Rational Design and Synthesis of Thioridazine Analogues as Enhancers of the Antituberculosis Therapy

Article abstract of DOI:10.1021/acs.jmedchem.5b00428

Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis.

Full text of DOI:10.1021/acs.jmedchem.5b00428

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Rational Design and Synthesis of Thioridazine
Analogues as Enhancers of the AntiꢀTB Therapy
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Marco Pieroni,†,#,* Diana Machado, §,# Elisa Azzali, † Sofia Santos Costa, § Isabel Couto, §
Gabriele Costantino† and Miguel Viveiros§,*
†Dipartimento di Farmacia, P4T group, University of Parma, Parco Area delle Scienze 27/A,
Parma, 43124
§Grupo de Micobactérias, Unidade de Microbiologia Médica, Global Health and Tropical
Medicine (GHTM), Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
(IHMT, UNL), Rua da Junqueira, 100, 1349ꢀ008 Lisbon, Portugal
KEYWORDS. Efflux pumps, Thioridazine, Tuberculosis, Antimicrobial Resistance, Medicinal
Chemistry.
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Abstract
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Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious
diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are
known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has
shown good antiꢀTB properties both in vitro and in vivo, likely due to its capacity to inhibit
efflux mechanisms. Here we report the design and synthesis of a number of putative efflux
inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro
and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of
the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a
remarkable synergistic effect with antiꢀTB drugs both in vitro and in human macrophages,
demonstrating their potential to be used as coꢀadjuvants for the treatment of tuberculosis.
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Introduction
Tuberculosis (TB) is a highly contagious airborne disease resulting from infection with
Mycobacterium tuberculosis. The World Health Organization (WHO) currently estimates that
oneꢀthird of the world’s population is infected with M. tuberculosis, and in 2013, approximately
210 000 human lives were lost to TB.1 The global health impact of M. tuberculosis is
exacerbated by HIV coꢀinfection, making TB one of the major AIDSꢀrelated opportunistic
diseases.2 This shocking failure to control TB is due to the combination of many factors, among
which: the nonꢀcompliance of the patients to the required six months of multidrug therapy; the
difficulty to provide it in a sustained and properlyꢀdosed regimen, particularly in those parts of
the world that are the least equipped to deal with the disease.3 These issues have altogether led to
the emergence of multidrug resistant (MDR) TB and extensively drugꢀresistant (XDR) TB. As
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such, to address this global health crisis, the process of drug discovery must rely both on novel
chemical antiꢀTB entities4,5 and on an innovative approach that rationally prevents the raise of
drug resistance.
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After many years without delivering any new antiꢀTB drug, the discovery of bedaquiline for
the treatment of MDRꢀTB, and a nourished pipeline of compounds already in clinical trials
(http://www.newtbdrugs.org/pipeline.php) have renewed hope for the treatment of TB and
especially MDRꢀTB. However, the lack of innovation in the overall therapeutic approach makes
quite predictable that mutationꢀbased resistant strains will inexorably emerge also for these novel
drugs. The discovery of bedaquiline6 and pretomanid7 has showed that the whole cell phenotypic
assay of large libraries of compounds, and the wise medicinal chemistry efforts toward the
improvement of these molecules, are reliable methodological approaches to drive the research
towards novel antiꢀTB agents.8 Indeed, most of the efforts of some of the authors of this work
has moved through this path.9–19 While this approach has proved to be correct, it is nevertheless
incomplete. It does not consider that, besides mutations, other mechanisms such as efflux play an
important role in the development of resistance in M. tuberculosis.20–22
Efflux pumps are involved in several physiological processes such as detoxification of
intracellular metabolites and cellular homeostasis.23 Simultaneously, they contribute to the
development of intrinsic and acquired drug resistance in many bacterial pathogens. Usually they
confer lowꢀtoꢀintermediate level of resistance, however, the constant pressure of subinhibitory
concentrations of the antibiotic promotes the selection of spontaneous mutants.20 Therefore: a)
efflux pumps are effectors of the innate drugꢀresistance machinery; b) they are crucial in
conferring a lowꢀlevel of drug resistance; c) they contribute to lower the concentration of the
drug inside the mycobacterial cell, enabling the emergence of a highꢀlevel of resistance; d) it has
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been demonstrated that efflux inhibitors (EIs) are able to improve or restore the activity of old
drugs towards which the bacteria had become resistant.24
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In spite of this knowledge, the extent to which an antimicrobial compound is a substrate of
efflux is seldom measured, and it is not considered an important parameter in the hitꢀtoꢀlead
process for the optimization of novel antiꢀTB drugs. The recent discovery by Lee and colleagues
of spectinamides as antiꢀTB drug candidates,25 also by virtue of their scarce tendency to be
extruded by efflux pumps, confirms the importance of taking into account efflux when the design
of new drugs is planned.26 In addition to these findings, although the mechanism of adaptation of
M. tuberculosis to the hostile environment is still a matter of debate, Adams and colleagues have
recently reported that bacterial efflux pumps mediate multiꢀdrug tolerance and the bacterial
survival in macrophages, leading to a longꢀlasting therapy.27,28
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It is therefore possible to claim that inhibition of efflux would lead to a number of beneficial
effects: a) increasing the activity of the antiꢀTB drugs subject to efflux; b) keeping the
concentration of a given drug at the therapeutic dose, minimizing the possibility to select
mutants; c) drastically shorten the duration of treatment by reducing multiꢀdrug tolerance. This
could be priceless in order to repurpose old drugs and preserve the efficacy of those novel
compounds, such as bedaquiline, that are either next to be introduced in the antiꢀTB arena or that
already are.
