Phenothiazine: A Better Scaffold against Tuberculosis

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several types of mycobacteria including, hypoxic, nutrient starved, replicating ..... should be within a spell of few weeks, like other microbial infections. However ...

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REVIEW ARTICLE

Phenothiazine: A Better Scaffold against Tuberculosis Satheeshkumar Sellamuthu, Mohammad Faizan Bhat, Ashok Kumar and Sushil Kumar Singh* Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (BHU), Varanasi-221005, India

ARTICLE HISTORY Received: February 00, 2016 Revised: June 00, 2016 Accepted: September 00, 2016 DOI: 10.2174/1389557517666170220152651

Abstract: The incidence of Tuberculosis (TB) is baffling in developing countries due to the increase in multidrug-resistant and extensively drug-resistant TB. Therefore, drugs acting through different mechanisms are in dire need to counter the resistant strains. Various chemical scaffolds are being investigated against tuberculosis, among them the molecules containing phenothiazine nucleus are found to be more effective against both susceptible and resistant strains of M. tuberculosis. In addition, the efficacy of first-line drugs has been found to be enhanced on supplementary treatment with phenothiazines. The present review provides an overview of the phenothiazine based molecules which were investigated during the last ten years for their anti-tubercular activity.

Keywords: Phenothiazine, psychotropic, repurposing, resistance, SAR, tuberculosis. 1. INTRODUCTION Tuberculosis (TB) has been a curse for the human race. The magnitude of morbidity and mortality of the disease is not matched with any other disease in the world. Among all the communicable diseases, it is the most miserable [1, 2]. In 2014, an estimated 9.6 million new TB cases and 1.5 million deaths from the disease were reported. Out of 1.5 million deaths, 0.4 million were immune compromised HIV-positive patients [3]. The transmission of pulmonary TB to Bacille Calmette Guerin (BCG) vaccinated patients is a threat to mankind in absence of any new TB vaccine [4, 5]. Although, 15 vaccine candidates entered the clinical trials but none of them reached the market [6]. The control of TB is hampered due to the increased incidence of multidrug resistant and extensively drug-resistant TB (MDR/XDR-TB) and thus leading to life-threatening situations. Although, clinical management of TB is becoming a challenging task, an estimated 37 million lives were saved between 2000 and 2013 by effective diagnosis and treatment [7, 8]. But, the death toll is still significantly high. Hence, accelerated efforts are needed to eradicate this deadly disease from the roots. Globally, 5% of new and 20 % of previously treated TB cases were estimated to have MDR-TB in 2014. This has translated to an estimated 0.48 million people having MDRTB. An estimated 111 000 people started treatment for MDR-TB in 2014, which is an increase of 14% from the *Address correspondence to this author at the Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (BHU), Varanasi-221005, India; Tel: +91-542-6702736; Fax: +91-542-368428; E-mail: [email protected] 1389-5575/17 $58.00+.00

previous year. Further, 9.0% of MDR-TB victims have been converted into extensively drug-resistant TB (XDR-TB) patients [9]. One of the major concerns in the current treatment regimens of MDR-TB is the non-availability of new drugs to replace the existing ones which have already developed resistance. Phenothiazine based compounds were reported for potent anti-TB activity [10]. They were also found to show significant activity against MDR-TB. This review is focused on phenothiazine derivatives having antitubercular activity and deals with their structure activity relationship (SAR). Many mechanisms of action have been proposed for this class of compounds including the inhibitory action on Type-2 NADH dehydrogenase enzyme (NDH-2) [7]. 2. WHY NEW ANTITUBERCULAR DRUGS? The four most effective first-line oral drugs i.e. rifampicin, isoniazid, pyrazinamide, and ethambutol are required to be taken together for the first two months. Subsequently, the treatment should be continued with rifampicin and isoniazid for another four months. This results in patient noncompliance and ultimately leading to chronic cases of drug-resistant tuberculosis and increased mortality [11]. Therefore, potent and novel drugs are required to shorten the duration of treatment to improve patient compliance and also to reduce programme supervision and distribution costs. Mycobacterium tuberculosis (Mtb) is able to develop a dormancy phenotype under anaerobic and nutrient deprived conditions [11]. This phenotype of mycobacteria causes latent tuberculosis which in turn may become active disease anytime during their © 2017 Bentham Science Publishers

