Structure mechanism insights and the role of nitric oxide donation guide the development of oxadiazole-2-oxides as therapeutic agents against schistosomiasis

Article abstract of DOI:10.1021/jm901021k

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901021k

6474 J. Med. Chem. 2009, 52, 6474–6483
DOI: 10.1021/jm901021k
Structure Mechanism Insights and the Role of Nitric Oxide Donation Guide the Development of
Oxadiazole-2-Oxides as Therapeutic Agents against Schistosomiasis
Ganesha Rai,† Ahmed A. Sayed,‡,§ Wendy A. Lea,† Hans F. Luecke, Harinath Chakrapani,^ Stefanie Prast-Nielsen,#
†
†
†
†
†
^
#
ꢀ
Ajit Jadhav, William Leister, Min Shen, James Inglese, Christopher P. Austin, Larry Keefer, Elias S. J. Arner,
Anton Simeonov,† David J. Maloney,† David L. Williams,*,‡,3 and Craig J. Thomas*,†
†NIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive, MSC 3370, Bethesda,
Maryland 20892-3370, ‡Department of Biological Sciences, Illinois State University, Normal, Illinois 61790, §Department of Biochemistry,
Ain Shams University, Cairo, Egypt, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
NIH, Bethesda, Maryland 20892-3370, ^Laboratory of Comparative Carcinogenesis, National Cancer Institute, NIH, Frederick, Maryland
21702-1201, #Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institutet, Stockholm SE-17177,
Sweden, and 3Department of Immunology/Microbiology, Rush University Medical Center, 1735 West Harrison Street, Chicago, Illinois 60612
Received July 10, 2009
Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide.
Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant
parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently
identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing
flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic
evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore,
establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this
chemotype versus related reductase enzymes, and present evidence that these agents can be modified to
possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between
exogenous NO donation and parasite injury is expanded and better defined. The results of these studies
verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal
agents.
Introduction
species has led to its widespread use (tens of millions of annual
treatments).7 Because individuals do not acquire immunity
after treatment and are rapidly reinfected, treatment must be
administeredannuallyorbiannually. Although PZQ’s mecha-
nism of action is not fully understood, research suggests that
PZQ alters homeostasis of calcium, resulting in a state of
schistosomal paralysis that leads to exposure and increased
susceptibility to the host immune system.8 A recent study has
demonstrated that RNAi knockdown of the voltage-operated
calcium channel β subunit ablated praziquantel efficacy in
Dugesia japonica.9 The dependence and extensive use of PZQ
has triggered concerns about the emergence of drug resistant
parasites, and evidence of drug resistance has been reported.10
Given the solitary and precarious reliance on PZQ for the
control of such a widespread disease, there is an urgent need to
define novel targets and drugs for the treatment of schistoso-
miasis.
The schistosome life cycle requires a snail intermediate host
and a mammalian definitive host. Schistosome development
in humans is complex and begins with cercarial penetration of
human skin. Following entry into the circulatory system,
parasites travel through the lungs and ultimately situate in
the liver where they grow, differentiate, mate, and then
migrate to the urogenital system or mesenteric veins of the
hepatoportal system to commence egg production. Given their
wide tissue distribution and long lifespan (up to 30 years),
schistosomes have evolved robust defense mechanisms to
mitigate oxidative damage and the host immune response.
The chronic infectious disease schistosomiasis is a major
neglected disease caused by trematode flatworms of the genus
Schistosoma.1 Estimates suggest that a staggering 200þ mil-
lion individuals suffer with this disease worldwide, and the
sub-Saharan Africa region experiences an estimated 280000
deaths annually from schistosomiasis.2,3 Further, schistoso-
miasis causes altered immune responses to infections and
vaccinations, increased perinatal transmission of HIV, and
increased susceptibility to and transmission of malaria, tuber-
culosis, hepatitis C, and HIV.1,4 Schistosomiasis ranks among
the most prominent neglected diseases due to the lack of
interest from commercial pharmaceutical enterprises. It is
rampant in impoverished populations due to inadequate
sanitation and tainted water supplies, and the burden of
infection perpetuates these conditions. There is currently no
vaccine for schistosomiasis, and treatment of the disease is
accomplished solely through the use of praziquantel (PZQa)
(1) (Figure 1).5,6 The low cost of this agent (<$0.25 USD/
dose) and its efficacy against the adult form of all schistosome
*To whom correspondence should be addressed. For C.J.T.: phone,
301-217-4079; fax, 301-217-5736; E-mail, craigt@nhgri.nih.gov.
For D.L.W.: phone, 312-942-1375; fax,
david_l_williams@rush.edu.
a Abbreviations: TGR, thioredoxin glutathione reductase; NO, nitric
oxide; PZQ, praziquantel; GR, glutathione reductase; TrxR, thioredox-
in reductase; Prx2, peroxiredoxin2; GSH, glutathione.
