Phenotypic‐Based Discovery and Exploration of a Resorufin Scaffold with Activity against Mycobacterium tuberculosis

Abstract

Tuberculosis remains a leading cause of death by infectious disease. The long treatment regimen and the spread of drug‐resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) necessitates the development of new treatment options. In a phenotypic screen, nitrofuran‐resorufin conjugate 1 was identified as a potent sub‐micromolar inhibitor of whole cell Mtb. Complete loss of activity was observed for this compound in Mtb mutants affected in enzyme cofactor F₄₂₀ biosynthesis (fbiC), suggesting that 1 undergoes prodrug activation in a manner similar to anti‐tuberculosis prodrug pretomanid. Exploration of the structure‐activity relationship led to the discovery of novel resorufin analogues that do not rely on the deazaflavin‐dependent nitroreductase (Ddn) bioactivation pathway for their antimycobacterial activity. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

Tuberculosis remains a leading cause of death by infectious disease. In this paper, we discuss the phenotypic‐based discovery and exploration of a resorufin scaffold with activity against Mycobacterium tuberculosis. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

Introduction

Tuberculosis (TB) is currently the second leading cause of death by infectious disease, only behind COVID‐19. ^[1] The World Health Organisation (WHO) reported a total of 1.3 million people succumbing to the disease, with an estimated 10.6 million people falling ill with TB worldwide in 2022. ^[1] The causative agent behind this disease is Mycobacterium tuberculosis (Mtb) and can be fatal if left untreated. First‐line treatments for TB infections include the standard 6‐month combination therapy regimen comprising of isoniazid, rifampicin, ethambutol, and pyrazinamide. ^[1] While effective, the long treatment duration, multi‐component nature and drug side effects can lead to poor patient compliance and poor treatment outcomes. ^[2] Furthermore, the emergence of resistant strains to first‐line and second‐line treatment options requires longer treatment duration with more expensive drugs, as well as more side effects and lower success rates, making this disease an ongoing public health concern. ^[3] Despite the approval of the new anti‐tuberculosis drugs bedaquiline (2012), delamanid (2014) and pretomanid (2019) for the treatment of drug‐resistant TB in the past decade, resistance to bedaquiline and delamanid has already been reported. ^[4] Therefore, the ongoing development of new treatment regimens, repurposing of existing drugs as well as the discovery of new drugs are necessary to expand our drug discovery pipeline to combat the disease.[ ³ , ⁵ , ⁶ ]

Tuberculosis drug resistance typically occurs due to the nature of tuberculosis treatments itself: the lengthy treatment duration, poor patient compliance, as well as treatment availability especially in less wealthy populations contribute to less than optimal drug usage. ^[2] These resistance mechanisms arise due to mutations in the drug target, drug transporters or enzymes involved in bioactivation pathways. ^[7] Furthermore, mutations arising from genetic drift as opposed to acquired/selective drug pressure in the bacteria have been reported in strains that have had no exposure to pretomanid, reaffirming the notion of using approved drugs selectively to reduce resistance. ^[8] To combat these problems, there has been a particular focus on discovering drugs with novel/unexploited modes of actions to circumvent existing mutations, decrease the spread of resistance and shorten treatment durations. While several drug discovery paradigms exist, the strategy that has historically been most successful for the treatment of TB and other antimicrobial agents is phenotypic drug discovery.[ ⁹ , ¹⁰ ] An advantage of using phenotypic screening is that identified hits possess favourable attributes allowing them to permeate the cell walls of Mtb, complex structures that present significant obstacles in the translation of target‐based hits to whole cell activity. ^[11] Moreover, this strategy allows the potential discovery of novel targets or pathways, as well as bioactivatable drugs that would otherwise not be discovered through the target‐based route.