Thioridazine (TZ) is a neuroleptic with efflux inhibitory properties,29 that has attracted the
attention of many researchers due to its ability to hit drug resistant strains.30 In a clinical trial in
Argentina, TZ was used for the treatment of patients with XDRꢀTB in combination with antiꢀTB
drugs, with good results.31 It has been demonstrated that TZ has a dual effect against M.
tuberculosis: it is an inhibitor of bacterial efflux pumps, and, also, it induces the acidification of
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the phagosome, contributing to restriction of intracellular M. tuberculosis growth.30,32–34 TZ is
not the only inhibitor of efflux investigated for the treatment of TB. Recently chlorpromazine,
another neuroleptic with efflux inhibitory properties, was tested along with its metabolites
against M. smegmatis and exhibited synergistic activity when administered in combination with
known antiꢀTB drugs.35,36 Also the antiarrhythmic verapamil is an efflux pump inhibitor that has
been shown to have an antimicrobialꢀpotentiating effect in the treatment of M. tuberculosis both
in vitro and in vivo.37 It was shown that inhibition of the efflux pumps of M. tuberculosis by
verapamil reduces the bacterial drug tolerance induced in the intracellular compartment inside
the macrophages and in zebrafish granulomaꢀlike lesions. Unfortunately, the use of TZ in the
therapy of TB is severely limited by its neuroleptic properties and a number of severe toxicity
issues. Inspired by these considerations, we herein report the design and synthesis of a number of
TZ derivatives, and their biological evaluation as efflux inhibitors and synergistic effect with
known antiꢀTB drugs both in vitro and on infected human monocyteꢀderived macrophages.
Among the novel compounds, when compared to the parent molecule TZ, compound 10 showed
higher efflux inhibitory ability, less toxicity, and a remarkable synergistic effect with firstꢀ and
secondꢀline drugs, especially rifampicin. As such, it may represent an interesting starting point
for the development of more potent and less toxic efflux inhibitors to be used in combination
with the current firstꢀ and secondꢀline antiꢀTB drugs.
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Rational design
Since the lack of detailed information about the structure of the efflux systems, the new
compounds were prepared following a LigandꢀBased Drug Design approach. TZ was used as
template, given its wellꢀknown and deeply investigated antiꢀTB properties when administered in
combination with firstꢀline drugs, as mentioned above.29,32–34,38,39 The aim of this work was
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therefore to chemically modify TZ in order to obtain a compound with improved adjuvant
properties while limiting the onset of side effects, especially at the CNS level. We reasoned that,
in first place, it was necessary to escape from the phenothiazine core because, according to the
SAR of antipsychotic drugs,40 it is crucial in conferring the desired neuroleptic activity.
Moreover, although with a few exceptions,41 a series of TZ analogues in which the
phenothiazine core was kept intact has already been reported as inhibitors of efflux in other
bacteria, but with scarce results.38,42 In addition to this, efflux systems are known to be affine to
charged quaternary ammonium salts such as ethidium bromide (EtBr) and berberine, and to
molecules bearing basic amino groups (TZ, VP, reserpine), that likely occur to be protonated at
the acidic pH of the sites infected by M. tuberculosis. Given these premises, chemical
manipulation was carried out keeping intact the Nꢀmethylpiperidine moiety, whereas modifying
the nature of the heterocyclic attached to it and the length of the spacer.
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Several heterocycles, embodying a variable number of rings, were attached by the nitrogen
atom to an Nꢀmethylꢀ2ꢀpiperidinyl appendage through an ethylene linker (compounds 9ꢀ15, chart
1).
