Metronidazole-triazole conjugates: Activity against Clostridium difficile and parasites

Article abstract of DOI:10.1016/j.ejmech.2015.06.019

Abstract Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window.

Full text of DOI:10.1016/j.ejmech.2015.06.019

Accepted Manuscript
Metronidazole-triazole conjugates: activity against Clostridium difficile and parasites
Angie M. Jarrad, Tomislav Karoli, Anjan Debnath, Chin Yen Tay, Johnny X. Huang,
Geraldine Kaeslin, Alysha G. Elliott, Yukiko Miyamoto, Soumya Ramu, Angela M.
Kavanagh, Johannes Zuegg, Lars Eckmann, Mark A.T. Blaskovich, Matthew A.
Cooper
PII:
S0223-5234(15)30094-5
10.1016/j.ejmech.2015.06.019
EJMECH 7946
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 March 2015
Revised Date: 4 June 2015
Accepted Date: 7 June 2015
Please cite this article as: A.M. Jarrad, T. Karoli, A. Debnath, C.Y. Tay, J.X. Huang, G. Kaeslin, A.G.
Elliott, Y. Miyamoto, S. Ramu, A.M. Kavanagh, J. Zuegg, L. Eckmann, M.A.T Blaskovich, M.A. Cooper
Metronidazole-triazole conjugates: activity against Clostridium difficile and parasites, European Journal
of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.019.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Metronidazole-triazole conjugates: activity against Clostridium
difficile and parasites
Angie M. Jarrada, Tomislav Karolia,e, Anjan Debnathb, Chin Yen Tayd, Johnny X. Huanga, Geraldine
Kaeslina, Alysha G. Elliotta, Yukiko Miyamotoc, Soumya Ramua, Angela M. Kavanagha, Johannes Zuegga,
Lars Eckmannc, Mark A.T Blaskovicha and Matthew A. Coopera,*
aDivision of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of
Queensland, Brisbane, Queensland, 4072, Australia.
c
bSkaggs School of Pharmacy and Pharmaceutical Sciences and Department of Medicine, University of
California, San Diego, La Jolla, California, 92093, USA.
dMarshall Centre for Infectious Diseases Research and Training, University of Western Australia, Perth,
Western Australia, 6099, Australia.
eCurrent address: Progen Pharmaceuticals Limited, Darra, Queensland, 4076, Australia.
Corresponding author. Tel: +61-7-3346-2044; e-mail: m.cooper@uq.edu.au

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Abstract
Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial
agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has
been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed
Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of
metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been
identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium
difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against
stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles,
the activity remained in a therapeutically relevant window.
Keywords
Nitroimidazole
Click Chemistry
Antibiotic
Anaerobe

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1
Introduction
For over 50 years the 5-nitroimidazole antibiotic metronidazole 1 has been in clinical use as a broad-
spectrum agent for treatment of Gram-positive and Gram-negative anaerobic bacterial infections as well as
parasitic infections.[1] Reduction of the nitro group to the nitro radical anion by electron carriers in an
anaerobic environment leads to decomposition to form toxic metabolites, which cause DNA damage and
nonspecific macromolecular damage leading to cell death.[2] However, metronidazole resistance has been
observed in both parasites[3,4] and anaerobic bacteria.[5,6] Over the last ten years metronidazole has also
been extensively used to treat Clostridium difficile infection (CDI), an intestinal infection that causes life-
threatening severe diarrhea, abdominal pain and fever.[7] The spores produced by C. difficile are resistant to
heat and alcohol based disinfectants, complicating eradication and promoting hospital-based outbreaks.
Metronidazole is one of three antibiotics routinely prescribed to treat the disease.[8] Metronidazole is
effective against mild to moderate C. difficile infection, but, while not wide-spread, resistance has been
observed in clinical isolates.[9] To this end, the development of next generation metronidazole analogues
that can overcome resistance is therapeutically important.
Beena et al. previously reported the synthesis of metronidazole-triazole conjugates (Mtz-triazoles) with
potent activity (reported as IC50 values 0.06-0.35 µg/mL) against the facultative anaerobic bacteria
Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli and Pseudomonas aeruginosa.[10]
Metronidazole is generally not active against Gram-negative and Gram-positive facultative anaerobic
bacteria, so the potent activities of Mtz-triazoles observed by Beena et al. are suggestive of a different mode
of action to metronidazole. Given that the active 5-nitroimidazole core (“warhead”) in metronidazole is
maintained in Mtz-triazoles, and that the hydroxyl group of metronidazole is amenable to modification[11]
(Figure 1), we reasoned that this class might also exhibit potent activity against the anaerobic bacteria and
parasites for which metronidazole is used as a treatment. Therefore, we believed Mtz-triazoles warranted
further investigation for both antimicrobial and antiparasitic activity under anaerobic conditions.
Independently, during the course of this work, Miyamoto et al.[12] reported a similar approach to Mtz-
triazoles, prepared by reaction of six different 5-nitroimidazole azides with a library of alkynes. The
compounds were prepared for testing by dilution of the crude reaction mixtures (>85% purity by LCMS)

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with dimethyl sulfoxide. This methodology bypassed the bottleneck of compound purification and
characterization and allowed for the rapid assessment of activity. The solutions of crude material were tested
against the parasites Giardia lamblia and Trichomonas vaginalis, the microaerophilic bacteria Helicobacter
pylori, the anaerobes C. difficile and Bacteroides fragilis and the facultative anaerobic bacteria E. coli. It is
possible that the crude mixtures contained unreacted starting azide 3, which we have shown in this study
retains activity, and may influence the reported crude compound potency. A key finding by Miyamoto was
that Mtz-triazoles were often active against metronidazole resistant (MtzR) strains of T. vaginalis (47% of
compounds) and G. lamblia (100% of compounds), but not against H. pylori frxA and rdxA double mutant
(1.4% of compounds).[12] From this result, we hypothesized that Mtz-triazoles may possess activity against
metronidazole resistant Clostridium difficile. More recently Beena et al. described the activity of Mtz-
triazoles against the anaerobic protozoan Entamoeba histolytica.[13] They tested a library of 10 Mtz-
triazoles and found 4a, 4h, 4p and 4q to have potent activity (IC50 = 0.008- 2.36 µM) against E. histolytica
HM1: IMSS.[13]
We now report an expansion and advancement of these approaches with the synthesis of a small library of
purified Mtz-triazoles 4a-t (>95 % purity, Scheme 1), including ten novel derivatives (4b, 4c, 4f, 4g, 4i, 4j,
4l, 4o, 4s and 4t). We also explore the antimicrobial and antiparasitic properties of the set of Mtz-triazoles
and the parent azide 3 (>95% purity) and determine their activity against additional microbial targets that
have not previously been examined. We evaluated the activity of Mtz-triazoles against the parasites E.
histolytica and G. lamblia, multiple pathogenic strains of the anaerobe C. difficile, the microaerophile H.
pylori and the facultative anaerobic bacterial ESKAPE pathogens E. coli, methicillin resistant S. aureus
(MRSA), Klebsiella pneumoniae, Acinetobacter baumanni and P. aeruginosa. The antibacterial activity was
measured against a stable MtzR C. difficile strain (CD26A54_R), the parent C. difficile strain with elevated
metronidazole minimum inhibitory concentration (MIC) (CD26A54_S) and MtzR H. pylori clinical isolates,
while cytotoxicity of the compounds was evaluated against mammalian liver (HepG2) and kidney (HEK293)
cell lines.

