Substituted N-Phenyl-5-(2-(phenylamino)thiazol-4-yl)isoxazole-3-carboxamides Are Valuable Antitubercular Candidates that Evade Innate Efflux Machinery

Article abstract of DOI:10.1021/acs.jmedchem.7b00793

Tuberculosis remains one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extremely drug-resistant strains is a significant reason for concern. This makes the discovery of novel antitubercular agents a cogent priority. We have previously addressed this need by reporting a series of substituted 2-aminothiazoles capable to inhibit the growth of actively replicating, nonreplicating persistent, and resistant Mycobacterium tuberculosis strains. Clues from the structure-activity relationships lining up the antitubercular activity were exploited for the rational design of improved analogues. Two compounds, namely N-phenyl-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 7a and N-(pyridin-2-yl)-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 8a, were found to show high inhibitory activity toward susceptible M. tuberculosis strains, with an MIC₉₀ of 0.125-0.25 μg/mL (0.33-0.66 μM) and 0.06-0.125 μg/mL (0.16-0.32 μM), respectively. Moreover, they maintained good activity also toward resistant strains, and they were selective over other bacterial species and eukaryotic cells, metabolically stable, and apparently not susceptible to the action of efflux pumps.

Full text of DOI:10.1021/acs.jmedchem.7b00793

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Article
Substituted N-phenyl-5-(2-(phenylamino)thiazol-4-yl)isoxazole-3-carboxamides
Are Valuable Antitubercular Candidates that Evade Innate Efflux Machinery
Elisa Azzali, Diana Machado, Amit Kaushik, Federica Vacondio, Sara Flisi, Clotilde Silvia
Cabassi, Gyanu Lamichhane, Miguel Viveiros, Gabriele Costantino, and Marco Pieroni
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00793 • Publication Date (Web): 27 Jul 2017
Downloaded from http://pubs.acs.org on July 27, 2017
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Substituted Nꢀphenylꢀ5ꢀ(2ꢀ(phenylamino)thiazolꢀ4ꢀ
yl)isoxazoleꢀ3ꢀcarboxamides Are Valuable
Antitubercular Candidates that Evade Innate Efflux
Machinery
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Elisa Azzali,
Diana Machado, Amit Kaushik, Federica Vacondio, Sara Flisi, Clotilde
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Silvia Cabassi, Gyanu Lamichhane, Miguel Viveiros, Gabriele Costantino,
and Marco
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,5,*
Pieroni.
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P4T group, and Department of Food and Drug, University of Parma, Parco Area delle Scienze
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7/A, Parma, 43124; Global Health and Tropical Medicine, GHTM, Instituto de Higiene e
Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira, 100, 1349ꢀ008
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Lisbon, Portugal; Center for Tuberculosis Research, Department of Medicine, Johns Hopkins
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University, 1503 E. Jefferson St Baltimore, MD 21231ꢀ1002, USA; Taskforce to study Resistance
Emergence & Antimicrobial development Technology, Department of Medicine, Johns Hopkins
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University, Baltimore, MD 21287; University of Parma, Department of Veterinary Science, via
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del Taglio 10, 43126, Parma; Centro Interdipartimentale Misure (CIM) ’G. Casnati’, University of
Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.
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Abstract
Tuberculosis remains one of the deadliest infectious diseases in the world, and the increased
number of multidrugꢀresistant and extremely drugꢀresistant strains is a significant reason of
concern. This makes the discovery of novel antitubercular agents a cogent priority. We have
previously addressed this need by reporting a series of substituted 2ꢀaminothiazoles capable to
inhibit the growth of actively replicating, nonꢀreplicating persistent and resistant Mycobacterium
tuberculosis strains. Clues from the structure‒activity relationships lining up the antitubercular
activity were exploited for the rational design of improved analogues. Two compounds, namely Nꢀ
phenylꢀ5ꢀ(2ꢀ(pꢀtolylamino)thiazolꢀ4ꢀyl)isoxazoleꢀ3ꢀcarboxamide 7a and Nꢀ(pyridinꢀ2ꢀyl)ꢀ5ꢀ(2ꢀ(pꢀ
tolylamino)thiazolꢀ4ꢀyl)isoxazoleꢀ3ꢀcarboxamide 8a, were found to show high inhibitory activity
toward susceptible M. tuberculosis strains, with an MIC of 0.125‒0.25 µg/mL (0.33ꢀ0.66 µM) and
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.06‒0.125 µg/mL (0.16ꢀ0.32 µM), respectively. Moreover, they maintained good activity also
toward resistant strains, they were selective over other bacterial species and eukaryotic cells,
metabolically stable, and apparently not susceptible to the action of efflux pumps.
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Introduction
a
Tuberculosis (TB), a highly contagious respiratory disease that results from infection with
Mycobacterium tuberculosis (Mtb), was recently declared as the number one infectious disease as it
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killed more people than HIV/AIDS or malaria. According to the World Health Organization
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(
WHO), in 2015, there were an estimated 10.4 million new TB cases and 1.4 million people died
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from this illness. 11% of all the new cases occurred in HIV infected patients, among which TB is
the leading cause of death, especially in Africa. While it is clear from these statistics that existing
treatment for TB is inadequate, the growing number of drugꢀresistant Mtb strains is an additional
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concern that needs to be addressed. Resistant Mtb strains are generally grouped into three distinct
phenotypes: a) multiꢀdrug resistant TB (MDRꢀTB), resistant to at least isoniazid (INH) and
rifampicin (RIF), b) extensively drugꢀresistant TB (XDRꢀTB), resistant to INH, RIF,
fluoroquinolones and, at least, one of the three injectable secondꢀline drugs, and c) totally drugꢀ
resistant TB (TDRꢀTB), a nonꢀconsensual designation for strains that are virtually untreatable since
5–8
have lost susceptibility to all of the molecules belonging to the antiꢀTB arsenal. The WHO has
estimated that about 3.7% of the new cases, and 20% of those previously treated, are sustained by
MDRꢀTB strains and about 9% of the patients suffering from MDRꢀTB are eventually infected with
XDRꢀTB. India, China and the Russian Federation accounted for 45% of the combined 580,000
drugꢀresistant cases recorded in 2015. Since the tight trade ties among these countries, United States
and European Union, the spread of the disease also in the developed countries is a highly realistic
2
matter of concern.
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The existing TB treatment requires daily intake of multiple drugs for, at least, six months. The
longꢀterm use of these drugs is often associated with the onset of several and severe side effects that
lead to patient noncompliance. In turn, this leads to the appearance of resistant strains, which
require the use of less effective and more toxic drugs for a longer period, and drastically increase
the financial burden for healthcare, jamming the treatment of TB in a vicious circle of difficult
resolution.
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Since the introduction of RIF in 1967, bedaquiline
is the only new chemical entity (NCE)
developed for the treatment of TB that has reached the market, although its use is restricted to the
treatment of MDRꢀ and XDRꢀTB. Unfortunately, after its introduction in the clinical practice, an
unexpected number of abnormal deaths has been reported, probably due to the serious side effects
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associated with significant cardiac arrhythmia.
Delamanid, a nitroimidazole inhibiting mycolic
acid biosynthesis, is another novel drug that has received conditional approval for MDRꢀTB by the
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European Medicines Agency in 2014. Also in this case, severe side effects such as cardiac
arrhythmia and general central nervous system (CNS) toxicity, especially when used in combination
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with INH or fluoroquinolones, have cooled down the initial enthusiasm caused by the introduction
of this novel antiꢀTB agent in therapy. In addition, mutations in the Mtb genome causing resistance
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to bedaquiline and delamanid have been recently documented. Therefore, efforts to develop novel
antiꢀTB therapeutic options that are safe and effective against drugꢀresistant Mtb are still needed.
After decades of oblivion, the emerging TB drug pipeline is nowadays nourished with a number of
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novel molecules that were developed following different drug discovery approaches.
Some of
these compounds were prepared starting from known inhibitors of old TB targets such as RNA
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polymerase, as in the case of rifapentine from RIF. On a similar vein, secondꢀline antiꢀTB
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therapeutics such as fluoroquinolones,
although conceived to treat infections brought by Gramꢀ
positive and Gramꢀnegative microorganisms, have recently entered the clinical trials to evaluate
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their use as firstꢀline agents.