[Insert chart 1 here]
The carbazole (9) and the acridone (12), that are tricyclic flat structures, were chosen for their
close resemblance with the phenothiazine core. The indole (10), and the benzimidazole (11) are
among the most privileged scaffolds used in medicinal chemistry, and could afford extended
chemical manipulation of the series. The diphenylamines 13 and 14 were synthesized to check
whether the planar structure of the phenothiazine could be disrupted without loss of the
inhibitory activity. The pyrrolidine 15 was prepared to check whether a small ring could still be a
suitable substrate of efflux. Once the preliminary assays had corroborated the design of
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compound 9, other appendages were attached to the carbazole core in place of the Nꢀmethylꢀ2ꢀ
piperidinyl one. To start, a less polar and flatter substrate such as the 2ꢀethylpyridine (18) was
attached to the carbazole nitrogen; then, the distance between the nitrogen of carbazole and that
of the appendage was shortened by one methylene unit in compound 19.
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The synthesis of compounds 5 and 6 was accomplished in the attempt to couple a moiety that
is thought to be responsible for the EI activity to a potent antiꢀTB agent;11,16 this would have led
to the synthesis of the first rationally designed TBꢀGEI (Growth and Efflux Inhibitor), which
could represent a superior therapeutic option for the treatment of TB. Indeed, the recent finding
that bedaquiline, along with its antibacterial activity, is also a weak inhibitor of efflux
mechanisms, accounts for this reasoning.43 Finally, since the recent interest in the use of VP and
its derivatives in the treatment of TB,44,45 we have tried to merge the two structures of TZ and
VP in one hybrid molecule (Figure 1), and therefore compounds 7 and 8 (chart 1) were
synthesized and tested.
[Insert Figure 1 here]
Chemistry
The synthesis of derivatives 5 ̶ 15 was achieved through reaction of the suitable heterocyclic
precursor and 2ꢀ(2ꢀchloroethyl)ꢀNꢀmethylpiperidine hydrochloride, in dry DMF using NaH in
excess, either at room temperature or heating for several hours, when necessary (Scheme 1).
When not commercially available, the synthesis of the heterocyclic precursors was achieved in
good yields according to a wellꢀestablished procedure, as in the case of intermediates 1 and
2.13,16 Compound 346 was prepared reacting 3,4ꢀdimethoxyaniline with triethylamine and 2ꢀ
iodopropane in refluxing methanol, differently from what already reported. For the synthesis of
compound 18, first 2ꢀ(hydroxymethyl)ꢀpyridine was treated with pꢀtoluensulfonyl chloride and
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triethylamine in dichloromethane to give the tosyl derivative 16, and then the ameliorated
leaving group is displaced by carbazole according to the conditions reported by Cheng (Scheme
2).47 In the case of compound 19, carbazole was first treated with dichloroethane to give Nꢀ
(chloroethyl)carbazole 17, that was reacted with piperidine in DMF at room temperature using
K2CO3 as the base to give 19 (Scheme 2).48 For the synthesis of compound 8, 3,4ꢀ
dimethoxyphenyl acetonitrile was first treated with sodium hydride and 2ꢀbromopropane to give
intermediate 4;49 several attempts to couple this derivative with 2ꢀ(2ꢀchloroethyl)ꢀNꢀ
methylpiperidine hydrochloride with the use of various bases, performed according to different
literature protocols,49–51 failed to yield the desired product. The synthesis was finally
accomplished by heating the two reagents in DMF in a microwave oven, using an excess of
sodium amide as the base. Interestingly, the same synthetic conditions did not give any product
with conventional heating also after many days at 120 °C. Compound 8 was tested preliminarily
as a racemic mixture.
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[Insert Scheme 1 here]
[Insert Scheme 2 here]
Results and Discussion.
A total of 13 compounds were synthesized in this study (5ꢀ15, 18, 19, chart 1), that underwent
biological assays. In order to set up a faster and cheaper model for screening the activity of the
compounds, they were first evaluated for their antimycobacterial and efflux inhibitory activity
against M. smegmatis mc2155 reference strain. For those compounds showing inhibitory activity
higher than TZ, cytotoxicity on humanꢀmonocyte derived macrophages was assessed.
Compounds showing less cytotoxicity and higher efflux inhibitory activity than that of TZ, were
further evaluated on M. tuberculosis H37rv, as described herein.
