.
4. Conclusions
Our team reports a novel class of 1,3,4-oxadiazole containing compounds that inhibit Ca2+/calmodulin-
stimulated cAMP production mediated by AC1 with varying selectivity over AC8. An SAR study has identified
functional group substitution patterns that provide either greater potency toward AC1 or greater selectivity for
AC1 over AC8. Primarily, substitution at the meta-position of the R2 side were consistently more potent than
the respective para-substitution. In addition, and among the substitutions investigated, halogen containing
functional groups also seemed to be preferred. We found that R1 = 5,6,7,8-tetrahydronaphthalene (A-series)
was the most potent moiety on the left-hand side of the molecule and this may be attributed to increased non-
specific, hydrophobic binding rather than productive interactions with the protein surface. Future medicinal
chemistry on this class will focus on introducing more polar functionalities to the molecule. Because our initial
SAR maintained R1 substitutions that were present in the HTS hit deck, future diversification at this position
may provide improved physicochemical and inhibitory properties. Nonetheless, one molecule, 61, was tested
for efficacy in a mouse model of inflammatory pain and was observed to provide a modest, yet significant,
analgesic effect compared to controls consistent with the current hypothesis that AC1 is a viable target to treat
chronic pain. Since the series was not fully selective toward AC1 but also exhibited potency toward AC8 further
exploration into potential side effects in learning and memory may be warranted. Previously reported AC1
inhibitors, such as NB001 and ST034307, have exhibited either greater potency toward AC1 and/or selectivity
over other AC isoforms with greater efficacy in animal pain models than our molecules [22, 24]. However, both
molecules have drawbacks with respect to their potential for further development while the 1,3,4-oxadiazole
scaffold has proven to be amenable for optimization. Therefore, based on our initial SAR and in vivo results,

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our team remains enthusiastic about the potential of the 1,3,4-oxadiazole scaffold as a promising new class of
AC1 inhibitors for the treatment of chronic pain.
5.
Experimental
5.1.
Chemistry
5.1.1. General
The purity of all final compounds was >95 % purity as assessed by HPLC. Final compounds were
analyzed on an Agilent 1200 series chromatograph. The chromatographic method utilized as ThermoScientific
Hypersil GOLD C-18 4.6 x 250 mm, 3 µm column. UV detection wavelength = 220 nm; flow-rate = 1.0 mL/min;
gradient = 5 – 95% acetonitrile over 12 min and 3 min hold time at 95% acetonitrile. Both organic and aqueous
mobile phases contain 0.1% v/v formic acid. The mass spectrometer used is an AB Sciex 4500 QTrap triple-
quadrupole mass spectrometer with an ESI source or an Advion CMS-L Compact Mass Spectrometer with an
ESI or an APCI source. Samples are submitted for analysis solubilized in 1:1 acetonitrile:water solution or
1
using the atmospheric solids analysis probe (ASAP). H and 13C NMR spectra were recorded on either Bruker
DRX500 spectrometer (operating at 500 and 125 MHz, respectively) or Bruker AVIII (operating at 800 and 200
MHz, respectively) in DMSO-d6 or CDCl3 with or without the internal standard of TMS at 0.05% v/v. The
chemical shifts (δ) reported as parts per million (ppm) and the coupling constants are reported as s = singlet,
bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet. Compounds were
generally prepared according to scheme 1 and protocols are detailed below. Intermediate 4C was
commercially available.
5.1.2. General procedure for synthesis of hydrazine-carboxamide intermediates (3).
(E)-2-((5,6,7,8-tetrahydronaphthalen-2-yl)methylene)hydrazine-1-carboxamide (3A).
To a vial was added the semicarbazide hydrochloride (0.73 g, 6.5 mmol, 1.0 eq.) and sodium acetate
(1.07 g, 13.1 mmol, 2.0 eq.) followed by water (10 mL). To a second vial was added the 5,6,7,8-
tetrahydronaphthalene-2-carbaldehyde (2A, 1.05 g, 6.5 mmol, 1.0 eq.) and methanol (10 mL). The aldehyde

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solution was then added dropwise to the first solution while stirring at room temperature. The reaction stirred
for 30 min in which time it formed a suspension. The solid was filtered out by vacuum filtration to produce 3A
(1.36 g, 6.26 mmol, 96%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 10.13 (s, 1H), 7.77 (s, 1H), 7.42
(dd, J = 7.9, 1.8 Hz, 1H), 7.38 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.42 (s, 2H), 2.77 – 2.69 (m, 4H), 1.78 – 1.69
(m, 4H).
5.1.3. (E)-2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)hydrazine-1-carboxamide (3B).