However, it is debatable whether these approaches based on “drugꢀ
repositioning” might lead to longꢀterm results, as the occurrence of crossꢀresistance is rather
predictable. Targetꢀbased approaches have produced encouraging results, especially when directed
toward the soꢀcalled promiscuous targets, proteins whose functionality can be inhibited by more
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than one chemical entity. Two representative Mtb promiscuous targets, that have been intensively
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studied in the last years, are decaprenylphosphorylꢀβꢀDꢀribose 2'ꢀoxidase (DprE1), and one of the
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mycobacterial membrane transport proteins, large (MmpL3), both inhibited by several different
29–37
chemical classes of compounds.
Although the targetꢀbased approach is a valuable method for
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drug identification, it does not take under consideration the thick Mtb cell wall and the role of
efflux machinery, leading sometimes to disappointing discrepancies between the biochemical and
the wholeꢀcell phenotype assays. For these reasons, phenotypic highꢀthroughput screening (HTS),
followed by ligand‒based optimization, remains the most profitable strategy to identify novel
antibacterial agents in general, and novel antiꢀTB leads in particular.
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Recently, the wholeꢀcell phenotypic screening of an inꢀhouse chemical library led us to identify a
number of molecules embodying a 2ꢀaminothiazole scaffold endowed with an interesting antiꢀTB
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activity. Although the antiꢀTB activity of aminothiazoles and benzothiazoles was already reported
39,40
elsewhere,
no attempt was made to establish a reliable structure‒activity relationship (SAR) for
this chemical class. This prompted us to synthesize several structurally related derivatives, in which
the 2ꢀaminothiazole core was kept intact whereas different substituents were introduced at the
positions Cꢀ4, Cꢀ5, and at the 2ꢀamino group.
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Some of the 2ꢀaminothiazoles synthesized (Figure 1), 47 and 48, exhibited good inhibitory
activity toward both the actively replicating Mtb strains and the nonꢀreplicating persistent (NRP)
mycobacteria in a low oxygen recovery assay (LORA). More importantly, when evaluated against a
panel of singleꢀdrug resistant (SDR) Mtb strains, representative compounds maintained the same
inhibitory activity as toward the wildꢀtype, indicating that these substituted 2ꢀaminothiazoles were
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acting with a mechanism of action different from those of the currently marketed drugs, therefore
encouraging further investigation around this nucleus.
By leveraging the SAR data collected and the hypothetical pharmacophore designed, we herein
report the design and synthesis of a novel series of antiꢀTB derivatives based on the 2ꢀ
aminothiazole scaffold, with two compounds, 7a and 8a, showing a remarkable antiꢀTB activity in
the submicromolar range. To expand the set of information already reported, an inꢀdepth study that
included several other biological assays was carried out for these leads: in particular, their
cytotoxicity toward human monocyteꢀderived macrophage (HMDM), their selectivity over other
bacterial species and fungi, their metabolic profiles in human liver microsomes (HLM), and their
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capability to interact with Mtb efflux systems, were measured. More importantly, the activity
toward a set of resistant Mtb phenotypes (MDRꢀ and XDRꢀTB) was as well evaluated.
Results and Discussion
Rational design
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Compounds 47 and 48, previously reported by our group, are representative examples of a
series of substituted 2ꢀaminothiazoles endowed with leadꢀlike antiꢀTB characteristics. The main
structural feature of these molecules (Figure 1) is the presence of two aromatic rings, suitably
substituted, connected to each other through a fiveꢀmembered heterocycle such as the 2ꢀ
aminothiazole. The antiꢀTB efficacy of this structural pattern was corroborated by the activity of a
series of diarylimidazoles, reported by our group, in which the two aromatic rings were connected
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through a different fiveꢀmembered ring such as the imidazole. After several rounds of
modifications, we were able to establish that the phenyl ring attached to the 2ꢀamino group tolerated
the presence of lipophilic functional groups (i.e. halogens, small alkyls), whereas in the phenyl ring
attached at the Cꢀ4, the presence of polar substituents (i.e. the methoxy moiety) conferred good
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activity along with reduced cytotoxicity. With the aim of further validate the proposed
pharmacophore, we planned to synthesize a novel series of 2ꢀaminothiazoles in which the aromatic
ring at the position Cꢀ4 of the 2ꢀaminothiazole was replaced by polar heterocyclic structures, chosen
depending on the different physicochemical properties and the synthetic accessibility. Therefore, a
pyridine, an ethyl isoxazoleꢀ3ꢀcarboxylate, an ethyl 1ꢀmethylpyrazoleꢀ3ꢀcarboxylate, an 1,2,3ꢀ
triazole, and a 2ꢀamineꢀ5ꢀmethylthiazole were selected as Cꢀ4 appendages.
Pyridine was prepared as it is a known bioisostere of benzene and it is the simplest sixꢀmembered
aromatic ring containing a heteroatom. 4ꢀ(2ꢀpyridyl)ꢀ2ꢀaminothiazoles have already been disclosed
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as valuable antiꢀTB chemotypes, and partially developed with respect to their SAR.
In order to
add further information, in the small set of compounds prepared the pyridine nitrogen is placed at
the paraꢀposition, to conform to the position of the polar methoxy moiety in compounds 47 and 48.
The ethyl isoxazoleꢀ3ꢀcarboxylate moiety was chosen in first instance as it has already proved to
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be a valuable moiety in order to promote good antiꢀTB activity, when properly substituted.
Moreover, while this work was being prepared, GlaxoSmithKline disclosed a number of antiꢀTB
chemotypes suitable for further investigation, among which a singleton structurally similar to our
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derivatives 1a and 1b. Finally, the synthesis of the isoxazole nucleus can be easily achieved
through an efficient 1,3ꢀdipolar cycloaddition protocol, and the ethyl carboxylate obtained can be
further functionalized.
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On a similar way, also other heterocycles such as the ethyl 1ꢀmethylpyrazoleꢀ3ꢀcarboxylate and
the 1,2,3ꢀtriazole were selected because of their presence in a number of molecules recently
reported to have good antiꢀTB activity. A series of triazole derivatives was synthesized to target
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InhA, showing inhibition of Mtb growth in the low micromolar range. Also in the case of the
pyrazole, numerous derivatives embodying this structural motif were found to display a broad
spectrum of pharmaceutical activities, amongst which the inhibition of Mtb growth has been
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recently reviewed. In addition to that, the synthesis of these derivatives relies on the same
synthetic protocol employed for the preparation of isoxazoles, and starts from the same chemicals.
Finally, a substituted 5ꢀmethylꢀ2ꢀaminothiazole was attached to position Cꢀ4 to obtain a series of
bithiazoles. Although the biological properties of numerous 2ꢀaminothiazoles are well established,
little is known about the biological activity of bithiazoles in general, and as antiꢀTB agents in
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particular. Among other biological properties, bithiazoles are reported to be inhibitors of DNAꢀ
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gyrase, a well validated target for the treatment of TB. Indeed, along with the quinolones
mentioned above, many other molecules targeting DNAꢀgyrase, belonging to different chemical
series (aminopyrazinamides, thiazolopyridine ureas, thiazoleꢀaminopiperidine hybrid analogues),
have been recently reported as promising antiꢀTB chemotypes.
At the 2ꢀamino group of the 2ꢀaminothiazole, either the pꢀtolyl or the 3,5ꢀdichlorophenyl
appendages were maintained since they have previously demonstrated to promote good antiꢀTB
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characteristics (see compounds 47 and 48).
Chemistry
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All of the target compounds evaluated (1a‒b, 2a‒b, 3a‒b, 4a‒b, 5a‒b, 6‒9a, 10a‒b, 11‒13a, 14,
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5, 16a‒c, 17a, 18b, 19b, 20a‒b, 21a, 22a‒b), reported in Chart 1, are characterized by the
presence of the 2ꢀaminothiazole moiety, synthesized according to the established Hantzsch
protocol, refluxing the appropriate αꢀbromoketone with the proper thiourea in absolute ethanol
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(
Schemes 1 and 2). When not commercially available, thioureas are prepared refluxing the
corresponding anilines and ammonium thiocyanate in a solution of 1N hydrochloric acid.