Compound 1 (Figure 1, Table 1) was identified as a potent hit against whole‐cell Mtb and with an MIC₉₀ (the minimum concentration required to inhibit 90 % of Mtb growth) of 0.27 μM from a phenotypic screen using an academic library available from Compounds Australia (previously known as The Queensland Compound Library, for more information see SI). ^[12] Compound 1 is a reported fluorogenic probe that was developed for the detection of hypoxia and enzymatic nitroreductase (NTR) activity in cancer cells and bacteria.[ ¹³ , ¹⁴ , ¹⁵ ] Conjugation of a chemical handle, such as a reducible group like nitrofuran, to the phenolic group of fluorophore resorufin (2) results in a quenched fluorescent signal, with conjugation of stimuli‐responsive groups being the basis for fluorescent resorufin‐based probes.[ ¹⁶ , ¹⁷ ] Upon bioactivation by NTR in cancers and bacteria, fluorescence increases as a result of the self‐immolative release of resorufin (2). Outside of these applications, there have been no reports of compound 1 having antibacterial properties. In contrast, resorufin (2) and the structurally related resazurin (3) (Figure 1), commonly used as indicators of cell viability, ^[18] have been reported to have activity against Francisella tularensis and Neisseria gonorrhoeae.[ ¹⁹ , ²⁰ ] In comparison to compound 1, resorufin (2) and resazurin (3) were found to be significantly less active against Mtb (Table 1). Furthermore, both compounds 1 and 2 were inactive (Supporting Information, Table 1) when tested against a panel of bacteria consisting of S. aureus, E. faecium, E. coli, K. pneumoniae, A. baumannii and P. aeruginosa, suggesting selectivity of the resorufin scaffold towards these mycobacteria.

Figure 1.

The chemical structures of hit 1 and the structurally related resorufin (2) and resazurin (3).

Table 1.

Anti‐tuberculosis activity of analogues containing nitro‐reductive moieties.^[a]

Compound	R1	MIC90 (μM)
		Mtb WT	CFZ Resistant Mutant	PA824 Resistant Mutant	BDQ Resistant Mutant
1		0.27	>256	0.25	0.31
2 [b]		>256	>256	>256	235
3 [b]		243	>256	46	118
6b		>256	>256	>256	>256
6c		26	213	>256	>256
6d		>256	>256	>256	>256
6e		>256	>256	>256	>256
6f		>256	>256	>256	>256

^[a]MIC₉₀ were determined by incubating Mtb cultures with the compounds (11‐point, 2‐fold dilution). On day 7, OD₆₀₀ values were measured, and MIC₉₀ values are reported in micromolar final concentration and were determined in biological triplicates. WT=wild type Mtb mc²6206 strain. Mutant strains are based on the mc²6206 strain. [b]  These compounds were purchased from commercial vendors and were tested without further purification. PA824 was used as the positive control (MIC₉₀ 0.53 μM) and CFZ (MIC₉₀ 0.39 μM).

The anti‐tuberculosis activity of compound 1 compared favourably in comparison with previous reported and clinically approved TB drugs (MIC of TB drugs range from 0.003 – 10 μM). ^[21] The potent anti‐tuberculosis activity and selectivity of lead 1 for Mtb prompted the exploration of this scaffold to examine its potential as a novel agent for TB. In this study, we report the structure‐activity relationship of resorufin‐based compound 1, with a particular focus on the side chain (Figure 2). The compounds were tested for activity against Mtb, as well as selected drug‐resistant strains. Furthermore, a subset of compounds was evaluated for their ability to kill the intracellular population of the TB.

Figure 2.

Schematic of regions of compound 1 that were explored in this study.

Results and Discussion

Synthesis

Being identified through a phenotypic whole‐cell screen, there was no prior knowledge of the SAR of this scaffold against Mtb or the molecular target of this compound. An extensive set of compounds was synthesised to assess the role of the nitro‐reductive moiety (nitrofuran of compound 1) by replacing it with both alternative nitroaromatic heterocycles and other non‐reductive side chains (Scheme 1). Acetate 5 was prepared by the acylation of commercially available resorufin sodium salt (4) with acetyl chloride using K₂CO₃ as the base in CH₂Cl₂. To obtain the desired analogues, O‐alkylated product 1 and 6b–z, the resorufin sodium salt (4) was reacted with alkyl halides in the presence of Cs₂CO₃ and catalytic amounts of tetrabutylammonium iodide (TBAI) in DMF. Hydrolysis of ester 6z to obtain carboxylic acid 7z was achieved using LiOH in MeOH at 40 °C, the reaction temperature and reaction time was closely monitored to avoid decarboxylation followed by methylation side reactions leading to the formation of the methoxy product 6j.

