Design, Synthesis, and Evaluation of Novel Hybrid Efflux Pump

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Design, Synthesis and Evaluation of Novel Hybrid Efflux Pump Inhibitors for use against Mycobacterium tuberculosis Malkeet Kumar, Kawaljit Singh, Krupa Naran, Fahreta Hamzabegovic, Daniel F Hoft, Digby F. Warner, Peter Ruminski, Getahun Abate, and Kelly Chibale ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00111 • Publication Date (Web): 24 Aug 2016 Downloaded from http://pubs.acs.org on August 25, 2016

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ACS Infectious Diseases

Design, Synthesis and Evaluation of Novel Hybrid Efflux Pump Inhibitors for use against Mycobacterium tuberculosis

Malkeet Kumara, Kawaljit Singha, Krupa Naranb, Fahreta Hamzabegovicc, Daniel F. Hoftc,d, Digby F. Warnerb,e, Peter Ruminski,f*, Getahun Abate,c*, Kelly Chibalea,e,g* a

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa

b

MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology,

University of Cape Town, Rondebosch 7701, South Africa c

Department of Internal Medicine, Division of Infectious Diseases, Allergy and Immunology,

Saint Louis University, 1100 S. Grand Blvd, 63104, MO, USA d

Department of Molecular Biology, Saint Louis University, 1100 S. Grand Blvd, 63104, MO, USA e

Institute of Infectious Disease and Molecular Medicine, University of Cape Town,

Rondebosch 7701, South Africa f

Centre for World Health and Medicine, Saint Louis University, 1100 S. Grand Blvd, 63104,

MO, USA g

South African Medical Research Council Drug Discovery and Development Research Unit,

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa Abstract Efflux pumps are considered a major potential contributor to the development of various forms of resistance in Mycobacterium tuberculosis leading to the emergence of multi-drug resistant tuberculosis (TB). Verapamil (VER) and tricyclic chemosensitizers such as the phenothiazines are known to possess efflux pump inhibition properties, and have demonstrated significant efficacy in various TB disease models. Novel hybrid molecules 1 ACS Paragon Plus Environment


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based on fusion of the VER substructure with various tricyclic, as well as non-tricyclic, chemosensitizer cores or their structural motifs are described. These hybrid compounds were evaluated in vitro and ex vivo individually for their intrinsic activity, and in combination for their potentiating potential with the frontline anti-TB drugs, rifampin and isoniazid. In addition, efflux pump inhibition was assessed in an ethidium bromide assay. This study led to the identification of novel compounds, termed hybrid efflux pump inhibitors, with intrinsic antimycobacterial activities (MIC90 ≤ 3.17 µg/ml), and intracellular activity in macrophages at a low concentration (≤ 6.25 µg/ml). Introduction Tuberculosis (TB) is a contagious, airborne disease, that remains one of the major health problems world-wide.1 According to the World Health Organization 2015 report, about one-third of the world’s population is infected with the causative agent, Mycobacterium tuberculosis (M. tuberculosis). However, TB is primarily a disease of the poor with most cases occurring in low income countries where it is highly prevalent in densely populated areas with sub-optimal hygiene standards.2 There are approximately 1.5 million TB deaths every year, and the disease constitutes a significant social and financial burden in the most severely affected countries.3 In many regions, the Human Immunodeficiency Virus (HIV) pandemic has exacerbated the high burden of TB as the virus weakens the immune system, increasing not only the risk of reactivation, but also rapid progression to active TB disease soon after infection.4 There exists a regimen of first-line anti-TB drugs that is highly efficacious, and is estimated to have saved 35 million lives among HIV-negative patients between 2000 and 2014 (WHO 2015). However, the emergence of multi-(MDR) and extensively (XDR) drug resistant M. tuberculosis strains poses a major threat to global TB control efforts. M. tuberculosis acquires MDR through the sequential/serial accumulation of chromosomal mutations in genes encoding drug targets, enzymes required for drug activation, as well as efflux pumps and related systems whose activity can limit drug efficacy.5 Moreover, emerging evidence has highlighted the fact that the molecular mechanisms which perpetuate antibiotic resistance in the clinical setting are not limited to a single resistance mechanism, but depend on the interplay between both intrinsic and genetic (acquired) resistance mechanisms.6 These include the low permeability of the mycolic acid-containing cell wall, the operation of