312-942-2808; E-mail,
r
pubs.acs.org/jmc
Published on Web 09/17/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6475
Most eukaryotes have multiple enzymatic processes to detox-
ify reactive oxygen species. In humans, these detoxification
pathways depend upon two NADPH-dependent flavoen-
zymes that maintain glutathione and thioredoxin in their
reduced state, aptly named glutathione reductase (GR) and
thioredoxin reductase (TrxR).11,12 Biochemical, reverse gene-
tic, and genomic analysis of Schistosoma mansoni provide
evidence that reduction of both glutathione and thioredoxin
in schistosomes is dependent upon a single multifunctional
selenocysteine-containing flavoenzyme designated thioredox-
in glutathione reductase (TGR).13 A crystal structure of TGR
(which contains a total of 13 cysteine residues and one
selenocysteine) has been presented and offers a structural
rationale for this dual activity.14 The dependence of schisto-
somes on a single, biochemically unique redox system is
theorized to present an opportunity for drug development.
Williams and co-workers revealed TGR as the putative
molecular target of potassium antimonyl tartrate (a former
treatment option for schistosomiasis) and that RNA inter-
ference targeting TGR resulted in rapid larval death (>90%
in 4 days).15 Additionally, the disease-modifying antirheu-
matic drug auranofin (a gold containing sugar conjugate) was
found to be a potent inhibitor of TGR and killing of ex vivo
parasites by this agent paralleled inhibition of TGR.13,15
These results present convincing evidence that TGR is an
appropriate molecular target for pharmacological interven-
tion and potential chemotherapy for schistosomiasis. We
recently reported the development and validation of assays
based upon the redox cascade of TGR in concert with
peroxiredoxin2 (Prx2) using fluorescence intensity change
associated with the consumption of NADPH16 and via a
colorimetric approach utilizing reduction of Ellman’s re-
agent.17 A quantitative high-throughput screen18 of the NIH
Molecular Libraries19 Small Molecule Repository was per-
formed, and numerous small molecule actives were identi-
fied.16 The chemotypes identified from this screen showed a
range of potencies (including several in the low nanomolar
range) andwerevalidatedasagentscapableof inhibiting TGR
in both the screening (Prx2-coupled NADPH consumption)
and the follow-up (DTNB reduction) assay formats. Selected
compounds were then advanced to investigations involving ex
vivo worm cultures from each Schistosoma species that infects
humans (S. mansoni, S. japonicum, and S. hematobium). One
agent, the well-studied oxadiazole-2-oxide designated furoxan
(2) (Figure 1), was capable of potent ex vivo worm killing at
modest concentrations (10 μM) and, importantly, against
all developmental stages of worm (transformed skin larva,
lung-stage schistosomula, juvenile liver worms, and adult
worms).20 On the basis of this encouraging result, furoxan
(2) was entered into an in vivo evaluation in S. mansoni
infected mice. Five consecutive daily intraperitoneal treat-
ments of 10 mg/kg of 2 were performed in mice during the
skin-stage infection (day 1-5), the juvenile/liver-stage infec-
tion (day 23-27), and the adult/egg-laying stage infection
(day 37-41), and efficacy was quantified by adult worm
burdens at day 49.20 Significant reduction of worm burdens
and hepatomegaly and splenomegaly were noted for treat-
ment groups at each parasite life stage. Both male and female
worms were affected, and a lower number of egg-induced
granulomas were noted in comparison to control groups.
Taken as a whole, the activity of furoxan (2) far exceeds
the activity criteria set forth by the World Health Organi-
zation for candidate therapeutic development for schisto-
somiasis.21
Figure 1. Structures of praziquantel (1), furoxan(2), and bis(thiophen-
2-ylmethanone)-furoxan (3).
The oxadiazole-2-oxide core is a well-studied structural
motif explored by Ponzio in the early half of the 20th century
and more recently expanded upon by Gasco and co-workers
as a preeminent class of nitric oxide (NO) donating com-
pounds.22,23 NO [the nitrogen-oxygen radical (NO•) not to
be confused with the nitroxyl anion (NO-)]24,25 is a well-
studied physiological signaling agent best known for its ability
to relax smooth muscle tissue, resulting in vasodilatation and
increased blood flow.26 NO has been implicated in numerous
biochemical processes and, importantly, elevated and sus-
tained levels of NO can be toxic. Gasco first reported the
thiol mediated release of NO from furoxan (2) and the
resulting vasodilatory properties in 1994.27 Since that time,
the core oxadiazole-2-oxide moiety has been conjugated to
numerous bioactive compounds in attempts to make chimeric
small molecules with dual activities including NO-donating
β-adrenoceptor agonists,28 NO-donating aspirin conjugates,29
and NO-donating proton pump inhibitors.30
There is ample direct and indirect evidence that NO can act
as an antischistosomal and, more broadly, antiparasitic
molecule.31-33 NO produced by human white cells has been
shown to kill larval schistosome parasites.34 NO produced
through inducible NO synthase activation is a core compo-
nent of the immune response, and direct evidence of NO being
a primary aspect of host defense against Echinococcus gran-
ulosus and Trypanosoma congolense has been presented.35,36
Winberg et al. recently demonstrated that NO production
diminished Leishmania donovani promastigotes ability to in-
hibit periphagosomal F-actin breakdown, allowing normal
phagosomal maturation and parasite killing.37 Beyond innate
activation of the NO synthase, exogenous NO from small
molecule NO donors has shown promise in several arenas. A
topical formulation of S-nitroso-N-acetylpenicillamine (SNAP)
was reported as an effective treatment option for the control
of cutaneous leishmaniasis.38 A class of bicyclic nitroimida-
zoles was recently reported as a deazaflavin-dependent nitror-
eductase prodrug that releases oxidized nitrogen molecules
that are converted to NO and are capable of anaerobic killing
of Mycobacterium tuberculosis.39 Further, the mainstays of
Chagas disease chemotherapy are benznidazole and nifurti-
mox (agents with heteroaromatic nitro moieties).40 Recently,
Wilkinson et al. demonstrated that these prodrugs are acti-
vated in Trypanosoma cruzi by a NADPH-dependent type I
nitroreductase.41 It is entirely plausible that this process
results in the generation of NO.
