Synthesis and structure-activity relationships for a new class of tetrahydronaphthalene amide inhibitors of Mycobacterium tuberculosis

Drug resistant tuberculsosis (TB) is global health crisis that demands novel treatment strategies. Bacterial ATP synthase inhibitors such as bedaquiline and next-generation analogues (such as TBAJ-876) have shown promising efficacy in patient populations and preclinical studies, respectively, suggesting that selective targeting of this enzyme presents a validated therapeutic strategy for the treatment of TB. In this work, we report tetrahydronaphthalene amides (THNAs) as a new class of ATP synthase inhibitors that are effective in preventing the growth of Mycobacterium tuberculosis (M.tb) in culture. Design, synthesis and comprehensive structure-activity relationship studies for approximately 80 THNA analogues are described, with a small selection of compounds exhibiting potent (in some cases MIC90 <1 μg/mL) in vitro M.tb growth inhibition taken forward to pharmacokinetic and off-target profiling studies. Ultimately, we show that some of these THNAs possess reduced lipophilic properties, decreased hERG liability, faster mouse/human liver microsomal clearance rates and shorter plasma half-lives compared with bedaquiline, potentially addressing of the main concerns of persistence and phospholipidosis associated with bedaquiline.


Introduction
The rise of drug-resistant tuberculosis (TB) in recent times has become a major global health problem [1], and this resurgence of such a major infectious disease has also provided an impetus for the development of new classes of drugs. These are aimed at a wide variety of mycobacterial targets, including the control of gene expression [2], inhibition of drug efflux pumps [3], and of proteins in the mycobacterial electron transport chain [4]. In particular, the spectacular success of the drug bedaquiline in treating multi-drugresistant TB (MDR-TB) by inhibition of the mycobacterial enzyme ATP synthase has resulted in a largely curative regime (NIX-TB) [5] for this disease, despite bedaquiline's side effect of hERG channel inhibition. This success has led to the development of potentially safer analogues of bedaquiline [6] and a search for alternative classes of ATP synthase inhibitors.
We recently reported [7] the synthesis and anti-mycobacterial structureeactivity relationships (SARs) of a new class of Nsubstituted tetrahydroisoquinolines (THIQs, 1; Fig. 1) as effective inhibitors of Mycobacterium tuberculosis (M.tb) ATP synthase enzyme and growth.
Building on this work, we now report synthesis and structureactivity-relationship (SAR) studies on a further novel class of tetrahydronaphthalene amide (THNA) derivatives as mycobacterial inhibitors. While the tetrahydronaphthalene amide unit is not widely featured in drugs, the derivative AR-A000002 (2; Fig. 1) has been studied as a selective and high affinity 5-hydroxytryptamine (5-HT 1B ) receptor antagonist [8]. It has been shown to be effective in animal models of depression and anxiety [9], and details of a chiral large-scale synthetic route have been published [10]. The related compounds 3 and 4 have demonstrated sub-mM affinity for cloned rat D2L and D3 receptors expressed in HEK293 cells [11]. While a handful of THNA derivatives have been recently reported as M.tb inhibitors [12,13] by targeting the cytochrome bc 1 complex [14], the THNA unit did not form part of the central scaffold, and was not progressed into further studies. To the authors knowledge, this is the first systematic SAR report of 2-substituted THNA compounds as inhibitors of ATP synthase for the development of anti-TB drug candidates.