Scheme 1.

Synthesis of resorufin‐based analogues with changes to the side chain. Reagents and conditions: (i) AcCl, K₂CO₃, CH₂Cl₂, rt, 30 %; (ii) respective alkyl halide, Cs₂CO₃, cat. TBAI, DMF, 60 °C, 13–60 %; (iii) 6z, LiOH, MeOH, 40 °C, 25 %; (iv) 6h, NIS, CH₂Cl₂, reflux, 24 %; (v) 1, 6g, or 6i, I₂, DMSO, rt, 19–42 %; (vi) 6i, NBS or NCS, CHCl₃, reflux, 40–77 %.

Furthermore, we investigated the effects of halogenation of the resorufin scaffold in selected compounds by using late‐stage derivatisation. Iodination of 6h was performed according to a previous literature procedure using N‐iodosuccinimide (NIS) under reflux in CH₂Cl₂ to afford product 8h. ^[22] Since reactions did not proceed under these conditions, iodine in DMSO at room temperature was used to afford the iodinated products of compounds 1, 6g and 6i. Halogenation of 6i with N‐bromosuccinimide (NBS) or N‐chlorosuccinimide (NCS) under reflux in CH₂Cl₂ resulted in the corresponding brominated and chlorinated products 9i and 10i, respectively. The position of halogenation was determined by NMR characterisation and confirmed by single crystal X‐ray structural analysis of 8g, 8h and 9i (Supporting Information). ^[23]

To obtain amino‐derivative resorufamine 13 (Scheme 2), commercially available nitrobenzene 11 was first reacted with urea, K₂CO₃ and KOH in DMSO to afford aminonitrosobenzene 12. ^[24] Due to instability, intermediate 12 was immediately reacted further with resorcinol in the presence of zinc chloride in EtOH under reflux to give the desired compound 13.

Scheme 2.

Synthesis of resorufamine (13). Reagents and conditions: (i) Urea, K₂CO₃, KOH, DMSO, 90 °C, 35 %; (ii) resorcinol, ZnCl₂, EtOH, reflux, 17 %.

Lastly, replacement of the fluorescent resorufin core was achieved by varying the phenolic nucleophilic groups (17 and 21) and alkylation with 2‐(bromomethyl)‐5‐nitrofuran to obtain fluorescein 18 and coumarin 22, respectively (Scheme 3). The fluorescein precursor 17 ^[25] was synthesised from the commercially available hydroxyacetophenone 14, which was condensed under pressure to obtain xanthone 15. Protection of the phenolic groups with tert‐butylsilyl groups afforded 16 in 83 % yield. In parallel, 4‐iodo‐3‐methylbenzoic acid 19 was converted to the tert‐butyl ester 20, which was then subjected to a magnesium‐halogen exchange reaction with isopropyl magnesium chloride to form nucleophilic intermediate 20a. The two convergent synthetic pathways lead to the reaction between the protected xanthone 16 reacting with tert‐butyl ester 20a to afford fluorescein 17 in 40 % yield. To obtain 22, 7‐hydroxy‐2H‐chromen‐2‐one (21) was reacted with 2‐(bromomethyl)‐5‐nitrofuran using Cs₂CO₃ in DMF.

Scheme 3.

Synthesis of fluorescein analogue 18 (top) and coumarin analogue 22 (bottom). Reagents and conditions: (i) NaOAc, H₂O, 200 °C, 80 %; (ii) TBDMSCl, imidazole, DMF, rt, 83 %; (iii) 20a, THF, −10 °C – rt, 40 %; (iv) 2‐(bromomethyl)‐5‐nitrofuran, Cs₂CO₃, DMF, rt, followed by TFA, CH₂Cl₂, rt, 42 % over 2 steps; (v) Boc₂O, cat. 4‐DMAP, THF, reflux, 63 %; (vi) iPrMgCl, THF, −10 °C – 0 °C; (vii) 2‐(bromomethyl)‐5‐nitrofuran, Cs₂CO₃, DMF, rt, 38 %.