Given the relationship between NO and antiparasitic activ-
ity and the prior research on furoxan (2) as a NO donor
compound, it was sensible to assess the NO generating
capacity of these agents. In the presence of a free thiol
(cysteine), it was determined that furoxan (2) was an effective
NO donor compound (as judged by ABTS42 oxidation).20
Analysis of NO release by 2 in the presence of TGR was found
to be dependent on the presence of NADPH. Interestingly,

6476 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Rai et al.
several oxadiazole-2-oxide analogues with potent TGR in-
hibition, for instance bis(thiophen-2-ylmethanone)-furoxan
(3) (TGR IC50 of ∼60 nM), were capable of NO generation in
the presence of cysteine but not in the presence of TGR/
NADPH. This ultimately proved significant as only oxadia-
zole-2-oxides that generated NO in the presence of TGR/
NADPH were capable of ex vivo worm killing.20 The associa-
tion between NO release and worm killing was further
evidenced by the partial mitigation of furoxan (2) induced
parasite death in the presence of a NO scavenger molecule
C-PTIO.20,43 Clearly, there is a relationship between NO
release and the activity of this chemotype versus schistoso-
miasis. Here, we define the molecular pharmacophore of these
agents, optimize compounds in terms of NO generation and
potency versus TGR and worm killing efficacy, provide
mechanistic evidence for TGR inhibition and parasite death,
and discuss the selectivity and bioavailability of chosen ana-
logues.
along the scale of oxidation states (i.e., alkyl to acyl). Several
analogues were accessible intermediates of synthesis of 2
(including derivatives 7 and 8), while others required a separate
synthetic effort (derivatives 6, 9, and 10).44 The SAR of these
analogues provided the first indication of the complexity
surrounding the relationship between NO release, TGR inhibi-
tion, and worm killing (described through visual examination
of cultured worms treated with 10 μM of compound over a 72 h
time-course). The methyl analogue 6 was inactive as a NO
donor and a TGR inhibitor and was essentially inactive in the
worm killing evaluation (Table 1). The hydroxyl analogue 7
regained the ability to inhibit TGR (IC50 of 11.3 μM) but was
only a weak NO donor and possessed minimal ability to kill ex
vivo worms. The aldehyde 8and carboxylic acid 9were noted to
be potent inhibitors of TGR (IC50 of 0.11 and 0.63 μM,
respectively). Surprisingly, these analogues possessed only
modest NO donating abilities and were ineffective worm killing
agents. Interestingly, the aldehyde moiety in 8would be capable
of entering into electrophilic/nucleophilic reactive mechanisms
with cysteine (and selenocysteine) residues in a similar fashion
to the nitrile of 2. The only analogue in this SAR series that
retained both TGR inhibition and worm killing ability was
the amide 10, albeit at slightly lower potency and efficacy.
With these results in place, it seems reasonable to consider the
3-cyano-1,2,5-oxadiazole-2-oxide core as the pharmacophore
for these agents in terms of TGR inhibition, NO donating
potential, and worm killing efficacy.
With a verified and consistent definition of the pharmaco-
phore, we next turned to a systematic evaluation of the phenyl
ring through the appraisal of standard substitutions (including
methyl, methoxy, halo, trifluoromethyl, nitro, hydroxyl, and
phenyl) and replacements (including heterocycles furan and
thiophene). The results are detailed in Table 2 and show clear
evidence that the majority of analogues preserving the core
3-cyano-1,2,5-oxadiazole-2-oxide moiety (11-18, 20-29)
retain TGR inhibition and worm killing capacity (11-18,
20-28 were additionally noted to be capable NO donating
molecules). The SAR trends revealed that electron-withdraw-
ing substitutions (halo, trifluoromethyl, and nitro) provided
slight potency enhancements and the meta-position seemed
favored (in general, substitutions at the ortho position were
not synthetically viable (compound 21 was the lone ex-
ception)). Importantly, there was general agreement between
TGR inhibition and worm killing efficacy. For instance,
analogues 12-17 possessed TGR IC50 between 2 and 4 μM
and were at least as efficacious as furoxan (2) in terms of worm
killing. Conversely, several analogues with TGR IC50 values
between 9 and 18 μM and were generally less active against
worms in culture. From this phenyl ring scan, there were
several intriguing lessons. The unique difference between
Results and Discussion
Prior to any optimization effort to improve this chemo-
type’s ability to inhibit TGR and effectively act as an anti-
schistosomal agent, we needed to establish a mechanistic link
between NO donation and TGR inhibition. As such, our first
considerations involved the synthetic deconstruction of the
molecular structure of 2. Various synthetic pathways for
oxadiazole-2-oxides have been reviewed by Gasco and co-
workers.22,23 Additionally, Maloney and co-workers have
recently described the synthetic elaboration of the small
molecule oxadiazole-2-oxides described in this report.44 From
the outset, an examination of the N-oxide moiety was of
paramount importance. To better understand this key struc-
tural feature of the compound, we examined analogues that
lack (derivative 4) and regiochemically transpose (derivative 5)
the N-oxide (Figure 2). As opposed to furoxan (2), both 4 and 5
gave no appreciable NO release with TGR nor was either
analogue active versus TGR or an effective ex vivo worm killing
agent. Our next consideration was to determine the importance
of the nitrile functionality. To explore the structure-activity
relationships (SAR) of this moiety, we considered analogues
Figure 2. Structure-activity relationships of the N-oxide moiety.