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various antibiotic-modifying or -inactivating systems, and the activity of efflux pumps (EPs).7–9 Critically, intrinsic resistance mechanisms limit the antibiotics available for treatment and drive the emergence of strains with high levels of antibiotic resistance. EPs are known to protect M. tuberculosis against low intracellular concentrations of toxic substances and metabolites like dyes, bile salts and fatty acids, while maintaining homeostasis of the bacillus in the host environment.10 M. tuberculosis possesses a number of EPs,11 some of which are substrate specific while others transport functionally dissimilar drugs,12 and hence contribute to the development of M(/X)DR forms. Recently, EPs have been implicated in the ability of M. tuberculosis to overcome intracellular stress inside macrophages.13 Induction of EPs in macrophages simultaneously confers resistance in non-replicating M. tuberculosis colonies a phenotype which is retained even after resumption of growth.14,15 Strategies to counteract efflux-mediated resistance include the development of efflux pump inhibitors (EPIs), by improving the structural design of existing antibiotics to reduce/avoid efflux, or blocking pump function.16–18 However, despite promising results, countering drug efflux remains an under-explored area in TB drug discovery, and so presents a potentially attractive strategy to mitigate the significant challenges in TB treatment. A number of molecular entities such as verapamil (VER) and the phenothiazines, chlorpromazine and thioridazine (Figure 1), have been shown to increase the efficacy of anti-TB drugs in vitro, in vivo and in macrophages.19–25 Moreover, these adjunctive agents might be used to reduce the probability of resistance development against new drugs, or to revive previously abandoned anti-TB agents that were discarded owing to EP-mediated drug resistance.

Figure 1: Various known efflux pump inhibitors.19–22,26



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As a result, there is increasing interest in the rational design and synthesis of EPIs in TB. For example, Pieroni and co-workers showed that a hybrid of verapamil and thioridazine substructures (H-1), and thioridazine analogue (H-2) (Figure 1), enhance the activities of isoniazid (INH) and rifampin (RIF) against both extracellular and intracellular M. tuberculosis.26 A major liability of EPIs is that they have to be administered in combination with the antibiotic of choice, which poses an additional challenge from a pharmacological perspective. For example within the context of TB, major deleterious pharmacokinetic (PK) interactions of verapamil and rifampin have hindered further studies of this combination. To overcome this potential liability, structural hybrid efflux pump inhibitors (HEPIs), which ensure simultaneous delivery to the disease site of a pharmacologically optimized EPI, that may also be endowed with intrinsic anti-mycobacterial activity, are worth exploring. Where HEPIs are selected for use in combination with an antibiotic such as RIF, however, there is still the need to optimize PK parameters in such a way as to avoid deleterious PK interactions. Here, we report the design, synthesis and biological evaluation of a panel of novel structural HEPIs designed via fusion of the VER substructure with various tricyclic as well as nontricyclic chemosensitizer cores substructure motifs required for a molecule to sensitize a resistant strain to a drug) or their structural motifs derived from phenothiazines (1a-b and 2), dibenzazapine (1c), cyproheptadiene (3a), thioxanthene (3b-c), dibenzosuberyl (4) and diphenylmethane (5 and 6) (Figure 2).27,28 These chemosensitizers are known to sensitise resistant strains of Mtb and reverse anti-mycobacterial drug resistance by various mechanisms including efflux pump inhibition. It is noteworthy that these chemosensitizer may not have any intrinsic anti-mycobacterial activity. The initial objective was to identify HEPIs that displayed enhanced drug efflux inhibition properties, which might be further optimized.


