Antifungal benzo[b]thiophene 1,1-dioxide IMPDH inhibitors exhibit pan-assay interference (PAINS) profiles

Article abstract of DOI:10.1016/j.bmc.2018.09.004

Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor.

Full text of DOI:10.1016/j.bmc.2018.09.004

Accepted Manuscript
Antifungal Benzo[b]thiophene 1,1-dioxide IMPDH Inhibitors Exhibit Pan-As-
say Interference (PAINS) Profiles
Lalith K. Kummari, Mark S. Butler, Emily Furlong, Ross Blundell, Amanda
Nouwens, Alberto B. Silva, Ulrike Kappler, James A. Fraser, Bostjan Kobe,
Matthew A. Cooper, Avril A.B. Robertson
PII:
S0968-0896(18)31141-6
https://doi.org/10.1016/j.bmc.2018.09.004
BMC 14529
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2018
21 August 2018
3 September 2018
Please cite this article as: Kummari, L.K., Butler, M.S., Furlong, E., Blundell, R., Nouwens, A., Silva, A.B., Kappler,
U., Fraser, J.A., Kobe, B., Cooper, M.A., Robertson, A.A.B., Antifungal Benzo[b]thiophene 1,1-dioxide IMPDH
Inhibitors Exhibit Pan-Assay Interference (PAINS) Profiles, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.09.004
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Antifungal Benzo[b]thiophene 1,1-dioxide IMPDH Inhibitors Exhibit Pan-Assay
Interference (PAINS) Profiles
Lalith K. Kummari,1-3 Mark S. Butler,3 Emily Furlong,3 Ross Blundell,2 Amanda Nouwens,1
Alberto B. Silva,3, 5 Ulrike Kappler,4 James A. Fraser,2 Bostjan Kobe,2, 3 Matthew A. Cooper,3
Avril A. B. Robertson1, 3*
1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
2Australian Infectious Diseases Research Centre, School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland
4072, Australia
4Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, The University
of Queensland, Brisbane, Queensland 4072, Australia
5AC Immune SA, EPFL Innovation Park, CH-1015 Lausanne, Switzerland.
*Corresponding author:
Professor Avril A. B. Robertson PhD GCHEd
Biotechnology Program Director,
School of Chemistry and Molecular Biosciences,
Affiliate of the Institute for Molecular Bioscience,
The University of Queensland, St Lucia, QLD, 4072, AUSTRALIA
Phone: +61 7 3346 2204, Email: a.robertson3@uq.edu.au
1

ABSTRACT
Fungi cause serious life-threatening infections in immunocompromised individuals and current
treatments are now complicated by toxicity issues and the emergence of drug resistant strains.
Consequently, there is a need for development of new antifungal drugs. Inosine
monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic
pathway, is essential for growth and virulence of fungi and is a potential drug target. In this
study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus
neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-
yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed
whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits.
Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with
nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated
by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced
IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the
presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-
dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-
oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these
compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-
specific and covalent binding with IMPDH at multiple cysteine residues. These results support
recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay
interference compounds) contributor.
Keywords: High-throughput screening; IMPDH inhibitor; antifungal; PAINS; mass
spectrometry; Cryptococcus neoformans.
2

1. Introduction
The Fungal Infection Trust has estimated that approximately 300 million people worldwide are
afflicted with serious fungal infections.1 Many of these pathogenic fungi are opportunistic and
infect when the human immune system is compromised due to HIV/AIDS, anticancer
chemotherapy or immunosuppressant treatment during organ transplantation. Aspergillus
fumigatus, Candida albicans and Cryptococcus neoformans are among the most common
fungal pathogens and are associated with highest mortality rates in immunocompromised
populations.2,3
According to the Center for Disease Control and Prevention, nearly 220,000 new cases of
cryptococcal meningitis occur each year with a fatality rate of >50%.4 Cryptococcus infection
occurs via inhaled spores that cause pulmonary infection in the lung alveoli. Later, the infection
spreads to other parts of the body. Fungal cells that cross the blood brain barrier infect the
meningeal membrane that protects the spinal cord and the brain. Resulting inflammation of the
meninges causes cryptococcal meningitis, which is fatal unless treated.5,6 The recommended
treatment for cryptococcal meningitis is a combination antifungal therapy that uses
amphotericin B deoxycholate and flucytosine, which is followed by life-long maintenance
therapy with fluconazole to prevent reinfection. Amphotericin B therapy is associated with
nephrotoxicity and infusion-related reactions such as chest pain, dyspnea, hypoxia, flushing,
and urticaria.7 Flucytosine and fluconazole cause adverse gastrointestinal effects and are toxic
to bone marrow.8,9 Moreover, the increasing resistance acquired by C. neoformans is making
available treatments less effective. Given the limitations of existing antifungal therapeutics,
there is a need to consider new approaches and targets.10
One such potential target is inosine monophosphate dehydrogenase (IMPDH), an enzyme of
the purine metabolic pathway. IMPDH forms a homotetramer that catalyses the two-step
3

conversion of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP) using
nicotinamide adenine dinucleotide (NAD) as a cofactor (Figure 1).11 Deletion of the IMD1
gene encoding IMPDH in C. neoformans leads to guanine auxotrophy and virulence factor
production defects. Moreover, the mutant was avirulent in two animal infection models.12
While C. neoformans is susceptible to the human IMPDH inhibitor mycophenolic acid (MPA),
this well-known drug also targets the human IMPDH isoforms as an immunosuppressant. In
treating immunocompromised individuals, the immunosuppressant properties of known
IMPDH inhibitors, such as MPA, would clearly be detrimental to health and therefore
discovery of fungal specific IMPDH inhibitors is essential. Both the crystal structure of
Cryptococcus IMPDH, and kinetic data have revealed significant differences between this
fungal enzyme and its human counterparts, providing an opportunity to produce novel, species-
specific inhibitors.12
Figure 1. Two step mechanism for the conversion of IMP to XMP catalysed by IMPDH.
In this study, we highlight a set of inhibitors of Cryptococcus IMPDH identified using an
enzyme inhibition assay adapted for high-throughput screening conducted with 114,000 drug-
4