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Materials and Methods
2
2.1
Synthesis of Mtz-triazole library
Mtz-triazoles were synthesized from metronidazole via an azide intermediate by activation of the hydroxyl
group with methanesulfonyl chloride (Scheme 1). Displacement of methanesulfonate 2 with sodium azide
provided the desired azide 3 by nucleophilic substitution. The azide substituent was then reacted with a
library of alkynes to give 4a-t by copper-catalysed Huisgen 1,3-dipolar cycloaddition with copper sulfate and
sodium ascorbate in methanol, with heating to 45 ºC and/or additional reagents added if monitoring indicated
the reaction was incomplete. All alkynes utilized were commercially available with the exception of pyrazole
alkyne 6. Pyrazole alkyne 6 was prepared by reacting pyrazole 5 with propargyl bromide in the presence of
potassium carbonate and t-butylammonium bromide as a phase transfer catalyst (Scheme 1).[14] All
1
compounds were characterized by H and 13C NMR, LCMS and HRMS and detailed experimental
procedures and characterization are provided in the supplementary information.
2.2
MIC assays
All compounds were tested for activity against a set of anaerobic C. difficile bacteria (630 ATCC BAA-1382,
NAP1/027 M7404, NAP1/027 ATCC BAA-1803, VPI10463 ATCC 43255, CD26A54_S and CD26A54_R)
and representative ESKAPE pathogens S. aureus (MRSA ATCC 43300), E. coli (ATCC 25922), K.
pneumoniae (ATCC 700603), A. baumannii (ATCC 19606) and P. aeruginosa (ATCC 27853) using a
standard broth microdilution assay. Selected compounds were tested for activity against H. pylori strains
(26695, and clinical isolates MtzS 13/25, MtzR 98/285 and MtzR 13/61) using agar plate dilution, and for
antimicrobial activity against twelve Gram-positive bacterial strains: Enterococcus faecalis (VanA clinical
isolate), Enterococcus faecium (MDR VanA ATCC 51559), Streptococcus pneumoniae (MDR ATCC
700677), S. aureus (MRSA ATCC 43300, MRSA clinical isolate, MRSA/DRSA clinical isolate, GISA
NARSA NRS 1, GISA NARSA NRS 17, VRSA NARSA VRS1, VRSA NARSA VRS4 and VRSA NARSA
VRS10) using broth microdilution. C. difficile strains were grown at 37 °C in a COY anaerobic chamber (5
% H2, 10 % CO2, 85% N2). H. pylori strains were grown at 37 °C under a microaerobic atmosphere (5 % O2,
10 % CO2). All facultative anaerobic bacteria were grown at 37 °C with normal atmospheric oxygen levels.
All experiments were performed in duplicate with metronidazole, vancomycin, linezolid or colistin as

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positive controls for relevant strains (see Table 1). Positive growth control of bacteria and DMSO as well as
a negative control of only media were included for every plate. Full assay details are provided in the
supplementary information.
2.3
Antiparasitic assays
Compounds were tested for antiparasitic activity against E. histolytica (HM1:1MSS strain) and G. lamblia
(WB, BRIS/87/HEPU/713 (713),[15] BRIS/83/HEPU/106 (106)[16] and the metronidazole resistant
syngeneic line 713-M3[15,16]) using an ATP-bioluminescence based screen for cell growth and
survival.[17,18] Assay plates were inoculated with trophozoites (5x103 parasites/well) and incubated in the
GasPakTM EZ Anaerobe Gas Generating Pouch Systems (VWR, West Chester, PA) to maintain anaerobic
conditions throughout the incubation period. The assays were performed in triplicate using the CellTiter-Glo
Luminescent Cell Viability Assay.[17] Metronidazole was used as a positive control.
2.4
Cytotoxicity
Compounds were tested for cytotoxicity against mammalian HepG2 and HEK293 cell lines, as detailed in
the supplementary information.
3
Results and Discussion
3.1
Design of metronidazole-triazole conjugate library
The Mtz-triazole library was designed to contain a variety of structural groups with some compounds
identical to those reported by Beena et al. (4a, 4h, 4p and 4q).[10] Initial results against C. difficile showed
that hydrophobic 4a (R = phenyl) maintained activity relative to metronidazole 1 while more hydrophilic 4p
(R = hydroxyethyl) and 4q (R = hydroxymethyl) lost activity. Therefore the subsequent library was biased
towards exploring variance of the aromatic ring substitutions, extension of the position of the aromatic ring
relative to the triazole core and replacement of the phenyl group with various heterocycles, with several of
the latter selected due to their rating in terms of medicinal chemistry ‘developability’.[19] Amine 4t, and
acids 4r and 4s were included to explore the structure activity relationships of non-aromatic ionisable
groups. Compounds 4a, 4d, 4e, 4h, 4k, 4m, 4n, 4q and 4r were reported by Miyamoto[12], but 4a, 4h and

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4q were not tested against C. difficile. Compounds 4b, 4c, 4f, 4g, 4i, 4j, 4l, 4o, 4s and 4t are described for
the first time.
3.2
Antimicrobial and antiparasitic activity of metronidazole-triazole conjugates
The antimicrobial and antiparasitic spectrum of action of Mtz-triazoles was assessed against the anaerobic
bacteria C. difficile and the anaerobic parasites E. histolytica and G. lamblia (Table 1). Variations to the
phenyl group of 4a were well tolerated and activity of 4b-e was maintained against C. difficile, E. histolytica
and G. lamblia (Table 1). Phenyl derivatives 4a-e were several fold more active against G. lamblia than
metronidazole. Pyridine 4h and thiophene 4n heterocycles also possessed broad-spectrum activity against C.
difficile, E. histolytica and G. lamblia. However, we found that 4h was not as potent compared to
metronidazole in the ATP-bioluminescence parasite assay in contrast to the eosin-stain method used by
Beena et al.[13] The pyrazole 4g and pyrimidine 4i-j maintained activity against C. difficile within one to
two 2-fold dilutions of metronidazole. Compounds 4g and 4i-j were still active against G. lamblia compared
to metronidazole, but were several fold less potent than the phenyl derivatives 4a-e. The pyrazole 4g and
pyrimidines 4i-j did not inhibit E. histolytica at 25 µM, showing that the R group can be used as a handle to
tune the selectivity of this class towards different organisms. Compound 4k (benzyl) and 4m (CH2NMe-
benzyl) maintained broad-spectrum activity but 4l (CHOH-phenyl) was inactive against E. histolytica at 25
µM.
Several compounds were inactive, or weakly active, against all three microorganisms at the highest
concentration tested. These included the non-aromatic thiomorpholine dioxide 4o, amine 4t and the
carboxylic acids 4r and 4s. The methyl hydroxyl 4q and ethyl hydroxyl 4p were inactive against E.
histolytica and G. lamblia and weakly potent against C. difficile, continuing the trend of reduced activity
with more polar substituents.
We also demonstrate that the parent azide 3 possesses potent activity against C. difficile, E. histolytica and G.
lamblia. Therefore, when assessing combinatorial-like libraries of crude material for biological activity, the
activity of any unreacted parent compounds is an important consideration, particularly when the compound
warhead is maintained.