Prepared using the same procedure as molecule 3A with semicarbazide hydrochloride (0.13 g, 1.1
mmol, 1.0 eq.), sodium acetate (0.19 g, 2.3 mmol, 2.0 eq.) in water (1 mL) and 2,3-
dihydrobenzo[b][1,4]dioxine-6-carbaldehyde (2B, 0.19 g, 1.1 mmol, 1.0 eq.) in methanol (1 mL) to produce 3B
(0.23 g, 1.0 mmol, 92%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 10.08 (s, 1H), 7.71 (s, 1H), 7.29
(d, J = 1.8 Hz, 1H), 7.14 (dd, J = 8.3, 1.8 Hz, 1H), 6.85 (br s, J = 8.3 Hz, 1H), 6.44 (d, J = 17.6 Hz, 2H), 4.26 (s,
4H).
5.1.4. (E)-2-(4-fluorobenzylidene)hydrazine-1-carboxamide (3D).
Prepared using the same procedure as molecule 3A with semicarbazide hydrochloride (0.88 g, 7.9
mmol, 1.0 eq.), sodium acetate (1.3 g, 16 mmol, 2.0 eq.) in water (10 mL) and 4-fluorobenzaldehyde (2D, 0.98
g, 7.9 mmol, 1.0 eq.) in methanol (10 mL) to produce 3D (1.23 g, 6.8 mmol, 86%) as a white solid. 1H NMR
(500 MHz, DMSO-d6): δ 10.24 (s, 1H), 7.83 (s, 1H), 7.80 (d, J= 8.8 Hz, 1H), 7.79 (d, J= 8.9 Hz, 1H), 7.23 (d, J=
8.9 Hz, 1H), 7.22 (d, J= 8.8 Hz, 1H), 6.50 (s, 2H).
5.1.5. (E)-2-(cyclohexylmethylene)hydrazine-1-carboxamide (3E).
Prepared using the same procedure as molecule 3A with semicarbazide hydrochloride (0.57 g, 5.1
mmol, 1.0 eq.), sodium acetate (0.84 g, 10 mmol, 2.0 eq.) in water (10 mL) and cyclohexanecarbaldehyde (2E,
0.57 g, 5.1 mmol, 1.0 eq.) in methanol (10 mL) to produce 3E (0.74 g, 4.4 mmol, 85%) as a white solid. 1H
NMR (500 MHz, DMSO-d6): δ 9.70 (s, 1H), 7.01 (d, J = 5.2 Hz, 1H), 6.07 (s, 1H), 2.13 – 2.01 (m, 1H), 1.72 –
1.60 (m, 5H), 1.61 – 1.52 (m, 1H), 1.25 – 1.08 (m, 6H).
5.1.6. General procedure for synthesis of 2-amino-1,3,4-oxadiazole intermediates (4).

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5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-amine (4A).
To a vial was added 3A (0.18 g, 0.81 mmol, 1.0 eq.), sodium acetate (0.13 g, 1.62 mmol, 2.0 eq.) and
acetic acid (0.5 mL). To a second vial was added the bromine (0.14 g, 0.89 mmol, 1.1 eq.) in acetic acid (0.5
mL). The bromine solution was then added to the first vial while stirring in a dropwise manner at room
temperature. The reaction was then heated to 60 °C and stirred for 1 hour. The reaction was then cooled to
room temperature and poured onto ice water. A yellow precipitate formed and was filtered by vacuum filtration
then rinsed with DCM to provide 4A (0.16 g, 0.77 mmol, 95%) as a pale yellow solid. 1H NMR (500 MHz,
DMSO-d6): δ 7.53 – 7.45 (m, 2H), 7.31 (br s, 2H), 7.20 (d, J = 7.9 Hz, 1H), 2.83 – 2.70 (m, 4H), 1.81 – 1.70 (m,
4H).
5.1.7. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-amine (4B).
Prepared using the same procedure as molecule 4A with 3B (0.49 g, 2.24 mmol, 1.0 eq.) and sodium
acetate (0.37 g, 4.48 mmol, 2.0 eq.) in acetic acid (1.0 mL). A second vial of bromine (0.39 g, 2.46 mmol, 1.1
eq.) in acetic acid (0.5 mL). Produced 4B (0.38 g, 1.75 mmol, 78%) as a pale red solid. 1H NMR (500 MHz,
DMSO-d6): δ 7.67 (s, 2H), 7.29 (dd, J = 8.4, 2.1 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 4.34
– 4.29 (m, 4H).
5.1.8. 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (4D).
Prepared using the same procedure as molecule 4A with 3D (0.50 g, 2.7 mmol, 1.0 eq.) and sodium
acetate (0.44 g, 5.4 mmol, 2.0 eq.) in acetic acid (2.0 mL). A second vial of bromine (0.43 g, 2.7 mmol, 1.1 eq.)
1
in acetic acid (1.0 mL). Produced 4D (0.39 g, 2.2 mmol, 81%) as a white solid. H NMR (500 MHz, DMSO-d6):
δ 7.86 – 7.74 (m, 2H), 7.40 – 7.31 (m, 2H), 7.23 (s, 2H).
5.1.9. 5-cyclohexyl-1,3,4-oxadiazol-2-amine (4E).