Anti‐Tuberculosis Activity

All synthesised compounds were tested for their ability to inhibit the growth of actively replicating Mtb strain mc²6206 in vitro (Tables 1–2, 3), with pretomanid (PA824, MIC₉₀ 0.53 μM) and clofazimine (CFZ, MIC₉₀ 0.39 μM) used as positive controls. Furthermore, compounds were also tested against drug‐resistant strains of TB, including laboratory‐generated strains resistant to CFZ, PA824 and bedaquiline (BDQ). ^[21] The CFZ‐resistant strain contained a mutation in the gene fbiC which is involved in the biosynthesis of cofactor F₄₂₀, while the PA824‐resistant strain contained a mutation in the gene ddn encoding for Ddn activity. Both of these genes are involved in the activation pathway of the TB prodrug pretomanid, with mutations hindering the ability of the enzyme to activate the prodrug resulting in drug resistance. ^[26] The BDQ‐resistant strain contained a common mutation affecting MmpL5 efflux pump regulation resulting in resistance to TB drugs including CFZ. ^[21] MIC values were determined by light scattering measurements using optical density at 600 nm (OD₆₀₀), as opposed to absorbance or fluorescence, and no interference was seen for any of the tested compounds.

Table 2.

Anti‐tuberculosis activity of selected analogues with side‐chain and scaffold modifications.^[a]

Compound	R1	R2	MIC90 (μM)
			Mtb WT	CFZ Resistant Mutant	PA824 Resistant Mutant	BDQ Resistant Mutant
1		H	0.27	>256	0.25	0.31
6g		H	>256	>256	27	>512
6h		H	6.7	46	7.2	14.3
6i		H	7.1	19	46	51
8a		I	13	>256	>256	2.3
8g		I	216	242	96	>256
8h		I	12	16	13	58
8i		I	25	12	60	>256
9i		Br	18	20	29	35
10i		Cl	20	20	9.7	>256
18		–	240	242	44	24
22		–	>256	>256	14.2	15.6

^[a]MIC₉₀ were determined by incubating Mtb cultures with the compounds (11‐point, 2‐fold dilution). On day 7, OD₆₀₀ values were measured, and MIC₉₀ values are reported in micromolar final concentration were determined in biological triplicates. WT=wild type Mtb mc²6206 strain. Mutant strains are based on the mc²6206 strain. PA824 was used as the positive control (MIC₉₀ 0.53 μM) and CFZ (MIC₉₀ 0.39 μM).

Table 3.

Anti‐tuberculosis activity of analogues with side chain modifications.^[a]

Compound	R1	MIC90 (μM)
		Mtb WT	CFZ Resistant Mutant	PA824 Resistant Mutant	BDQ Resistant Mutant
5		>256	>256	>256	>256
6j		3.6	60	1.4	6.6
6k		24	18	29	33
6l		10	7.1	4.8	19
6m		10	6.7	4.8	7.0
6n		5.7	5.7	17	9.0
6o		28	39	69	88
6p		11	6.9	8.4	9.9
6q		14	9.9	18	13
6r		29	68	138	83
6s		>256	>256	>256	>256
6t		47	198	42	68
6u		9.3	12	>512	14
6v		34	25	44	21
6w		55	73	69	42
6x		113	225	>256	243
6y		>256	>256	>256	>256
6z		70	55	74	46
7z		>256	>256	>256	>256
13		2.2	5.3	4.8	3.6

^[a]MIC₉₀ were determined by incubating Mtb cultures with the compounds (11‐point, 2‐fold dilution). On day 7, OD₆₀₀ values were measured, and MIC₉₀ values are reported in micromolar final concentration and were determined in biological triplicates. WT=wild type Mtb mc²6206 strain. Mutant strains are based on the mc²6206 strain. PA824 was used as the positive control (MIC₉₀ 0.53 μM) and CFZ (MIC₉₀ 0.39 μM).