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The MICs of 1, 3 and 4a-t were >32 µg/mL against the representative ESKAPE pathogens (MRSA, E. coli,
K. pneumoniae, A. baumannii and P. aeruginosa) tested under aerobic conditions. In addition, the MIC
values of a subset of compounds (metronidazole 1, azide 3, 4a (phenyl) and 4q (CH2OH)) were all >64
µg/mL against a panel of 8 additional drug resistant S. aureus strains, vancomycin resistant E. faecalis,
vancomycin resistant E. faecium and multidrug resistant S. pneumoniae. This lack of activity is contrary to
Beena’s report, but consistent with the reported inactivity of 378 Mtz-triazoles against E. coli in Miyamoto’s
study, and the inactivity of metronidazole against facultative anaerobic bacteria.
3.3
Activity of metronidazole-triazole conjugates against C. difficile strain panel
The activity of the Mtz-triazoles did not vary significantly against multiple strains of C. difficile, including
two major pathogenic strains of NAP1/027 and a VPI10463 strain associated with epidemics (Table 1). The
MICs of individual compounds typically remained within one 2-fold dilution against the four strains of C.
difficile tested. This is important, as viable drug candidates must possess appropriate broad strain coverage.
A review of antibiotic drug candidates in development found that activity against multiple strains of C.
difficile could vary substantially.[20] This can be explained since C. difficile has a highly mobile, mosaic
genome and there is wide strain diversity between isolates.[21,22] Isolates can be divided across five main
phylogenetic clades, multiple ribotypes and toxinotypes.[23] Recently, genomic epidemiology studies
examining and tracking C. difficile outbreaks in Europe found that a high proportion of strains causing
infection were not related to prior infectious strains.[22] Therefore, there is a large pool of genetically
diverse strains in the community and selection and spread of intrinsically resistant strains could occur.
However, the broad-strain activity of Mtz-triazoles against C. difficile in this study encourages further
development of this class.
3.4
Activity of metronidazole-triazole conjugates against metronidazole resistant C. difficile
While metronidazole resistance has been reported in the clinic,[9,24] performing antimicrobial susceptibility
testing against resistant C. difficile isolates is problematic since the resistance phenotype is unstable and
often reported to be lost on freeze thaw cycles or on passaging of the isolates.[5] However, Lynch et al. have
reported a stable metronidazole resistant (MtzR) C. difficile clone.[25] The activity of Mtz-triazoles was

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assessed against this stable MtzR strain of C. difficile (CD26A54_R) and the parent metronidazole sensitive
(MtzS) (CD26A54_S) strain, which lost the metronidazole resistance on freeze thawing, but still retained a
slightly elevated MIC value to metronidazole compared to the control MtzS NAP1/027 strain. The MIC to
metronidazole in BHIS broth at 48 hr was lower than the metronidazole MIC determined by E-test on
BAKHS (4 µg/mL vs 48 µg/mL), similar to values reported by Chong et al. (8 µg/mL vs >32 µg/mL).[26]
The resistance to metronidazole became more apparent on incubation in broth for 48 hr (MIC = 2 µg/mL
after 24 hr incubation vs MIC = 4 µg/mL after 48 hr) (Supplementary Table 1). In contrast, MICs against the
control MtzS NAP1/027 strain ATCC 1803 did not change significantly between 24 hr and 48 hr
(Supplementary Table 1). Although several compounds (4a, 4e and 4n) were active against MtzR C. difficile
at 1 dilution lower than metronidazole (MIC = 2 µg/mL vs 4 µg/mL), none of the Mtz-triazoles were active
against MtzR C. difficile at levels comparable to the activity of metronidazole against MtzS strains (MIC =
0.5 µg/mL). Reduced activity against the parent C. difficile strain CD26A54_S with intermediary
metronidazole susceptibility was also observed.
3.5
Activity of metronidazole-triazole conjugates against additional G. lamblia strains
including metronidazole resistant G. lamblia
Selected compounds (4a-f, 4h, 4k-n) were tested against two additional strains of MtzS G. lamblia (106 and
713) and one MtzR strain (713-M3) derived from the parent MtzS strain 713 (Single column size:
Table 2). The Mtz-triazoles displayed similar activity against the additional MtzS strains (106 and 713)
compared to G. lamblia strain WB. Encouragingly, the Mtz-triazoles were all more potent than
metronidazole against MtzR G. lamblia 713-M3. However, with the exception of 4d, all of the Mtz-triazoles
lost activity against the MtzR G. lamblia 713-M3 when compared to the parent MtzS strain 713, similar to
the loss of activity against MtzR C. difficile. Mtz-triazoles of scaffold 4 were also all observed by Miyamoto
et al. to lose activity against MtzR G. lamblia 713-M3 and 106-2ID10. However, while activity diminished
against the MtzR 713M strain, the Mtz-triazoles were often still much more potent than metronidazole itself,
meaning that the EC50 remained within a therapeutically meaningful window.