like compounds from the Walter and Eliza Hall Compound Collection. Out of the screening, a
hit-set of three molecules was identified comprising 1,3,4-oxadiazole ring attached to a
benzothiophene-1,1-dioxide moiety via thioether group linkage. In addition to their unique
structure and under-explored biological activity, they had potent inhibitory activity against
Cryptococcus IMPDH and significant antifungal activity. Fifteen analogs were synthesized by
modification of the aromatic group at the fifth position of the 1,3,4-oxadiazole ring moiety, to
find selective Cryptococcus IMPDH inhibitors with potent antifungal activity. The observed
IC50 values against Cryptococcus IMPDH were consistent across all synthesized compounds
and displayed potent antifungal activity. Modification of the 1,3,4-oxadiazole ring to imidazole
or 1,3,4-triazole led to the loss of Cryptococcus IMPDH inhibition and antifungal activity.
Mass spectrometry studies of Cryptococcus IMPDH incubated with one of the compounds
showed nucleophilic attack of the cysteine thiolate anion at the C3-position of
benzo[b]thiophene 1,1-dioxide. This reactivity also caused compound breakdown when
performing kinetics studies in the presence of dithiothreitol (DTT). Consistent with our
findings, Dahlin et al., recently reported that benzo[b]thiophene 1,1-dioxides were assay
interfering compounds (PAINS).13-16
2. Results and discussion
2.1. Chemistry
To identify inhibitors of Cryptococcus IMPDH, the Walter and Eliza Hall Compound
Collection (114,000 drug-like compounds) was screened using a spectrophotometric assay to
measure the amount of NADH produced by IMPDH enzymatic activity. A 3-((5-substituted)-
1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxide series of hits was identified, with IC50
values between 1 and 2 µM. On the basis of in silico docking studies to improve enzyme
inhibition and antifungal activity, substitution at position 5 of the 1,3,4-oxadiazole ring was
investigated. The synthetic route involved coupling 5-substitued 1,3,4-oxadiazole-2-thiol
5

intermediates with 3-bromo benzo[b]thiophene 1,1-dioxide (2) with copper iodide catalyst and
benzotriazole under basic conditions (potassium tert-butoxide or Cs2CO3) in DMSO. These
conditions had not previously been investigated with heteroaromatic thiols but afforded the
desired compounds 5a-5o in good yields (Scheme 2).17 The N-acetyl modified compound 5o
was synthesized from compound 5n by acetylation of amino groups using acetyl chloride and
Et3N as the base, in THF as the solvent.
Scheme 1. Synthetic route of intermediate 2. Reagents and conditions: (i) m-CPBA, DCM, rt
3-Bromo benzo[b]thiophene 1,1-dioxide (2) was synthesized by oxidization of commercially
available 3-bromo benzo[b]thiophene (1) using m-CPBA (Scheme 1). Required 5-substitued
1,3,4-oxadiazole-2-thiol intermediates were purchased commercially or synthesized by
cyclization of the respective acid hydrazide using carbon disulfide in DMF, as reported by
Mohammad Soleiman-Beigi et al. (Scheme 2).18 However, an alternative method was needed
to cyclize the cyclohexane carbohydrazide (3e) using carbon disulfide in ethanol, followed by
addition of potassium hydroxide to give 5-(cyclohexyl)-1,3,4-oxadiazole-2-thiol (4e).19,20
6

Scheme 2. Synthetic route of final compounds. Reagents and conditions: i) CS2, DMF, 70 °C,
6 h. ii) 2, CuI, BtH, potassium tert-butoxide, 1,4-dioxane, 95 °C. *Reagents and conditions for
4e: KOH, CS2, ethanol, 70 °C, overnight; 2M HCl, pH 5.
†4m and 4n were purchased from Chem-Impex International Inc.
Analogs of 5k where the 1,3,4-oxadiazole ring was altered to 1,2,4 triazole (5p) and 1H-
imidazole (5q) were prepared according to the procedure outlined in Scheme 3. Compound 5p
was synthesized by coupling of commercially purchased thiol 4o with intermediate 3-bromo
benzo[b]thiophene 1,1-dioxide (2). Compound 5q was synthesized in a three-step reaction that
involved the initial conversion of 2-bromo-1-(3-methoxyphenyl)ethan-1-one (6) to 2-amino-1-
(3-methoxyphenyl)ethan-1-one (7) using Delepine reaction conditions. In the second step, the
corresponding thiol 4p was obtained by the treatment of compound 7 with potassium
thiocyanate and glacial acetic acid at 140 °C for 2 h. Finally, the thiol 4p was coupled with the
intermediate 3-bromo benzo[b]thiophene 1,1-dioxide (2) to give 5q.
7

Scheme 3. A) Synthetic route of compound 5p. Reagents and conditions: i) CuI, BtH,
potassium tert-butoxide, 1,4-dioxane, 95 °C; B) synthetic route of compound 5q. Reagents and
conditions: i) hexamine, DCM, rt, overnight; MeOH, HCl, 60 °C, 2h; ii) KSCN, glacial acetic
acid, 140 °C, 2 h; iii) CuI, BtH, potassium tert-butoxide, 1,4-dioxane, 95 °C.
2.2. Biological screening
The designed compounds 5a-5o were initially tested for their inhibitory activity against
Cryptococcus IMPDH in an enzymatic assay, with MPA as a positive control. MPA showed
Cryptococcus IMPDH inhibitory activity with an IC50 of 0.12 ± 0.02 µM. All compounds,
except compound 5l, exhibited high inhibitory activity on Cryptococcus IMPDH with IC50
values of ≤1 µM (Table 1). The relatively similar IC50 values of all compounds against
8

Cryptococcus IMPDH suggested that any substitution made at the fifth position of the 1,3,4-
oxadiazole ring did not influence the level of Cryptococcus IMPDH inhibition. Given the level
of similarity between Cryptococcus IMPDH and the human IMPDH isoforms, it was also
important to assess the selectivity of the compounds. Therefore, all compounds were also tested
for inhibitory activity against the human IMPDH isoforms I and II. Interestingly, compounds
5f and 5l with 4-methyl carboxylate phenyl and 4-methoxy phenyl, respectively, at the fifth
position of the 1,3,4-oxadiazole ring showed higher inhibitory selectivity (>30-fold) for
Cryptococcus IMPDH (5f = 0.34 ± 0.07 µM, 5l = 1.7 ± 0.38 µM) over the human IMPDH I
and II isoforms (5f and 5l = 30 µM).
Since IMPDH is evolutionarily conserved, these compounds might also inhibit other
pathogenic fungi such as C. albicans and A. fumigatus. As an initial effort we tested the
synthesized compounds at cellular level against C. neoformans (type strain H99; ATCC
208821) along with C. albicans (ATCC 90028), in order to evaluate their potential as broad-
spectrum antifungal agents. The in vitro antifungal screening was performed at a concentration
range of 256 µg/mL to 0.12 µg/mL. We observed that compounds 5a-5d exhibited good
inhibition against C. neoformans and C. albicans with MIC values ranging from 4 to 9 μM.
Most of the other compounds showed prominent antifungal activity against C. neoformans but
not C. albicans. These fungi have very different cellular structure for example C. neoformans
has a polysaccharide capsule absent in C. albicans and this may alter the compound ability to
penetrate the cell and subsequently inhibit IMPDH.
Finally, the cytotoxicity of 5a-5o was tested using human embryonic kidney 293 (HEK293)
and human liver hepatocellular carcinoma (HepG2) cell lines, with Tamoxifen as the positive
control. All of these compounds displayed potent cytotoxic activity against the tested cell lines,
9