Table 1. SAR of Nitrile Substitutions
a Data represent the results from three separate experiments. b Data collected by visual examination of worm movement and shape. n.e. (no effect): all
worms are scored as active in culture with typical appearance; curling: worms are seen to curl rather than stretch and contract; sluggish: worm movement
is significantly reduced; % D: % of worms dead as judged by lack of movement.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6477
Table 2. SAR of Phenyl Ring Substitutions and Substitutions on the 3 and 4 Carbons of the Oxadiazole Ring
a Data represent the results from three separate experiments. b Data collected by visual examination of worm movement and shape. n.e. (no effect): all
worms are scored as active in culture with typical appearance; curling: worms are seen to curl rather than stretch and contract; sluggish: worm movement
is significantly reduced; % D: % of worms dead as judged by lack of movement. ND = not determined.
substitutions at the meta and para positions was further
highlighted by the hydroxyl analogues 19 and 27. Analogue
19 possessed good TGR inhibition but was inactive as a worm
killing agent, while 27 was among the least potent analogues
for TGR inhibition yet retained respectable activity versus
worms. Replacement of the phenyl ring with heterocycles
furan and thiophene (analogues 28 and 29, respectively)
resulted in derivatives with slightly improved potencies for
TGR inhibition, good NO release profiles, and good worm
killing efficacy. Another important exploration involved the
substitution of the 4 position of the oxadiazole ring with a
substituted carbonyl. Data from our initial studies on com-
pounds with acyl substitutions on the oxadiazole ring system
(such as in 3) and analogues presented here (such as aldehyde
8) demonstrate that such functionality confers significant
potency enhancements in terms of TGR inhibition. These
analogues, however, are devoid of the key 3-cyano-1,2,5-
oxadiazole-2-oxide pharmacophore and were markedly less
active versus cultured worms. We were, therefore, eager to
examine an analogue that possessed both the nitrile moiety
and a ketone moiety. As such, we synthesized a novel oxadia-
zole-2-oxide (analogue 30) that satisfies these requirements
(it should be noted that 30 required an entirely novel synthetic
route spanning a total of nine transformations44). The inhibi-
tion of TGR by 30 was in alignment with our previous results
for 3 and 8 (potency of 63 nM). Unfortunately, like the
aforementioned analogues, 30 was devoid of activity versus
worms in culture.
in TGR inhibition and ultimately worm killing. That NO can
modify protein structure and function by the act of S-nitro-
sylation was first shown in the early 1990s.45,46 Becker et al.
subsequently showed that human GR can be inhibited by NO
donating compounds and an active site cysteine residue is
oxidized as a result.47 Additionally, S-nitrosylation of cysteine
residues on coxsackieviral protease 2A, 3C, and rhinovirus 3C
is recognized to affect coxsackievirus and rhinovirus, re-
spectively.48-50 Given the aforementioned precedence and
the intriguing presence of 13 cysteine residues and one sele-
nocysteine in TGR, we set out to examine the potential role of
S-nitrosylation (or Se-nitrosylation) during TGR inhibition
by 2. To explore this possibility, we took advantage of the
well-known biotin-switch assay introduced by Snyder and
co-workers.51,52 A cartoon representation of the principal
experiment is outlined in Figure 3A. Here, recombinant
TGR was incubated with and without NADPH, furoxan
(2), and glutathione (GSH). After incubation, the reactants
were removed from TGR on a spin column and by acetone
precipitation of TGR. All cysteine (or selenocysteine) residues
that remain in their reduced sulfhydryl form are then capped
by methyl methanethiosulfonate (MMTS). TGR was then
treated with reduced ascorbate to return all S-nitrosylated
cysteines (or Se-nitrosylated selenocysteines) to their reduced
form, followed by reaction with the thiol-modifying reagent
biotin-HPDP to allow visualization of labeled protein. After
purification of the protein on a spin column and acetone
precipitation, it was analyzed by native-Western blotting
using a HRP-streptavidin conjugate to identify biotinylated
proteins (Figure 3B). The results shown clearly indicate that
recombinant TGR is S-nitrosylated by 2 and that the nitro-
sylation depends on the protein activity (i.e., requires
NADPH) (Figure 3B). The experiment was performed with
The results of these studies provide solid evidence that
inhibition of TGR by oxadiazole-2-oxides must be coupled
with the ability of these small molecules to act as a NO
donating compound in order to effect parasite death. A major
question, however, concerns the role that molecular NO plays

6478 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Rai et al.