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The activities of purified Mtz-triazoles (>95% purity) against G. lamblia strains 106, 713 and 713M were
compared with the literature activities[12] determined with crude reaction mixtures (Supplementary Table 3).
The activities were generally in close agreement, supporting the methodology used by Miyamoto,[12]
although a 3.7 to 8 fold difference was observed for Mtz-triazoles 4d, 4e and 4m against 1 strain of G.
lamblia.
3.6
Activity of metronidazole-triazole conjugates against metronidazole resistant H. pylori
Selected compounds (4f-h, 4m and 4s) were tested against a panel of H. pylori strains using the CLSI agar
dilution susceptibility method (
Single column size:
Table 3).[27] These strains included the reference strain 26695 and three clinical isolates 13/25
(metronidazole E-test MIC = 2 µg/mL), 98/285 (E-test MIC = 24 µg/mL) and 13/61 (E-test MIC = 256
µg/mL) with a range of resistance levels to metronidazole determined by the E-test. The susceptibilities of
the strains to metronidazole determined by the agar dilution method differed from the susceptibilities
obtained from the E-test as has been described previously.[28] The methyl ester 4f was weakly active against
H. pylori, while the carboxylic acid 4s was inactive against all H. pylori strains as observed with E.
histolytica and G. lamblia as well. Compounds 4g, 4h and 4m were more active than metronidazole against
the MtzS strains (26695 and clinical isolate 13/25) and 1-2 dilutions more active than metronidazole against
the MtzR strains (clinical isolates 98/285 and 13/61) but they could not completely overcome metronidazole
resistance, similar to the loss of activity against metronidazole resistant C. difficile.
3.7
Cytotoxicity
All compounds, including metronidazole 1, azide 3, and 4a-t showed no cytotoxicity against HepG2 and
HEK293 cells at concentrations up to 100 µM (CC50). This is consistent with the findings by Miyamoto et al.
which did not observe cytotoxicity against HeLa cells at 50 µM.[12]

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3.8
Relationship of compound activity with compound properties
In order to understand the relationship between compound properties and activity, compound properties
including logP (or logD at pH = 7.4), molecular weight, topological polar surface area (tPSA), and predicted
solubility (logS) were examined for correlation with activity against MtzS strains of C. difficile, E.
histolytica and G. lamblia, with the activity expressed as pMIC (-log10MIC) or pEC50 (-log10EC50))
(Supplementary Table 4). Inactive compounds were included in the analysis at the highest concentration of
compound tested. A correlation can be observed between the logD values and the activity against G. lamblia
(with R2 (linear regression) = 0.84, Supplementary Figure 1) (Figure 2). The logD of the most potent
compounds (4a-e, 4h, 4k and 4n) against G. lamblia is between 1.5 and 3.0, with activity decreasing with
lower logD. In contrast, only a mild correlation could be detected between logD and the activity against C.
difficile (R2 = 0.68, Supplementary Figure 1) and no correlation for activity against E. histolytica (R2 = 0.48,
Supplementary Figure 1). This might explain why the compounds with aromatic phenyl, thiophene and
pyridine groups were more potent against G. lamblia than E. histolytica and C. difficile. Increasing the
hydrophobicity (logD) of compounds to improve potency is generally considered undesirable in drug
development due to the tendency for increased metabolism[29] and increased promiscuity[30]. However, in
this case the antibiotic metronidazole has a low logD to begin with, such that the relatively higher logD of
the compounds explored in this study does not place the compounds in an undesirable chemical space. On
the other hand, the site of infection of G. lamblia is the small intestine, where low (< 1) or high (> 3) logD
values,[29] associated with reduced systemic uptake, might be beneficial for efficacy. However,
metronidazole is 100 % oral bioavailable[31] and so the importance of bioavailability for in vivo efficacy
against G. lamblia infections is unclear.
4
Conclusion
Mtz-triazoles were synthesized and assessed for activity against the anaerobe C. difficile, microaerophile H.
pylori, the parasites E. histolytica and G. lamblia as well as facultative anaerobic Gram-positive and Gram-
negative bacteria. While no activity was observed against the facultative anaerobic bacteria, nine compounds
were identified with potent broad spectrum activity against anaerobic organisms, while having no
cytotoxicity against mammalian cell lines. Hydrophobic R groups such as the benzyl 4k, varyingly

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substituted phenyl derivatives 4a-e and heterocyclic R groups such as pyridine 4h and thiophene 4n were
well tolerated and favored broad spectrum anaerobic activity. In contrast, polar R groups including
pyrimidines 4i-j, carboxylic acids 4p and 4r and methyl amine 4t either resulted in loss of broad spectrum
activity or were inactive against all organisms tested.
While active Mtz-triazoles displayed a narrow MIC range against multiple strains of C. difficile and G.
lamblia, there was metronidazole cross resistance against the stable MtzR C. difficile strain CD26A54_R,
MtzR G. lamblia strain 713M and clinical isolates of MtzR H.pylori. Mtz-triazoles 4a-t generally lost
activity against MtzR C. difficile, MtzR G. lamblia and MtzR H. pylori. However, since the Mtz-triazoles
were often much more potent than metronidazole against G. lamblia, the EC50 remained within a
therapeutically meaningful window. It is possible that surveying a more diverse chemical space of R group
substituents or using alternative 5-nitroimidazole scaffolds could provide access to Mtz-triazoles that are
even more potent and therefore active against MtzR strains of C. difficile and G. lamblia. Future work will
focus on identifying such compounds and explore their in vivo efficacy against anaerobic pathogens.

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Supporting Information
5
The following file is available free of charge.
SI_Mtztriazole_EuJMedChem: Detailed descriptions of experimental procedure for chemical synthesis,
biological assays and compound activity analysis. Supplementary Tables 1-4, Supplementary Figure 1.
6
Abbreviations
MIC- minimum inhibition concentration
MtzS- metronidazole sensitive
MtzR- metronidazole resistant
Clin. Isol.- clinical isolate
7
Author Information
Corresponding author. Tel: +61-7-3346-2044; e-mail: m.cooper@uq.edu.au
a
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of
Queensland, Brisbane, Queensland, 4072, Australia.
b
c
Skaggs School of Pharmacy and Pharmaceutical Sciences and Department of Medicine, University of
California, San Diego, La Jolla, California, 92093, USA.
d
Marshall Centre for Infectious Diseases Research and Training, University of Western Australia, Perth,
Western Australia, 6099, Australia.
e Current address: Progen Pharmaceuticals Limited, Darra, Queensland, 4076, Australia.
8
Author Contribution
Planned experiments: AMJ, TK, AD, MAB, MAC
Performed experiments: AMJ, AD, CYT, JH, GK, AG, YM, SR, AK, JZ
Wrote paper: AMJ, TK, LE, MAB, MAC
9
Conflict of Interest
The authors declare no competing financial interest.