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Antimycobacterial and efflux inhibitory activity against M. smegmatis mc2155
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The compounds were first tested for their capability to inhibit the growth of M. smegmatis
strain mc2155 (Table 1). The compounds demonstrated extremely poor or absent
antimycobacterial activity against M. smegmatis, especially if compared with TZ, which was
used as positive control. This was considered a good result, as high bactericidal/bacteriostatic
activity could bias the following experiments of synergy with firstꢀ and secondꢀline antiꢀTB
agents. Due to the reduced solubility and the low antimycobacterial activity, it was not possible
to determine the exact MIC values for some compounds (i.e. 5ꢀ8, 11, 13, 15, 18, 19). As such,
when bacterial growth is still noticed at the maximum concentration tested, the MIC was
indicated by “>MIC”. For instance, >256 µg/mL means that the MIC of this compound is higher
than 256 µg/mL, as it was technically impossible to determine it. After determining their MICs,
the compounds were tested for their capability to inhibit efflux of EtBr from M. smegmatis
mc2155 cells by realꢀtime fluorometry, using TZ and VP as controls (Table 1, relative final
fluorescence (RFF) values). According to a widely used protocol for this kind of assay,52 in order
not to compromise the cellular viability, all of the compounds were tested at ½ of their MIC. For
those compounds for which the exact MIC could not be determined (above reported), the last
concentration value that could be technically determined was considered ½ of the MIC. Despite
the small set of compounds prepared for this preliminary study, a few SAR considerations can be
made. In general, the majority of the compounds bearing the Nꢀmethylꢀ2ꢀpiperidinyl appendage
had inhibitory effect on the efflux of EtBr. We were pleased to notice that compound 7, designed
as a hybrid molecule resembling both VP and TZ, had a very good inhibitory activity of EtBr
efflux. However, the same does not apply to the other hybrid compound 8. It might be then
speculated that the Nꢀmethylꢀ2ꢀpiperidinyl appendage must be attached, through an ethylene
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linker, to another nitrogen atom of the heterocyclic, yielding a more polar structure. When the Nꢀ
methylꢀ2ꢀpiperidinyl appendage is attached through an ethylene linker either to tricyclic
(compounds 9, 12), bicyclic (compounds 10, 11) or more flexible ring structures (compounds 13,
14), a good inhibitory activity, sometimes higher than that of the parent compound TZ, is
noticed. However, bulkier heterocyclic (compounds 5 and 6) or small rings (compound 15), have
a detrimental impact on the efflux inhibitory properties. Surprisingly, the benzimidazole
derivative 11 showed an exceptionally high activity, resulting by far more potent than VP. The
distance between the nitrogen of the appendage and that of the heterocyclic seems to have
pharmacological significance, since the activity of compound 19 is considerably low. Finally, the
presence of a tertiary aliphatic amino moiety seems to be determinant, being compound 18 the
least active of the series.
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Cytotoxicity evaluation of compounds 7, 9, 10, 11 and 12
Compounds 7, 9, 10, 11 and 12, that resulted more active than TZ in inhibiting the efflux of
EtBr out of M. smegmatis cells, were evaluated against humanꢀmonocyte derived macrophages
to assess their ex vivo cytotoxicity toward eukaryotic cells (Table 1). We were pleased to notice
that, with the exception of compound 12 (IC50 = 0.762 µg/mL) all of the tested compounds were
found to be less cytotoxic than TZ in this assay. Compounds 9 and 10, (IC50 = 8.8 ꢁg/mL and
10.5 ꢁg/mL, respectively) are twoꢀfold less cytotoxic than TZ (IC50 = 5.6 ꢁg/mL), whereas
compounds 7 and 11 (IC50 = 84.8 ꢁg/mL and 170.8 ꢁg/mL) were found to be safe towards human
macrophages. Overall, these preliminary data clearly suggest that the replacement of the
phenothiazine core with other ring systems allows maintaining the efflux inhibitory activity with
the possibility to decrease the cytotoxicity.
[Insert Table 1 here]
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[Insert Figure 2 here]
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Compounds 7, 9, 10 and 11, that resulted more active than TZ in inhibiting the efflux of EtBr
from M. smegmatis, and less cytotoxic against humanꢀmonocyte derived macrophages, were
selected for additional biological studies against M. tuberculosis H37Rv strain. Compound 12,
although presenting higher RFF values than that of TZ, was excluded based on its higher
cytotoxicity.