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course of life. New therapeutics are required to improve the life expectancy of the patients suffering from dormant/latent tuberculosis, before it is converted into active disease [12]. In addition to latent TB, the treatment of MDR and XDR-TB is also very challenging. In case of MDR-TB, mycobacteria developed resistance to first line drugs isoniazid and rifampicin whereas in XDR-TB, it developed resistance to any of the flouroquinolones and at least one of the three injectable second-line drugs i.e. amikacin, kanamycin, or capreomycin along with rifampicin and isoniazid (Fig. 1). The second-line or the third-line anti-tubercular drugs used in the treatment of MDR-TB are associated with high toxicity, high cost and are less effective[13]. In addition, the efficacy of antitubercular drugs is diminished mainly in immune compromised patients with increased side effects. So, there is huge need for a kind of drug which is compatible with antiretroviral and immuno suppressive drugs [14, 15]. The drugs present in the current treatment are inadequate to address the problems associated with clinical presentation. Therefore, safe and potent antitubercular drugs are in definite O

3. WHY PHENOTHIAZINE SCAFFOLD AGAINST TUBERCULOSIS? By the end of the 19th century, Paul Ehrlich had proclaimed that methylene blue, a phenothiazine dye effectively rendered the bacteria immobile [16, 17]. But the antimicrobial potency of phenothiazines was overshadowed by their anti-psychotic effect, even though there was huge demand for antimicrobials rather than anti-psychotic drugs during those days [18, 19]. Thus, phenothiazine derivatives eventually found their place in the management of psychosis. Chlorpromazine (CPZ), the first phenothiazine, used for the treatment of psychosis, displayed broad spectrum activity against bacteria, mycobacteria, viruses, and protozoa [20]. Due to this, there is a renewed interest to develop phenothiazines as antimicrobial agents. In addition, thioridazine, the mildest antipsychotic phenothiazine, was also found to be active against all the S

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necessity to reduce the treatment duration and for effective control of latent, MDR and XDR-TB.

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Clarithromycin

Fig. (1). Drugs used in the management of tuberculosis.

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Phenothiazine: A Better Scaffold against Tuberculosis

treated strains of Mtb irrespective of whether they were drug-susceptible or drug-resistant [11, 21, 22]. These factors emphasise the need to further investigate the phenothiazine scaffold for the development of antitubercular drugs. 4. REPURPOSING PSYCHOTROPIC PHENOTHIAZINE DRUGS FOR THE MANAGEMENT OF TUBERCULOSIS The drugs developed initially to treat certain ailment may interact with unrelated targets and exhibit secondary therapeutic effect, which may offer positive therapeutic windows to cure other ailments [23]. Phenothiazines are one such class of drugs, which were found to exhibit antitubercular activity as secondary therapeutic effect. Chlorpromazine, the first psychotropic phenothiazine drug, exhibited in-vitro antimicrobial activity against number of gram positive and gram negative microorganisms including M. tuberculosis [24]. The cardio toxicity and extrapyramidal side-effects (EPS) associated with the phenothiazines, at plasma concentration required for bactericidal activity, prevented their potential to transform into antitubercular agents. Interestingly, phenothiazines showed remarkable in-vitro activity against both drug susceptible and drug resistant Mtb strains. In addition, these agents exhibited synergistic effect with first line anti-TB drugs. Therefore, these drugs can be used to reduce the dosing and toxicity associated with high doses of rifampicin and streptomycin. Phenothiazines can be used as an adjunct along with the regular therapy till the antibiotic vulnerability data is available against Mtb [7]. The researcher across the globe have recognised the need of repurposing the older drugs to accomplish the dream of eliminating TB by 2050 [25]. This has spurred a renewed interest in developing less toxic phenothiazines as anti-TB agents. Chlorpromazine inhibited intracellular mycobacteria effectively at concentrations ranging from 0.23 to 3.6µg/mL [21] [26] and exhibited antitubercular effect by blocking the efflux pump involved in the expulsion of xenobiotic substances [27]. In spite of the poor in-vitro activity, it has the potency to act in-vivo against drug-susceptible and drug-resistant Mtb strains. The antitubercular potency of rifampicin and streptomycin increased by the introduction of chlorpromazine as an adjunct at concentrations that were feebly effective against multi-drug resistant strains of Mtb [28]. Even the metabolite of chlorpromazine, 7-hydroxychlorpromazine showed synergistic effect with kanamycin, streptomycin, spectinomycin and 25desacetylrifampicin with fractional inhibitory concentration index (FICI) in the range 0.19–0.5 [23]. The combination of chlorpromazine and spectinomycin was associated with the greatest synergy at FICI of 0.31[11]. The psychiatric phenothiazine drug, thioridazine, was more effective in comparison to chlorpromazine [29] and also showed lowest EPS side-effects. However, this property was not used for clinical presentation due to its cardiac side-effects. In contrary, thioridazine did not produce such effect. It also did not produce any in-vitro toxicity to the macrophages and hence could be used in the treatment of intracellular Mtb infections. Further, the cardio toxicity of thioridazine is associated with its molecular structure, whereas, the antitubercular property is shared amongst the different molecular structures of phenothiazine derivatives [30]. The (-) thioridazine was more active and was also devoid of the associated toxic effects