with CC50 values in the range of 2-6 µM for the HEK293 cell line and 14-31 µM for HepG2
cell line (Table 1). Our attempts to reduce the cytotoxicity by the modification of the 1,3,4-
oxadiazole ring to 1,2,4 triazole in compound 5p and 1H-imidazole in compound 5q decreased
Cryptococcus IMPDH inhibitory activity (5p: 14.6 ± 1.4 µM; 5q: 24.3 ± 3 µM) and antifungal
activity (5p and 5q >172 µM) against both C. neoformans and C. albicans. This suggested that
the 1,3,4-oxadiazole moiety was important for Cryptococcus IMPDH inhibitory activity but
also lead to cytotoxicity. As IMPDH functions biologically as a homotetramer, with each
monomer consisting of the active site for binding the substrate IMP and the cofactor NAD, the
two most probable ways to inhibit IMPDH would be through binding to the active site or at
subunit homotetrameric interfaces.
10

Table 1: Biological activities of 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide derivatives.
IMPDH inhibition (IC50)a (µM)
Antifungal activity (MIC)b
(µM)
Cytotoxicity
(CC50)c (µM)
Compound
R
Cryptococcus
Human
Human
IMPDH II C. neoformans C. albicans HEK293 HepG2
IMPDH
IMPDH I
-
-
-
-
0.12 ± 0.02
0.26 ± 0.01 0.11 ± 0.01
-
0.25
0.3
-
-
0.18
8
-
-
MPA
-
-
-
-
-
-
-
-
-
-
-
-
-
Amphotericin B
Fluconazole
Tamoxifen
-
39.4
24.7
1.06 ± 0.03
0.59 ± 0.14
13.2 ± 0.6
6.3 ± 1
4.2 ± 0.6
6
4
4
4
5
4
24
14
5a
5b
2.6 ± 0.4
11

0.76 ± 0.07
0.37 ± 0.06
0.67 ± 0.16
0.34 ± 0.07
8.6 ± 1
5.6 ± 1
7.6 ± 0.2
>30
2.1 ± 0.2
2.1 ± 0.1
2.2 ± 0.7
>30
9
6
6
6
4
3
3
4
17
20
15
18
5c
5d
5e
5f
7
46
60
>160
0.23 ± 0.05
7.2 ± 1
1.2 ± 0.5
6
167
4
25
5g
5h
0.40 ± 0.19
0.49 ± 0.23
10.1 ± 3.4
11.1 ± 3.6
1.6 ± 0.7
1.5 ± 0.5
6
167
167
6
3
27
22
16
5i
12

0.48 ± 0.10
0.15 ± 0.05
9.9 ± 0.6
4.3 ± 0.4
3.2 ± 1
64
8
172
172
3
3
16
20
5j
1.2 ± 0.2
5k
1.7 ± 0.38
>30
>30
172
172
2
22
5l
0.41 ± 0.06
1.01 ± 0.14
20.3 ± 1.4
13.8 ± 0.6
5.7 ± 0.6
4.5 ± 0.1
22
33
90
3
4
31
24
5m
5n
>179
0.16 ± 0.05
20.4 ± 1.6
4.4 ± 0.6
>160
>160
4
31
5o
aIC50 values of the compounds against Cryptococcus IMPDH and human IMPDH isoforms I and II. The values represent the average of at least
three independent experiments and include standard deviations.
13

bAntifungal assays were carried out in duplicate with DMSO as the negative control, whereas amphotericin B and fluconazole were used as
positive controls.
cCytotoxic activities of the compounds with DMSO as the negative control and tamoxifen as the positive control
14

2.3. Enzyme inhibition kinetics
To further understand the mechanism of enzyme inhibition, a Michaelis-Menten enzyme
kinetic analysis was performed. Initial experiments to determine the Michaelis constant (Km)
were undertaken using the assay conditions [buffer containing 50 mM Tris-HCl (pH 8.0), 100
mM KCl, 3 mM EDTA, 1 mM DTT] reported by Morrow et al. for their kinetic studies.12
Similar to all other IMPDHs, Cryptococcus IMPDH also exhibited substrate inhibition at
higher concentrations of NAD.11,12,21-24 Therefore, the Km value of NAD (Km(NAD) = 402 ± 91
µM) was calculated by fitting kinetic data to a modified Michaelis-Menten equation that takes
substrate inhibition into account (Equation 5). The apparent Km value of IMP (Km(IMP) = 94 ±
18 µM) was determined with sub-saturating concentrations of NAD (700 µM) due to substrate
inhibition and fitted into the classical Michaelis-Menten equation (Equation 4). The inhibition
kinetics were also performed with MPA and compounds 5d and 5j by varying the concentration
of one substrate, while the other substrate was maintained at a fixed concentration (Table 2).
Consistent with previous reports, MPA showed uncompetitive inhibition (both Vmax and Km
decreased) against both substrates, because it binds at the NAD site of the enzyme-substrate
covalent complex (E-XMP*) formed after NADH leaves in the first step of the enzymatic
reaction.11
Initially, there was no inhibitory activity observed for compounds 5d and 5j when tested in the
DTT-containing buffer, which was used to determine the Km values for the substrates. To
investigate this, liquid chromatography-mass spectrometry (LC-MS) analysis of samples with
compounds 5a-5o dissolved in a DTT-containing buffer was employed and showed their
breakdown due to the attack of the free thiol group of DTT at the C3-position of the
benzo[b]thiophene 1,1-dioxide moiety; 5-substituted-1,3,4-oxadiazole thiols were detected in
the mass spectra (Figure 2).
15