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Evaluation of compounds 7, 9, 10 and 11 antimycobacterial and efflux inhibitory activity
against M. tuberculosis H37Rv
Compounds 7, 9, 10 and 11 were tested against the panꢀsusceptible reference strain H37Rv,
following the same rationale used for M. smegmatis, i.e. the determination of antimycobacterial
activity and the evaluation of their effect on EtBr efflux (Table 2). As observed for M.
smegmatis, the selected derivatives showed no antimycobacterial activity against M.
tuberculosis, therefore excluding the possibility of bias during the following assays. With regard
to their activity as inhibitors of EtBr efflux, the compounds were tested with the same procedure
used as in the case of M. smegmatis. Compound 9 showed similar activity to that of TZ, whereas
compounds 7, 10 and 11 were found to be 2ꢀ4 fold more potent than TZ, although less active
than VP (Table 2, Figure 2). Although quite active, the benzimidazole derivative 11 failed to
reproduce the extraordinary efflux inhibitory effect observed against M. smegmatis. This is not
surprising and can be somehow explained on the basis of the faster metabolism of M. smegmatis
compared to that of M. tuberculosis, that accounts for the higher susceptibility to the efflux
inhibitory effect. Our own experience shows that RFF values are always scaled down in M.
tuberculosis in comparison to those observed for the same compounds in M. smegmatis, although
maintaining the inhibitory relation.53,54 Regardless, we can claim that, in the conditions reported,
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all of the tested compounds have maintained their superior capability of inhibiting efflux
compare to TZ against M. tuberculosis.
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[Insert Table 2 here]
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Evaluation of synergistic effect of the selected TZ analogues in combination with first-
and second-line anti-TB drugs and determination of the fractional inhibitory concentration
(FIC).
Once that the inhibition of efflux was confirmed, the study went ahead evaluating compounds
7, 9, 10 and 11 for their effect in combination with the current firstꢀline (isoniazid, INH;
rifampicin, RIF) and secondꢀline (amikacin, AMK; ofloxacin, OFX) drugs used for the treatment
of TB. Their effect was also tested in combination with EtBr, to correlate its efflux with the
efflux of the antiꢀTB drugs.55 The MICs of INH, RIF, AMK and OFX were calculated in the
absence and in the presence of scalar subꢀinhibitory concentrations of each compound against M.
tuberculosis H37Rv, that is known to have intrinsic and readable efflux activity of these antiꢀTB
drugs22,54 (Table 3).
[Insert Table 3 here]
Initially, compounds 7, 9, 10 and 11 were tested at ½ of their MIC (see Table 3 for details),
and then, according to the protocol widely used for this sort of investigations,52 they were tested
by twoꢀdimensional broth microdilution checkerboard assay, to evaluate the extent of the
synergism. Also in this case, as already explained in the case of the efflux inhibition assays, for
those compounds for which the exact MIC could not be determined, ½ of the MIC was
considered the concentration that was equal to the highest concentration where it was obtained
growth. When tested at these concentrations, all of the newly synthesized compounds and the
controls demonstrated very high synergistic effect with both the antibiotics and EtBr. In this
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case, the MIC values of INH, RIF, AMK, OFX and EtBr could be reduced more than 64ꢀfold
(Table 3). Compound 10, for which the inhibitory effect on efflux was already assessed, had in
general a very high synergistic effect with the tested drugs and, in particular, with RIF. In fact,
compound 10 was able to improve the activity of RIF by 4ꢀfold at concentrations as low as 8
µg/mL (approx. 1/64 of its MIC), demonstrating a potent synergistic effect. Moreover, at a
concentration of 64 µg/mL (approx. 1/8 of its MIC), compound 10 was also able to improve the
activity of AMK and OFX by more than 128ꢀfold, and EtBr by 8ꢀfold (Table 3). Importantly, we
have noticed that the synergistic activity demonstrated by the compounds in combination with
EtBr correlates well with the results obtained with the inhibition of EtBr efflux by RFF.
One may argue that TZ, considering the absolute concentrations at which the synergistic
activity is measured, is slightly more active than compound 10. However, seen under a more
widespread perspective, compound 10 is less toxic, more active with regard the inhibition of
efflux, and unlikely to possess CNSꢀrelated properties. This can be considered a good
achievement, in particular considering that the aim of this paper was the selection of compounds
devoid of antimycobacterial activity, with reduced toxicity to the human macrophage and with
higher efflux inhibitory capability, so as to promote the antimycobacterial activity of the antiꢀTB
drugs and be considered as a future adjuvant in the fighting against TB.