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[31]. Therefore, developing the phenothiazine based lead compounds may be fruitful for development of antitubercular drugs. In combination therapy, phenothiazine derivatives have shown tremendous potential to cure totally drug resistant (TDR) TB [7]. They act through the inhibition of Type-2 NADH dehydrogenase, leading to the prevention of aerobic respiration, which is essential for the endurance of Mtb [27]. The control of tuberculosis is only possible by the effective treatment of multidrug-resistant tuberculosis. Fortunately, thioridazine is able to target mycobacteria which are resistant to first line drugs, viz. streptomycin, rifampicin, isoniazid, ethambutol and pyrazinamide and also produces enhanced activity against intracellular MDR and XDR-TB [21]. Further, thioridazine produced 50% inhibition of Mtb H37Rv at MIC of 2.5 µg/mL [11]. The potency of thioridazine against Mtb has renewed the attention of fellow researchers due to the increased incidence of MDR-TB [22]. In addition to this, thioridazine also exhibited synergistic effect with rifampicin and streptomycin at concentrations that were least effective when used without thioridazine against poly-drug resistant strains of Mtb [28]. Trifluoperazine, a known calmodulin antagonist, completely inhibited the growth of mycobacteria [32]. In a synthetic medium containing 0.2% Tween 80, the MIC of trifluoperazine was between 5 to 8 µg/mL against Mtb H37Rv and isoniazid resistant strains. In spite of poor in-vitro activity, it showed potent in-vivo activity against both drug-vulnerable and drug-resistant Mtb strains [32, 33]. Levomepromazine, the low potent antipsychotic drug inhibited Mtb H37Rv at MIC of 10µg/mL. While the anticholinergic drug, diethazine and the antihistaminic drug, promethazine showed inhibition at MIC of 20 µg/mL against Mtb H37Rv [34]. When phenothiazine was given as adjunct with rifampicin and streptomycin at concentrations that were least effective when used otherwise against the Mtb resistant strains, the in-vivo activity increased steadily [28]. Further, the commercially available phenothiazines inhibited recombinant Type-2 NADH dehydrogenase (NDH-2) with IC50 values ranging between 10 and 160 µM. The potency of these phenothiazines increased in the following order i.e. promazine32!g/mL

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14a ; R=4-N(CH3)2 ;MIC=>25!g/ml 14b ;R=4-NO2 ;MIC=3.125!g/ml 14c ;R=3,4,5-OCH3 ;MIC=>12.5!g/ml 14d ;R=4- CH3 ;MIC=>25!g/ml 14e ;R=2-Cl;MIC=>3.125!g/ml 14f ;R=4-OCH3 ;MIC=12.5!g/ml 14g ; R=H ; MIC=>6.25 !g/ml