Figure 2. A) LC-MS of compound 5d in buffer containing DTT. B) Mechanism of breakdown
of compound 5d by the attack of a free thiol of DTT at the C3-position of benzo[b]thiophene
1,1-dioxide.
The degradation of the benzo[b]thiophene 1,1-dioxide moiety supported the recent findings of
Dahlin et al.,13 who reported that the compounds with this chemotype as PAINS that formed
covalent adducts when treated with glutathione (GSH); these adducts were detected using mass
spectrometry (MS). Thus, inhibition kinetics for compounds 5d and 5j were performed using
a buffer without DTT, and with different concentrations of one substrate and fixed
concentrations of the other, with inhibitor concentrations from 0 to 20 µM. The plots obtained
with the best fit of kinetic data from both compounds showed a decrease in the Vmax value and
increase in the Km value (Figure 3 and Table 2). However, based on our studies (with DTT)
and the observed thiol reactivity of these compounds,13 it could be possible that free thiol
groups of cysteine residues of other proteins, including Cryptococcus IMPDH, could be
modified. Cryptococcus IMPDH has a cysteine residue in the active site that makes a covalent
interaction with IMP during initial step of biological catalysis, as well as other free cysteines.
To confirm that the inhibitory activity shown by synthesized compounds was due to the
modification of cysteine residues in IMPDH we performed MS studies.
16

Figure 3. Inhibition kinetics at different concentrations of compound 5d in conditions: A) by
varying the concentration of NAD, while holding IMP at a fixed concentration (500 µM) B)
by varying the concentration of IMP, while holding NAD at a fixed concentration (700 µM).
The data were fitted to Equation 4 shown in experimental section.
17

Table 2: Kinetic parameters for Cryptococcus IMPDH reaction and inhibition by compounds 5d and 5j. The concentrations of fixed substrate
are 500 µM for IMP and 700 µM for NAD, with NAD concentration varied from 31.25 µM to 1300 µM and IMP from 25 µM to 800 µM. The
data were fitted to Equation 4 shown in experimental section.
Substrate
Inhibition kinetics
5d (µM)
Kinetic
parameters
Fixed Varied
No
inhibitor
MPA (µM)
0.078
5j (µM)
2.5
0.039
0.156
0.3
2.5
10
0.3
10
1.5
1.1
0.6
0.26
1.5
1.3
1
1.5
1.5
1
Vmax
(µM/min)
NAD
IMP
IMP
257.5 ±
23
200 ±
15
137 ±
12
37
307 ± 22 336 ±
17
386 ± 23 278 ± 21.5 358.5 ± 36 355
± 20
Km (µM)
Kcat (s-1)
1.5
1.1
0.9
0.6
0.6
0.25
0.3
1.5
1.1
1.3
0.9
1
1.5
1.4
1.5
1
1
1.45
0.6
0.7
Vmax
(µM/min)
NAD
90 ± 12 64 ± 13 53 ±
10
32 ± 7.5 92.5 ± 19 187 ± 315.5 ± 95
44
93 ± 11
1.4
165 ± 26
1
258
± 47
Km (µM)
Kcat (s-1)
1.38
0.9
0.6
0.3
1.1
0.9
0.6
0.7
18

2.4. Mass spectrometry studies
Intact MS studies were conducted for samples of His-tagged Cryptococcus IMPDH in the
absence and presence of compound 5d. Deconvoluted MS data showed that the intact mass of
His-tagged Cryptococcus IMPDH as 59308 Da (theoretical mass = 59289 Da), and His-tagged
Cryptococcus IMPDH incubated with compound 5d were determined as 59471 Da, 59633 Da
and 59794 Da, which corresponded to single, double and triple modifications of cysteine
residues from the addition of 164 Da (from attack of a thiol group in cysteine at the C3-position
of benzo[b]thiophene 1,1-dioxide of 5d) (Figure 4). To investigate and characterize the
modification of cysteine residues and confirm sites of cysteine modification in the sequence of
Cryptococcus IMPDH, our mass spectrometry studies were extended to peptide sequencing.
Figure 4. A) Mass spectrometry of His-tagged Cryptococcus IMPDH incubated with
compound 5d. B) The deconvoluted mass spectrum showing intact masses of unmodified
(59308.71 Da) and modified protein (59471.85 Da, 59634 Da and 59794.23 Da).
The PeptideMass tool on the Expasy server25,26 predicted 35 tryptic peptide fragments
generated from IMPDH, with the seven cysteines in seven unique peptides (Table S1). The
catalytic cysteine (Cys 357) is present in fragment 5 (residue 349-382, M = 3455.7046 Da),
which corresponded to the active site loop in Cryptococcus IMPDH. The fragments identified
from the His-tagged Cryptococcus IMPDH sample showed good coverage of 97% with 95%
confidence. The peptide fragment 5 produced an ion at m/z 1153.9 [(M + 3H)3+], which agreed
19

with the theoretical m/z value for 3+ protonated peptide (Figure S1). All cysteine containing
peptide fragments were also detected by the database search (Figure S3-S7).
Tryptic digestion of His-tagged Cryptococcus IMPDH incubated with compound 5d on ice
and room temperature resulted in 81% coverage (at a 95% confidence) of the protein sequence
and modification of 5 cysteines with the 164 Da from benzo[b]thiophene 1,1-dioxide from
compound 5d (Figure S2 and S8-S11). This was consistent with the multiple modifications
observed in the mass spectra of the undigested His-tagged Cryptococcus IMPDH incubated
with compound 5d. Interestingly, the catalytic cysteine (Cys 357) containing peptide fragment
5 (residues 349-382) was not detected in this sample, neither in the unmodified or modified
form. Some differences with the enzyme-only digest were also detected, where peptide
fragment 1 (residue 108-135) was found as a mis-cleaved form containing residues 125-135,
with cyano group modification on cysteine.
3. Conclusions
3-((5-Substituted)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxides identified by
high-throughput screening of Cryptococcus IMPDH were investigated as potential antifungals.
All analogs 5a-5o had similar Cryptococcus IMPDH in vitro activities (≤ 1 µM) but exhibited
potent human cell line cytotoxicities. Modifications to the 1,3,4-oxadiazole ring led to
decreased cytotoxicity but also impaired Cryptococcus IMPDH inhibition and antifungal
activities. The inhibition kinetics of compounds 5d and 5j using previously reported conditions
that incorporated DTT did not show Cryptococcus IMPDH inhibitory activity due to the adduct
formation of the benzo[b]thiophene 1,1-dioxide with DTT. Further investigation through MS
studies confirmed that these compounds made covalent bound to multiple cysteine residues in
Cryptococcus IMPDH in a promiscuous fashion. This reinforces the importance of eliminating
PAINS from screening libraries.
20