Chemistry
Initial synthesis of analogues (compounds 5e35; Fig. 2) in Table 1 focused on synthesis of analogues with 5-methyl and 8-Nmethylpiperidyl substituents for the tetrahydronaphthalene unit. This substitution pattern was on the basis of our previous work [7] which showed near-optimal anti-M.tb properties with the structurally related tetrahydroisoquinoline compounds (Fig. 1). With this constant THNA core in place, attention was initially turned to SAR studies on the optimal substituents for the heterocyclic linker and the terminal benzene ring and any chiral preferences for activity.
Once the optimal linker and terminal groups had been identified from the study outlined in Table 1, efforts next focused on replacing the 5-methyl substituent on the tetrahydronaphthalene unit with groups of various steric bulk, electronic and lipophilic properties (Compounds 53e61, Fig. 2, Table 2) while modification of the 8-Nmethylpiperidyl motif with more weakly basic heterocycles and cyclic amines (compounds 62e79, Fig. 2, Table 3) were carried out in attempt to address any potential hERG liabilities.
The urea-linked compounds 36e40 of Table 1 (B; Scheme 1) were prepared by the formation of an activated 4-nitrophenyl sidechain carbamate intermediate followed by coupling with tetrahydronaphthalen-2-amine 80. This bromo-intermediate underwent further Suzuki cross-coupling with the appropriately substituted phenylboronic acids to furnish compounds 36, 37, 39 and 40. This route proved to be a superior route to access urealinked compounds as compound 38 which was accessed via reaction between preformed 4-nitrophenyl sidechain 81 and 80 gave a lower overall yield.
Urea-linked compounds 41e52 of Table 1 (C; Scheme 1) were prepared by the formation of piperizine/piperidine-benzene ring linker which were coupled to tetrahydronaphthalen-2-amine 80 using 4-nitrophenyl chloroformate and triethylamine in DCM.
Schemes 2e4 outline the synthesis of analogues with varied groups at the tetrahydronaphthalene 5-position in an attempt to explore SAR aspects such as steric bulk, electronics and lipophilicity at this site ( Table 2). With carboxylic acid side chain 83 established as the favored side chain (rationale for selection discussed in detail in section 2.2.), Scheme 2 describes the formation of compound 53 via well-established amide coupling between 83 and tetrahydronaphthalenamine 82. THNA analogue 53 could be further elaborated using bromination conditions to yield compound 57. Nitration of tetrahydronaphthalenamine 82 afforded an inseparable mixture of 7-nitro and desired 5-nitro intermediate 84. The mixture could be used crude for the next step after which the desired 5-nitro isomer could be isolated using silica chromatography to yield 58. Bromo-functionality in 57 was used as a synthetic handle to access further diverse analogues. Buchwald-Hartwig amination reaction between 57 and N,N-dimethylethane-1,2diamine required prolonged reaction time of 6 h to afford tethered amine 59 and cyanation reaction of 57 using zinc cyanide led to successful formation of nitrile 61. Tetrahydronaphthalene 5-position was further elaborated with para-substituted phenyl groups (compounds 54 and 55, Scheme 3). Synthesis begins with 8-bromo-3,4-dihydronaphthalen-2(1H)-one, which undergoes reductive amination with (R)-N-ethylphenylamine to yield amine 86, which is benzyl protected to yield 87 in 82% yield. Buchwald-Hartwig amination reaction with N-methylpiperazine gave 88 which underwent selective bromination on the tetrahydronaphthalene 5-position to yield bromide 89. Suzuki coupling with the appropriate boronic acids gave tetrahydronaphthalenes with para-substituted phenyl groups 91 and 92, which underwent deprotection followed by amide coupling gave analogues 54 and 55.
Scheme 4 depicts the preparation of 5-benzyl 56 and more hydrophilic 5-(N-methylpiperidyl) 60 analogues. Commencing with common intermediate bromide 89 (prepared in Scheme 3), lithium-halogen exchange followed by quenching with benzaldehyde led to alcohol 95, which was then reduced to 96. Final amide coupling of amine 96 with acid 83 led to analogue 56. Buchwald-Hartwig amination reaction between bromide 89 and N-methylpiperazine followed by reduction gave amine 85 and subsequent amide coupling with 83 furnished analogue 60.
To explore the effect of altering the pKa of the 8-methyl-piperazine unit on activity, a range of cyclic amines and heterocyclic amine analogues were prepared as described in Schemes 5e9. Scheme 5 reports the synthesis of the simple des-methyl piperidine analogue 62, while Scheme 6 replaces it with a range of 6membered aliphatic and aromatic ring systems (compounds 63, 64 and 66e70). Scheme 7 outline the syntheses of the NH and NMe piperidinyl analogues (65 and 66) of the corresponding piperazinyl analogues 62 and 5 respectively. Schemes 8 and 9 show the synthesis of compounds 71e79, which have a range of six-and fivemembered nitrogen heterocycles in place of the 8methylpiperazine group.