Depending on the nature of the heterocycle, the desired αꢀbromoketone intermediates were obtained
with different procedures. The other synthetic pillar for the compounds reported in this study is the
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,3ꢀdipolar cycloaddition, used to prepare the isoxazole, triazole and 1ꢀmethylpyrazole
intermediates. The isoxazole (Scheme 1) was prepared reacting the nitrile oxide generated in situ by
the properly substituted chloroxime, and 3ꢀbutynꢀ2ꢀone. When not commercially available, the
chloroꢀoximes were prepared by condensation of the proper aldehyde with hydroxylamine
hydrochloride, followed by the treatment of the resulting aldoxime with Nꢀchlorosuccinimide, to
give intermediates 23 and 24 (Scheme 2). On a similar way, the synthesis of the pyrazole ring was
carried out according to the procedure of Oh et al., reacting a nitrileꢀimine dipole, generated by
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baseꢀpromoted dehydrohalogenation of hydrazonoyl halide, and the 3ꢀbutynꢀ2ꢀone. The
hydrazonoyl halide 24 was prepared by condensation of methylhydrazine and ethyl glyoxylate,
followed by the bromination of the resulting hydrazine with Nꢀbromosuccinimide in a mixture of
ethyl acetate and dichloromethane (Scheme 1). Finally the triazole ring 37 was prepared by “click”
reaction of 3ꢀbutynꢀ2ꢀone with sodium azide stirred in DMF at 60 °C. All of the above reported
ketones (14, 15, 25, 26, 35‒38, 40) were then brominated with bromine and glacial acetic acid in
chloroform at 50 °C, affording the αꢀbromoketones (27, 28, 41‒46) in good yields, although
sometimes with traces of the corresponding αꢀdibromoketones as collateral products. The synthesis
of the bithiazoles started with the reaction of 3ꢀchloropentaneꢀ2,4ꢀdione and the appropriate
thiourea in absolute ethanol at reflux. The reaction afforded in high yields the ketones 14 and 15,
that were brominated according to a procedure slightly different from that reported, that is using
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bromine and HBr solution in 1,4ꢀdioxane at 50 °C. The ethyl ester moiety was hydrolysed to
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carboxylic acid with LiOH in a solution of THF/MeOH/H O at room temperature. For the
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synthesis of amides, the suitable carboxylic acids were first activated with 1ꢀethylꢀ3ꢀ(3ꢀ
(dimethylamino)propyl)carbodiimide
(EDC)
and
2ꢀ(1Hꢀbenꢀzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀ
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tetramethyluronium tetrafluoroborate (TBTU), and then coupled with the appropriate amine at room
temperature using trimethylamine as the base, to yield the corresponding amides 5, 7‒12 in good
57
overall yields. Amide 6a was obtained stirring the corresponding ethyl ester derivative 3a and
NH OH overnight at room temperature whereas, starting from the same parent compound, alcohol
3a was obtained after reduction with sodium borohydride.
4
1
Evaluation of the antimycobacterial activity.
A total of 32 compounds (chart 1, 1a‒b, 2a‒b, 3a‒b, 4a‒b, 5a‒b, 6‒9a, 10a‒b, 11‒13a, 14, 15,
1
6a‒c, 17a, 18b, 19b, 20a‒b, 21a, 22a‒b) were synthesized and tested for their ability to inhibit the
growth of actively replicating Mtb strain H37Rv in a microdilution assay (see biological methods).
The whole set of compounds prepared can be ideally divided into two groups. In the “firstꢀ
generation”, the aim was to identify those heterocycles conferring good activity; feedback from this
preliminary evaluation drove the synthesis of improved “secondꢀgeneration” analogues. Finally,
most promising compounds were evaluated for their stability in human liver microsomes (HLM),
toxicity against human monocyte derived macrophage (HMDM), interaction with efflux pumps, and
selectivity over other microorganisms, including MDR and XDRꢀTB strains. While 21 compounds
failed to inhibit the growth of Mtb at concentrations up to 64 µg/mL, the minimum inhibitory
concentration (MIC) of 12 compounds were within a therapeutically interesting range, with some of
them exhibiting MIC lower than 1 µg/mL.
90
Firstꢀgeneration compounds.
The first round of synthetic efforts led to 10 derivatives (1a‒b, 2a‒b, 16a‒c, 17a, 18b, 19b, 22a‒
b) that were evaluated toward actively replicating Mtb. The 4ꢀ(pyridinꢀ4ꢀyl)ꢀ2ꢀaminothiazoles 16a‒
c and the 4ꢀ(triazolꢀ5ꢀyl)ꢀ2aminothiazole 19b were found to be devoid of any antiꢀTB activity
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(Table 1, MIC >64 µg/mL). It is well known that a decrease in the ClogP, especially in the case of
90
antiꢀTB agents, is often accompanied by the loss of activity because of poor cell membrane
penetration. However, since the remarkable activity of some of the derivatives described in this
work, for these compounds the lack of activity is probably due to weak interactions with the
molecular target. The substituted 4ꢀ(isoxazolꢀ5ꢀyl)ꢀ2ꢀaminothiazole derivatives 1a and 1b were
found to show good activity (Table 1, MIC < 4 µg/mL and 4‒8 µg/mL, respectively). Substitution
0
1
2
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8
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90
of the oxygen atom with an Nꢀmethyl moiety led to a sharp reduction of the antiꢀTB potency, as in
the case of compounds 2a and 2b (MIC90 = 16‒32 µg/mL and > 64 µg/mL, respectively). Regarding
the bithiazole derivatives, the symmetric compounds 17a and 18b showed conflicting results, in that
the first had an encouraging MIC90 of 4‒8 µg/mL, whereas the latter, a strictly close analogue, was
found to be completely inactive (MIC > 64 µg/mL). A similar trend is maintained by compounds
90
2
2a and 22b (MIC = 8‒16 µg/mL and 16‒32 µg/mL, respectively), in which the symmetry of the
90
molecule is lost due to removal of the phenyl ring attached at the 2ꢀamino group. To some extent,
and based on the biological feedback from compounds 1a‒b and 2a‒b, it can be speculated that the
pꢀtolyl moiety performs better than the 3,5ꢀdichlorophenyl one in order to provide the best antiꢀTB
activity. However, it is highly arguable whether the different substitution pattern might be
accountable for the large difference in the potency showed by compounds 17a and 18b. Due to
these inconsistencies, bithiazoles were put aside, and our attention was focused on the synthesis of
“secondꢀgeneration” 4ꢀ(isoxazolꢀ5ꢀyl)ꢀ2ꢀaminothiazole derivatives and a few 4ꢀ(1ꢀmethylpyrazolꢀ5ꢀ
yl)ꢀ2ꢀaminothiazoles.
Secondꢀgeneration derivatives.
Although derivatives 1a and 1b showed good activity, the ester moiety represented an evident
weakness, as it can be easily hydrolysed in the biological systems to give the corresponding acid.
Indeed, experimental evidences were obtained by assessing the metabolic stability of compounds 1a
and 1b in human liver microsomes (HLM). As expected, compounds 1a and 1b had only poor
metabolic stability (1a t
½
< 1 min; CL’int > 400 ml/min/kg; 1b t
½
= 3.3±0.3 min; CL’int = 188.8
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ml/min/kg) and the HPLC‒MS analysis clearly revealed that the ester moiety was rapidly cleaved to
give the corresponding acids. Since sometimes biotransformations are beneficial to improve the
activity of molecules, and are employed as a strategy for the activation of the soꢀcalled prodrugs,
we synthesized and tested the corresponding acids derived from 1a and 1b. Unfortunately, these
derivatives were found to be devoid of any antiꢀTB activity (Table 1, 3a, 3b MIC90 >64 µg/mL),
likely due to permeability issues. The same results applied to the pyrazolꢀ5ꢀylꢀ2ꢀaminothiazoles
counterparts (Table 1, 4a, 4b MIC > 64 µg/mL). As such, we reasoned that improving the stability
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90
of the compounds was a priority matter in order to make meaningful any further investigation.