10
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Surprisingly, despite the high activity in inhibiting the efflux, compound 11 was found to be
not active in the combination assay. Compounds 7 and 9 were found to be slightly less active
than 10 in the combination assay; however, in the case of compound 7, it must be noticed that it
is able to improve the activity of RIF by 4ꢀfold also at very low concentrations. It is intriguing to
notice that compounds 7 and 9, as compound 10, demonstrated to have a significant synergistic
effect in particular with RIF. The reason of this preferred synergism is worth of further
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investigation but, at this moment, it can only be speculated that 10, as well as the other
derivatives belonging to this series, although to a lesser extent, have high affinity for interfering
with efflux systems that extrude RIF. However, it cannot be ruled out that the two derivatives
interfere with other mechanism of resistance used by the cell in the presence of RIF. Finally, it is
worth of consideration the fact that compounds 7, 9 and 10 were more active in the combination
assay that VP, another drug often investigated for it potential role as adjuvant in the TB
treatment.
10
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The synergistic activity between the compounds was also represented as the fractional
inhibitory concentration (FIC), that is a value expressing the synergistic activity of the
compounds in combination with the antibiotics and EtBr (Figure 3). FIC was determined for
each antiꢀTB drug by dividing the MIC of each drug when used in combination, by the MIC of
each drug used alone. Since a drug cannot interact with itself, the effect of a selfꢀdrug
combination will always be additive, with an FIC index of 1. As such, when the FIC value is
lower than 0.25, a synergistic effect is noticed between the modulator and the antimicrobial.
When above 2, there is antagonism between the compounds tested, whereas within these two FIC
values, indifference is noticed.24
[Insert figure 3 here]
Isobolograms were constructed, by plotting changes in the MIC of antibiotics as a function of
the tested compounds concentration (Figure 3 and S1 – example for compound 10). As it can be
seen, for the majority of the combinations of compound 10 with the antibiotics synergy was
observed, with FIC values ranging from 1 to not possible to determine (ND) due to the very high
synergistic effect, depending on the antibiotic combination (see legend of Figure 3 supporting
information for details).
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Evaluation of the intracellular anti-TB activity of the selected TZ analogues.
Finally, we evaluated the intracellular antimycobacterial activity of compounds 7, 9, 10 and 11
alone and in combination with INH or RIF against M. tuberculosisꢀinfected human monocyteꢀ
derived macrophages (Figure 4). This assay is a fair reproduction of what actual happens during
the TB infection, and therefore it can help in obtaining a clearer idea of the potential of our
compounds. INH and RIF were used at ½ of its MIC (0.05 and 0.5 µg/mL, respectively),
therefore having no direct inhibitory effect on the bacteria at these concentrations. The
compounds 7, 9, 10 and 11 were tested at nontoxic concentrations (16 ꢁg/mL, 0.078 ꢁg/mL; 1.25
ꢁg/mL, and 40 ꢁg/mL, respectively) determined against humanꢀmonocyte derived macrophages
(Figure S2). Above these concentrations, macrophage viability is reduced below 90%, nullifying
the assay. At these concentrations, the compounds tested showed to be devoid of any
antimycobacterial activity against intracellular M. tuberculosis. Consistent with the findings
above reported, we were pleased to notice that the coꢀadministration of the compounds with INH
or RIF (at a nonꢀantibiotic inhibitory concentration) led to a strong enhancement of the killing
activity of the human macrophages against the H37Rv strain. This is comparable with the results
previously obtained with TZ and other efflux inhibitors that has been shown to have a dual
bacterialꢀhost boosting effect against M. tuberculosis.56
10
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Conclusion
The contribution of efflux systems to M. tuberculosis drug resistance and tolerance has been
increasingly recognized, although only a few medicinal chemistry efforts have been made
towards the synthesis of M. tuberculosis efflux inhibitors. Despite the amount of literature
establishing TZ as a valuable agent against TB, the numerous toxicity issues have always
hampered its safe administration as an adjuvant of TB therapy. To our knowledge, this is the first
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medicinal chemistry campaign aimed at modifying TZ with regard to its potential as adjuvant of
TB therapy, with specific assays such as the determination of the efflux inhibition and the
synergistic activity in combination with known antiꢀTB drugs both in vitro and ex vivo.
Compared to TZ, the representative derivatives tested were found to be less toxic toward human
macrophages, ranging from twoꢀfold (compound 9 and 10) to more than 50ꢀfold (compound 11).