4. Experimental section
4.1. Chemistry
4.1.1. General Methods and Materials
All starting materials were purchased from standard commercial sources and used without
further purification, unless otherwise stated. All reactions were carried out with magnetic
stirring using magnetic bars under an inert nitrogen atmosphere with anhydrous solvents unless
otherwise stated. The progress of reactions was monitored by either thin-layer chromatography
(TLC) on 0.25 mm silica gel plates (TLC) on 0.25 mm silica gel plates (60F-254) and visualized
with UV light or a KMnO4 dip. Analytical 1H and 13C NMR spectra were recorded in deuterated
solvents at 298 K on a Varian Unity 400 MHz or Bruker Advance 600 MHz spectrometers.
The chemical shifts are reported as a part per million (ppm) downfield from tetramethylsilane
(TMS) and coupling constants (J) were calculated in hertz (Hz). All final compounds were
purified to ≥95% by the Grace Reveleris chromatography instrument using UV detection. High
resolution mass spectrometry (HRMS) was performed on the Bruker MicroTOF mass
spectrometer.
4.1.2. General procedure for synthesis of 5-substituted 1,3,4-oxadiazole-2-thiols
To a solution of acid hydrazide in anhydrous 5-15 mL of DMF, carbon disulfide (2.5 mL/mmol)
was added at room temperature and under a nitrogen atmosphere. The reaction mixture was
then heated to 40 °C for 15 minutes and then to 70 °C for 4-8 h until the reaction was completed.
After completion, the reaction mixture was cooled to room temperature and poured dropwise
into ice cold water. The solids formed were separated by filtration, washed with water and dried
in vacuo.
5-(Pyridin-2-yl)-1,3,4-oxadiazole-2-thiol (4a) Prepared from pyridine-2-carbohydrazide 3a
(0.3 g, 21.8 mmol) and CS2 (0.5 g, 6.50 mmol) according to general procedure A. Yield 170
21

mg (43%); yellow solid; MS (ESI) m/z (%): 180.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ
14.8 (s, 1H), 8.76 (dd, J = 1.7, 1.1 Hz, 1H), 8.09-7.97 (m, 2H), 7.64 (dd, J = 4.8, 1.9 Hz, 1H).
5-(Pyridin-3-yl)-1,3,4-oxadiazole-2-thiol (4b) Prepared from pyridine-3-carbohydrazide 3b
(0.3 g, 21.8 mmol) and CS2 (0.5 g, 6.50 mmol) according to general procedure A. Yield 120
mg (30%); yellow solid; MS (ESI) m/z (%): 180.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ
9.04 (dd, J = 2.3, 0.9 Hz, 1H), 8.79 (dd, J = 4.9, 1.6 Hz, 1H), 8.31-8.20 (m, 1H), 7.68-7.54 (m,
1H).
5-(Pyridin-4-yl)-1,3,4-oxadiazole-2-thiol (4c) Prepared from pyridine-4-carbohydrazide 3c
(0.3 g, 21.8 mmol) and CS2 (0.5 g, 6.50 mmol) according to general procedure A. Yield 180
mg (50%); yellow solid; MS (ESI) m/z (%): 180.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ
9.05-8.92 (m, 2H), 8.09 - 7.97 (m, 2H).
5-(Phenyl)-1,3,4-oxadiazole-2-thiol (4d) Prepared from benzohydrazide 3d (1.0 g, 5.6 mmol)
and CS2 (1.6 g, 21.0 mmol) according to general procedure A. Yield 0.52 g (40%); yellow
solid; MS (ESI) m/z (%): 179.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 14.7 (s, 1H), 7.93-
7.85 (m, 2H), 7.69-7.55 (m, 2H).
Methyl 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzoate (4f) Prepared from methyl 4-
(hydrazinecarbonyl)benzoate 3f (0.15 g, 0.77 mmol) and CS2 (0.12 g, 1.50 mmol) according to
general procedure A. Yield 120 mg (66%); yellow solid; MS (ESI) m/z (%): 237.2 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.15- 8.09 (m, 2H), 8.06-8.00 (m, 2H), 3.90 (d, J
= 1.4 Hz, 3H).
5-(2-Chlorophenyl)-1,3,4-oxadiazole-2-thiol (4g) Prepared from 2-chlorobenzohydrazide 3g
(2.0 g, 11.7 mmol) and CS2 (2.6 g, 34.2 mmol) according to general procedure A. Yield 1.53 g
(63%); yellow solid; MS (ESI) m/z (%): 213.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.88
(ddd, J = 7.8, 1.7, 0.5 Hz, 1H), 7.67 (dd, J = 1.4, 0.5 Hz, 1H), 7.61 (dd, J = 1.4, 0.5 Hz, 1H),
7.52 (dd, J = 1.4, 0.5 Hz, 1H).
22