Prepared using the same procedure as molecule 4A with 3E (0.18 g, 1.1 mmol, 1.0 eq.) and sodium
acetate (0.17 g, 2.1 mmol, 2.0 eq.) in acetic acid (1.0 mL). A second vial of bromine (0.19 mL, 1.2 mmol, 1.1
1
eq.) in acetic acid (0.5 mL). Produced 4E (0.15 g, 0.87 mmol, 83%) as a pale yellow solid. H NMR (500 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): δ 6.82 (s, 2H), 2.71 (tt, J = 10.9, 3.6 Hz, 1H), 1.95 – 1.87 (m, 2H), 1.75 – 1.68 (m, 2H), 1.67 – 1.58
(m, 1H), 1.48 – 1.29 (m, 4H), 1.28 – 1.20 (m, 1H).
5.1.10. *General procedure “A” using a commercially available acyl chloride for amide coupling.
N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)benzamide (1).
To a vial was added the 4A (0.030 g, 0.14 mmol, 1.0 eq.) followed by pyridine (0.6 mL) and 4-benzoyl
chloride (0.020 g, 0.14 mmol, 1.0 eq.) at room temperature. The reaction stirred for 18 hours then was poured
into ice water and a precipitate formed. This precipitate was filtered by vacuum filtration and was further
purified by reverse-phase flash chromatography (5 – 100% MeCN:water). All fractions containing the desired
1
product were isolated and concentrated to produce 1 (0.028 g, 0.084 mmol, 40%) as a pale yellow solid. H
NMR (500 MHz, CDCl3): δ 12.21 (br s, 1H), 8.26 (d, J = 7.7 Hz, 2H), 7.84 – 7.74 (m, 2H), 7.62 (t, J = 7.3 Hz,
1H), 7.53 (t, J = 7.5 Hz, 2H), 7.19 (d, J = 7.8 Hz, 1H), 2.89 – 2.79 (m, 4H), 1.89 – 1.79 (m, 4H). 13C NMR (200
MHz, DMSO-d6): δ 165.0, 161.5, 157.7, 141.1, 137.9, 132.9, 132.2, 130.0, 128.6, 128.3, 126.5, 123.1, 120.5,
28.84, 28.58, 22.35, 22.30. ESI-MS: m/z 319.7 [M + H]+; HPLC retention time: 12.940 min. HPLC purity 96.5%.
5.1.11. *General procedure “B" using the commercially available carboxylic acid for amide coupling.
N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-3-(trifluoromethyl)benzamide (6).
Step 1: To a vial was added the 3-trifluoromethylbenzoic acid (0.044 g, 0.23 mmol, 1.0 eq.) in DCM (0.5
mL) followed by 1 drop of DMF. The reaction was then cooled to 0 °C followed by slow addition of oxalyl
chloride (0.023 mL, 0.27 mmol, 1.2 eq.). The reaction then was allowed to warm to room temperature and stir
for 1 hour. After 1 hour the reaction was concentrated in vacuo and carried into the amide coupling reaction
without purification or characterization.
Step 2: To a vial was added the 4A (0.050 g, 0.23 mmol, 1.0 eq.) followed by pyridine (0.3 mL). To the
vial containing the 3-trifluoromethylbenzoyl chloride (carried over from previous step) was add pyridine (0.3
mL) to form a suspension. The acyl chloride suspension was added to the first vial while stirring at room
temperature. The reaction stirred for 18 hours then was poured into ice water and a precipitate formed. This
precipitate was filtered by vacuum filtration and was further purified by normal phase flash chromatography (0
– 5% MeOH:DCM). All fractions containing the desired product were isolated and concentrated to produce 6

ACCEPTED MANUSCRIPT
(0.14 g, 0.035 mmol, 15%).1H NMR (500 MHz, CDCl3): δ 12.72 (s, 1H), 8.41 (s, 1H), 8.34 (d, J = 7.9 Hz), 8.04
(d, J = 7.9 Hz), 7.82 (t, J = 7.8 Hz, 1H), 7.74 – 7.62 (m, 2H), 7.29 (d, J = 7.9 Hz, 1H), 2.85-2.78 (m, 4H), 1.85 –
1.72 (m, 4H). 13C NMR (200 MHz, DMSO-d6): δ 167.8, 166.4, 160.6, 141.3, 138.4, 132.9, 132.0, 130.5, 130.3,
130.1, 129.6 (q, J = 32.0 Hz), 129.1, 126.8, 126.1 (q, J = 270.0 Hz), 123.5, 121.3, 29.3, 29.1, 22.9, 22.8. ESI-
MS: 387.9 [M + H]+. HPLC retention time: 13.813 min. HPLC purity 95.5%.
5.1.12. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(bromo)benzamide (7).