In the first instance, the nitrofuran group of hit 1 was replaced with alternative nitroaromatic heterocycles, comprising of nitrobenzene (6b), nitrothiophene (6c), and various nitroimidazole groups (6d–f). A complete loss of activity (MIC₉₀>256 μM) was observed for these analogues, with only the nitrothiophene (6c) retaining of activity (MIC₉₀ 26 μM), although with an 86‐fold loss in activity compared to hit 1. These results indicated a sharp preference for the nitrofuran ring. Similar preferences for the nitrofuran ring have been reported for other anti‐tuberculosis scaffolds containing nitroaromatic heterocycles. ^[27] Nitroaromatic groups in drugs are generally accepted to be activated enzymatically, acting through a bioactivation mechanism to generate the active metabolite or reactive species. The redox potential of the nitro group is a measure of its reducibility, thereby indirectly contributing to the biological activity of compounds. In particular, nitrofurans are known to have higher reduction potentials (−0.33 V) than nitrothiophene (−0.39 V), nitroimidazoles (−0.39 to −0.56 V, depending on the position of the nitro group) and nitrobenzenes (−0.49 V). ^[28] These results might indicate that only the nitrofuran containing analogue 1 was able to be reduced successfully, although the resulting released product resorufin (2) showed no anti‐tuberculosis activity either. Despite this rationalisation, it was surprising that analogues 6b–f with alternative nitroaromatic heterocycles were mostly inactive, considering nitroimidazole‐containing compounds, such as the approved drug pretomanid and other pre‐clinical compounds are highly active against TB. ^[29]

Next, we evaluated the effects of changes to the side chain by introducing non‐reductive moieties, representative examples included the benzyl, methoxymethyl ether and glycolic ether analogues 6g–i (Table 2). Additionally, modifications to the resorufin scaffold were explored including analogues with different halogens (Cl, Br and I) introduced by late‐stage halogenation of the core. The introduction of halogen atoms was inspired by previous work by Huigens et al., which demonstrated that halogenation of a structurally similar phenazine core improved antibacterial activity. ^[30] Fluorescein and coumarin compounds with antibacterial properties have been reported previously.[ ³¹ , ³² ] Hence, the resorufin scaffold was replaced with the structurally related fluorescein and coumarin fluorophores (compound 18 and 22) to test their activity against Mtb.

Benzyl analogue 6g, like its nitrobenzyl counterpart 6b, showed no anti‐tuberculosis activity. In contrast, the methoxymethyl ether 6h and glycolic ether 6i analogues exhibited significantly reduced potency with MIC₉₀ of approximately 7 μM compared with compound 1 (0.27 μM). Given the perceived importance of the nitrofuran moiety for activity, it was intriguing that these compounds were able to retain some activity unlike previous nitroaromatic analogues 6b–f. Furthermore, compounds 6h and 6i were significantly more potent than the nitrothiophene analogue 6c (MIC₉₀=26 μM).

Iodination of the resorufin core generally resulted in similar or slightly reduced activity for compounds 8g–i. The iodinated analogue of nitrofuran 1 was an exception, which resulted in a large 43‐fold reduction in activity (compound 8a MIC₉₀=13 μM). Similar to iodo analogue 8i, bromination (9i) and chlorination (10i) of the glycolic ether analogue resulted in slightly reduced activity. Replacement of the resorufin core of hit 1 with either a fluorescein (18) or a coumarin (22) core abolished activity (MIC₉₀>256 μM) suggesting the important role of the resorufin core.

Overall these results suggest that the nitro‐reductive moiety of 1 can be replaced with non‐reducible groups to obtain resorufin‐based analogues with anti‐tuberculosis activity. Hence, a wider range of side chain substituents were explored (Table 3). Compound 5, containing an acetyl group was completely inactive, whereas analogues with linear alkyl chains (6j–m) resulted in low micromolar activity (MIC₉₀ 3.6 – 24 μM). No clear trends were observed with respect to the carbon length, for instance, the methyl analogue 6j was the most potent (MIC₉₀ 3.6 μM) compound from this subset, whereas ethyl analogue 6k exhibited significantly reduced activity (MIC₉₀ 24 μM). On the other hand, the propyl 6l and n‐butyl 6m analogues regained some of the anti‐tuberculosis activity, with both exhibiting a MIC₉₀ of 10 μM. Compound 6n, with a 4‐fluoro‐n‐butyl side chain, was nearly as potent (MIC₉₀ 5.7 μM) as the methyl compound 6j. Branched chains (6o+p) or cyclic alkyl chains (6q+r) were tolerated (MIC₉₀ 11 – 29 μM), with the isopropyl 6o and cyclohexane 6r compounds being less active than the isobutyl 6p and cyclopentane 6q analogues.