5-(3-Chlorophenyl)-1,3,4-oxadiazole-2-thiol (4h) Prepared from 3-chlorobenzohydrazide 3h
(2.0 g, 11.7 mmol) and CS2 (2.6 g, 34.2 mmol) according to general procedure A. Yield 1.4 g
1
(58%); yellow solid; MS (ESI) m/z (%): 213.3 [M+H]+. H NMR (400 MHz, DMSO-d6) δ
14.79 (s, 1H), 7.84-7.80 (m, 1H), 7.80 (dd, J = 1.6, 1.1 Hz, 1H), 7.67 (dd, J = 2.1, 1.1 Hz, 1H),
7.58 (dd, J = 1.6, 0.6 Hz, 1H).
5-(4-Chlorophenyl)-1,3,4-oxadiazole-2-thiol (4i) Prepared from 4-chlorobenzohydrazide 3i
(2.0 g, 11.7 mmol) and CS2 (2.6 g, 34.2 mmol) according to general procedure A. Yield 1.4 g
(58%); yellow solid; MS (ESI) m/z (%): 213.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.94-
7.81 (m, 2H), 7.67 - 7.58 (m, 2H).
5-(2-Methoxyphenyl)-1,3,4-oxadiazole-2-thiol
(4j)
Prepared
from
2-
methoxybenzohydrazide 3j (2.0 g, 12.0 mmol) and CS2 (2.74 g, 36.0 mmol) according to
1
general procedure A. Yield 1.7 g (68%); yellow solid; MS (ESI) m/z (%): 209.2 [M+H]+. H
NMR (400 MHz, DMSO-d6) δ 7.70 (dd, J = 7.7, 1.8 Hz, 1H), 7.62-7.48 (m, 1H), 7.22 (dd, J =
8.5, 0.9 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.86 (s, 3H).
5-(3-Methoxyphenyl)-1,3,4-oxadiazole-2-thiol
(4k)
Prepared
from
3-
methoxybenzohydrazide 3k (2.0 g, 12.0 mmol) and CS2 (2.74 g, 36.0 mmol) according to
1
general procedure A. Yield 1.6 g (64%); yellow solid; MS (ESI) m/z (%): 209.2 [M+H]+. H
NMR (400 MHz, DMSO-d6) δ 7.50-7.41 (m, 2H), 7.31 (dd, J = 1.5, 0.4 Hz, 1H), 7.17 (dd, J =
2.7, 1.3 Hz, 1H), 3.81 (s, 3H).
5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-thiol
(4l)
Prepared
from
4-
methoxybenzohydrazide 3l (2.0 g, 12.0 mmol) and CS2 (2.74 g, 36.0 mmol) according to
1
general procedure A. Yield 1.5 g (60%); yellow solid; MS (ESI) m/z (%): 209.2 [M+H]+. H
NMR (400 MHz, DMSO-d6) δ 7.83-7.74 (m, 2H), 7.17-7.05 (m, 2H), 3.81 (s, 3H).
Synthesis of 5-(Cyclohexyl)-1,3,4-oxadiazole-2-thiol (4e) To solution of synthesized
cyclohexyl hydrazide 3e (0.5 g, 3.52 mmol) in ethanol, potassium hydroxide (0.25 g, 4.46
23

mmol) and carbon disulfide (1.3 g, 17.1mmol) were added at room temperature and under a
nitrogen atmosphere. The reaction mixture was then heated to 70 °C overnight. The reaction
mixture was evaporated in vacuo and acidified to pH 5 with 2 M HCl. The aqueous layer was
extracted with ethyl acetate (3 × 50 mL) and organics were dried (MgSO4) and concentrated in
vacuo to yield 5-(cyclohexyl)-1,3,4- oxadiazole-2-thiol (4e) as colorless oil; yield 0.25 mg
(39%) MS (ESI) m/z (%): 185.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 2.77 (tt, J = 10.9,
3.7 Hz, 1H), 1.94-1.87 (m, 2H), 1.71-1.55 (m, 3H), 1.45-1.16 (m, 5H).
4.1.3. Synthesis of 5-(3-Methoxyphenyl)-1H-imidazole-2-thiol (4p)
Step A. To a solution of 2-bromo-1-(3-methoxyphenyl)ethan-1-one) (2.0 g, 8.70 mmol) in 20
mL of dichloromethane, hexamine (1.46 g, 10.4 mmol) was added and the mixture was stirred
at room temperature overnight. The white solids formed were separated by filtration and dried
in vacuo. Then solids were dissolved in 15 mL of methanol and 2 mL of c. HCl and heated at
60 °C for 3 h. The reaction completion was monitored by LC-MS and then reaction mixture
was cooled and filtered to separate the inorganics. Excess solvent from the filtrate was
evaporated in vacuo and solids were neutralized with aqueous solution of sat. NaHCO3,
separated by filtration and dried in vacuo to give 2-amino-1-(3-methoxyphenyl)ethan-1-one as
a dark brown solid. Yield: 0.8 g, 57%; MS (ESI) m/z (%): 166.2 [M+H]+. 1H NMR (400 MHz,
DMSO-d6) δ 7.58 (dt, J = 7.8, 1.2 Hz, 1H), 7.51 – 7.45 (m, 2H), 7.27 (dd, J = 2.6, 0.9 Hz, 1H),
4.55 (s, 2H), 3.81 (s, 3H).
Step B. To a solution of 2-amino-1-(3-methoxyphenyl)ethan-1-one (0.2 g, 1.21 mmol) in 4 mL
of glacial acetic acid, potassium thiocyanate (0.15g, 1.57 mmol) was added at room
temperature and heated to 140 °C for 2 h. The reaction mixture was then diluted with water (25
mL) and extracted with ethyl acetate 2 × 50 mL. The organics were dried (MgSO4) and
evaporated in vacuo to give 5-(3-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (4p) as a dark
1
brown solid. Yield: 70 mg, 29%; MS (ESI) m/z (%): 207.2 [M+H]+. H NMR (400 MHz,
24

DMSO-d6) δ 12.47 (s, 1H), 12.09 (s, 1H), 7.38 (t, J = 2.3 Hz, 1H), 7.28 - 7.18 (m, 3H), 6.78
(dd, J = 2.5, 1.6 Hz, 1H), 3.74 (s, 3H).
4.1.4. Synthesis of 3-bromobenzo[b]thiophene 1,1-dioxide (2)
To solution of 1 (1.0 g, 4.70 mmol) in dichloromethane, mCPBA (4.4 g, 25.0 mmol) was added
at 0 °C and the mixture was warmed to room temperature and left stirring for 24 h. TLC was
spotted with a small aliquot of the reaction mixture, which indicated that the reaction was
completed. Excess DCM was added and washed with sat. NaHCO3 (aq) (50 mL × 2), followed
by brine solution (50 mL × 1). The organics were dried (MgSO4) and evaporated in vacuo. The
residue was then recrystallized from absolute ethanol to get product in good yield. White
crystalline solid; Yield: 0.65 mg, 57%; MS (ESI) m/z (%): 247 [M+H]+; 1H NMR (400 MHz,
CDCl3) δ 7.76-7.53 (m, 4H), 6.98 (s, 1H).
4.1.5. General procedure for coupling of 3-bromobenzo[b]thiophene 1,1-dioxide with 5-
substituted 1,3,4-oxadiazole-2-thiols
To a solution of 3-bromobenzo[b]thiophene 1,1-dioxide 2 dissolved in 1,4-dioxane (5
mL/mmol), CuI (0.5 mol %) and benzotriazole (1.0 mol %) were added and the mixture was
stirred for 10 min at room temperature. Then thiols 4a-4p and potassium tert-butoxide (1.5 eq)
were added to the reaction mixture, which was stirred at 95 °C overnight. The reaction
completion was monitored by LC-MS analysis. Then reaction mixture was cooled to room
temperature and excess solvent was evaporated in vacuo. The residue obtained was dissolved
in DMSO/acetonitrile (3:1) and loaded onto C18 column (GRACE Flash) for purification using
water/acetonitrile as the mobile phase. The fractions that contained product were collected and
lyophilized to provide 4a-4q in good yields.
3-((5-(Pyridin-2-yl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5a) Prepared
from thiol 4a (50.0 mg, 0.28 mmol) and 2 (82 mg, 0.33 mmol) according to general procedure
1
E. Yield 16 mg (17%); light yellow solid; MS (ESI) m/z (%): 344.1 [M+H]+. H NMR (600
25