Figure 3. (A) Cartoon representation of the Biotin-switch assay (see refs 49 and 50). (B) Detection of S-nitrosylation in recombinant
S. mansoni TGR after reaction with furoxan (2) 20 μM (lane 1) and 10 μM (lane 2) ( NADPH. The molecular weight of unmodified
recombinant TGR is 64880 Da.
Table 3. SAR of Bis Oxadiazole-2-Oxides
a Data represent the results from three separate experiments. b Data collected by visual examination of worm movement and shape. n.e. (no effect):
all worms are scored as active in culture with typical appearance; curling: worms are seen to curl rather than stretch and contract; sluggish: worm
movement is significantly reduced; % D: % of worms dead as judged by lack of movement. ND = not determined.
pure, recombinant TGR and analyzed on a native gel; addi-
tional bands are likely the result of both monomeric and
dimeric proteinsandmultiple S-nitrosylationevents. No TGR
labeling was observed when incubation was with GSH or in
the absence of NADPH.
profiles also demonstrated similarity to furoxan (2) and in
some cases modest improvements (analogues 33, 34, and 35
were examined in a separate experiment). Worm death will
not occur until the GSH/GSSG ratio reaches a critical point
and the overall worm redox balance is unrecoverable, making
it difficult to derive correlations between worm killing and
TGR inhibition below a certain level.
The results from this experiment strongly suggest that NO
plays a significant rolein TGR inhibition and likely in parasite
killing. On the basis ofthis knowledge, our thoughts regarding
the optimization of the oxadiazole-2-oxide chemotype fo-
cused on ways to optimize the NO donating qualities of
individual compounds. Our previous attempts to optimize
the compound through a standard phenyl ring scan (Table 2)
provided evidence that the electron-withdrawing nature of the
substituent at the 4-position of the oxadiazole ring was
beneficial. While this makes mechanistic sense, even the most
electron deficient phenyl rings provided only modest potency
enhancements in terms of TGR inhibition and no discernible
benefit in worm killing. Our next consideration was to explore
analogues capable of delivering two equivalents of NO. To
this end, we synthesized and evaluated several bis-oxadiazole-
2-oxide derivatives linked through substituted phenyl rings
and heterocycles. The results for these agents are shown in
Table 3 and demonstrate the validity of the theory. All bis-
oxadiazole-2-oxide analogues were capable of TGR inhibi-
tion at potencies exceeding furoxan (2) and worm killing
With a growing understanding of this chemotype’s poten-
tial and the advancements of several analogues with improved
properties, it was important to consider the mechanism of NO
donation from oxadiazole-2-oxides. A mechanistic rationale
for NO donation that is initiated through nucleophilic attack
(presumably by the sulfhydryl moiety of a cysteine residue or
by selenocysteine) at either the 3 or 4 position of the oxadia-
zoleringand subsequent rearrangementof the heterocyclein a
manner that allows release of the nitroxyl anion (NO-) has
been presented.22,23 An enzymatic oxidation is posited to
transform this agent to NO (NO•). It is important to note
thatthe nitroxyl anion(NO-) exists as a triplet thatreactsvery
rapidly with triplet dioxygen to form peroxynitrite ion. Thus,
it is unlikely that the transformation from NO- to NO• occurs
via oxidation by molecular oxygen. Additionally, the pre-
viously reported mechanism27 suggests a permanent ligation
between the attacking nucleophile and the rearranged carbon
scaffold of furoxan (2). While this mechanism is plausible,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6479
there are several aspects that are not consistent with the data
from our study. Foremost, this mechanism does not account
fortherelease of NO(ratherthanNO-) duringareaction with
cysteine (i.e., in an environment lacking an enzyme capable of
oxidizing NO- to NO•). A second issue is the stoichiometry of
the processes. Our previous data suggest that TGR acts
catalytically on furoxan (2) in terms of NO release. A 10 μM
concentration of 2 in the presence of 15 nM of TGR and
NADPH resulted in the generation of ∼15 mol % of NO
relative to 2; i.e., ∼1500 nM NO from only 15 nM of TGR or
100 equiv of NO per enzyme equivalent. Obviously, TGR has
the ability to attack furoxan (2), effect molecular rearrange-
ment and release of NO, and subsequently release from the
heterocycle scaffold in such a manner that it retains the ability
to re-enter the cycle with another molecule of furoxan (2).
Presumably, this cycle continues until a critical concentration
of NO is present and the enzyme is sufficiently S-nitrosylated as
to lose activity. Given these inconsistencies, we sought evidence
of alternate mechanisms.