Although inactive, derivative 3a was tested with regard to its metabolic stability in order to
46
ascertain whether the ester moiety was the only metabolic softꢀspot in the molecule. We were
pleased to notice a drastic improvement in the metabolic stability of this compound (3a, t =
½
1
4.3±1.1 min; CL’int = 43.7 ml/min/kg), proving that the ester functionality was the only issue
hampering the use of these molecules in the biological systems. Considering these findings, we
reasoned that preparing acid isosteres, with improved stability to hydrolysis, could couple good
activity and reasonable stability. The first round of modification was focused on the synthesis of
various carboxamides that are generally more resistant than esters to hydrolysis. The amide nitrogen
was either left unsubstituted, mono‒ or disubstituted with moieties different for size and
physicochemical characteristics, in order to evaluate their contribution to activity and stability. A
primary amide led to a complete loss of activity (Table 1, 6a, MIC90 > 64 µg/mL) as in the case of
the acid. Secondary amide 11a, that is a close analogue of ester 1a, showed the same unpleasant
result (MIC > 64 µg/mL). A diethyl substitution led to compound 9a, that showed some hint of
90
activity (MIC90 = 16‒32 µg/mL), although still unsatisfactory compared to that of the corresponding
ethyl ester. It can be concluded that smallꢀsized aliphatic substituents at the amide nitrogen are not
suitable to convey the desired potency. To see the effect of bigger aliphatic groups, the 1ꢀadamantyl
and the 1ꢀpiperidinyl moieties were selected as their lipophilic structures can enhance the
penetration through the Mtb cell wall. In addition, a number of preclinical and clinical antiꢀTB
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candidates such as NꢀAdamantanꢀ2ꢀylꢀN'ꢀ((E)ꢀ3,7ꢀdimethylꢀoctaꢀ2,6ꢀdienyl)ꢀethaneꢀ1,2ꢀdiamine
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36
dihydrochloride (SQ‒109), [1ꢀ(2ꢀadamantyl)ꢀ3ꢀ(2,3,4ꢀtrifluorophenyl)urea] (AU‒1235), and
some indolecarboxamides show the 1ꢀadamantyl functional group in their structure. However,
compounds 5a, 5b and 12a were found to be inactive up to a concentration of 64 µg/mL. Therefore,
it can be proposed that aliphatic groups, either bulky or smallꢀsized, resulting in secondary or
tertiary amides, are not suitable to confer the desired antiꢀTB activity. On the other hand, those
compounds bearing an aromatic or heteroaromatic ring attached at the amide nitrogen were found to
be active toward the replicating Mtb phenotype in the sub µg/mL range (7a, MIC90 = 0.125‒0.250
µg/mL, 0.33‒0.66 µM ; 8a, MIC90 = 0.06‒0.125 µg/mL, 0.16‒0.32 µM). As reported in Table 2,
considering the different molecular weights, compounds 7a and 8a are therefore slightly more
active than isoniazid, that is one of the most potent antiꢀTB drug ever discovered. To evaluate
whether abolishing the amide linker was detrimental for the activity, compounds 20a and 20b, in
which an aromatic ring was directly attached at the Cꢀ3 position of the isoxazole, were synthesized
and tested. In this case, the 4ꢀmethoxyphenyl moiety was selected because inspired by the structure
0
1
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9
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8
of compounds 47 and 48. Although it can be clearly seen that the amide moiety is crucial to confer
high potency, this modification led to contradictory results, (20a, MIC = 4‒8 µg/mL; 20b, MIC
90
90
>
64 µg/mL), making difficult the assessment of their collocation within the SAR. Compound 21a
was synthesized as the 2ꢀthiazole is considered a bioisostere of the ester moiety, but unfortunately it
did not show any activity up to the highest concentration tested (MIC90 > 64 µg/mL), further
establishing the amide as favourable functional group for the antiꢀTB activity. Finally, alcohol 13a,
although being more active than the acid counterpart, showed to be severalꢀfold less active than the
corresponding ester 1a (MIC = 16‒32 µg/mL). All of these findings are summarized in Figure 2,
9
0
where a visual representation of the SAR is reported.
Further biological characterization
Since our first concern was to improve the metabolic stability of the hit compounds, 7a and 8a
were evaluated for their t (min) and Cl’ (mL/min/kg) in HLM. We were pleased to notice that,
½
int
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compared to the esters, both the hit derivatives showed a severalꢀfold improvement in the metabolic
stability (7a, t
½
= 35.1±1.4 min; CL’int = 17.8 ml/min/kg; 8a, t = 16.1±0.2 min; CL’int = 38.8
½
ml/min/kg), with halfꢀlives and clearance values in the range of those of many marketed drugs. In
addition, compounds 7a and 8a were also found to be also more stable than the acid counterpart 3a
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(t = 14.3 ± 1.1 min; CL’int = 43.7 mL/min/kg). Then we wanted to check whether the strong
½
activity was specific for Mtb or it was due to general toxicity. Thus, compounds 7a and 8a were
evaluated in both eukaryotic and other prokaryotic cells. Although we have already reported that 2ꢀ
aminothiazoles were found to be devoid of cytotoxicity toward VERO cells, we have further
confirmed the safety of this class in HMDM, that are the cells where Mtb mostly resides during the
59
infection, and where antiꢀTB drugs exert their action. Also in this case, we were pleased to notice
that both the compounds resulted toxic at concentrations up to >500‒fold the MIC (7a, IC = 70
5
0
µg/mL; 7b, IC50 = 53 µg/mL). Compounds 7a and 8a were also tested against a panel of bacteria
other than mycobacteria (Gramꢀpositive, Gramꢀnegative, and fungi) to assess their selectivity of
action, resulting highly specific for Mtb, as they did not inhibit the growth of P. aeruginosa, E. coli,
E. faecium, S. aureus, and C. albicans (see Table 2 for MICs). To summarize, the activity of
compounds 7a and 8a toward Mtb is highly specific, as they neither are toxic toward the human
cells where the Mtb harbours, nor toward the most common bacterial strains, preventing the
occurrence of antimicrobial resistance.
Derivatives 7a and 8a were also tested against a panel of Mtb resistant strains. Some of the 2ꢀ
aminothiazoles previously reported were reported to maintain their activity against a panel of SDRꢀ
38
TB strains. In this work, we expanded the set of information by testing the novel 2ꢀaminothiazoles
synthesized against two MDRꢀTB and one XDRꢀTB strains. We were very pleased to notice that
also in this case compounds 7a and 8a maintained, toward the resistant strains, the same activity as
toward the wildꢀtype strain (Table 3).
On a similar vein, compounds were tested against a model of persistent Mtb strain. Molecules
bearing a 2ꢀaminothiazole scaffold have already demonstrated to possess a moderate activity
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against nonꢀreplicating Mtb strains in a carbon starvation model, a behaviour confirmed also in a
38
Low Oxygen Recovery Assay (LORA) as previously reported by us. Since the introduction of an
Nꢀarylisoxazoleꢀ3ꢀcarboxamide moiety, we have investigated whether the activity towards nonꢀ
replicating strains was maintained. Compounds 7a and 8a, tested using the Wayne model, were
found to maintain good activity, although slightly lesser than toward the activelyꢀreplicating
phenotypes, also towards nonꢀreplicating bacteria (Table 2), further confirming the versatility of
this scaffold.
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Finally, compounds were also evaluated for their susceptibility to be substrates of the efflux
systems. After the exposure to an antibiotic, the activity of bacterial efflux pumps increases and this
increased activity results in the reduction of the intracellular levels of the antibiotic, which enable
the survival of a lowꢀlevel resistant subpopulation. During this period, mutants with alterations in
the genes that favour drug resistance can be selected, therefore insuring the establishment of an
antibiotic resistant population presenting clinically significant, highꢀlevel resistance. In spite of this
knowledge, the effects of an antimicrobial compound toward efflux is seldom measured, and it is
not considered an important parameter in the hitꢀtoꢀlead process for the optimization of novel antiꢀ
61,62
TB drugs.
The recent discovery by Lee and colleagues of spectinamides as antiꢀTB drug
candidates, also by virtue of their scarce tendency to be extruded by efflux pumps, confirms the
63
importance of taking into account efflux when the design of new drugs is planned. Taking into
account all of these considerations, we tested some of our newly synthesized derivatives to
investigate their effect toward efflux. In particular, from one side we wanted to investigate whether
these compounds could be considered GEIs (growth and efflux inhibitors), a concept already
59
reported by some of us, aimed at discovering dual action antiꢀTB chemotypes. On the other side,
we wanted to preliminarily evaluate the tendency of derivatives 7a and 8a to be suitable substrates
of the mycobacterial efflux machinery, so as to predict the likeliness of resistance development by
Mtb. First, compounds 7a and 8a were evaluated for its activity as efflux inhibitors by EtBr realꢀ
time fluorometry and showed to be considerably less active (RFF = 0.22 and 0.17, respectively)
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than VP and TZ (Table 3), two of the most potent and characterized mycobacterial efflux pump
inhibitors reported so far. These results demonstrate that none of these compounds is capable of
inhibiting EtBr efflux by Mtb cells at subꢀlethal concentrations. Then, we have determined the
MICs of compounds 7a and 8a alone and in the presence of VP and TZ at concentrations of 64
µg/mL and 3.75 µg/mL, respectively. Indeed, at these concentrations, VP and TZ have already
demonstrated to exhibit strong synergistic effect with many first‒ and second‒line antiꢀTB drugs
that are substrate of efflux systems, considerably lowering their absolute MICs also toward wildꢀ
type strains. The results showed no significant changes in the MIC values of both compounds
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(Table 3), indicating that they have little probability to be extruded by the Mtb efflux pumps.