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10 Acknowledgement
We would like to acknowledge Prof. Dena Lyras and Ms Kate Mackin for training in C. difficile
microbiology and for providing C. difficile M7404. We thank Prof. George Golding for kind provision of C.
difficile CD26A54_S and CD26A54_R. We thank Prof. David Patterson for kind provision of Gram positive
clinical isolates S. aureus (MRSA clinical isolate, MRSA DRSA clinical isolate), Enterococcus faecalis
(VanA Clinical Isolate). The following isolates were obtained through the Network of Antimicrobial
Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/NIH Contract
#HHSN272200700055C: GISA NARSA NRS 1, GISA NARSA NRS 17, VRSA NARSA VRS1, VRSA
NARSA VRS4 and VRSA NARSA VRS10. This research was supported financially by a UQ CIEF grant,
NHMRC grants APP631632, APP1026922 and AF511105, and NIH grants AI075527, DE020607 and
DK080506. AMJ is supported by an Australian Postgraduate Award (APA) and QLD Government Smart
Futures PhD scholarship, TK, SR, AK, MAB and MSB by a Wellcome Trust Seeding Drug Discovery
Award (094977/Z/10/Z), and MAC by an NHMRC Principal Research Fellowship (APP1059354). CYT and
his Helicobacter Research team are supported by the Vice-Chancellor of the University of Western Australia
and NHMRC Sir McFarlane Burnett Fellowship grant (No. 572723).
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12 Figures
Single column size:
Figure 1. Metronidazole contains the 5-nitroimidazole warhead and a hydroxyl group amenable to modification
1.5 column size:
Scheme 1: Synthesis of metronidazole-triazole conjugate library. i) MsCl, TEA, DCM, 2 ºC to rt, 80 min, 85%
yield ii) NaN3, DMF, 50 ºC, 21 hr, quant. yield iii) CuSO4, sodium ascorbate, MeOH, rt or 45 ºC, 20 min – 48 hr,
9 – 97% iv) propargyl bromide (80% w/v in toluene), K2CO3, TBAB, rt, 3 days, 16%.

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Single column size:
Figure 2: Correlation of activity against G. lamblia, E. histolytica and C. difficile with logD. Compounds with
activity against parasites (EC50 < 25 µM) and C . difficile (MIC ≤ 8 µg/mL) have pEC50 or pMIC > 5.0.

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13 Tables
Double column size:
Table 1: Activity of Mtz-triazoles against C. difficile, ESKAPE bacteria, E. histolytica and G. lamblia.
MIC (µg/mL)
EC50 (µM)
C. difficile
ESKAPE E. histolytica G. lamblia
bacteriah
630b
VPI
NAP1/
027c
NAP1/
027d
NAP1/
027f
NAP1/
027g
HM1:IMSS
WB
Compound
10463e
MtzS
0.5
MtzS
0.5
MtzS
0.5
MtzS
0.5
MtzS
1
MtzR
4
MtzS
5
MtzS
7.9
1
3
metronidazole
azide
4a phenyl
>32
1
0.5
0.5
0.5
0.25-0.5
0.25-0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
1
1
8
2
4
>32
>32
>32
2.1
4.2
4.8
1.5
0.25
0.27
4b p-OMe-phenyl
4c p-Cl-phenyl
4d p,m-Cl2-phenyl
4e p-Me-phenyl
4f methyl ester
4g pyrazole
0.5
0.5
0.5
1
0.25
0.5-1
0.25
1
0.25
1
0.25
1
2
1
16
8-16
2
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
3.6
5.2
0.20
0.39
0.18
3.0
0.5
1
0.5
1
1
3.7
2
4
>25
>25
4.6
1
1
1
1
2
4
4.9
4h pyridine
0.5
1
0.5
0.5
0.5-1
2
0.5
1
1
4
0.74
3.3
4i 5-pyrimidine
4j 2-pyrimidine
4k benzyl
1
1
4
>25
>25
1.9
2
2
2
2-4
1
4
1.7
0.5
1
0.25-0.5
0.5
0.5
1
0.5
0.5-1
2
4
0.70
4.4
4l CHOH-phenyl
4m CH2NMe-benzyl
4n thiophene
2
4
>25
4.6
2
1-2
0.25-0.5
>64
8
1
2
8
1.6
0.5
>64
8
0.5
>64
16
0.5
>64
8
1
2
3.8
0.37
>25
>25
>25
>25
>25
>25
4o thiomorpholine
4p CH2CH2OH
4q CH2OH
>64
8
>64
16
16
>64
>64
>64
>25
>25
>25
>25
>25
>25
16
>64
64
>64
16
16-32
>64
64
16
>64
64
>64
16-32
>64
>64
>64
4r CH2CH2COOH
4s COOH
>64
64
4t CH2NH2
>64
>64
aMIC results determined against CD26A54_S and CD26A54_R after 48 hr growth, all other C. difficile
strain MIC results determined at 24 hr. See Supplementary Table 1 for MIC at 24 hr for CD26A54_S and
CD26A54_R and at 48 hr for ATCC BAA-1803. C. difficile MICs are the median of at least n = 4, except for
CD26A54_R where n = 8. ESKAPE pathogen MICs were performed in a single concentration screen with n
= 3. EC50 results n = 3.
bATCC BAA-1382, cATCC 43255, dM7404, eATCC BAA-1803, fCD26A54_S, gCD26A54_R
hS. aureus MRSA (ATCC 43300), E. coli (ATCC 25922), K. pneumoniae (ATCC 700603), A. baumannii
(ATCC 19606) and P. aeruginosa (ATCC 27853).

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Single column size:
Table 2. EC50 of selected Mtz-triazoles against G. lamblia strains. The EC50 values for G. lamblia WB strain are
shown from Table 1 for comparison.
EC50 (µM)
G. lamblia
Compound
WB
MtzS
7.9
106
MtzS
2.8
713
MtzS
2.3
713M
MtzR
17
1
metronidazole
phenyl
4a
4b
4c
4d
4e
4f
0.25
0.27
0.20
0.39
0.18
3.0
0.28
0.64
0.89
1.1
0.16
0.64
1.2
2.3
1.0
3.5
1.2
1.0
3.1
0.95
1.1
3.0
2.2
0.9
p-OMe-phenyl
p-Cl-phenyl
p,m-Cl2-phenyl
p-Me-phenyl
methyl ester
pyridine
2.5
0.29
2.0
0.29
1.8
4h
4k
4l
0.74
0.70
4.4
0.38
0.51
2.2
0.28
0.32
2.4
benzyl
CHOH-phenyl
CH2NMe-benzyl
thiophene
4m
4n
1.6
1.1
0.71
0.18
0.37
0.34
Single column size:
Table 3. MIC of selected Mtz-triazoles against H. pylori strains
MIC (µg/mL)
Compound
H. pylori
26695
Clin. Isol. Clin. Isol. Clin. Isol.
13/25
MtzS
4-8
98/285
MtzR
64
13/61
MtzS
8
MtzR
32-64
64->64
1
metronidazole
4f methyl ester
4g pyrazole
32-64
32-64
64->64
2-4
2
16-32
16-32
4h pyridine
4m CH2NMe-benzyl
4s COOH
2
4
>64
1
2
>64
32
16->64
>64
16
16
>64
Clin. Isol. = clinical isolate