Recently, Chakrapani et al. presented experimental evi-
dence of NO donation from C-nitroso compounds through a
first-order homolytic C-N bond scission event.53 Interest-
ingly, this mechanism required a geminal nitrile moiety to
allow homolysis of the C-N bond. Moreover, the reactivity
between nitrile groups and free thiols is well established and
explored.54,55 To further explore the mechanistic release of
NO, we examined selected analogues ability to donate NO
as judged by the aforementioned ABTS method (see the
Supporting Information section for these data). The results
confirmed that analogues must be capable of generating NO
in the presence of TGR and NADPH to be effective worm
killing agents. The ABTS method provides a measure of the
oxidation products of NO rather than NO itself. To more
exactly quantify the release of NO, we examined the release of
NO quantitatively through a well-known chemiluminescent
assay that relies upon the reaction between NO and ozone to
produce an activated NO2 species that produces an intense
chemiluminescence emission.54 Additionally, selected analo-
gues were characterized for their ability to react with free
cysteine (as judged by consumption of starting material
detected across an HPLC gradient). For these experiments,
we selected a number of compounds with specific SAR in
terms of TGR inhibition and worm killing ability (Figure 4).
Furoxan(2) was noted toreact fully withcysteine and produce
approximately 0.6 equivalents of NO per equivalent of 2.
Gratifyingly, bis-oxadiazole-2-oxide analogues 33 and 34
were both cysteine reactive and produced more NO equiva-
lents relative to furoxan (2) (1.55 mol equiv for 33 and 1.19
mol equiv for 34). Analogue 6, with the replacement of the
nitrile with a methyl group, was not capable of reacting with
cysteine nor was 6 capable of NO donation. Most interest-
ingly, derivatives3 (withthe bis-thiophene ketonemoiety) and
5 (with a transposed N-O moiety, but retaining the nitrile)
did react with cysteine but did not generate NO. An addition-
ally interesting result was that compound 30 (with both
thiophene ketone and nitrile moieties) reacted with cysteine
and was capable of releasing NO. From this result, the failure
of 30 to produce worm death remains unexplained.
Mechanistically, it is not possible to identify the location of
the initial nucleophilic attack, but the ultimate result is addi-
tion into the oxadiazole ring system alpha to the nitrile
(Figure 5). The ring system would likely open resulting in a
quaternary alkyl-thio-nitroso-nitrile species that is highly
similar to the C-nitroso compounds shown by Chakrapani
et al. to be capable of homolytic C-N bond cleavage and
release of NO (path 1). Alternatively, this intermediate could
undergo transnitrosylation and release of R-SNO and a
byproduct of the original oxadiazole-2-oxide heterocycle
(path 2). It is further possible that this intermediate could
rearrange to form an isoxazol-5(4H)-imine type intermediate
prior to homolytic C-N bond cleavage and release of NO
(path 3). A second mechanistic issue is the ultimate release of
the attacking nucleophile from the core oxadiazole scaffold.
Figure 4. Evaluation of furoxan (2) and selected analogues for
reactivity with free cysteine as judged by consumption of reagent
and the molar ratio of NO release per stoichiometric equivalent of
reagent.
Figure 5. Schematic description of potential mechanisms for NO release by oxadiazole-2-oxides via reaction with reactive thiols (or with
selenol).

6480 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Rai et al.
Table 4. Comparative Inhibition of Selected Analogues vs TGR,
Human GR, and Rat TrxR
and co-workers have found that forms of selenium compro-
mised thioredoxin reductase-derived apoptotic proteins
(SecTRAPs) mayhave prooxidanttoxic propertiesinaddition
to a loss of thioredoxin reducing capacity.63
TGRa,
hGRb,
rTrxRb,
analogue
IC50 (μM)
IC50 (μM)
IC50 (μM)
While there is not yet any direct evidence, it seems reason-
able to assume that the enhanced nucleophilicity associated
with the selenocysteine residues (including the one found in
TGR) makes this moiety a likely candidate for the initial
attack on furoxan (2) and other oxadiazole-2-oxides. As such,
it was important to explore the selectivity of this chemotype
with regards to its ability to act as a general electrophile for
other selenocysteine containing enzymes. Additionally, it was
essential to consider if these compounds inhibit related host
enzymes, most significantly GR and TrxR. To resolve both
questions, we assayed several analogues versus human GR
and rat TrxR (rat TrxR is a selenoprotein64 while human GR
is not). We found that furoxan (2) does not inhibit recombi-
nant human GR (IC50 > 50 μM) or recombinant rat TrxR
(IC50 > 400 μM) (Table 4). Of interest was the discovery that
three of the agents tested (the aldehyde 8 and phenyl ring
analogues 15 and 18) were found to inhibit both hGR and rat
TrxR, albeit with reduced affinity. The aldehyde derivative 8
may yield insight into the mechanism of these agents, and the
discovery of inhibitors of both hGR and rat TrxR is an
important finding on its own accord. More important for this
study, however, is the fact that several of the antischisotoso-
mal analogues were found not to be inhibitors of the TGR-
related mammalian host enzymes. Taken as a whole, the data
presented here provide strong evidence supporting the selec-
tive action by oxadiazole-2-oxides against schistosomes and
suggests that the interaction of this chemotype with TGR is
dependent upon both binding affinity and appropriately
aligned reactivities.