Compound 7, intended as a hybrid of TZ and VP, showed a higher inhibitory activity than that of
TZ itself and much less toxicity, therefore confirming the rationale of the design. More
importantly, we have escaped from the phenothiazine scaffold, thus assuming a reduction of the
onset of side effects at the CNS level. The newly synthesized compounds showed high
synergistic activity when tested in combination with some of the most important firstꢀline (INH,
RIF) and secondꢀline (AMK, OFX) drugs used for the treatment of TB. In particular compound
10 was able to improve the activity of RIF at concentration as low as approximately 1/64 of its
MIC. Additionally, these compounds were able to improve the activity of INH and RIF against
intracellular M. tuberculosis. Considering the data on inhibition of EtBr efflux, coupled to the
low antimycobacterial activity, it might be claimed that the synergetic activity is due to their
ability to hamper the intrinsic mycobacterial drug efflux.
10
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Finally, we confirmed M. smegmatis mc2155 as a suitable surrogate of M. tuberculosis for the
preliminary assessment the efflux inhibitory activity, at a lower cost and time consuming rate.
Altogether, these findings provide a solid base to further investigate compound 10 as a booster of
the antimycobacterial chemotherapy when associated with firstꢀ and secondꢀline drugs, by virtue
of its capability to block the intrinsic efflux activity of mycobacteria. This might represent a
lateral approach toward the cure of TB. Studies expanding this versatile series and its mechanism
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of action and activity toward M. tuberculosis drug resistant strains, especially resistant to
5
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isoniazid and rifampicin (MDRꢀTB), are currently underway in our research groups.
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EXPERIMENTAL PART
Chemistry
7
8
9
General information. All the reagents were purchased from SigmaꢀAldrich, AlfaꢀAesar and
Enamine 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. MCRs were performed using CEM Microwave
SynthesizerꢀDiscover model. 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. When indicated, intermediates and final products were purified
through silica gel flash chromatography (silica gel, 0.040ꢀ0.063 mm), using appropriate solvent
mixtures.
10
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1HꢀNMR and 13CꢀNMR spectra were recorded on a BRUKER AVANCE spectrometer at 300,
1
400 and 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% purity samples or higher (by HPLC/MS).
The synthesis and chemical characterizations of intermediates 1ꢀ4, 16, 17 is reported in the
Supporting Information.
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General procedure for the synthesis of compounds 5, 6, 9-15, 18. To a suspension of
sodium hydride (60% suspension in mineral oil, 3 eq) in DMF (5 mL/mmol) was added the
suitable heterocyclic compound (1 eq) at 0 °C. Reaction mixture was stirred for 15 minutes at the
same temperature and then 2ꢀ(2ꢀchloroethyl)ꢀpiperidine hydrochloride (1.3 eq) was added
portion wise. The mixture was allowed to react at room temperature until consumption of the
starting material. In some cases (5, 6, 13) reflux heating was necessary. After quenching with
water, the mixture was extracted with ethyl acetate (3 × 10 mL), and the organic layers were
washed with water and brine, dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude material was purified through flash chromatography. Reaction times and
conditions, yields, purification methods and other analytical data are reported in the supporting
information.
10
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N-isopropyl-3,4-dimethoxy-N-(2-(1-methylpiperidin-2-yl)ethyl)aniline (7). To a solution of
3 (60 mg; 0.38 mmol) in dry toluene (2 mL) was added sodium amide (120 mg; 3.07 mmol) and
2ꢀ(2ꢀchloroethyl)piperidine hydrochloride (244 mg; 1.23 mmol). The reaction mixture was
stirred and heated in a microwave oven at 140 °C for 30 minutes. After quenching with brine (25
mL), the mixture was extracted with ethyl acetate (3 × 10 mL), and the combined organic layers
were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude material
was then purified through flash chromatography eluting with dichloromethane/methanol 9:1.
1
Yield: 16%. HꢀNMR (400 MHzꢀCDCl3): 1.10ꢀ1.15 (m, 6 H); 1.20ꢀ1.87 (m; 8H), 2.30ꢀ2.35
(bs; 5H); 2.95ꢀ3.10 (m; 2H), 3.15ꢀ3.25 (m, 1H); 3.65ꢀ3.75 (m, 1H); 3.84 (s, 3H); 3.87, (s, 3H);
6.41 (d, J = 9 Hz, 1H); 6.49 (s, 1H); 6.79 (d, J = 9 Hz, 1H). 13CꢀNMR (100.6 MHzꢀCDCl3):
19.4, 20.2, 23.3, 28.3, 41.3, 53.8, 55.9, 56.3, 105.6, 111.9, 142.6, 143.7, 149.5. HRMS (ESI)
calculated for C19H32N2O2 [M + H]+ 321,2464, found 321.25365.