64
Although aware that 2ꢀaminothiazoles may cause a false positive interacting with every protein,
and taking into consideration the call made by some editors of the ACS journals with regard to the
65
early identification of PAINS, we would like to point out that the data in our hand rule out the
possibility of an unselective and unspecific mechanism of action. Indeed, the compounds in this
study were tested toward several cellular lines (Mtb H37Rv, MDRꢀTB, XDRꢀTB, HMDM, P.
aeruginosa, E. coli, E. faecium, S. aureus, and C. albicans), according different methods, and also
in the presence of liver subcellular fractions (microsomes), yielding a wide range of activities and
allowing an analysis of their SAR. Moreover, some of these derivatives were not recognized as
PAINS using the software “False Positive Remover” (http://www.cbligand.org/PAINS/) nor as
aggregator according to the software “Aggregator Advisor” (http://advisor.bkslab.org/).
Often, the description of the complete mechanism of action for the majority of antibacterials has
been unravelled long after their introduction in the market, and this applies also to many currently
used antiꢀTB compounds. This happens because their antimycobacterial properties and good
tolerability for the patient overcome the need for an exhaustive search for the description of their,
multiꢀtarget activity. In the case of the molecules reported in our manuscript, we can assert that the
molecular target is very likely different from that of the first and secondꢀline antiꢀTB drugs, as they
are active against MDR and XDRꢀTB strains. However, the actual enzyme(s) toward which
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compounds 7a and 8a exerts their inhibitory action is/are still unknown. The isoxazoleꢀ2ꢀ
aminothiazole scaffold, that is assumed to be the pharmacophore for these derivatives, is quite an
unexplored moiety in the field of medicinal chemistry. In the literature, only a few examples of
enzymes targeted by structurally related molecules can be retrieved. Analogues were reported to
inhibit the activity of the antigenꢀinduced slow reacting substance of anaphylaxis (SRSꢀAꢀlike
0
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material), and proved to be efficient in preventing anaphylaxis in guinea pigs. In another work,
similar compounds were tested as correctors by targeting the cystic fibrosis transmembrane
conductance regulator (CFTR), that is a chloride channel present in the membrane of epithelial
67
cells. In another study, analogues were also found to be active against histone deacetylase 8
68
(HDAC8), an anticancer target. Although HDAC inhibitors are known to possess a plethora of
69,70
biological activities, spanning from the anticancer to the antiparasitic,
and although the presence
of potential ortholog(s) in Mtb cannot be excluded, to hypothesize that the molecules herein
reported might be HDAC inhibitors is highly speculative and, as such, of no use. If the analysis is
limited to the 2ꢀaminothiazole scaffold alone, a couple of works have attempted to identify the
71,72
suitable molecular target. In both the works,
the synthesis of 2ꢀaminothiazoles took the cue from
the structure of thiolactomycin (TLM), that is an inhibitor of βꢀKetoacyl Synthase (KAS). As such,
the authors have reasoned that also the 2ꢀaminothiazoles should possess inhibitory activity toward
this enzyme. However, since the general lack of cellular activity, the drastic difference in the
structures of TLM and 2ꢀaminothiazoles, and, in particular, since the remarkable discrepancies
between biochemical and cellular results, it is reasonable to doubt that KAS might be the target, or
the only one, also for our derivatives. Recently, Chiarelli et al. have reported a phenotypic based
target screening approach in which a set of substituted 2ꢀaminothiazoles were found to be inhibitors
7
3
of the CTP synthetase PyrG, a validated bactericidal target in Mtb. Also in this case, the
substantial structural differences with the derivatives reported by Chiarelli et al. and our molecules
make it highly unlikely that they share the same mechanism of action.
Conclusion
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The final aim of this work was to identify a novel valuable antiꢀTB chemotype, to be submitted for
advanced in vivo studies. To this end, we have synthesized a total of 32 compounds focused on the
2
ꢀaminothiazole core, with various heterocycles rationally attached at the Cꢀ4 position. Based on
the activity against wildꢀtype Mtb, the most promising derivatives were selected and evaluated
especially with regard to those characteristics that are highly desirable in an antiꢀTB candidate.
Compared to the phenyl ring, heterocycles such as the isoxazole and the thiazole ring exhibited
remarkable antiꢀTB potency. In particular, when the isoxazoleꢀ3ꢀcarboxylate moiety is suitably
functionalized, as in the case of compounds 7a and 8a, antiꢀTB activity in the sub µg/mL range
could be obtained. It is worthwhile to highlight that among the antibiotics that are listed in WHO’s
recommendation for treatment of all forms of TB, the activity of our compound 8a would rank
among the best. Along with the improved inhibitory activity, these modifications also led to a
noticeable improvement of the metabolic stability. To further promote the advancement of these
compounds, additional biological assays were carried out. Both 7a and 8a maintained remarkable
activity against a panel of MDRꢀTB and XDRꢀTB strains, indicating that the mechanism of action
by which they exert their inhibitory activity is different from that of the currently used antiꢀTB
agents. Also, the mechanism of action is highly specific for Mtb, as compounds 7a and 8a failed to
show any inhibitory activity either against eukaryotic cells and against a panel of Gramꢀpositive and
Gramꢀnegative microorganisms. Finally, although the extent to which an antimicrobial compound is
a substrate of efflux is seldom measured, we consider it an important parameter in the hitꢀtoꢀlead
process and we have demonstrated that both these compounds are likely not subject to the effect of
mycobacterial efflux pumps. Along with the specific mechanism of action, this increases the bar for
the development of resistance to these molecules. In addition to these promising biological
characteristics, it must be considered that these 2ꢀaminothiazoles have physicochemical properties
that indicate great potential for absorption and permeation when used as orally available
compounds, as they do not show any violation of the four physicochemical characteristics defined
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by the Lipinski rule of five. Indeed, their molecular weight is lower than 500 g/mole, the total
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number of oxygen and nitrogen atoms is lesser than 10, the number of hydrogen bond donor is
lesser than 5 and, also, the ClogP is lower than 5 (calculated at http://www.molinspiration.com/), a
considerable improvement over that of bedaquiline (ClogP = 7.25) and some indoleꢀ2ꢀ
75
carboxyamides in clinical studies. Considering all of this information, the ease of synthesis and
the promising physicochemical characteristics, compounds 7a and 8a can be considered mature
leads for further characterization, especially with regard to their in vivo activity and the
determination of their mechanism of action. This investigation is currently ongoing in our
laboratories.
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Experimental section
Chemistry
General information. All the reagents were purchased from SigmaꢀAldrich and AlfaꢀAesar at
reagent purity and, unless otherwise noted, were used without any further purification. Dry solvents
used in the reactions were obtained by distillation of technical grade materials over appropriate
dehydrating agents. Reactions were monitored by thin layer chromatography on silica gelꢀcoated
aluminium foils (silica gel on Al foils, SUPELCO Analytical, SigmaꢀAldrich) at both 254 and 365
nm wavelengths. Where indicated, intermediates and final products were purified through silica gel
flash chromatography (silica gel, 0.040ꢀ0.063 mm), using appropriate solvent mixtures.
1
13
HꢀNMR and CꢀNMR spectra were recorded on a BRUKER AVANCE spectrometer at 400 and
1
100 MHz respectively, with TMS as internal standard. HꢀNMR spectra are reported in this order:
multiplicity and number of protons. Standard abbreviation indicating the multiplicity was used as
follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet, m = multiplet
and br = broad signal. HPLC/MS experiments were performed with HPLC: Agilent 1100 series,
equipped with a Waters Symmetry C18, 3.5 µm, 4.6 mm x 75 mm column and MS: Applied
Biosystem/MDS SCIEX, with API 150EX ion source. HRMS experiments were performed with
LTQ ORBITRAP XL THERMO.
All compounds were tested as 95‒100% purity samples (by HPLC/MS).
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General procedure for Hantzsch synthesis. The suitable αꢀbromoketone (1 equiv) and the proper
thiourea (1 equiv) were solubilized in anhydrous ethanol (20 mL/mmol) and reacted at 70° C until
consumption of the starting materials as indicated by TLC. After cooling, the solvent was
evaporated and the crude material was purified by flash column chromatography or in a few cases,
by precipitation. Purification conditions, yields and analytical data are reported in the supporting
information. TLC for control (7:3 Petroleum ether/Ethyl Acetate)
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60
αꢀBromination of the ketones: method A. Acetic acid (0.1 mL/mmol) was added to a solution of
the ketone (1 equiv) in chloroform (1.5 mL/mmol), and reaction mixture was heated at 50 °C. Then
2
a solution of Br (1.05 equiv) in chloroform (0.4 mL/mmol) was added dropwise, and the mixture
was stirred at the same temperature for 1 hour. After consumption of the starting material according
to TLC (9:1 Petroleum ether/Ethyl Acetate), NaHCO (aq sat solution, 5 mL/mmol) was added and
3
the mixture was extracted with dichloromethane (3 times). The combined organic layers were
2 4
washed with brine, dried over Na SO , and, after removal of the solvent in vacuo, the crude
material was purified by flash column chromatography. Purification conditions, yields and
analytical data are reported in the supporting information.