Prepared using general procedure B with 4-bromobenzoic acid (0.051 g, 0.26 mmol, 1.1 eq.), oxalyl
chloride (0.023 mL, 0.27 mmol, 1.15 eq.), 4A (0.050 g, 0.23 mmol, 1.0 eq.), and pyridine (1.0 mL) to produce 7
1
(0.049 g, 0.117 mmol, 50%) as a light pink solid. H NMR (500 MHz, CDCl3): δ 12.04 (s, 1H), 8.12 (d, J = 8.5
Hz, 2H), 7.78 – 7.71 (m, 2H), 7.70 – 7.59 (m, 2H), 7.21 (d, J = 8.1 Hz, 1H), 2.89 – 2.78 (m, 4H), 1.86 – 1.83
(m, 4H). HPLC retention time: 13.657 min. HPLC purity 95.3%.
5.1.13. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(isopropoxy)benzamide (8).
Prepared using general procedure A with 4A (0.050 g, 0.23 mmol, 1.0 eq.), 4-isopropoxybenzoyl
1
chloride (0.062 g, 0.32 mmol, 1.4 eq.), and pyridine (1.0 mL) to produce 8 (0.010 g, 0.027 mmol, 12%). H
NMR (800 MHz, DMSO-d6): δ 8.00 – 7.98 (m, 2H), 7.66 – 7.64 (m, 1H), 7.64 – 7.62 (m, 1H), 7.27 – 7.24 (m,
1H), 7.05 – 7.03 (m, 2H), 4.75 (p, J = 6.0 Hz, 1H), 2.82 – 2.75 (m, 4H), 1.77 – 1.73 (m, 4H), 1.29 (d, J = 6.0 Hz,
6H). 13C NMR (200 MHz, CDCl3):
δ 165.2, 161.7, 158.7, 141.4, 138.3, 130.9, 130.5, 126.9, 124.6, 123.5, 121.1,
115.51, 70.1, 29.3, 29.1, 22.8, 22.8, 22.2. ESI-MS: m/z 378.0 [M + H]+; HPLC retention time: 13.629 min.
HPLC purity 97.9%.
5.1.14. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-3-(thiomethoxy)benzamide (9).
Prepared using general procedure B with 3-thiomethoxybenzoic acid (0.026 g, 0.26 mmol, 1.1 eq.),
oxalyl chloride (0.015 mL, 0.17 mmol, 1.2 eq.), 4A (0.030 g, 0.14 mmol, 1.0 eq.), and pyridine (1.0 mL) to
1
produce 9 (0.015 g, 0.047 mmol, 26%). H NMR (800 MHz, CDCl3): δ 8.07 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H),
7.79 (s, 2H), 7.53 – 7.41 (m, 2H), 7.20 (d, J = 8.4 Hz, 1H), 2.89 – 2.82 (m, 4H), 2.55 (s, 3H), 1.88 - 179 (m,
4H). 13C NMR (200 MHz, CDCl3): δ 142.0, 140.0, 138.2, 130.8, 129.9, 129.0, 127.3, 126.2, 125.3, 123.7, 120.2,

ACCEPTED MANUSCRIPT
29.6, 29.3, 22.9, 22.8, 15.6. ESI-MS: m/z 366.0 [M + H]+, 388.0 [M + Na]; HPLC retention time: 13.511 min.
HPLC purity 95.8%.
5.1.15. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-3-(methoxy)benzamide (10).
Prepared using general procedure B with 3-methoxybenzoic acid (0.042 g, 0.28 mmol, 1.2 eq.), oxalyl
chloride (0.027 mL, 0.32 mmol, 1.4 eq.), 4A (0.049 g, 0.23 mmol, 1.0 eq.), and pyridine (2.0 mL) to produce 10
(0.031 g, 0.088 mmol, 39%) as a white solid. 1H NMR (800 MHz, DMSO-d6 ): δ 12.11 (s, 1H), 7.76 – 7.53 (m,
4H), 7.46 (t, J = 7.9 Hz, 1H), 7.30 – 7.15 (m, 2H), 3.83 (s, 3H), 2.83 – 2.68 (m, 4H), 1.81 – 1.61 (m, 4H). 13C
NMR (200 MHz, DMSO-d6): δ 165.4, 161.6, 159.7, 158.3, 141.5, 138.4, 134.2, 130.4, 130.2, 126.9, 123.5,
121.0, 121.0, 119.4, 113.5, 55.9, 29.3, 29.1, 22.8, 22.8. ESI-MS: m/z 350.1 [M + H]+, 372.1 [M + Na]+; HPLC
retention time: 13.034 min. HPLC purity 95.2%.
5.1.16. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(methyl)benzamide (11).