2
6.3
>50
11.2
0.11
0.63
17.8
2.2
>50
>50
>50
35.2
>400
>400
>400
<12.5
>400
>400
ca. 150
ca. 250
ca. 250
ca. 150
<12.5
ca. 250
>400
<12.5
ca. 300
ca. 150
>400
>400
>400
>400
ca. 350
ca. 150
6
7
8
9
>50
>50
>50
>50
>50
>50
35.7
10
11
12
13
14
15
16
17
18
20
22
23
24
25
27
28
31
2.5
2.8
2.8
3.5
3.5
4.0
>50
>50
45.3
7.1
7.9
>50
>50
>50
>50
>50
>50
>50
>50
8.9
8.9
10.0
11.2
17.9
2.8
3.5
a Data represent the results from three separate experiments. b Methods
for determining inhibitory potential versus hGR and rTrxR are presented
in the Supporting Information.
While plausible mechanisms can be envisioned for this deliga-
tion event, there is currently no experimental evidence for how
it would proceed. Interestingly, we tested this point through
the synthesis of a biotinylated version of furoxan (2)
(conjugated through an ether linkage at the 4-position of the
phenyl ring) and subsequent treatment of this analogue with
recombinant TGR and NADPH. Gel analysis of the resulting
mixture provided evidence that TGR was not cross-linked to
biotin, further suggesting that TGR reacts with oxadiazole-2-
oxides in a catalytic rather than terminally reactive mechan-
ism (data not shown).
We were further interested in determining if the oxadiazole-
2-oxide scaffold has suitable properties for oral bioavailabil-
ity. Because therapies for schistosomiasis are intended to be
single dose agents (as opposed to chronic treatments), we were
initially and primarily interested in determining selected ana-
logues’ potential for passive membrane permeation and sta-
bility in liver microsomes. Profiles of these agents binding to
These insights into the interaction of oxadiazole-2-oxides
withTGR suggestthat the preeminentmechanistic eventisthe
nucleophilic attack by either cysteine or selenocysteine resi-
dues on TGR. Selenium has a unique role in biology and the
biosynthesis of selenocysteine residues and, ultimately, sele-
noproteins have been the subject of numerous reviews.56 To
date, 25 human proteins have been characterized that contain
selenocysteine residues and each retain a specified cellular
function (over 300 selenoprotein families have been found in
nature).57 A minimalist view of atomic sulfur and selenium
highlight several divergent properties that translate into im-
portant biochemical differences for selenocysteine and cys-
teine residues. Primary among these variations are the relative
pKa values for both amino acids (8.5 for cysteine versus 5.2 for
selenocysteine) and the stronger nucleophilicity of seleno-
cysteine.58-60 Evolutionary analyses clearly suggest that sele-
nocysteine supports unique roles in selenoproteins that can
not easily be maintained by cysteine.61 The strikingly low pKa
of selenocysteine results in full anionic character for this
residue at physiological pH. In several instances, the divergent
activities of selenocysteine and cysteine residues have been
exploited. For instance, Filipovska and co-workers recently
reported the rational design of gold(I) containing N-hetero-
cyclic carbene complexes that target the selenocysteine-con-
ꢁ
the human ether-a-go-go related gene (hERG) potassium
channel and inhibition of selected members of the cytochrome
P450 class of enzymes were also of interest. Our own investi-
gations of these agents suggested highly variable solubilities in
water. We were interested in going beyond conjectural evalua-
tions of this property (i.e., HPLC retention times) and estab-
lish more reliable data for selected analogues. Consequently,
solubility measurements (in both μM and μg/mL) and LogD
determinations were ascertained. The results of these profiles
are shown in Table 5. Several compounds had only modest
water solubility including furoxan (2), 16, 31, 32, and 34.
Analogues containing the thiophene ring were noted ashaving
better solubility profiles, including 29 and 35. Interestingly,
the addition of a single fluorine moiety to the bis-oxadiazole-
2-oxide analogue 31 resulted inan impressive gain in solubility
(see derivative 33).