MHz, DMSO-d6) δ 8.82 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.25 (dt, J = 7.9, 1.1 Hz, 1H), 8.10 (td,
J = 7.8, 1.8 Hz, 1H), 8.02-7.99 (m, 1H), 7.84-7.80 (m, 1H), 7.79-7.75 (m, 2H), 7.73 (s, 1H),
7.69 (dd, J = 4.8, 1.2 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ 167.1, 157.6, 150.8, 142.9,
138.7, 138.4, 136.8, 135.0 ,132.5, 129.4, 127.3, 127.0, 123.8, 123.3, 121.8; HRMS calculated
for C15H10N3O3S2: 344.0158, found 344.0153.
3-((5-(Pyridin-3-yl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5b) Prepared
from thiol 4b (50.0 mg, 0.28 mmol) and 2 (82 mg, 0.33 mmol) according to general procedure
1
E. Yield 14 mg (15%); light yellow solid; MS (ESI) m/z (%): 344.1 [M+H]+. H NMR (600
MHz, DMSO-d6) δ 9.22 (d, J = 2.1 Hz, 1H), 8.86 (dd, J = 5.0, 1.6 Hz, 1H), 8.43 (dt, J = 8.0,
13
2.0 Hz, 1H), 8.00 (d, J = 7.4 Hz, 1H), 7.85-7.74 (m, 4H), 7.69 (dd, J = 8.0, 5.0 Hz, 1H). C
NMR (150 MHz, DMSO-d6) δ 166.2, 157.3, 153.4, 147.9, 138.7, 136.8, 135.0, 134.9, 132.5,
129.4, 126.9, 124.8, 123.3, 121.8, 120.2; HRMS calculated for C15H10N3O3S2: 344.0158, found
344.0164.
3-((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5c) Prepared
from thiol 4c (50.0 mg, 0.28 mmol) and 2 (82 mg, 0.33 mmol) according to general procedure
1
E. Yield 12 mg (13%); light yellow solid; MS (ESI) m/z (%): 344.1 [M+H]+. H NMR (600
MHz, DMSO-d6) δ 8.89 - 8.86 (m, 2H), 8.01 (t, J = 0.9 Hz, 1H), 7.99-7.98 (m, 2H), 7.84-7.75
(m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 166.3, 158.0, 151.4, 138.51, 136.8, 134.9, 132.5,
130.6, 129.4, 127.1, 123.4, 121.8, 120.8; HRMS calculated for C15H10N3O3S2: 344.0158, found
344.0168.
3-((5-Phenyl-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5d) Prepared from
thiol 4d (50.0 mg, 0.28 mmol) and 2 (82 mg, 0.33 mmol) according to general procedure E.
Yield 35 mg (37%); light yellow solid; MS (ESI) m/z (%): 343.2 [M+H]+. 1H NMR (600 MHz,
DMSO-d6) δ 8.06-8.04 (m, 2H), 8.00-7.96 (m, 1H) 7.83-7.80 (m, 1H), 7.78-7.74 (m, 3H), 7.70-
7.67 (m, 1H), 7.65-7.62 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 167.9, 156.6, 138.8, 136.8,
26

134.9, 133.0, 132.5, 129.9, 129.9, 129.4, 126.8, 123.5, 23.3, 121.8; HRMS calculated for
C16H11N2O3S2: 343.0205, found 343.0211.
3-((5-Cyclohexyl-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5e) Prepared
from thiol 4e (50.0 mg, 0.27 mmol) and 2 (79 mg, 0.32 mmol) according to general procedure
1
E. Yield 18 mg (16%); light yellow solid; MS (ESI) m/z (%): 349.5 [M+H]+. H NMR (600
MHz, DMSO-d6) δ 7.98 (dd, J = 7.5, 4.6 Hz, 1H), 7.82 - 7.74 (m, 2H), 7.70 (dd, J = 7.6, 4.6
Hz, 1H), 7.60 (d, J = 4.9 Hz, 1H), 3.04 (td, J = 11.1, 5.0 Hz, 1H), 2.06 - 2.01 (m, 2H), 1.74
(dt, J = 13.3, 4.3 Hz, 2H), 1.67-1.60 (m, 1H), 1.55 (q, J = 11.9 Hz, 2H), 1.43 - 1.34 (m, 2H),
1.27 (td, J = 12.2, 4.1 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ 174.0, 156.4, 138.9, 137.1,
135.2, 132.8, 129.7, 127.1, 123.5, 122.1, 35.1, 30.00, 25.88, 25.3; HRMS calculated for
C16H17N2O3S2: 349.0675, found 349.0686.
Methyl 4-(5-((1,1-dioxidobenzo[b]thiophen-3-yl)thio)-1,3,4-oxadiazol-2-yl)benzoate (5f)
Prepared from thiol 4f (50.0 mg, 0.21 mmol) and 2 (61.0 mg, 0.25 mmol) according to general
procedure E. Yield 5 mg (6%); light yellow solid; MS (ESI) m/z (%): 401.4 [M+H]+. 1H NMR
(600 MHz, DMSO-d6) δ 8.24-8.16 (m, 4H), 8.00 (dt, J = 7.5, 1.0 Hz, 1H), 7.87- 7.73 (m, 4H),
3.91 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 167.2, 165.8, 157.3, 138.7, 136.8, 134.9, 133.1,
132.5, 130.6, 129.4, 127.8, 127.4, 126.9, 123.3, 121.8, 53.0; HRMS calculated for
C18H13N2O5S2: 401.0260, found 401.0260.
3-((5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5g)
Prepared from thiol 4g (50.0 mg, 0.23 mmol) and 2 (67 mg, 0.27 mmol) according to general
1
procedure E. Yield 24 mg (32%); dark brown solid; MS (ESI) m/z (%): 377.2 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 8.07 (dd, J = 7.9, 1.7 Hz, 1H), 8.01 - 7.99 (m, 1H), 7.84 - 7.80
13
(m, 2H), 7.78 - 7.73 (m, 3H), 7.69 (dd, J = 7.4, 1.7 Hz, 1H), 7.62 - 7.59 (m, 1H). C NMR
(150 MHz, DMSO-d6) δ 165.8, 157.5, 138.0, 136.8, 134.9, 134.1, 132.5, 132.3, 131.9, 131.7,
27