Prepared using general procedure A with 4A (0.040 g, 0.19 mmol, 1.0 eq.), 4-methylbenzoyl chloride
(0.032 g, 0.20 mmol, 1.1 eq.), and pyridine (1.0 mL) to produce 11 (0.028 g, 0.084 mmol, 45%) as a white
solid.1H NMR (500 MHz, DMSO-d6): δ 12.06 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.71 – 7.66 (m, 2H), 7.38 (d, J =
8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 2.91 – 2.76 (broad m, 4H), 2.40 (s, 3H), 1.85 – 1.73 (broad m, 4H). 13C
NMR (126 MHz, DMSO-d6): δ 167.4, 158.0, 143.3, 141.1, 138.0, 130.1, 129.4, 129.3, 129.2, 128.4, 126.5,
123.1, 120.6, 28.9, 28.6, 22.4, 22.4, 21.2. ESI-MS: 333.5 [M]+. HPLC retention time: 13.244 min. HPLC purity
100%.
5.1.17. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(methoxy)benzamide (12).
Prepared using general procedure A with 4A (0.050 g, 0.23 mmol, 1.0 eq.), 4-methoxybenzoyl chloride
(0.040 g, 0.24 mmol, 1.1 eq.), and pyridine (1.0 mL) to produce 12 (0.047 g, 0.13 mmol, 58%) as a white solid.
1H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.69 – 7.59 (m, 2H), 7.23
(dd, J = 52.5, 7.9 Hz, 1H), 7.08 (dd, J = 23.4, 8.4 Hz, 2H), 3.83 (s, 3H), 2.78 (s, 4H), 1.77 (d, J = 4.4 Hz, 4H).
13C NMR (200 MHz, DMSO-d6): δ 166.4, 165.8, 163.5, 162.5, 138.4, 137.2, 129.8, 129.5, 128.6, 126.9, 124.9,

ACCEPTED MANUSCRIPT
123.6, 114.2, 56.0, 29.3, 29.3, 23.0, 22.9. APCI-MS = m/z 350.3 [M + H]+. HPLC retention time: 12.242 min.
HPLC purity 100%.
5.1.18. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(fluoro)benzamide (13).
Prepared using general procedure A with 4A (0.045 g, 0.21 mmol, 1.0 eq.), 4-fluorobenzoyl chloride
(0.040 g, 0.25 mmol, 1.2 eq.), and pyridine (1.0 mL) to produce 13 (0.020 g, 0.059 mmol, 28%) as a white
1
solid. H NMR (500 MHz, DMSO-d6): δ 12.10 (br s, 1H), 8.13 - 8.09 (broad m, 2H), 7.70-7.64 (broad m, 2H),
7.46 – 7.35 (broad m, 2H), 7.30-7.26 (broad m, 1H), 2.83-2.78 (broad m, 4H), 1.81-1.74 (broad m, 4H). 13C
NMR (200 MHz, DMSO-d6): δ 170.6, 166.1, 165.3 (d, J = 248.0 Hz), 158.2, 141.7, 138.4, 131.7 (d, J = 8.0 Hz),
130.5, 129.5, 127.0, 123.6, 120.9, 116.2 (d, J = 22.0 Hz), 29.3, 29.1, 22.8, 22.8. ESI-MS: 338.2 [M + H]+. HPLC
retention time: 12.785 min. HPLC purity 97.9%.
5.1.19. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-3-(fluoro)benzamide (14).
Prepared using general procedure A with 4A (0.042 g, 0.20 mmol, 1.0 eq.), 3-fluorobenzoyl chloride
(0.031 g, 0.20 mmol, 1.0 eq.), and pyridine (1.0 mL) to produce 14 (0.024 g, 0.071 mmol, 36%) as a white
solid.1H NMR (800 MHz, DMSO-d6): δ 7.90 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 9.8 Hz, 1H), 7.66 (d, J = 7.8 Hz,
1H), 7.65 (s, 1H), 7.62 - 7.59 (m, 1H), 7.52 - 7.48 (m, 1H), 7.28 (d, J = 7.9 Hz, 1H), 2.82 - 2.79 (broad m, 4H),
1.78-1.76 (m, 4H). 13C NMR (200 MHz, DMSO-d6): δ 165.5, 162.4 (d, J = 256.0 Hz), 160.0, 159.6, 141.4,
138.4, 136.5, 131.2 (d, J = 8.0 Hz), 130.5, 126.9, 125.0, 123.5, 121.2, 119.8 (d, J = 20.0 Hz), 115.5 (d, J = 24.0
Hz), 29.3, 29.1, 22.9, 22.8. ESI-MS: 338.2 [M + H]+. HPLC retention time: 13.239 min. HPLC purity 97.7%.
5.1.20. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(chloro)benzamide (15).
Prepared using general procedure A with 4A (0.050 g, 0.23 mmol, 1.0 eq.), 4-chlorobenzoyl chloride
(0.049 g, 0.28 mmol, 1.2 eq.), and pyridine (1.0 mL) to produce 14 (0.047 g, 0.13 mmol, 57%) as a white
solid.1H NMR (500 MHz, DMSO-d6): δ 8.07 (d, J = 8.1 Hz, 2H), 7.69 – 7.62 (m, 4H), 7.29 (d, J = 7.9 Hz, 1H),
2.81 (broad m, 4H), 1.77 (m, 4H). 13C NMR (200 MHz, DMSO-d6): δ 167.0, 165.3, 158.7, 141.6, 138.4, 138.1,
+
131.6, 130.8, 130.5, 129.9, 129.2, 126.9, 123.6, 29.3, 29.1, 22.8, 22.8. ESI-MS: 355.3 [M + H] . HPLC
retention time: 13.573 min. HPLC purity 99.0%.