The introduction of more polar functional groups (compound 6s‐6z) generally resulted in noticeably less active compounds, with the exception of trifluoropropyl‐2‐ol‐containing analogue 6u exhibiting a MIC₉₀ of 9.3 μM. Carbonyl‐containing analogues (5, 6v, 6z, 7z) were not well tolerated and compounds 6s and 6y, with an ethanol and an oxetanemethanolic side chain respectively, were completely inactive. Additionally, the observed drop in activity from ester 6z to the carboxylic acid 7z further reinforces the observation that polar side chains are not favoured for anti‐tuberculosis activity of resorufin‐based analogues. This may be explained by the waxy bacterial cell wall of Mtb, which prevents more hydrophilic compounds from being taken up by the bacteria. This correlation between activity and lipophilicity has been reported for other classes of Mtb inhibitors. ^[33] The amino‐analogue of resorufin, resorufamine (13), was surprisingly amongst the most potent compounds in this series with an MIC₉₀ of 2.2 μM, which is in stark contrast to the inactive resorufin (2).

From the presented SAR data in wild type Mtb, it was established that the nitrofuran analogue 1 was the most potent anti‐tuberculosis agent. Isosteric replacement with other nitroaromatic heterocycles, surprisingly, resulted in significant loss of activity. Compounds 1, 6c and 8a, which all showed some activity against wild type Mtb, were inactive against the CFZ‐resistant strain. In contrast, compounds 6c and 8a were both ineffective against the PA824‐resistant strain, while compound 1 retained its potent activity. These results highlight that subtle structural changes, in this instance the absence or presence of an iodo‐functional group, can result in different biological profiles. As these strains contained mutations in genes involved in the bioactivation of bicyclic 2‐nitroimidazole TB prodrug pretomanid, these results allude to a similar bioactivation mechanism of action. Pretomanid is activated by the F₄₂₀‐dependent nitroreductase Ddn and targets the enzyme DprE2 involved in the synthesis of cell wall component arabinoglycan.[ ³⁴ , ³⁵ ] The ability of compound 1 to retain its activity against the PA824‐resistant strain points to potentially an alternative activating enzyme.

Furthermore, we identified eight analogues (6h, 6i, 6j, 6l, 6m, 6n, 6u and 13) exhibiting potent anti‐tuberculosis activity with a MIC₉₀ ≤10 μM, albeit not as potent as 1 (0.27 μM). These compounds are devoid of nitroaromatic groups suggesting that the nitroaromatic group is non‐essential for anti‐tuberculosis activity of resorufin analogues. Importantly, these results demonstrated that subtle modifications to the resorufin phenol group result in biological activity compared to the inactive resorufin (2). Overall, compounds listed in Tables 2 and 3, exhibited a range of different activity profiles against the three tested resistant strains. Most compounds showed none to low‐level loss of activity against the BDQ‐resistant strain, which suggested that these compounds were either not substrates or only weak substrates for the MmpL5 drug efflux pump. Iodinated and chlorinated analogues, 8h, 8i and 10i, lost activity against the BDQ‐resistant strain, compared to the corresponding non‐halogenated compounds. In contrast, brominated compound 9i had improved activity against the BDQ‐resistant strain relative to compound 6i. These varied results suggested that halogenation may have mixed effects on compound binding to the MmpL5 drug efflux pump, though a more extensive dataset of compounds is required to establish meaningful SAR trends.