2.2. Structure-activity relationships for the compounds of Table 1 Since the 5-hydroxytryptamine receptor antagonist 3 is a chiral compound, with much effort previously expended in its synthesis to obtain the pure R enantiomer for the production of compound 3 [10], we were initially interested to determine if the chirality was also of importance in our related series targeted against M.tb. To evaluate this, twelve sets of R/S enantiomer pairs of THNAs (Table 1 Fig. S1). In contrast, there were no significant differences between the isomers for mammalian toxicity, as measured by IC 50 values in VERO cell cultures (Table 1).
Consequently, the remaining SAR studies were conducted using only the more active S enantiomers. For these 48 compounds, there is a modest correlation between the overall lipophilicity of the compounds and their potency of inhibition of bacterial growth in the MABA assay (logMIC 90 values for inhibition of bacterial growth under aerobic (replicating) conditions) (equation (1)).
The general trend suggested that higher overall compound lipophilicity correlated with more potent bacterial survival inhibition (Supplementary Data-Fig. S2). This relationship of the antiproliferative potency of compounds against cultures of live M.tb being correlating with increasing overall lipophilicity of the agents has been previously observed across agents with differing mechanisms of action, and has been attributed to drug distribution, with lipophilic drugs needed to efficiently cross the very lipophilic cell walls of mycobacteria [19e21].
In the present case, the THNAs studied were comprised of a  constant N-(5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4tetrahydronaphthalen-2-yl)acetamide unit with side chains made up of two variable but distinct "linker" and "terminal" regions. There was thus an opportunity to see whether variation in lipophilicity within these regions made differing contributions to MIC potency. The results (equation (2)) suggest that the lipophilicity of the linker unit does have a slightly larger influence on LORA potency than the lipophilicity of the terminal unit.
A wide variety of both aromatic and cyclic aliphatic linker groups were explored. The most effective linkers in terms of compound potency were the aromatic 1,4-benzene (compounds 5, 6) and the 1,4-(2-pyridyl (compounds 18e27). The angular pyridine 29 was less effective, while the linear 2,5-pyrimidine (30) and the 2,5-pyrazine (31) were among the most active compounds in the set (especially when allowing for their considerably lower lipophilicity). In contrast, the linear aminopyridine (36), aminopyrazine (37) and aminopyrimidine (39) linkers were less effective. The 2,5-thiophene 32 was also among the most potent compounds, but a number of the five-membered aromatic linkers in compounds 33e35 (admittedly of much lower overall lipophilicity) were less effective. Finally, a series of 1,4-piperazines (41e49), N-piperazin-1amines (50e52) and piperazin-1-amides (53, 54) were also less effective than the above compound with linear aromatic linkers. A number of the different linker units were then paired with a variety of terminal units to evaluate the comparable efficacies of the latter. Comparison of compounds 5, 7, 11, 15, 22, 28, 30, 48 with their counterparts bearing other terminal substitution show that the 2-methyl-4-chloro terminal unit consistently resulted in better activity.
Having established that the optimal sidechain in the THNAs in this study for anti-tubercular potency was the S-configuration with a 2-pyridyl linker group and a 2-methyl-4-chloro terminal ring, we then fixed this side chain and explored variations at the 5-position of the tetrahydronaphthalene unit in compounds 53e61 (Table 2).
Compounds 5, 2-pyridyl analogues 18 and 27, pyrimidine 30, pyrazine 31 and thiophene 32, which displayed unusually superior anti M.tb potency for their lipophilicity profiles (Supplementary Data-Figs. S2B and C) were subject to further study. Table 2 The results in Table 2 show there is considerable bulk tolerance at the 5-position of the tetrahydronaphthalene unit, with substituents varying in size from H (molar refractivity; MR 1.03; compound 53) to benzyl (MR 31.2; compound 56) having no effect on antibacterial potency. This is supported by the MIC/overall lipophilicity relationship for this group (equation (3)) being very similar to that of equation (1), suggesting that, for 5-substituted compounds the primary determinant of MIC potency is again overall lipophilicity, with the 5-substituent not making substantial target interactions.