ACCEPTED MANUSCRIPT
5.1.21. N-(5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(ethyl)benzamide (16).
Prepared using general procedure A with 4A (0.050 g, 0.23 mmol, 1.0 eq.), 4-ethylbenzoyl chloride
(0.039 g, 0.23 mmol, 1.0 eq.), and pyridine (1.0 mL) to produce 16 (0.032 g, 0.093 mmol, 40%) as a white
solid.1H NMR (500 MHz, DMSO-d6): δ 7.93 (m, 2H), 7.62 (m, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 8.0 Hz,
1H), 2.82-2.74 (m, 4H), 2.66 (q, J = 7.6 Hz, 2H), 1.74 – 1.71 (br m, 4H), 1.18 (t, J = 7.5 Hz, 3H). 13C NMR (126
MHz, DMSO-d6): δ 160.9, 158.7, 149.1, 141.0, 138.0, 130.1, 128.6, 128.1, 126.5, 123.1, 120.8, 28.9, 28.7,
28.2, 22.5, 22.4, 15.3. APCI-MS: 348.3 [M + H]+. HPLC retention time: 13.633 min. HPLC purity 100%.
5.1.22. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)benzamide (17).
Prepared using general procedure A with 4B (0.052 g, 0.24 mmol, 1.0 eq.), benzoyl chloride (0.037 g,
0.26 mmol, 1.1 eq.), and pyridine (1.0 mL) to produce 17 (0.032 g, 0.099 mmol, 42%) as a white solid. 1H NMR
(500 MHz, DMSO-d6): δ 12.22 (s, 1H), 8.05 (d, J = 7.6 Hz, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H),
7.44 (dd, J = 8.4, 2.1 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 4.34 (q, J = 5.6 Hz, 4H). 13C
NMR (200 MHz, DMSO-d6): δ 146.3, 143.8, 132.3, 128.4, 128.3, 119.4, 118.1, 116.7, 114.5, 64.4, 64.1. ESI-
MS: m/z 323.6 [M]+; HPLC retention time: 11.191 min. HPLC purity 99.6%.
5.1.23. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-3-(thiomethoxy)benzamide (18).
Prepared using general procedure B with 3-thiomethoxybenzoic acid (0.025 g, 0.25 mmol, 1.1 eq.),
oxalyl chloride (0.014 mL, 0.16 mmol, 1.2 eq.), 4B (0.030 g, 0.14 mmol, 1.0 eq.), and pyridine (2.0 mL) to
1
produce 18 (0.002 g, 0.005 mmol, 4%) as a white solid. H NMR (800 MHz, DMSO-d6): δ 7.90 (t, J = 1.8 Hz,
1H), 7.79 (d, J = 7.3 Hz, 1H), 7.50 – 7.39 (m, 3H), 7.36 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.35 –
4.29 (m, 4H), 2.54 (s, 3H). 13C NMR (200 MHz, DMSO- d6): δ 163.2, 146.0, 143.8, 138.4, 128.9, 128.8, 125.3,
124.9, 119.2, 118.1, 117.2, 114.3, 64.4, 64.1, 40.0, 14.6. ESI-MS: m/z 339.7 [M + H]+, HPLC retention time:
11.946 min. HPLC purity 95.0%.
5.1.24. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-3-(trifluoromethyl)benzamide (19).

ACCEPTED MANUSCRIPT
Prepared using general procedure B with 3-trifluoromethylbenzoic acid (0.038 g, 0.20 mmol, 1.1 eq.),
oxalyl chloride (0.018 mL, 0.21 mmol, 1.2 eq.), 4B (0.040 g, 0.18 mmol, 1.0 eq.), and pyridine (2.0 mL) to
produce 19 (0.013 g, 0.032 mmol, 18%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 12.59 (s, 1H), 8.40
(s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.45 (dd, J = 8.5, 2.1 Hz,
1H), 7.40 (t, J = 2.2 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 4.34 (q, J = 5.0 Hz, 4H). 13C NMR (200 MHz, DMSO-d6)
δ 146.6, 143.9, 133.3, 132.5, 130.2, 130.0, 129.4 (q, J = 32.2 Hz), 125.0 (q, J = 4.0 Hz), 124.9, 123.9 (q, J =
271.4 Hz), 119.7, 118.3, 116.3, 114.7, 64.4, 64.2. ESI-MS: 391.6 [M]+. HPLC retention time: 12.513 min. HPLC
purity 99.1%.
5.1.25. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-4-(isopropoxy)benzamide (20).
