Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide

Article abstract of DOI:10.1016/j.bmcl.2010.04.126

Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreductase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route for the synthesis of NTZ-related antibacterial analogues.

Full text of DOI:10.1016/j.bmcl.2010.04.126

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3537–3539
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biological activity of modified and exchanged 2-amino-5-nitrothiazole
amide analogues of nitazoxanide
b
b
a
a
T. Eric Ballard a, , Xia Wang , Igor Olekhnovich , Taylor Koerner , Craig Seymour ,
*
Paul S. Hoffman b,c, Timothy L. Macdonald a
a Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA
b Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, VA 22908-0734, USA
c Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908-0734, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A
library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter
pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreduc-
tase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over
their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route
for the synthesis of NTZ-related antibacterial analogues.
Received 1 April 2010
Revised 23 April 2010
Accepted 27 April 2010
Available online 18 May 2010
Keywords:
Nitazoxanide
Ó 2010 Elsevier Ltd. All rights reserved.
Antibacterial
Small-molecule drug target
Pyruvate:ferredoxin oxidoreductase
Helicobacter pylori
Campylobacter jejuni
Clostridium difficile
Nitazoxanide 1 (NTZ—Fig. 1) is a broad-spectrum FDA approved
drug utilized for the treatment of anaerobic intestinal parasites
Giardia lamblia and Cryptosporidium parvum but is efficacious in
the treatment of other anaerobic bacteria and parasites residing
in the human gut.1–4 NTZ has also been shown to have activity
against microbial biofilms, rotavirus, influenza A/B/HIN1, hepatitis
B/C and Mycobacterium tuberculosis.5–10 Although structurally sim-
ilar to other nitro-drugs like metronidazole 2 (Fig. 1), the 5-nitro
group on the thiazole ring of NTZ is not metabolically reduced.
As a result, nitro reduction is not responsible for NTZ activity as
is the case for most nitro-containing drugs.3,11,12 Resistance to
NTZ has not been observed clinically or through in vitro generation
methods which further supports a different mechanism of action
(MoA).13
Campylobacter jejuni), while mammals and eubacteria oxidize
pyruvate by the NTZ insensitive pyruvate dehydrogenase (PDH)
(Fig. 2).14–19 Therefore, PFOR of H. pylori, C. jejuni and Clostridium
difficile is a logical small-molecule drug target and we postulated
that modification of the 2-amino-5-nitrothiazole portion of NTZ
would potentiate analogue activity.
The key interaction responsible for biological activity is be-
lieved to occur between NTZ and the TPP vitamin co-factor
(Fig. 3).14 NTZ was shown to completely prevent the production
of both acetyl-CoA and CO2 indicating that inhibition occurs very
early in the catalytic cycle. In this system only the anion of NTZ
is biologically active and becomes inactive upon protonation. Addi-
tionally, NTZ is not chemically modified during the enzymatic reac-
tion and is only protonated, allowing NTZ to be utilized again after
equilibration.14 NMR analysis revealed four resonance structures
The MoA for NTZ has been elucidated to occur through the an-
ion of NTZ. The NTZ anion abstracts a proton from the thiamine
pyrophosphate vitamin co-factor of the essential enzyme pyru-
vate:ferredoxin oxidoreductase (PFOR), thereby inhibiting produc-
tion of acetyl-coenzyme
A and CO2 necessary for energy
metabolism.14,15 PFOR is present in all strictly anaerobic bacteria,
anaerobic parasites and e-proteobacteria (Helicobacter pylori and
* Corresponding author.
E-mail address: teb2m@virginia.edu (T.E. Ballard).
Figure 1. Nitro heterocyclic drugs.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.126