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Figure 2: HEPI templates and structural motifs covalently linked to the VER substructure.

Chemistry: The synthesis of various HEPIs, as summarized in Schemes 1-3, was achieved by covalently linking the VER substructure intermediate 11 with various EPI moieties. Scheme 1 outlines the synthesis of intermediate 11, which was derived from 9 by reacting this previously reported intermediate with 2-(methylamine)ethanol in the presence of K2CO3 in DMF followed by chlorination with thionyl chloride in DCM to obtain 11 in 83% yield.29 Scheme 1: Synthesis of VER sub-structure intermediate.

Reagents and reaction conditions: (i) 2-Bromopropane, n-BuLi, THF, 0-25 °C, 2 h, 60%; (ii) 1-Bromo-3-chloropropane, LDA, THF, -78-25 °C, 2 h, 76%; (iii) 2-(Methylamine)ethanol,



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K2CO3, DMF, 80 °C, 12 h, 60%; (iv) SOCl2, DCM, 25 °C, 14 h, 83%.

Phenothiazines 12a-b, dibenzazapine 12c, and diphenyl moieties 17 are commercially available. The synthesis of intermediates 14 and 16 is summarised in Scheme 2. Dibenzosuberylchloride 13 was reacted with piperazine in the presence of DBU in toluene to generate 14,30 while 16 was synthesized by reacting biphenylbromide 15 with piperazine using K2CO3 in acetonitrile. The target compounds 1, 4, 5 and 6 were obtained by coupling these structural motifs with intermediate 11.


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Scheme 2: Synthesis of EPI moieties and HEPIs.

Reagents and reaction conditions: (i) Piperazine, DBU, MS-4A°, 0-25 °C, toluene, 12 h, 67%; (ii) Piperazine, K2CO3, NaI, acetonitrile, reflux, 8 h, 65%; (iii) NaH, DMF, 25 °C, 6-8 h, 16-22%; (iv) NaNH2, toluene, reflux, 12 h, 22%; (v) K2CO3, EtOH, reflux, 12 h, 30%.

Intermediates 22 and 26 were synthesized as summarized in Scheme 3. Synthesis of 22 commenced with Boc-protection of 4-hydroxypiperidine 18, followed by mesylation of the hydroxyl group with methanesulfonyl chloride to yield intermediate 20. Phenothiazine 12 was reacted with intermediate 20 in the presence of NaH in DMSO to generate 21. This intermediate was further deprotected using TFA to obtain the desired analogue 22. The dibenzosuberenone analogue (26a) and thioxanthene analogues (26b and 26c) were obtained by

coupling

N-Boc-protected

piperidinone

24

with

dibenzosuberenone (25a)

and

thioxanthenes (25a and 25b), respectively, using the McMurry reaction. The conditions of this reaction also facilitiated in situ N-Boc-deprotection of intermediate formed by the coupling of 24 and 25 to afford intermediate 26. The target compounds 2 and 3 were obtained by reacting intermediates 22 and 26, respectively, with compound 11 using K2CO3 in DMF (Scheme 3).



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Scheme 3: Synthetic approaches for the phenothiazine analogues 22 and 26.

Reagents and reaction conditions: (i) (Boc)2O, Et3N, DCM, 0-25 °C, 1.5 h, 90%; (ii) Methanesulfonylchloride, Et3N, DCM, 0-25 °C, 6 h, 95%; (iii) NaH, DMSO, 25-60 °C, 10-12 h, 30%; (iv) TFA (50% v/v in DCM), 1N-NaOH, 1-1.5 h, 95%; (v) (Boc)2O, Et3N, DMAP, DCM, 025 °C, 6 h, 95%; (vi) Zn, TiCl4 (1M in toluene), 1-4-dioxane, 60 °C-reflux (115 °C), 10-12 h, 43-45%; (vii) K2CO3, DMF, 80 °C, 12 h, 20-35%.