Prepared using general procedure A with 4B (0.050 g, 0.23 mmol, 1.0 eq.), 4-isopropoxybenzoyl
chloride (0.068 g, 0.34 mmol, 1.5 eq.), and pyridine (1.0 mL) to produce 20 (0.020 g, 0.052 mmol, 23%) as a
1
white solid. H NMR (500 MHz, DMSO-d6): δ 12.00 (s, 1H), 8.01-7.99 (m, 2H), 7.44 – 7.35 (m, 2H), 7.08-7.01
(m, 3H), 4.77 – 4.72 (broad m, 1H), 4.36 – 4.32 (broad m, 4H), 1.30 (d, J = 6.0 Hz, 6H). 13C NMR (126 MHz,
CDCl3): δ 160.9, 146.2, 143.9, 143.1, 130.5, 122.2, 119.4, 118.2, 116.9, 114.9, 114.5, 69.7, 64.4, 64.1, 21.8.
ESI-MS: 382.1 [M + H] +. HPLC retention time: 12.247 min. HPLC purity 100%.
5.1.26. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-4-(bromo)benzamide (21).
Prepared using general procedure B with 4-bromobenzoic acid (0.053 g, 0.26 mmol, 1.1 eq.), oxalyl
chloride (0.025 mL, 0.28 mmol, 1.2 eq.), 4B (0.053 g, 0.26 mmol, 1.0 eq.), and pyridine (2.0 mL) to produce 21
1
(0.002 g, 0.005 mmol, 2%) as a white solid. H NMR (800 MHz, DMSO-d6): δ 12.27 (s, -1H), 7.97 (d, J = 8.1
Hz, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.42 (dd, J = 8.4, 2.1 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.06 (d, J = 8.4 Hz,
1H), 4.37 – 4.24 (m, 4H). 13C NMR (200 MHz, DMSO-d6): δ 146.8, 144.3, 132.0, 131.2, 130.9, 126.8, 119.9,
118.6, 117.0, 115.0, 64.9, 64.6. ESI-MS: m/z 401.8 [M]+, 403.8 [M + 2]+. HPLC retention time: 12.191 min.
HPLC purity 96.7%.
5.1.27. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-3-(methoxy)benzamide (22).

ACCEPTED MANUSCRIPT
Prepared using general procedure B with 3-methoxybenzoic acid (0.034 g, 0.23 mmol, 1.1 eq.), oxalyl
chloride (0.021 mL, 0.24 mmol, 1.2 eq.), 4B (0.045 g, 0.21 mmol, 1.0 eq.), and pyridine (2.0 mL) to produce 22
1
(0.025 g, 0.071 mmol, 35%) as a white solid. H NMR (500 MHz, DMSO-d6): δ 12.36 (s, 1H), 7.69 – 7.64(m,
2H), 7.46-7.38 (m, 2H), 7.18-7.09 (m, 2H), 6.62 (s, 1H), 4.41 – 4.34 (m, 4H), 3.86 (s, 3H). 13C NMR (200 MHz,
DMSO-d6): δ 169.9, 167.6, 162.1, 159.5, 146.4, 144.2, 136.1, 129.8, 121.2, 119.7, 118.6, 114.8, 113.6, 113.1,
110.5, 64.9, 64.6, 55.7. ESI-MS: 354.1 [M + H]+. HPLC retention time: 11.457 min. HPLC purity 95.6%.
5.1.28. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-4-(methyl)benzamide (23).
Prepared using general procedure A with 4B (0.040 g, 0.18 mmol, 1.0 eq.), 4-methylbenzoyl chloride
(0.034 g, 0.22 mmol, 1.2 eq.), and pyridine (1.0 mL) to produce 23 (0.015 g, 0.044 mmol, 24%) as a white
1
solid. H NMR (500 MHz, DMSO-d6): δ 12.02 (s, 1H), 7.97-7.90 (m, 2H), 7.84 – 7.76 (m, 1H), 7.45 (t, J = 8.3
Hz, 1H), 7.41 – 7.35 (m, 2H, 7.09 (t, J = 8.3 Hz, 1H), 4.34 (bs, 4H), 2.50 (s, 3H). 13C NMR (201 MHz, DMSO-
d6): δ 146.5, 143.9, 141.1, 131.5, 129.4, 129.2, 128.8, 128.4, 127.5, 119.7, 118.3, 116.4, 114.7, 64.5, 64.2,