3538
T. E. Ballard et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3537–3539
Figure 2. Pyruvate:ferredoxin oxidoreductase (PFOR) enzymatic reaction. PFOR
catalyzes the oxidative decarboxylation of pyruvate producing acetyl-CoA and CO2
requiring the electron acceptors ferredoxin (Fd) or flavodoxin (Fld). Oxidation of Fd/
Fld is accomplished by NADP oxidases or hydrogenases, respectively. Solid arrows
indicate the forward reaction; hollow arrows indicate reverse reaction.
existing simultaneously for the anion involving the nitro, N20 and
N3 nitrogens and the amide oxygen.14 The amide (pKa 6.18) anion
(Fig. 3) or the other resonance forms are predicted to interact with
the N40 of the pyrimidine ring and prevent pyruvate binding. NTZ
has a Ki of ꢀ5 ꢁ 10ꢂ6 M with PFOR which is roughly two orders
of magnitude better than pyruvate (Km = ꢀ3 ꢁ 10ꢂ4 M) providing
evidence that NTZ is a competitive inhibitor of PFOR.14
Figure 4. Composition of library separated as head group and tail groups.
Owing to the structural simplicity of NTZ we postulated that
modifications of the benzene ring moiety (tail region) and thiazole
(head group) could yield increased antibacterial properties. Previ-
ous research in this area has determined that the 2-amino-5-nitro-
thiazole ring 3 is well suited for activity yet has been problematic
to replace or modify to obtain increased activity. In an effort to fur-
ther test this conclusion, we aimed to synthesize a small library of
NTZ-head group analogues based on five simple amide tail region
analogues. Through the synthesis of a library of ꢀ100 NTZ-tail re-
gion analogues, five diverse tail regions were chosen to screen new
head groups for comparison against NTZ and the 2-amino-5-nitro-
thiazole 3 analogues, respectively (Fig. 4).
The head groups to be tested were mainly comprised of elec-
tron-withdrawing substituted heterocycles in hopes of mimicking
either the electronic or spatial characteristics of 3. Previous work
by Rossignol, Hemphill and Appelbaum has clearly shown that
the 5-nitro group is necessary for activity and its removal and
modification tend to reduce activity against PFOR organisms but
may increase activity against other organisms or viruses.4,20–22
Unsubstituted 2-aminothiazole 4 was planned as a control head
group that should possess no activity while the other head groups
in question had not been previously reported.
All of the head groups were available through commercial
sources except for 6 which was synthesized as previously reported
(Fig. 4).23,24 Synthesis of the NTZ-head group analogue library was
accomplished through amide bond coupling of the acid chloride/
carboxylic acid of tail regions a–e with the respective head group
3–12 (Fig. 4). As most of the head groups are strongly electron
withdrawing, nucleophilicity of the amine head group was reduced
resulting in low to moderate yields (Supplementary data). Follow-
ing synthesis, the head group library was subjected to in vitro min-
imum inhibitory concentration (MIC) testing against H. pylori, C.
jejuni and C. difficile followed by in vitro direct PFOR enzyme
inhibition.
Helicobacter pylori is a microaerophilic Gram-negative bacterium
that resides in the gastric mucosa. It has been linked to duodenal
and peptic ulcer disease and is a risk factor for gastric cancer.25Cam-
pylobacter jejuni is also a microaerophilic Gram-negative bacterium
and causes severe lower gastrointestinal infections in mammals.26
Clostridium difficile is a Gram-positive bacterium that naturally re-
sides in gut flora.27,28 Many broad-spectrum antibiotics deplete
natural floral enabling C. difficile to dominate and produce severe
enterocolitis. Recently, C. difficile infections have become more
challenging to treat due to increased antibiotic resistance worsened
by increased recurrence rates of infection.27 Current therapies to
treat these infections are problematic and new drugs acting
through different MoAs are necessary for continued treatment.
All library members were screened for activity against H. pylori
and C. jejuni using a standard dilution procedure to determine MIC
values. Escherichia coli MIC screening was also incorporated to pre-
clude activity against PDH utilizing organisms. Selected analogues
were then further tested for their ability to inhibit C. difficile and
directly inhibit the PFOR enzyme. Activity for selected active ana-
logues, their respective 3a–e counterparts and NTZ (1) is summa-
rized in Table 1. Activity for all analogues synthesized is
summarized in Supplementary Table S1.
Overall, most head group analogues generated were inactive
against the target organisms with high double to triple digit micro-
molar MIC values. This result was not unexpected as this trend for
inactivity has been reported.20–22 We did note minor activity with
5 and 12 but the activity was not consistent across the tail regions
(Supplementary data). Although many of the analogues were inac-
tive, we did discover two head groups (6a–d and 9a–e) that pos-
sessed notable activity against the target organisms with all tail
Figure 3. Pyruvate binding to activated vitamin co-factor thiamine pyrophosphate
(TPP) which then is consumed in the production of acetyl-CoA. NTZ is thought to
bind to TPP in an analogous fashion to pyruvate and prevent its binding thereby
inhibiting the enzymatic reaction.