αꢀBromination of the ketones: method B. A solution of Br (0.8 equiv) in 1,4ꢀdioxane (4
2
mL/mmol) was added to a solution of the appropriate ketone (1 equiv) solubilized in HBr (48%
solution, 4 mL/mmol) preheated to 50 °C. After consumption of the starting material according to
3
TLC (9:1 Petroleum ether/Ethyl Acetate), saturated NaHCO aq. solution (5 mL/mmol) was added
and the mixture was extracted with chloroform (3 times). The combined organic layers were
washed with brine, dried over Na SO , and, after removal of the solvent in vacuo, the crude
2
4
material was purified by flash column chromatography. Purification conditions, yields and
analytical data are reported in the supporting information.
General procedure for the hydrolysis of the esters. The appropriate ester (1 equiv) and
2 2
LiOH·H O (4 equiv) were dissolved in a solution of THF/MeOH/H O (3:1:1, 1 mL/mmol) and
stirred at room temperature until consumption of the starting material as indicated by TLC (7:3
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Petroleum ether/Ethyl Acetate, then 9:1 Dichloromethane/methanol). The reaction mixture is then
evaporated under reduced pressure, and the crude obtained is taken up with H O, acidified with 2N
2
HCl and extracted with ethyl acetate (3×10 mL). After evaporation of the solvent, the product is
used for the next reaction step without further purification.
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5ꢀ(2ꢀ(pꢀtolylamino)thiazolꢀ4ꢀyl)isoxazoleꢀ3ꢀcarboxamide (6a). A suspension of compound 1a
(50 mg, 0.15 mmol) in NH OH (2 ml) was stirred overnight at room temperature, then H O (8 mL)
4
2
was added and the mixture extracted with ethyl acetate (3×8 mL). The organic layers were treated
with water, washed with brine and dried over Na SO . After filtration, the solvent was removed in
2
4
vacuo and the crude material was purified by flash column chromatography eluting with petroleum
1
ether/ethyl acetate 7:3 to give the title compound as a white powder in 82% yield. HꢀNMR (400
MHz, DMSOꢀd
6
): δ = 2.27 (s, 3H); 7.02 (s, 1H); 7.16 (d, J=8 Hz, 2H); 7.55 (d, J=8 Hz, 2H); 7.62
13
(
s, 1H); 7.90 (s, 1H); 8.20 (s, 1H); 10.30 (s, 1H). CꢀNMR (100.6 MHz, DMSOꢀd ): δ = 20.86;
6
100.49; 109.72; 117.88; 129.95; 131.22; 138.43; 138.78; 159.83; 160.49; 164.95; 166.73. HRMS
+
(ESI) calculated for C16
H N
15 3
O
3
S [M + H] 301.0681found 301.0660
General procedure for the synthesis of amides. Oꢀ(Benzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀ
tetramethyluronium tetrafluoroborate (TBTU, 1 equiv) and Nꢀ(3ꢀDimethylaminopropyl)ꢀN′ꢀ
ethylcarbodiimide hydrochloride (EDC·HCl, 1 equiv) were added to a solution of the proper
carboxylic acid (1 eq) in dry DMF (4 mL/mmol). The reaction mixture was stirred at room
2
temperature under N for 15 minutes, then triethylamine (1.5 equiv) and the suitable amine (1
equiv) were added to the mixture that was stirred at the same temperature until the complete
consumption of the starting material, as indicated by TLC (usually 7:3 Petroleum ether/Ethyl
Acetate). Water (10 mL) was added and the mixture extracted with ethyl acetate (3×10 mL). The
2 4
organic layers were treated with water, washed with brine and dried over Na SO . After filtration,
the solvent was removed in vacuo and the crude material was purified by flash column
chromatography to give the title compounds. Purification conditions, yields and analytical data are
reported in the supporting information.
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(5ꢀ(2ꢀ(pꢀtolylamino)thiazolꢀ4ꢀyl)isoxazolꢀ3ꢀyl)methanol (13a). Sodium borohydride (14 mg;
.36 mmol) was added to solution of 1a (20 mg; 0.06 mmol) in anhydrous methanol at 0 °C (1 mL)
0
and the reaction mixture was stirred at room temperature for 30 minutes. After the complete
consumption of the starting material, as indicated by TLC (8:2 Petroleum ether/Ethyl Acetate), the
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mixture was cooled to 0 °C and NH Cl (aq sat sol, 5 mL) was added. The aqueous layers were
extracted with ethyl acetate (3×8 mL), and the combined organic layers were washed with brine and
dried over Na SO . After filtration, the solvent was removed in vacuo and the crude material was
2
4
purified by silica gel flash chromatography column eluting with dichloromethane/methanol from
1
9
8:2 to 95:5 to give compound 13a as a white powder in 93% yield. HꢀNMR (400 MHz, CDCl ):
δ
3
13
= 2.38 (s, 3H); 4.84 (s, 2H); 6.66 (s, 1H); 7.14 (s, 1H); 7.23ꢀ7.27 (m, 2H); 7.29ꢀ7.31(m, 2H). Cꢀ
NMR (100.6 MHz, DMSOꢀd
6
):
δ
= 20.86; 55.45; 100.49; 108.58; 117.78; 129.92; 131.12; 138.87;
+
1
39.10; 164.78; 165.30; 165.49. HRMS (ESI) calculated for C16
H
15
N
3
O
3
S [M + H] 288.0728,
found 288.0699
General procedure for the 1,3ꢀdipolar cycloaddition. Triethylamine (1 equiv) was added
dropwise to a solution of the suitable halogenated oxime (1 eq) and 3ꢀbutynꢀ2ꢀone (2 equiv) in
benzene (1.8 mL/mmol) at 0 °C. The reaction mixture was stirred either at room temperature or at
6
0 °C for 1 hour. After this period, 1N HCl (5 mL/mmol) was added and the mixture extracted with
ethyl acetate (3 × 10 mL). The organic layers were washed with water, brine and dried over
Na SO . After filtration, the solvent was removed in vacuo and the crude material was purified by
2
4
flash column chromatography.
With this procedure, starting from intermediates 23, 24, 33, 34, the isoxazole derivatives 25, 35
and 36 and the pyrazole derivative 26, were synthesized. Detailed purification conditions, yields
and analytical data are reported in the supporting information.
Synthesis of (Z)ꢀethyl 2ꢀbromoꢀ2ꢀ(2ꢀmethylhydrazono)acetate (24).
A
solution of
methylhydrazine (228 µL; 4.34 mmol) in methanol (0.33 mL/mmol) was heated at 40 °C, then ethyl
glyoxalate (50% solution in toluene, 400 µL; 4.03 mmol) was added while maintaining the
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temperature below 50 °C. The mixture was allowed to stir at 50 °C until consumption of the starting
material as revealed by TLC (8:2 Petroleum ether/Ethyl Acetate, stained with KMnO ). Solvents
4
and excess methylhydrazine were distilled off and the crude material was purified by flash column
chromatography eluting petroleum ether/ethyl acetate from 75:25 to 50:50. Yield 50%. The product
obtained (50 mg, 0.38 mmol) was solubilized in dichloromethane (1.38 mL) and added to a slurry
of Nꢀbromosuccinimide (68 mg; 0.38 mmol) in ethyl acetate (1.38 mL) maintained at 0 °C. The
reaction mixture was stirred for 1 hour at 5 °C, then the solvents were evaporated under reduced
pressure and the crude material used for the next reaction step without purification, as the product is
instable and easily decomposes when left at the air.
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General procedure for the synthesis of the oximes. NaOH 10% (0.5 mL/mmol) was added to a
solution of hydroxylamine hydrochloride (1.2 equiv) in water (0.7 mL/mmol). Then a solution of
the suitable aldehyde (1 equiv) in ethanol (3 mL/mmol) was added dropwise. The reaction mixture
was stirred at room temperature for 30 minutes, then the solvents were evaporated under reduced
pressure and the residue solubilized in water and extracted with ethyl acetate. The combined
2 4
organic layers were washed with brine and dried over Na SO . After filtration, the solvent was
removed in vacuo and the crude material was used for the next reaction step without further
purification.