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H 2N 17a ; R=H; MIC=n.d 17b ; R=2-OH; MIC=2.23!g/mL 17c ; R=3-OH; MIC= n.d 17d ; R=4-OH; MIC=n.d 17e ; R=2-Cl; MIC=1.73 !g/mL 17f ; R=3-Cl; MIC=n.d 17g ; R=4-Cl; MIC=0.02 !g/mL 17h ;R=2-NO2; MIC=1.73 !g/mL 17i ; R=3-NO2; MIC= n.d 17j ;R=4-NO2; MIC=1.73 !g/mL 17k ; R=4-OCH3; MIC=2.23!g/mL

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Fig. (3). Phenothiazine derivatives possessing antitubercular potency.

the optimum logP value of 4.29 and was used as a drug functionality to increase the permeability of the compounds through the cell membrane of the Mtb. Compounds (2a-2e) (Fig. 3) on varying R1 and keeping R2 as H have provided interesting results. The addition of electron withdrawing groups i.e. chlorine (Cl) and trifluromethane (CF3) increased the LogP value and also the anti-TB activity. The presence of a bulky tridecane group at R2 also enhanced the LogP value, while N,N-dimethylpropan-1-amine substitution decreased the LogP value appreciably. The value of LogP

also increased in compound 3 (Fig. 3) in presence of electron withdrawing groups like chlorine (Cl) and trifluromethane (CF3) but the change was relatively meagre than that of other compounds. Compound 3a was found to be the most active with MIC of 0.5µg/mL in the series [36]. It is well established fact that the side chain of phenothiazine having an amino group is essential for anti-psychotic activity [11, 22] but the same may not be true for anti-tubercular activity. He et al., changed the side chain at tenth position and also the amino group tethered at one end of the side chain and

Phenothiazine: A Better Scaffold against Tuberculosis

made further modifications on tricyclic phenothiazine ring. Among the various compounds synthesized and tested against Mtb H37Rv, compound 8e showed the most promising activity with MIC90 value of 4µg/mL (Fig. 3).Further, compound 8e did not show any cytotoxicity against normal human cell lines at the concentration of 32µg/mL. Compound 8e seems to be a promising lead for further exploration and development. The phenothiazine derivatives possessing modified side chain with terminal carboxyl (4), hydroxyl (6), alkoxy (7) and phenoxy (8b) through an alkyl connector did not show any activity (Fig. 3). No obvious improvement in anti-tubercular potency was observed when a minor change in the phenothiazine core was made (9a, 9b, 11 & 12) (Fig. 3). It was also observed that the change in the side chain at the N-group of the phenothiazine barely causes any improvement in the activity with MIC remaining >32µg/mL. However, addition of a hydrophobic benzene ring at R in compound 8e showed a dramatic decrease in MIC. There was no change in activity on changing the R group substitution (compound 11). Interestingly, an amazing increase in activity was observed in compound 12 due to bulky group substitution with fluoro-benzene and tertiary amino group (Fig. 3) [46]. He et al., noticed that the modification of phenothiazine core with thioxanthenes or 9H-thioxanthene produced no obvious improvement in the anti-tubercular activity. Further, non-basic aryl substituents at the position 10 of phenothiazine core were found necessary to maintain activity and also eliminated the anti-psychotic side effects. The substitutions at second position of the phenothiazine nucleus and the N,N-dimethyl amino group of phenothiazine drugs were essential for the optimal neuroleptic activity but were not essential to maintain the anti-TB activity [46]. Structure activity correlation of some new triazole based phenothiazine compounds (13a-c) revealed that on increasing the alkyl chain length attached to 1,2,3-triazole nucleus, there was also increase in the antitubercular activity (Fig. 3). It should be noted that alkyl chain of the compound with hydroxyl group (13d) and phenyl group (13e) has displayed reduced Mtb inhibition activity. Among the phenothiazinetriazole hybrids having phenyl substituted 1,2,3-triazole ring 13 (h–v), compounds 13l and 13o possessing electron donating methoxy group and two fluoro substituents respectively on the phenyl ring were found to be more active (Fig. 3) [38]. In a series of some novel phenothiazine based pyrazole[3,4d]pyrimidine derivatives, the substitutions in the phenyl ring have significantly affected the activity. The compounds having methoxy (16b), chloro (16d), and nitro group (16f) at second position showed excellent anti-tubercular activity with MIC of