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Highlights:
Metronidazole-triazole conjugates: activity against Clostridium difficile and
parasites
Angie M. Jarrada, Tomislav Karolia,e, Anjan Debnathb, Chin Yen Tayd, Johnny X. Huanga, Geraldine
Kaeslina, Alysha G. Elliotta, Yukiko Miyamotoc, Soumya Ramua, Angela M. Kavanagha, Lars Eckmannc,
Mark A.T Blaskovicha and Matthew A. Coopera,*
•
•
•
•
•
A series of 20 metronidazole (Mtz)-triazole conjugates were synthesised
Several Mtz-triazoles had increased potency against pathogenic anaerobes
The compounds were not cytotoxic at 100 µM against mammalian cell lines
Cross-resistance was observed against Mtz-resistant bacteria and parasites
Therapeutically relevant activity retained against Mtz-resistant G. lamblia

Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
Supplementary Information
Metronidazole-triazole conjugates: activity against Clostridium difficile
and parasites
Angie M. Jarrad, Tomislav Karoli, Anjan Debnath, Chin Y. Tay, Johnny X. Huang, Geraldine
Kaeslin, Alysha G. Elliott, Yukiko Miyamoto, Soumya Ramu, Angela M. Kavanagh, J. Zuegg, Lars
Eckmann, Mark A.T. Blaskovich and Matthew A. Cooper
Table of Contents
1
2
3
4
General Methods..................................................................................................................2
Synthesis..............................................................................................................................3
Spectra...............................................................................................................................12
Biological Methods.............................................................................................................61
4.1 Determination of Minimum Inhibition Concentration (MIC) against facultative anaerobic
bacteria......................................................................................................................................... 61
4.2 Maintenance of C. difficile5 ................................................................................................... 61
4.3 Determination of Minimum Inhibition Concentration (MIC) against C. difficile....................... 62
4.4 Determination of Minimum Inhibition Concentration (MIC) by E-test against C. difficile........ 62
4.5 Determination of Minimum Inhibition Concentration (MIC) against H. pylori ......................... 62
4.6 Maintenance of E. histolytica and G. lamblia........................................................................ 63
4.7 Determination of EC50 against E. histolytica and G. lamblia trophozoites ............................. 63
4.8 Cytotoxicity........................................................................................................................... 63
4.9 Correlation analysis of compound activity and properties..................................................... 64
5
Supplementary Results: Tables S1-3, Figure S1....................................................................65
5.1 MIC results of Clostridium difficile at 24 and 48 hr................................................................ 65
5.2 Metronidazole E-test ............................................................................................................ 66
5.3 Comparison of purified and crude Mtz-triazole activity against G. lamblia strains................. 66
5.4 Correlation matrix of compound activity and properties........................................................ 66
6
References .........................................................................................................................68
S1

Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
1
General Methods
Reagents and anhydrous solvents (dimethylformamide, dichloromethane, and acetonitrile) were
used as received. Reactions requiring anhydrous conditions were performed under an inert
atmosphere of nitrogen. Reactions were monitored by thin layer chromatography (TLC) or LCMS.
Analytical TLC was performed on Merck TLC aluminium sheets pre-coated with Silica Gel 60 F-
254 and compounds were visualised using UV lamp and potassium permanganate stain. Melting
points were determined using a Gallenkamp melting point apparatus and are corrected to a
standard curve of the measured and literature melting points of vanillin, acetyl salicylic acid, 3-
phenoxybenzoic acid and caffeine standards. Analytical LCMS was performed on Shimadzu LCMS
using 0.05% formic acid in water as solvent A and 0.05% formic acid in acetonitrile as solvent B.
LCMS conditions (solvent A = H2O + 0.05% formic acid, solvent B = acetonitrile + 0.05% formic
acid): Standard (unspecified): Column Zorbax Eclipse XDB-Phenyl, 3.0×100mm, 3.5 ꢀ: Flow: 1
mL/min: Gradient timetable: 0.00 min, 5% B; 3.00 min, 100% B; 3.7 min, 100% B; 5.00 min, 5% B.
Waters Column: Waters Atlantis T3, 2.1×50mm, 5 ꢀ: Flow 1 mL/min, Gradient timetable: 0.00 min,
0% B; 3.30 min, 25% B; 3.50 min, 100% B; 4.00 min 100% B; 5.00 min, 0% B; 13.00 min, 5.0% B.
Detection: UV at 254 nm, ELSD and electrospray MS. Compounds were purified using Biotage
Isolera, Gilson or Grace Reveleris X2 chromatography systems. Commercially available cartridges
were used for MPLC chromatography: Biotage SNAP cartridge HP-Silica 10, 25 or 50 g, Reveleris
4 g Silica (40 µm) cartridge or Reveleris C18 Reversed-Phase 12 g cartridge. All final products
were obtained in >95% purity as determined by HPLC using UV at 254 nm, ESIMS and ELSD
detection. NMR data were collected and calibrated in DMSO-d6 or CDCl3 at 298K on a Varian Unity
1
400 MHz or Bruker Avance-600 MHz spectrometers. Where appropriate, H-coupling constants
were examined using resolution enhancement with MestReNova software. Data are presented as
follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, t= triplet, q = quartet, quin =
quintet, m = multiplet, br = broad), coupling constant (Hz) and integration. High resolution mass
spectrometry (HRMS) was performed on a Bruker MicroTOF mass spectrometer using (+)-ESI
calibrated to HCOONa. For compounds purified by reverse phase chromatography, the exact
concentration of the compounds was determined by the quantitative NMR integration ‘PULCON’
experiment.1 These settings were used for all PULCON experiments: relaxation delay of 30 s, 8
scans, 2 dummy scans, 90° pulse and temperature at 298 K. The formula weight obtained from
PULCON experiments was also used to calculate the percentage yields.
S2

Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
2
Synthesis
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl methanesulfonate; 2
Methanesulfonyl chloride (2.7 mL, 35.0 mmol) was added dropwise to a stirring suspension of
metronidazole (4.99 g, 29.2 mmol) and TEA (6.1 mL, 43.7 mmol) in anh. DCM (25 mL) cooled to 2
°C. The reaction was warmed to room temperature and stirred for 1 hr 20 min. Volatiles were
removed in vacuo. The solid was washed with water (40 mL), triturated with pet. spirits (20 mL)
and washed with DCM (15 mL) and then dried under vacuum. An off-white solid was obtained
1
(6.18 g, 85%). LCMS: Rt = 2.28 min, 99 A% @ 254 nm, [M + H]+ = 249.8. H NMR (600 MHz,
DMSO-d6) δ 8.06 (s, 1H), 4.65 (dd, J = 5.4, 4.5 Hz, 2H), 4.55 (dd, J = 5.4, 4.4 Hz, 2H), 3.15 (s,
3H), 2.46 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 150.7, 138.4, 133.1, 68.4, 45.1, 36.7, 14.0.
HRMS calcd for C7H11N3NaO5S [M + Na]+, 272.0312; found, 272.0312.
1-(2-azidoethyl)-2-methyl-5-nitro-1H-imidazole; 3
To stirring suspension of mesylate (4.50 g, 18.1 mmol) in anh. DMF (45 mL) was added NaN3
(1.48 g, 22.8 mmol). The reaction was stirred under N2 at 50 ˚C overnight before pouring into H2O
(450 mL) and extracting with EtOAc (3 x 360 mL). The combined organic layers were washed with
brine, dried over MgSO4, filtered and volatiles removed in vacuo to give an orange solid (3.60 g,
100%). LCMS: Rt = 2.50 min, 99 A% @ 254 nm, [M + H]+ = 197.1. 1H NMR (600 MHz, DMSO-d6)
δ 8.06 (s, 1H), 4.48 (dd, J = 6.1, 5.3 Hz, 2H), 3.79 (dd, J = 6.1, 5.3 Hz, 2H), 2.48 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) δ 151.5, 138.3, 133.1, 50.0, 44.9, 14.0. HRMS calcd for C6H8N6NaO2 [M +
Na]+, 219.0601; found, 219.0590.
Triazole General Procedure A:
To a stirring solution of 3 (1 eq.) in MeOH (5 vol) was added alkyne (1 eq.) followed by 100 mM aq.
CuSO4 (5 mol %) and 100 mM aq. sodium ascorbate (10 mol %).
Triazole General Procedure B:
As for General procedure A, except that if the reaction was not proceeding when monitored by
LCMS, the reaction was warmed to 45 °C and an addit ional portion of methanol (5 vol) was added.
S3

Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
Triazole General Procedure C:
As for General procedure A, except that if the reaction was not proceeding when monitored by
LCMS, further portions of 100 mM aq. CuSO4 (5 mol %) and 100 mM aq. sodium ascorbate (10
mol %) were added.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl-1H-1,2,3-triazole; 4a
General procedure A. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 75- 100% EtOAc in pet. spirits then 0-10% MeOH in EtOAc) to give a
colourless solid (21 mg, 68%). LCMS: Rt = 2.51 min, 99 A% @ 254 nm, [M+ ACN + H]+ = 340.0.
1H NMR (600 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.07 (s, 1H), 7.86 – 7.62 (m, 2H), 7.49 – 7.41 (m,
2H), 7.39 – 7.30 (m, 2H), 4.88 (dd, J = 7.1, 6.1 Hz, 2H), 4.77 (dd, J = 6.9, 5.8 Hz, 2H), 1.92 (s, 3H).
13C NMR (150 MHz, DMSO-d6) δ 151.2, 146.5, 138.4, 133.3, 130.3, 128.9, 128.0, 125.1, 122.2,
48.9, 46.1, 12.9. HRMS calcd for C14H14N6NaO2 [M + Na]+, 321.1070; found, 321.1063.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(4-methoxyphenyl)-1H-1,2,3-triazole; 4b
General procedure A. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Gilson, 100% EtOAc to 30% MeOH) to give a colourless solid (71 mg, 42%). LCMS:
Rt = 2.50 min, 99 A% @ 254 nm, [M + H]+ = 329.3. 1H NMR (600 MHz, DMSO-d6) δ 8.31 (s, 1H),
8.06 (s, 1H), 7.71 – 7.65 (m, 2H), 7.04 – 6.98 (m, 2H), 4.85 (dd, J = 6.5, 4.5 Hz, 2H), 4.76 (dd, J =
13
6.6, 4.4 Hz, 2H), 3.78 (s, 3H), 1.91 (s, 3H). C NMR (150 MHz, DMSO-d6) δ 159.1, 151.2, 146.5,
138.4, 133.3, 126.5, 122.9, 121.3, 114.3, 55.1, 48.8, 46.1, 12.9. HRMS calcd for C15H16N6NaO3 [M
+ Na]+, 351.1176; found, 351.1178.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(4-chlorophenyl)-1H-1,2,3-triazole; 4c
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Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
General procedure B. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 0- 10% MeOH in DCM) to obtain a colourless solid (82 mg, 48%). LCMS:
Rt = 2.69 min, 99 A% @ 254 nm, [M + H]+ = 332.9. 1H NMR (600 MHz, DMSO-d6) δ 8.49 (s, 1H),
8.06 (s, 1H), 7.81 – 7.75 (m, 2H), 7.54 – 7.49 (m, 2H), 4.88 (dd, J = 6.4, 4.7 Hz, 2H), 4.77 (dd, J =
6.6, 4.6 Hz, 2H), 1.94 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 151.2, 145.4, 138.3, 133.3, 132.4,
129.2, 129.0, 126.8, 122.6, 48.9, 46.0, 12.9. HRMS calcd for C14H13ClN6NaO2 [M + Na]+, 355.0681;
found, 355.0682.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(3,4-dichlorophenyl)-1H-1,2,3-triazole; 4d
General procedure B. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 0-10% MeOH in DCM) to obtain a colourless pearlescent solid (129 mg,
69%). LCMS: Rt = 2.86 min, 99 A% @ 254 nm, [M + H]+ = 366.9. 1H NMR (600 MHz, DMSO-d6) δ
8.59 (s, 1H), 8.06 (s, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 8.4, 2.0 Hz, 1H), 7.72 (d, J = 8.3
Hz, 1H), 4.89 (dd, J = 6.5, 4.7 Hz, 2H), 4.77 (dd, J = 6.5, 4.7 Hz, 2H), 1.96 (s, 3H). 13C NMR (150
MHz, DMSO-d6) δ 151.2, 144.3, 138.3, 133.3, 131.7, 131.3, 131.0, 130.3, 126.7, 125.1, 123.3,
49.0, 45.9, 13.0. HRMS calcd for C14H12Cl2N6NaO2 [M + Na]+, 389.0291; found, 389.0292.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(p-tolyl)-1H-1,2,3-triazole; 4e
General procedure A. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 0- 10% MeOH in DCM gradient) to give a colourless solid (129 mg, 81%).
LCMS: Rt = 2.62 min, 99 A% @ 254 nm, [M + H]+ = 313.0. 1H NMR (600 MHz, DMSO-d6) δ 8.37
(s, 1H), 8.06 (s, 1H), 7.67 – 7.61 (m, 2H), 7.27 – 7.22 (m, 2H), 4.86 (dd, J = 6.5, 4.5 Hz, 2H), 4.76
(dd, J = 6.6, 4.5 Hz, 2H), 2.32 (s, 3H), 1.92 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 151.2, 146.6,
138.3, 137.3, 133.2, 129.4, 127.6, 125.0, 121.8, 48.8, 46.0, 20.8, 12.9. HRMS calcd for
C15H16N6NaO2 [M + Na]+, 335.1227; found, 335.1222.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(methoxycarbonyl)- 1H-1,2,3-triazole; 4f
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Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
General procedure A. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 2-10% MeOH in DCM gradient and then Grace Reveleris X2, 2-10%
MeOH in DCM gradient). The product crystallised from a fraction and was collected by filtration to
give a colourless solid (43 mg, 30%). Mp = 197 - 198 °C (decomposed). LCMS: R t = 2.02 min, 99
A% @ 254 nm, [M + H]+ = 280.8. 1H NMR (600 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.05 (s, 1H), 4.90
13
(dd, J = 6.5, 4.4 Hz, 2H), 4.77 (dd, J = 6.6, 4.5 Hz, 2H), 3.82 (s, 3H), 1.93 (s, 3H). C NMR (150
MHz, DMSO-d6) δ 160.4, 151.0, 138.6, 138.4, 133.2, 130.0, 51.8, 49.1, 45.7, 12.9. HRMS calcd for
C10H12N6NaO4 [M + Na]+, 303.0812; found, 303.0814.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-((1H-pyrazol-1-yl)methyl)-1H-1,2,3-triazole; 4g
General procedure A. The reaction mixture was concentrated to dryness and the residue was
purified by MPLC over silica gel (Biotage, 2- 15% MeOH in DCM gradient). The product was
obtained as a colourless solid (57 mg, 55%). LCMS: Rt = 2.03 min, 99 A% @ 254 nm, [M + H]+ =
1
302.9. H NMR (600 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.95 (s, 1H), 7.71 (dd, J = 2.3, 0.7 Hz, 1H),
7.42 (dd, J = 1.9, 0.7 Hz, 1H), 6.24 (t, J = 2.1 Hz, 1H), 5.37 (s, 2H), 4.81 (dd, J = 6.5, 4.4 Hz, 2H),
13
4.69 (dd, J = 6.5, 4.5 Hz, 2H), 1.79 (s, 3H). C NMR (150 MHz, DMSO-d6) δ 151.2, 143.3, 138.9,
138.3, 133.2, 129.6, 124.6, 105.4, 48.7, 46.2, 46.1, 12.7. HRMS calcd for C H N NaO [M +
12 14
8
2
Na]+, 325.1132; found, 325.1129.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(2-pyridinyl)-1H-1,2,3-triazole.xTFA; 4h
General procedure A. The crude mixture was diluted with water and the organics were extracted
with DCM (3 x 50 mL). The combined organics were washed with brine, dried over anh. MgSO4
and filtered. Volatiles were removed in vacuo and the residue was purified by MPLC over C18
silica gel (Grace Reveleris X2, A: H2O + 0.1% TFA, B: ACN + 0.1% TFA, 5-30% B). The eluent
was removed by lyophilisation to give a yellowish solid (148 mg, 97%). LCMS: Rt = 2.08 min, 99
A% @ 254 nm, [M + H]+ = 299.8. 1H NMR (600 MHz, DMSO-d6) δ 8.59 (ddd, J = 5.0, 1.8, 1.0 Hz,
1H), 8.55 (s, 1H), 8.06 (s, 1H), 8.03 (dt, J = 8.0, 1.1 Hz, 1H), 7.92 (td, J = 7.7, 1.8 Hz, 1H), 7.38
(ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 4.92 (dd, J = 6.5, 4.4 Hz, 2H), 4.80 (dd, J = 6.5, 4.4 Hz, 2H), 1.93
13
(s, 3H). C NMR (150 MHz, DMSO- d6) δ 158.5, 158.3, 158.0, 157.8, 151.1, 149.3, 147.0, 138.4,
137.7, 133.1, 124.4, 123.2, 119.6, 49.0, 46.0, 12.9. HRMS calcd for C13H14N7O2 [M + H]+,
300.1203; found, 300.1194.
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Supplementary Material:
Metronidazole-triazole conjugates
ACCEPTED MANUSCRIPT
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(5-pyrimidinyl)-1H-1,2,3-triazole; 4i
General procedure A. The reaction was diluted with DCM (10 mL). The tan ppt was then collected
by filtration. The organic layer filtrate was evaporated to give a crude crystalline solid. The ppt and
crystals were combined and purified by MPLC over C18 silica gel (Grace Reveleris X2, A: H2O +
0.1% TFA, B: ACN + 0.1% TFA, 5-30% B). The eluent was removed by lyophilisation to give a
1
colourless powder (86 mg, 56%). LCMS: Rt = 1.99 min, 99 A% @ 254 nm, [M + H]+ = 300.8. H
NMR (600 MHz, DMSO-d6) δ 9.19 (s, 2H), 9.18 (s, 1H), 8.70 (s, 1H), 8.07 (s, 1H), 4.94 (dd, J = 6.5,
13
4.7 Hz, 2H), 4.79 (dd, J = 6.5, 4.8 Hz, 2H), 2.00 (s, 3H). C NMR (150 MHz, DMSO-d6) δ 157.7,
153.3, 151.3, 140.8, 138.4, 133.3, 124.7, 123.7, 49.1, 45.9, 13.0. HRMS calcd for C12H12N8NaO2
[M + Na]+, 323.0975; found, 323.0966.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(2-pyrimidinyl)-1H-1,2,3-triazole; 4j
General procedure A. The reaction was poured into water (20 mL) and extracted with DCM (4 x 50
mL). The combined organics were washed with brine (50 mL), dried over anh. MgSO4 and filtered.
The volatiles were removed in vacuo and the crude was purified by MPLC over C18 silica gel
(Grace Reveleris X2, A: H2O + 0.1% TFA, B: ACN + 0.1% TFA, 5-25%) then repurified (Grace
Reveleris X2, A: H2O + 0.1% TFA, B: ACN + 0.1% TFA, 5-100% B) to give a cream powder (16
mg, 9%). LCMS: Rt = 2.21 min, 99 A% @ 254 nm, [M + H]+ = 300.8. 1H NMR (600 MHz, DMSO-
d6) δ 8.88 – 8.84 (m, 2H), 8.69 (s, 1H), 8.06 (s, 1H), 7.44 (t, J = 4.9 Hz, 1H), 4.93 (dd, J = 6.5, 4.4
Hz, 2H), 4.80 (dd, J = 6.6, 4.4 Hz, 2H), 1.93 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 158.3, 157.8,
151.1, 146.5, 138.4, 133.2, 127.3, 120.2, 48.9, 46.0, 12.9. HRMS calcd for C12H12N8NaO2 [M +
Na]+, 323.0975; found, 323.0976.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)- 4-benzyl-1H-1,2,3-triazole; 4k
General procedure A. The reaction mixture was concentrated to dryness and purified by MPLC
over silica gel (Biotage, 0- 10% MeOH in DCM gradient) to give a colourless solid (34 mg, 21%).
S7