129.5, 128.3, 127.9, 123.4, 122.3, 121.8; HRMS calculated for C16H10ClN2O3S2: 376.9816,
found 376.9802
3-((5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5h)
Prepared from thiol 4h (50.0 mg, 0.23 mmol) and 2 (67 mg, 0.27 mmol) according to general
1
procedure E. Yield 26 mg (33%); dark brown solid; MS (ESI) m/z (%): 377.2 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 8.06-7.99 (m, 3H), 7.84-7.81 (m, 1H), 7.79-7.75 (m, 4H), 7.68
(t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ 166.8, 157.2, 138.6, 136.8, 134.9, 134.5,
132.8, 132.5, 132.0, 129.4, 127.0, 126.8, 126.1, 125.4, 123.3, 121.8; HRMS calculated for
C16H10ClN2O3S2: 376.9816, found 376.9812.
3-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5i)
Prepared from thiol 4i (50.0 mg, 0.23 mmol) and 2 (67.0 mg, 0.27 mmol) according to general
1
procedure E. Yield 20 mg (26%); dark brown solid; MS (ESI) m/z (%): 377.2 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 8.09-8.03 (m, 2H), 7.99 (dd, J = 7.1, 1.6 Hz, 1H), 7.83-7.74 (m,
4H), 7.73-7.70 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 167.2, 156.9, 138.7, 137.7, 136.8,
134.9, 132.5, 130.1, 129.4, 129.2, 126.8, 123.3,122.4, 121.8; HRMS calculated for
C16H10ClN2O3S2: 376.9816, found 376.9801.
3-((5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5j)
Prepared from thiol 4j (50.0 mg, 0.24 mmol) and 2 (67.0 mg, 0.28 mmol) according to general
1
procedure E. Yield 18 mg (18%); light yellow solid; MS (ESI) m/z (%): 373.1 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 8.00 (d, J = 7.4 Hz, 1H), 7.92 (dd, J = 7.9, 1.7 Hz, 1H), 7.81 (t,
J = 7.5 Hz, 1H), 7.78-7.73 (m, 2H), 7.71 (s, 1H), 7.66-7.62 (m, 1H), 7.28 (d, J = 8.6 Hz, 1H),
7.16 (t, J = 7.6 Hz, 1H), 3.88 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 166.6, 157.9, 156.5,
138.3, 136.8, 134.9, 134.6, 132.5, 130.7, 127.4, 123.3, 123.3, 121.8, 121.2, 113.2, 112.0, 56.4;
HRMS calculated for C17H13N2O4S2: 373.0311, found 373.0309.
28

3-((5-(3-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5k)
Prepared from thiol 4k (50.0 mg, 0.24 mmol) and 2 (67.0 mg, 0.28 mmol) according to general
1
procedure E. Yield 10 mg (10%); light yellow solid; MS (ESI) m/z (%): 373.1 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 7.99 (dd, J = 7.8, 1.2 Hz, 1H), 7.84-7.79 (m, 1H), 7.79-7.72 (m,
3H), 7.62 (dt, J = 7.7, 1.2 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.51 (dd, J = 2.7, 1.6 Hz, 1H),
7.26 (dd, J = 2.7, 1.0 Hz, 1H), 3.85 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 167.6, 160.1,
156.8, 138.7, 136.8, 134.9, 132.5, 131.3, 129.4, 127.1, 124.6, 123.3, 121.8, 119.7, 119.0, 112.0,
55.9; HRMS calculated for C17H13N2O4S2: 373.0294, found 373.0311.
3-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5l)
Prepared from thiol 4l (50.0 mg, 0.24 mmol) and 2 (67.0 mg, 0.28 mmol) according to general
1
procedure E. Yield 15 mg (15%); light yellow solid; MS (ESI) m/z (%): 373.1 [M+H]+. H
NMR (600 MHz, DMSO-d6) δ 8.02-7.96 (m, 3H), 7.81 (td, J = 7.5, 1.2 Hz, 1H), 7.79-7.74 (m,
13
2H), 7.72 (s, 1H), 7.20-7.15 (m, 2H), 3.86 (s, 3H). C NMR (150 MHz, DMSO-d6) δ 167.8,
162.9, 155.7, 138.9, 136.8, 134.9, 132.5, 129.4, 129.3, 126.6, 123.2, 121.8, 115.7, 115.3, 56.0;
HRMS calculated for C17H13N2O4S2: 373.0311, found 373.0293.
3-((5-(p-Tolyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5m) Prepared
from thiol 4m (50.0 mg, 0.25 mmol) and 2 (73.0 mg, 0.3 mmol) according to general procedure
1
E. Yield 13 mg (18%); light yellow solid; MS (ESI) m/z (%): 157.4 [M+H]+. H NMR (600
MHz, DMSO-d6) δ 8.01-7.99 (m, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.84- 7.80 (m, 1H), 7.79-7.75
(m, 2H), 7.74 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 2.42 (s, 3H). 13C NMR (150 MHz, DMSO-d6)
δ 168.0, 156.2, 143.3, 138.8, 136.8, 134.9, 132.5, 130.4, 129.4, 127.3, 126.8, 123.3, 121.8,
120.7, 21.6; HRMS calculated for C17H13N2O3S2: 357.0362, found 357.0376.
3-((5-(4-Aminophenyl)-1,3,4-oxadiazol-2-yl)thio)benzo[b]thiophene 1,1-dioxide (5n)
Prepared from thiol 4n (50.0 mg, 0.25 mmol) and 2 (73.0 mg, 0.3 mmol) according to general
1
procedure E. Yield 30 mg (26%); Dark yellow solid; MS (ESI) m/z (%): 358.2 [M+H]+. H
29