Structure-activity relationships for the compounds of
This region did appear to be quite sensitive to changes in electronics contributed to the aromatic system (as measured by the Hammett constant), with both electron donating (59 and 60) and electron withdrawing (58 and 61) groups detrimental to anti-M.tb activity.
Finally, we evaluated variations in the 8-position of the tetrahydronaphthalene unit (Table 3). Table 3 Compounds 62e66, bearing a range of cyclic aliphatic strong bases, all showed activity similar to the original N-methylpiperazine analogue 5, whereas the weaker aliphatic (67 and 68) and aromatic (69 and 70) bases were inactive, despite having high overall lipophilicity. The concept that the pKa rather than the nature of the base is more important is reinforced by the pyridinetype bases 71e74; the stronger bases 71 and 72 were active Table 2 Structures and biological activity of (S) 5-substituted tetrahydronaphthalenes.   inhibitors of bacterial growth, whereas ones with weaker bases (73e76) were not, despite retaining high lipophilicity. Finally, the pyrazole analogues 77e79 had weak activity. Overall this suggests an important role for an ionisable base at the 8-position.

Preclinical evaluation
2.5.1. Mammalian cell toxicity of THNA compounds In order to assess safety and selectivity in humans, all compounds were also screened for mammalian cell toxicity in VERO (green monkey kidney cell) [17] cultures (Tables 1e3). For the  compounds of Tables 1 and 2 there was no clear overall relationship between their mammalian and mycobacterial cell potencies, but for compounds of Table 3, the weakly basic compounds 67e79 were much less toxic in both assays than those (62e66) bearing more basic side chains off the tetrahydronaphthalene chromophore. Based on the best MABA, LORA potencies, and superior selectivity profiles with respect to mammalian cell toxicity (based on the ratio of MABA or LORA inhibition vs VERO), a subset of THNAs were selected for further evaluation (Fig. 3).  [23,24] the ability of the clinicallyapproved tuberculosis drug bedaquiline (BDQ) to inhibit the hERG cardiac potassium channel, with the concomitant risk of QT prolongation, has been a significant concern. Selected tetrahydronaphthalenes from Tables 1e3 were also evaluated for hERG channel blockade (Table 4) (Table 3) with aromatic heterocycles in that position, were much less hERG-inhibitory, suggesting that structural variations in this position are influential.

Microsome stability of THNA compounds
Selected compounds from Tables 1e3 were also evaluated for their stability against mouse and human liver microsome preparations, as a guide to their likely in vivo stability (Table 6). BDQ is known to be cleared very slowly, leading to a very long in vivo halflife in humans and concomitant concerns about long-term accumulation [22]. The results show that most of the tetrahydronaphthalenes evaluated had desirably faster clearance rates by both mouse liver microsomes (MLM) and human liver microsomes (HLM) than did BDQ.
Finally, a small number of representative compounds from Tables 1e3 were evaluated for their PK properties in a mouse model following a single dose of drug at 100 mg/kg, and the results are shown in Table 7. BDQ is very lipophilic (clogP 7.25) which has been suggested to contribute to its very long terminal half-life in humans (164 days after 8 weeks of dosing) [23]. The THNAs evaluated were considerably less lipophilic than BDQ and had desirably shorter in vivo half-lives without compromising on total plasma drug exposure (AUC).

Conclusions
In summary, we show that tetrahydronaphthalene amides, a      varying in size from hydrogen to benzyl having similar antibacterial potencies. An ionisable substituent at the 8-position of the tetrahydronaphthalene unit was important for anti-microbial activity.
Results from a representative group of compounds also showed that weak aromatic bases (pyridines and pyrazoles) off the 8positon of the tetrahydronaphthalene unit could desirably suppress the hERG inhibition activity. A smaller panel of these compounds (  min. (S)-5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4tetrahydronaphthalen-2-amine 80 (0.259 g, 0.679 mmol) was added to the reaction mixture and stirred at r.t. for 40 h. The reaction mixture was diluted with EtOAc and washed with water and 2 M NaOH solution. The organic layer was dried over anhydrous Na 2 SO 4 and filtered through a pad of Celite. The solvent was removed to give the crude product, which was purified by silica column chromatography using MeOH (0e5% v/v) in EtOAc as eluent to give 5 (0.287 g, 87%) as a white foam. HPLC 99.1%. 1
HPLC 97.4%. 1   To a suspension of 5-bromopyridin-2-amine (1.00 g, 5.78 mmol) and pyridine (0.56 mL, 6.94 mmol) in DCM (10 mL) in an ice bath was added 4-nitrophenyl carbonochloridate (1.40 g, 6.94 mmol) portionwise. The mixture was stirred at room temperature overnight. The resulting precipitate was collected by filtration, washed with DCM, and dried under vacuum to give the product 165 as a white solid (1.97 g, 100%) which was used crude for the next step.