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2-(3,4-Dimethoxyphenyl)-2-isopropyl-4-(1-methylpiperidin-2-yl)butanenitrile (8). To a
solution of 4 (70 mg; 0.31 mmol) in dry toluene (2 mL) was added sodium amide (124 mg; 3.19
mmol) and 2ꢀ(2ꢀchloroethyl)piperidine hydrochloride (252 mg; 1.25 mmol). The reaction
mixture was stirred and heated in a microwave oven at 140 °C for 30 minutes. After quenching
with brine (25 mL), the mixture was extracted with ethyl acetate (3 × 10 mL), and the combined
organic layers were dried over anhydrous Na2SO4. The crude material was then purified through
flash column chromatography eluting with dichloromethane/methanol 9:1. Yield: 50%. 1HꢀNMR
(300 MHzꢀCDCl3): 0.8 (dd, J1 = 4 Hz, J2 = 7 Hz, 3 H); 1.23ꢀ1.80 (m; 11H), 1,85ꢀ2.40 (m; 8H);
2.80ꢀ2.95 (m; 1H), 3.88 (s, 3H); 3.90 (s, 3H); 6.80ꢀ7.00 (m, 3H). 13CꢀNMR (100.6 MHzꢀCDCl3):
18.7, 19.0, 24.0, 28.2, 29.9, 32.1, 32.5, 37.9, 53.4, 55.9, 56.0, 63.0, 109.1, 109.4, 111.0, 118.8,
121.1, 130.5, 148.3, 149.0. HRMS (ESI) calculated for C21H32N2O2 [M + H]+ 345,2464, found
345.25365.
10
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9-(2-(Piperidin-1-yl)ethyl)-9H-carbazole (19). To a solution of piperidine (33 µl; 0.33 mmol)
in DMF (15 mL/mmol) was added potassium carbonate (138 mg, 1 mmol), this mixture was
stirred at room temperature for 20 minutes and then the intermediate 17 (10 mg, 0.43 mmol) was
added. Reaction mixture was stirred to 60 °C overnight. After cooling, brine (25 mL) was added
and the mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic layers were
washed with water and brine, dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude material was purified through flash chromatography eluting with petroleum
ether/ethyl acetate 95/5, yielding the title compound as a white solid. Yield: 19%. 1HꢀNMR (400
MHzꢀCDCl3): 1.24ꢀ1.60 (m, 6H); 3.07 (bs, 2H); 3.39 (bs, 2H); 4.49 (t, J = 8 Hz, 2H); 4.62 (t, J
= 8 Hz, 2H); 7.23ꢀ7.28 (m, 2H); 7.46ꢀ7.79 (m, 4H), 8.12 (d, J = 8 Hz, 2H). 13CꢀNMR (100.6
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MHzꢀCDCl3):
24.2, 25.4, 42.1, 44.7, 62.8, 108.7, 119.1, 120.3, 123.0, 125.7, 140.5, 155.0.
5
6
7
8
HRMS (ESI) calculated for C19H22N2 [M + H]+ 279,1783, found 279.39258
Biology
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Evaluation of the novel synthesized compounds against mycobacteria
Reagents and mycobacterial strains
Thioridazine (TZ), verapamil (VP), isoniazid (INH), rifampicin (RIF), amikacin (AMK),
ofloxacin (OFX), EtBr, phosphateꢀbuffered saline (PBS), and glucose were purchased from
SigmaꢀAldrich (St. Louis, MO, USA). All solutions were prepared in deionized water, except
rifampicin and the TZ analogues 5-15, 18 and 19, which were prepared in DMSO. All solutions
were prepared on the day of the experiment. Middlebrook 7H9 (MB7H9) and the OADC
supplement (oleic acid/albumin/dextrose/catalase) were purchased from Difco (Madrid, Spain)
and Becton and Dickinson (Sparks, MD, USA) respectively. The mycobacterial reference strains
M. smegmatis mc2155 ATCC700084 and M. tuberculosis H37Rv ATCC27294T were used to
evaluate the biologic activity of TZ analogues.
The determination of the minimum inhibitory concentration (MIC), the evaluation of the
synergistic effect of TZ analogues 7, 9, 10 and 11 with selected antibiotics and EtBr by broth
microdilution checkerboard assay, the evaluation of efflux inhibitory activity of the compounds
by realꢀtime fluorometry and the cytotoxicity evaluation are reported in detail in the Supporting
Information.
References
(1) WHO
|
Global
tuberculosis
report
2014
http://www.who.int/tb/publications/global_report/en/ (accessed Feb 5, 2015).
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(2) CDC
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Resistant Mycobacterium Tuberculosis. Nat. Commun. 2013, 4, 2907.
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