T. E. Ballard et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3537–3539
3539
Table 1
analogues. We also identified 2-amino-5-nitrothiazole analogues
(3a–e) that displayed better activity than the parent compound
NTZ (1) against PFOR utilizing microorganisms. While most of
the library members were inactive, head group 6 was discovered
to be only slightly less active while analogues of 9 were equipotent
or more active compared to NTZ or 3. Direct PFOR enzyme inhibi-
tion was also noteworthy for 9 although some activity may be re-
lated to nitroreduction activity. Overall we have been able to show
that head group modification is a viable route for the synthesis of
NTZ-related antibacterial analogues. Further modifications of 6 and
9 are currently under investigation as well as linkage of tail regions
with increased activity profiles. Disclosure of the full tail region li-
brary and biological activity will be presented in due course.
Selected NTZ-head group analogue activity
Analogue
MIC’s (
l
M)
PFOR inhibition
[Drug] = 40 M (%)
H. pylori
C. jejuni
C. difficile
l
Nitazoxanide (1)
13.0
0.5
1.3
3.5
2.9
0.8
0.9
8.8
5.7
6.9
2.6
1.3
0.5
2.5
2.6
39.1
2.1
3.0
4.7
2.9
12.5
36
0.8
8.2
32.1
0.4
1.0
2.1
28.8
>28.2
>22.7
>13.8
27.8
27.3
4.2
54
70
53
47
50
56
63a
59
27
51
7
8
3a
3b
3c
3d
3e
6a
6b
6c
6d
9a
9b
9c
9d
9e
7.0
34.1
13.8
0.9
0.9
2.1
1.0
2
80 8a
55
Acknowledgment
47
20.0
3.5
77
0.4
66 13a
This work was supported by U01 Grant AI075520 from the Na-
tional Institute of Allergy and Infectious Diseases to P.S.H.
a
Complex pattern of inhibition with two different rates.
Supplementary data
regions. All library members were also completely inactive against
E. coli indicating that PDH is not being targeted (data not shown).
The head group analogues 6a–d are direct derivatives of 3 with
6 bearing an additional 4-chloro substituent. The 4-chloro substi-
tuent imparts both steric and electronic effects on the thiazole,
notably the 5-nitro group. A slight reduction in activity for 6 com-
pared to 3 (H. pylori, C. jejuni MIC’s and PFOR) is most likely attrib-
utable to the steric interaction with the 5-nitro group forcing the
nitro slightly out-of-plane with the thiazole ring, lowering the res-
onance stability of the amide anion. More pronounced loss of activ-
ity against C. difficile for these analogues may be attributed to
minor differences in enzyme and binding pocket structure. Further
studies to support substitution at the 4-position employing a 4-
methyl or 4-bromo substituent are currently under investigation.
Recapitulation of bio-activity compared to 3a–e with 9a–e was
unanticipated but appreciated. Di-nitrothiophene 9 replaced thia-
zole 3 and improved activity for all analogues compared to 3
against H. pylori and C. jejuni with comparable activities against
C. difficile. Thiophenes 9a–e were subsequently assayed for their
ability to directly inhibit PFOR compared to 3a–e, and NTZ. Nearly
all of the thiophene analogues 9a–e inhibited PFOR to an equal or
greater extent then NTZ or 3a–e, respectively. Of note, several of
the 9a–e analogues inhibited PFOR at different inhibition rates pos-
sibility indicating that nitroreduction via PFOR may be occurring.
To test this, thiophenes 9a–e were subjected to an NfsB nitroreduc-
tase assay to determine if these analogues were substrates for
nitroreduction. Compared to NTZ, analogues 9b–e were only
slightly more susceptible to nitroreduction compared to a good
substrate (nitrofurazone), however analogue 9a showed higher
rates of nitroreduction (Supplementary data).
Complete analogue activity table, full biochemical and chemical
experimental procedures, spectral data for all new compounds and
representative spectra associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.126.
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Through this study we discovered two unique and active 2-ami-
no-5-nitrothiazole surrogate head groups from a library of 39