21.1. ESI-MS: 337.9 [M]+. HPLC retention time: 11.666 min. HPLC purity 98.1%.
5.1.29. N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-3,4-(dimethyl)benzamide (24).
Prepared using general procedure B with 3,4-dimethylbenzoic acid (0.038 g, 0.25 mmol, 1.1 eq.), oxalyl
chloride (0.023 mL, 0.26 mmol, 1.2 eq.), 4B (0.050 g, 0.23 mmol, 1.0 eq.), and pyridine (2.0 mL) to produce 24
(0.030 g, 0.085 mmol, 37%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 11.93 (s, 1H), 7.80 (s, 1H), 7.73
(d, J = 8.0 Hz, 1H), 7.40 (dd, J = 8.4, 2.1 Hz, 1H), 7.35 (bs, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 8.5 Hz),
4.30 (m, 4H), 2.27 (s, 3H), 2.05 (s, 3H). 13C NMR (200 MHz, DMSO-d6): δ 146.8, 144.3, 137.0, 130.1, 129.8,
126.34, 120.0, 118.7, 117.1, 115.0, 64.9, 64.6, 20.0, 19.9. ESI-MS: 351.8 [M]+. HPLC retention time: 12.094
min. HPLC purity 100%.
5.1.30. N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)benzamide (25).
Prepared using general procedure A with 4C (0.055 g, 0.33 mmol, 1.0 eq.), benzoyl chloride (0.051 g,
0.36 mmol, 1.1 eq.), and pyridine (2.0 mL) to produce 25 (0.055 g, 0.199 mmol, 60%) as a white solid. 1H NMR
(800 MHz, DMSO-d6): δ 12.17 (s, 1H), 8.05 (d, J = 7.7 Hz, 2H), 7.94 (d, J = 4.9 Hz, 1H), 7.78 (d, J = 3.7 Hz,

ACCEPTED MANUSCRIPT
1H), 7.67 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.30 (dd, J = 5.0, 3.6 Hz, 1H). 13C NMR (200 MHz,
DMSO-d6): δ 165.0, 157.5, 157.4, 132.9, 132.2, 131.1, 129.7, 128.7, 128.6, 128.3, 124.3. ESI-MS: m/z 271.6
[M]+; HPLC retention time: 10.957 min. HPLC purity 98.4%.
5.1.31. N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(bromo)benzamide (26).
Prepared using general procedure B with 4-bromobenzoic acid (0.071 g, 0.35 mmol, 1.1 eq.), oxalyl
chloride (0.031 mL, 0.37 mmol, 1.2 eq.), 4C (0.053 g, 0.22 mmol, 1.0 eq.), and pyridine (2.0 mL) to produce 26
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(0.055 g, 0.199 mmol, 60%) as a white solid. H NMR (800 MHz, DMSO-d6): δ 12.23 (s, 1H), 7.98 – 7.91 (m,
3H), 7.80 – 7.74 (m, 3H), 7.28 (dd, 1H). 13C NMR (200 MHz, DMSO-d6): δ 164.55, 158.07, 157.58, 132.17,
131.65, 130.82, 130.16, 129.19, 127.39, 124.69. ESI-MS: m/z 349.9 [M]+, 351.9 [M + 2]+. HPLC retention time:
11.964 min. HPLC purity 97.6%.
5.1.32. N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)-3-(thiomethoxy)benzamide (27).
Prepared using general procedure B with 3-thiomethoxybenzoic acid (0.033 g, 0.20 mmol, 1.1 eq.),
oxalyl chloride (0.019 mL, 0.22 mmol, 1.2 eq.), 4C (0.030 g, 0.18 mmol, 1.0 eq.), and pyridine (2.0 mL) to
produce 27 (0.006 g, 0.016 mmol, 12%) as a white solid. 1H NMR (800 MHz, DMSO-d6): δ 12.19 (s, 1H), 7.94
(dd, J = 5.0, 1.2 Hz, 1H), 7.87 (s, 1H), 7.79 – 7.76 (m, 2H), 7.54 (t, J = 7.7, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.30
(dd, J = 5.0, 3.7 Hz, 1H), 2.56 (s, 3H). 13C NMR (200 MHz, DMSO-d6): δ 164.5, 157.5, 157.2, 139.2, 132.9,
131.1, 130.0, 129.7, 129.2, 128.7, 124.9, 124.7, 124.3, 14.5. ESI-MS: m/z 317.7 [M]+, 340.0 [M + Na]+. HPLC
retention time: 11.811 min. HPLC purity 97.3%.
5.1.33. N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)-4-(isopropoxy)benzamide (28).
Prepared using general procedure A with 4C (0.049 g, 0.29 mmol, 1.0 eq.), 4-isopropoxybenzoyl
chloride (0.064 g, 0.32 mmol, 1.1 eq.), and pyridine (2.0 mL) to produce 28 (0.058 g, 0.174 mmol, 59%) as a
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white solid. H NMR (800 MHz, DMSO-d6): δ 11.91 (br s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.92 – 7.90 (m, 1H),
7.75 – 7.73 (m, 1H), 7.28 – 7.26 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H), 4.74 (p, J = 6.0 Hz, 1H), 1.28 (d, J = 6.1 Hz,
6H). 13C NMR (200 MHz, DMSO-d6): δ 164.7, 161.8, 158.1, 157.9, 131.5, 131.0, 130.0, 129.1, 124.8, 124.1,