Beyond solubility profiling, several analogues were found
to have modest ability to permeate artificial Caco-2 cell
monolayers (apical to basolateral (A f B) permeability was
typically greater than basolateral to apical (BfA)). Furoxan
(2) was not found to pass through Caco-2 cells, however,
several analogues (including 16, 29, 33, and 35) were noted to
possess adequate Caco-2 profiles. While these data do not
guarantee successful realization of these analogues as orally
taining TrxR and are selectively toxic to cancer cells.62 Arner
ꢀ

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6481
Table 5. Solubility and Selected AD ME Properties for Chosen Analogues
Caco-2
permeabilityc
mean A f B
Caco-2
permeabilityc
mean B f A
solubilitya
solubilitya
microsomal
stabilityd
hERG
FastPatche
analogue
(μM)
(μg/mL)
LogDb
2
30.1
18.6
240.4
5.0
5.6
5.0
2.42
3.1
1.0
7.9
0
8/63
10/67
1/64
13.2%
5.5%
3.4%
7.8%
ND
16
29
31
32
33
34
35
4.8
9.0
ND*
ND
2.7
1.8
3.4
46.4
1.5
2.54
ND*
ND*
2.91
2.92
2.92
2.54
ND*
ND
7.9
ND*
ND
3.7
1.1
89.1
11.5
122.2
28.0
3.5
20/51
3/56
12/52
29.4%
83.9%
57.2%
4.0
9.4
36.9
a Kinetic solubility analysis was performed by Analiza Inc. and are based upon quantitative nitrogen detection as described (www.analiza.com). The
data represent results from three separate experiments with an average intraassay %CV of 4.5%. b LogD analysis was performed by Analiza Inc. and are
based upon octanol/buffer partitioning and quantitative nitrogen detection of sample content as described (www.analiza.com). The data represent
results from three separate experiments and is corrected to calibration standards and adjusted to account for fixed amounts of DMSO. c Caco-2
permeability analysis was performed by Apredica Inc. and are based upon monolayer Caco-2 cells applied to collagen-coated BioCoat Cell Environment
at 24500 cells per well as described (www.apredica.com). Data are expressed in Papp (apparent permeability) in 10-6 cm s-1. Atenolol was used as a low
permeability control (mean AfB=1.2 and mean BfA=1.2) and propanolol was used as a high permeability control (mean AfB=20.3 and mean
BfA = 17.4). d Microsomal stability analysis was performed by Apredica Inc. and are based upon duplicate incubations of test reagent with liver
microsomes (human and rat; only human data is shown) over 60 min at 37 ꢀC as described (www.apredica.com). LC/MS/MS is utilized to quantitate
remaining test reagent and the data is reported as % remaining. Analysis in the absence and presence of NADPH was performed to assess NADPH free
degradation. Data is listed as NADPH(þ) degradation/NADPH(-) degradation. e hERG FastPatch analysis was performed by Apredica Inc. using an
automated patch clamp electrophysiological procedure using HEK293 cells stably transfected with hERG cDNA as described (www.apredica.com).
Test reagents were applied at standard concentrations (10 μM) and data is recorded as mean % inhibition from three separate experiments.
available agents, it does dispel concerns that the oxadiazole-2-
oxide scaffolds are incapable of passive membrane permea-
tion. Analysis of these analogues in liver microsomes (over 1 h
in the presence/absence of NADPH) suggests that these
compounds are only moderately stable and may possess short
half-lives in intact animal models. However, the variable data
(for instance compound 29 versus 33) suggests that more
robust analogues may be found through additional modifica-
tion. Further, the hERG and Cyp profiles for these agents
were generally reasonable. Taken as a whole, these data
suggests that properly substituted oxadiazole-2-oxides may
possess efficacy and ADMET properties adequate for clinical
application.
Acknowledgment. We thank Allison Peck for critical read-
ing of this manuscript. This research was supported by the
Molecular Libraries Initiative of the National Institutes of
Health Roadmap for Medical Research, the Intramural Re-
search Program of the National Human Genome Research
Institute, National Cancer Institute, and Center for Cancer
Research. Schistosome life stages used in this research were
supplied in part by the National Institute of Allergy and
Infectious Diseases Schistosomiasis Resource Center at the
Biomedical ResearchInstitute(Rockville, Maryland) through
NIAID contract N01-AI-30026. This work was partially
supported by grant AI065622.
Supporting Information Available: An extended table of TGR
inhibition and NO release profiles for selected analogues and
experimental methods for TGR biochemical assay protocol,
ex vivo worm assay protocol, GR assay protocol, biotin-switch
assay protocol, TrxR1 assay protocol (DNTB), NO release pro-
tocol, reactivity of oxadiazole-2-oxides with Cys (HPLC Assay),
and characterization of compounds are detailed. This material is
available free of charge via the Internet at http://pubs.acs.org.
Conclusion
Given the widespread morbidity and mortality associated
with schistosomiasis and the precarious reliance on PZQ as
the exclusive antischistosomal agent, development of new
treatments for the disease is critical. Here, we present data
from our continued evaluation of the oxadiazole-2-oxide class
of reagents as inhibitors of the multifunctional selenocysteine-
containing flavoenzyme TGR. Through chemical modifica-
tion of the lead agent (furoxan (2)), we have defined the
pharmacophore as the 3-cyano-1,2,5-oxadiazole-2-oxide moi-
ety and established SARs surrounding the phenyl ring of the
core structure. Through use of the biotin-switch assay, we
have shown that incubation of TGR with furoxan (2) leads to
S-nitrosylation (or Se-nitrosylation) of TGR. These data
allowed us to focus our attention on analogues capable of
dual release of NO, resulting in agents with superior activity
versus ex vivo parasites. We have further defined the selectiv-
ity of these agents as compared to other reductases including
human GR and rat TrxR (also a selenoprotein). Finally,
a preliminary evaluation of selected in vitro pharmaco-
kinetic properties, including water solubility, Caco-2 cell per-
meability, and microsomal stability, suggest that the oxadia-
zole-2-oxide chemotype can possess the properties required
for an orally bioavailable clinical reagent. We are currently
developing models to examine the oral efficacy of selected
agents.
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