Results and discussion A total of 12 HEPIs were synthesized and screened in various biological assays. Compounds found to display an acceptable cytotoxicity profile were then screened in vitro against M. tuberculosis H37Rv, individually as well as in combination with the first-line anti-TB drug, RIF. Compounds which displayed weak anti-mycobacterial activity (MIC90 > 50 µM) on their own, but which reduced the RIF MIC90 by at least 4-fold when tested in combination, were further evaluated in macrophages, individually as well as in combination with RIF and INH. VER was used as positive control in all experiments. Cytotoxicity and in vitro anti-mycobacterial activity The THP-1 cell line was used for cytotoxicity evaluation of compounds and for determining the activity against intracellular M. tuberculosis. In order to select non-cytotoxic compounds and to achieve selective activity against 8 ACS Paragon Plus Environment

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M. tuberculosis in infected THP-1 monocyte macrophages, a strict cytotoxicity cut-off of 20% inhibition of THP-1 cells (IC20 >10 µg/ml) was adopted. Only four compounds - 1a, 1b, 2b and 3a - satisfied this criterion, and were selected for further analysis. Of these, 1b and 3a were found to be the least cytotoxic (IC20 > 100 µg/ml) while 1a and 2b showed IC20 values in the range 50-100 µg/ml, which was comparable to VER. The four selected compounds - 1a, 1b, 2b and 3a - were evaluated against M. tuberculosis H37Rv individually as well as in combination with RIF using the classic checkerboard 96well plate assay in which a range of concentrations was used for both the EPI and RIF. VER was used as a reference and, together with 1a and 1b, demonstrated poor activity against axenically grown M. tuberculosis (MIC90 > 100 µg/ml) (Table 1). In contrast, 2b and 3a displayed intrinsic anti-mycobacterial activity with MIC90 values of 3.17 µg/ml and 1.47 µg/ml, respectively. Combination studies showed that three compounds (1b, 2b and 3a) increased the susceptibility of M. tuberculosis to RIF as demonstrated by a 4-fold reduction in the RIF MIC90, which is comparable to the potentiation shown by VER, while 1a potentiated RIF by 8-fold. This is presumed to result from more efficient inhibition of EPs by 1a. Whereas 1a and 1b delivered a 4-fold and 8-fold reduction in the RIF MIC90, respectively, at approximately 2-fold lower concentration (< 35 µg/ml) than that required by VER (61.4 µg/ml), even lower concentrations of 2b and 3a (< 0.8 µg/ml) delivered a 4-fold potentiation of RIF. In addition to their low MIC90, the ability of 2b and 3a to enhance the anti-TB activities of RIF makes them unique hybrids. The potential synergistic interactions between these compounds and RIF were determined by calculating the fractional inhibitory concentration index (FICI). According to the strict definition, a combination of two compounds is said to be synergistic when the FICI ≤ 0.5, with FICI ≥ 4 indicating an antagonistic effect, and any value falling in between reported as indicating no interaction.31,32 The combination of VER with RIF resulted in a synergistic interaction with a FICI value of 0.5. HEPIs 1a, 1b and 2b also demonstrated a synergistic interaction with RIF, yielding FICI values of 0.4, 0.5 and 0.5, respectively. However, compound 3a did not display any interaction with RIF (FICI = 0.75).



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Table 1: Cytotoxicity and antimycobacterial activity of HEPIs in combination with RIF against

M. tuberculosis (H37Rv).

a

IC20 (µg/ml) THP-1 cell lines

MIC90 (µg/ml)

Fold reduction in RIF MIC90 4

VER

>50

245.5

61.4

RIF in comb with HEPIs 0.00175

1a

>50 100

137.5

34.4

0.00175

4

0.50

Comp

R

HEPI

HEPI in comb with RIF

FICI 0.50

S

1b

N

Cl

1c

Design, Synthesis, and Evaluation of Novel Hybrid Efflux Pump

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