1
(
E)ꢀ4ꢀmethoxybenzaldehyde oxime (31). Yield 99%. HꢀNMR (400 MHzꢀCDCl
3
):
δ
= 3.86 (s,
3H); 6.92 (d, J=8 Hz, 2H) 7.53 (d, J=8 Hz, 2H); 8.11 (s, 1H).
1
(
E)ꢀthiazoleꢀ4ꢀcarbaldehyde oxime (32). Yield 91%. HꢀNMR (400 MHzꢀDMSOꢀd
6
):
δ
= 7.95
(s, 1H); 8.23 (s, 1H); 9.15 (s, 1H); 11.3 (s, 1H).
(Z)ꢀNꢀhydroxyꢀ4ꢀmethoxybenzimidoyl chloride (33). NꢀChlorosuccinimide (103 mg; 0.77
mmol) was added to a stirred solution of compound 31 (100 mg, 0.64 mmol) in DMF (2 mL), and
the reaction mixture was reacted at room temperature for 1 hour. After the complete consumption of
the starting material according to TLC (95:5 Dichloromethane/methanol), water (5 mL) was added
and the mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were
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washed with brine and dried over Na SO . After filtration, the solvent was removed in vacuo and
2
4
the crude material was used for the next reaction step without further purification.
Z)ꢀNꢀhydroxythiazoleꢀ4ꢀcarbimidoyl chloride (34). Following a similar procedure, but using
E)ꢀthiazoleꢀ4ꢀcarbaldehyde oxime 32 as the starting material, and a mixture of pyridine (6 µL,
.078 mmol) and dichloromethane (1.5 mL) as the solvent, compound 34 was prepared and used for
the next reaction step without further purification.
ꢀ(1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)ethanone (37). A solution of 3ꢀbutynꢀ2ꢀone (114 µL, 1.46 mmol) in
DMF (0.4 mL/mmol) was added over a period of 30 minutes to a suspension of NaN (143 mg, 2.20
(
(
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60
0
1
3
mmol) in DMF (1 mL/mmol) preheated at 60 °C. The reaction mixture was stirred at the same
temperature for 1 hour, and then water (10 mL) was added and the slurry obtained was washed with
dichloromethane (2×10 mL). The solvent was separated and the aqueous layers were acidified with
3
N HCl and extracted with ethyl acetate (3×10 mL). The combined organic layers were washed
with brine and dried over Na SO . After filtration, the solvent was removed in vacuo and the crude
2
4
material was purified by flash column chromatography eluting petroleum ether/ethyl acetate from
1
9
:1 to 7:3. Yield 30%. HꢀNMR (400 MHz, DMSOꢀd
6
):
δ
= 2.56 (s, 3H); 8.53 (s, 1H).
2
ꢀBromoꢀ1ꢀ(pyridinꢀ4ꢀyl)ethanone (44). A solution of Br (294 µL; 5.76 mmol) in diethyl ether
2
(
2.8 mL) was added dropwise over a period of 30 minutes to a solution of 4ꢀacetylpyridine (456 µL;
.10 mmol) in 1,4ꢀdioxane/diethyl ether 1/1 (4.8 mL) cooled to 0° C. The reaction mixture was
stirred overnight, then saturated NaHCO aq. solution (20 mL) was added and the mixture extracted
4
3
with ethyl acetate (3×10 mL). The combined organic layers were treated with water (3×10 mL),
washed with brine and dried over Na SO . After filtration, the solvent was removed in vacuo and
2
4
the crude material was purified by flash column chromatography eluting petroleum ether/ethyl
1
acetate 7:3. Yield 50%. HꢀNMR (300 MHz CDCl ):
δ = 3.10 (s, 2H); 8.53 (d, J=6 Hz, 2H); 8.62
3
(d, J=6 Hz, 2H). TLC for control (8:2 Petroleum ether/Ethyl Acetate).
Biology
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Inhibition of M. tuberculosis H37Rv. Minimum Inhibitory Concentration (MIC ) for Mtb was
90
determined using standard broth macrodilution in 15ꢀml sterile conical tubes containing 2.5 ml of
7
H9 broth. Standard broth microdilution method (using 96ꢀwell plates) was used for other
76
organisms as per Clinical and Laboratory Standard Institute (CLSI) recommendations.
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Middlebrook 7H9 broth was used for Mtb growth as per CLSI guidelines (Desmond, 2011). In
5
summary, 10 bacilli grown to exponential phase in liquid medium were inoculated into each well
containing drug at two fold dilutions ranging from 64 µg/ml to 0.03 µg/ml. Growth medium alone
5
and without drug but inoculated with 10 bacilli were included as negative and positive controls,
respectively. Appropriate drugs (INH and RIF) were included as positive control for growth
inhibition. Growth was evaluated by visual inspection for presence of bacterial pellet following
incubation for fourteen days at 37 °C. The first well in which bacterial pellet is absent and therefore
growth is not observable is considered the MIC90 as per the standard CLSI guidelines. MIC90 is
expressed as a range spanning two concentrations: the higher concentration represents the lowest
concentration at which bacterial growth could not be observed.
Inhibition of resistant Mtb Strains. Mtb drugꢀresistant clinical isolates (Mtb1, Mtb2 and Mtb3)
77
were obtained from patients diagnosed with active drugꢀresistant TB in Lisbon in 2008 and 2009.
MICs of compounds 7a and 8a was conducted by the 96ꢀwell broth microdilution method using a
7
8
tetrazolium microplateꢀbased assay with slight modifications. Mtb strains were grown in MB7H9
plus 10% OADC supplement at 37 ºC until an OD600nm of 0.8. The inoculum was prepared by
5
79
diluting the bacterial cultures in MB7H9/OADC to a final density of approximately 10 cells/ml.
Briefly, aliquots of 0.1 ml of inoculum were transferred to each well of the plate that contained 0.1
ml of each compound at concentrations prepared from twoꢀfold serial dilutions in MB7H9/OADC
medium. Growth controls and a sterility control were included in each assay. The inoculated plates
were sealed in plastic bags and incubated at 37°C during seven days. After the seven days of
incubation, MTT was added to each well to a final concentration of 2.5% and the plates incubated
overnight. The bacterial viability was registered for each well based on the MTT colour change and
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the MIC was defined as the lowest concentration of compound that totally inhibited bacterial
growth (no colour change). The assays were performed in triplicate. MICs of the compounds in
presence of the inhibitors verapamil or thioridazine were performed as described above with the
exception that each inhibitor was added to each drugꢀcontaining well at ¼ MIC (VP, 64 µg/ml and
TZ, 3.75 µg/ml). The results were interpreted as described above.
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Inhibition of nonꢀreplicating Mtb Strains. Mtb H37Rv ATCC27924 cultures were adapted to
8
0
hypoxic conditions as described earlier with modifications. Briefly, Mtb cells were grown in tubes
containing 8 mL MB7H9. The tubes were tightly capped, sealed with parafilm and incubated
standing at 37°C during 8 weeks. Methylene blue was added as a redox indicator (final
concentration of 1.5 µg/mL) to all tubes to monitor oxygen depletion. MIC determination of the
compounds 7a and 8a against the NRP Mtb cells was determined in 96ꢀwell microtiter plates in an
anaerobic jar by exposing the hypoxic cells to varying concentrations of compounds for 5 days at
o
3
7 C. An anaerobic indicator strip was placed inside the jar to visually confirm the removal of
oxygen during the incubation. After this period the plates were transferred to normal atmosphere for
o
recovery during 2 days at 37 C. Then, MTT was added to each well to a final concentration of 2.5%
and the plates incubated overnight at room temperature. The bacterial viability was registered for
each well based on the MTT colour change and the MIC was defined as the lowest concentration of
compound that totally inhibited bacterial growth (no colour change). The assays were performed in
triplicate.
Cytotoxicity assays toward human monocyteꢀderived macrophage (HMDM). Cellular toxicity
was assayed against human monocyteꢀderived macrophages. Blood was collected from healthy
volunteers and peripheral blood mononuclear cells isolated by FicollꢀPaque Plus (GE Healthcare,
Freiburg, Germany) density gradient centrifugation. Monocytes were differentiated into
macrophages during 7 days in macrophage medium containing RPMIꢀ1640 medium with 10% fetal
calf serum (FCS), 1% GlutaMAX™, 1 mM sodium pyruvate, 10 mM HEPES at pH 7.4, 100 IU/ml
penicillin and 100 µg/ml streptomycin (Gibco, Life Technologies), and 20 ng/ml MꢀCSF
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(Immunotools, Friesoythe, Germany) and incubated at 37ºC with 5% CO . Fresh medium was
2
added at day 4 post isolation. The effect of the compounds 7a and 8a was evaluated using
AlamarBlue (Molecular Probes, Life Technologies) according to the manufacturer’s instructions.
4
Briefly, 5x10 cells were seeded in 96ꢀwell microplates, treated with the compounds and then
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incubated at 37ºC in a 5% CO atmosphere. After 3 days of exposure, cell viability was assessed.
Briefly, 10% AlamarBlue was added to each well and incubated at 37ºC and 5% CO . Fluorescence
2
was measured with a 540/35 excitation filter and a 590/20 emission filter in a Synergy HT multiꢀ
mode microplate reader (BioTek Instruments, Inc, Vermont, USA). The IC50 value corresponds to
8
1
the highest concentration of compound at which 50% of the cells are viable relative to the control.
Antimicrobial activity of compounds 7a and 8a. Bacterial and fungal reference strains (E. coli
ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC25923, E. faecium ATCC 35667,
Candida albicans ATCC 11006) were purchased from Mast Diagnostic (Germany). The
82,83
antimicrobial activity of 7a and 8a was evaluated as reported elsewhere
following the CLSI
guidelines (2008). Powder compounds were dissolved in sterile water with 5% DMSO. DMSO
concentration were increased to 55% to dissolve macroscopic aggregation of the compounds and
solutions were sonicated for about 30 minutes before use. The final concentration of 7a and 8a
stock solution was 500 µg/ml. The bacterial suspension was standardized following the Clinical and
Laboratory Standards Institute guidelines. Briefly, the logꢀgrowing phase was reached by
incubating each strain in Mueller Hinton broth (MH) (Difco, USA) at 37 °C in a shaker at 225 rpm
for 3–4 h. After being pelleted at 1000 g for 20 min, the bacterial suspension was adjusted
spectrophotometrically at 600nm to an optical density value in the range 0.08–0.13, containing
8
approximately 10 colony forming unit (CFU)/ml in phosphate buffer (PB) 10mM pH 7. Fifty
6
microliters of bacterial suspension containing 10 CFU/ml were inoculated into each well, to obtain
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a final concentration of approximately 5x10 CFU/ml. For C. albicans susceptibility tests were
performed by broth microdilution methodology in RPMI according to the CLSI M27ꢀA3 guidelines
(2008). Briefly, C. albicans culture was adjusted by adding PB to a 0.5 McFarland standard which
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correspond to a concentration of 1ꢀ5 x 10 CFU/ml. Fungal suspension was further diluted in broth
3
to obtain a final concentration of about 10 CFU/ml and used within 30 minutes after its preparation
for microdilution assay. Compounds were serially diluted into 96 U bottomed microtiter plate wells
from 200 µg/ml to 0,4 µg/ml concentration, in a volume of 50 µl. In each well 50 µl of
bacterial/fungal suspension was added (final volume 100 µl /well). Therefore, the final
concentrations were ranging from 100 µg/ml to 0.2 µg/ml. For every bacterial/fungal strain, a
growth and sterility control were set. Plates were incubated at 37°C for 24 hours (48 hours for
fungal strains) in aerobic atmosphere. The Minimum Inhibitory Concentration (MIC) is defined as
the lowest concentration of antimicrobial at which there is no visible growth of the organism.
Because of the turbidity of the suspension at higher concentrations, the test was also evaluated
through viability staining with resazurin. In this case, MIC was considered as the lower
concentration of the compounds with no colour change from blue to pink (no cells metabolic
activity).
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Evaluation of efflux inhibitory activity of compounds by realꢀtime fluorometry. The EtBr
accumulation by the mycobacterial strains was assessed on a realꢀtime basis using a fluorometric
61,84
method, as previously described.
Mtb H37Rv was grown in MB7H9 supplemented with 10%
OADC and 0.05% Tween 80 until OD600nm of 0.8. For the accumulation of EtBr, the cells were
collected by centrifugation at 2940 x g for three minutes, the pellet washed in PBS, and the OD600nm
of the suspension adjusted to 0.8 with PBS. To assess the effect of compounds 7a and 8a on EtBr
accumulation, the assays were performed in a final volume of 0.1 mL containing 0.05 mL of the
cellular suspension (final OD600nm of 0.4) and 0.05 mL of a solution of EtBr to a final concentration
of 0.25 µg/ml, and the compound to be tested to a final concentration of half MIC, in order to not
compromise the cell viability. Verapamil and thioridazine were included as controls and used at
1
8.5 µM. The assays were conducted in a RotorꢀGene 3000 (Corbett Research, Sydney, Australia)
at 37ºC, and the fluorescence acquired at 530/585 nm at the end of every 60 seconds, during 60
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minutes. The activity of the compounds was evaluated by the relative final fluorescence (RFF)
85
index as previously described.
Stability studies in Human Liver Microsomes. Stability of selected compounds in the presence
of pooled HLM (X200 pooled, Xenotech LLC, USA) was assessed by incubation of a 1 µM
0
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concentration for 60 min in the presence of HLM (1 mg protein mL ), at 37 °C, in the presence of a
+
ꢀ1
NADPHꢀregenerating system (2 mM NADP , 10 mM glucoseꢀ6ꢀphosphate, 0.4 U mL glucoseꢀ6ꢀ
phosphate dehydrogenase, 5 mM MgCl ) in 100 mM PBS buffer solution pH 7.4. The reaction
2
mixtures were preheated (37 °C) for 5 min before adding the parent compound. At fixed time points
(t = 0; 15; 30; 60; min), aliquots of samples were withdrawn, deproteinized with two volumes of
acetonitrile, centrifuged (9,000 g, 4 °C, 10 min) and the supernatant analyzed by injection in HPLCꢀ
MS/MS system. The chromatographic separation was performed employing a gradient elution
starting from 70% water + 0.1% formic acid (solvent A):30% methanol (solvent B) to 90%B:10%A
in 10 min; 90%B:10%A was kept for further 5 min; then back to 70%A:30%B and further 5ꢀmin of
reconditioning time. HPLCꢀMS/MS analysis employed a Thermo Quantum Access Max TSQ triple
quadrupole mass spectrometer (Thermo, USA) equipped with an HꢀESI (HeatedꢀElectroSpray
Ionization) interface and coupled to an Accela UHPLC system (Thermo, USA) constituted of a
quaternary pump, a degasser and a thermostated autosampler. Compounds were analyzed in
positive ion mode using both total ion monitoring mode, over a mass range from 50 to 500 amu,
and single ion monitoring mode. Data were acquired and analyzed employing Thermo Excalibur 1.4
software (Thermo, USA).
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Supporting Information Available: The HꢀNMR spectra of the intermediates and the HꢀNMR,
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CꢀNMR, HRMS and the molecular formula strings (CSV) of the title compounds are available in
the supporting information. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Corresponding Author
Marco Pieroni +39 0521 905054; Eꢀmail marco.pieroni@unipr.it
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Acknowledgment
Maria Giovanna Massanova is kindly acknowledged for the help in the synthesis of derivatives.
The Centro Interdipartimentale Misure ‘‘G. Casnati’’ is kindly acknowledged for the contribution in
the analytical determination of the molecules synthesized. DM was supported by grant
SFRH/BPD/100688/2014 from Fundação para Ciência e a Tecnologia (FCT), Portugal. DM and
MV are thankful to FCT for funding to the Global Health and Tropical Medicine (GHTM) Research
Center (Grant UID/Multi/04413/2013).
a
Abbreviations
AMK, amikacin ; CAP, capreomycin, DMF, N,Nꢀdimethyl formamide ; DOTS, directly observed
therapy shortꢀcourse; EDC, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide; EDG, electronꢀdonor
group; EWG, electronꢀwithdrawing group; EI, efflux inhibitor; INH, isoniazid; LORA, low oxygen
recovery assay; MIC, minimum inhibitory concentration; MDRꢀTB, multidrugꢀresistant
tuberculosis; MOX, moxifloxacin; Mtb, Mycobacterium tuberculosis; NRPꢀTB, nonꢀreplicating
persistent tuberculosis; OFX, ofloxacin ; RIF, rifampin; SAR, Structure Activity Relationships; SM,
streptomycin; TB, tuberculosis; TBTU, 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; TEA, triethylamine; THF, tetrahydrofuran; XDRꢀTB, extensively drugꢀresistant
tuberculosis
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