Of importance, all eight of the most potent non‐nitroaromatic anti‐TB analogues except 6u showed little to no loss of activity against the CFZ‐, PA824‐ and BDQ‐resistant strains. These findings support the hypothesis that the nitroaromatic compounds act through a prodrug mechanism of action, while the non‐nitroaromatic compounds act through an alternate unknown mode‐of‐action. Additionally, analogues 1 and 6j, were both inactive in a time‐kill kinetic assay against Mtb strain mc²6206 under hypoxic non‐replicating culture conditions (Supporting Information, Figure S1).

Intracellular Anti‐Tuberculosis Activity and Cytotoxicity

Selected compounds were assessed for their potential as preclinical leads by testing their cytotoxicity in macrophages and their activity against intracellular Mtb (Table 4 and Figure 3). The tested compounds were generally well tolerated in human macrophage THP‐1 cells and had IC₅₀ values above their Mtb MIC₉₀ values (where active), with the lowest IC₅₀ being 37 μM for 6j. When tested against intracellular Mtb, 4 out of the 6 compounds (at concentrations 10 x MIC₉₀) displayed appreciable intracellular anti‐tuberculosis activity. The intracellular TB activity of compound 6j might be due to non‐selective toxicity, as the concentration used was similar to its THP‐1 IC₅₀. In contrast, compounds 1, 6i and 8h demonstrated intracellular TB killing at concentrations below their THP‐1 IC₅₀. In particular, compounds 6i demonstrated remarkable killing with only approximately 10 % intracellular Mtb survival. These early results highlight the potential utility of this scaffold for use as anti‐TB agents.

Table 4.

THP‐1 Macrophage cytotoxicity of selected compounds.^[a]

Compound	THP‐1 IC50 (μM)
1	50.0±18.2
2	94.0±10.9
6f	141±6.5
6g	90.0±10.9
6h	138±1.8
6i	149±2.4
6j	37.1±4.9
8h	275±1.8
18	96.7±7.6

^[a]Compound cytotoxicity was tested against THP‐1 human macrophages utilising MTT cell viability assay. Confluent transformed THP‐1 cells (1x10⁵) were treated with compound dilutions in a 96 well micro titre plate made up in complete RPMI‐1640 medium with 10‐point 2‐fold dilutions for 24 h. Compounds were then washed off and after adding fresh media, 10 μL of 5 mg/mL solution of MTT was added and further incubated for 4 h. MTT was then dissolved in DMSO and read in a micro titre plate reader at 570 nm. IC₅₀ values were determined in biological triplicates. The errors are reported as standard error of the mean.

Figure 3.

The intracellular activity of select compounds against Mtb‐infected human THP‐1 macrophages. Compounds were dosed at 10 x their MIC for wild‐type Mtb (values shown in brackets). Cells were infected with Mtb and incubated for 24 h. Treated THP‐1 macrophages were then lysed with filtered distilled water +0.1 % tyloxapol, and colony forming units (CFU/mL) was obtained by plating on 7H11 agar with supplements. Error represents standard error of the mean of three biological triplicates.

Conclusions

The widespread use of the resorufin (2) and resazurin (3) scaffolds as indicators, e. g. in cell viability and cytotoxicity assays,[ ¹⁸ , ³⁶ ] may lead to underappreciation of these scaffolds potential for biological activity. Compound 1, a previously reported resorufin‐based fluorogenic probe for nitroreductase activity in cancer and bacteria,[ ¹³ , ¹⁴ , ¹⁵ ] was identified as a potent inhibitor of Mtb through phenotypic assays. Exploration of the resorufin scaffold against Mtb included modifications to the side chain as well as halogenation and replacement of the resorufin core.

The nitrofuran group of 1 was found to be required for potent anti‐tuberculosis activity (MIC₉₀ 0.27 μM), with other nitroaromatic heterocycle replacements (analogues 6b–f) resulting in a sharp loss of activity. On the other hand, replacement of the nitrofuran moiety with linear and aliphatic rings resulted in low micromolar compounds 6h, 6i, 6j, 6l, 6m, 6n, and 13 that showed little to no loss of activity against the CFZ‐, PA824‐ and BDQ‐resistant strains. These results indicate that the non‐nitroaromatic analogues (6h, 6i, 6j, 6l, 6m, 6n, and 13) likely act through a different mode‐of‐action. The loss of activity against Mtb strains containing mutations linked to the biosynthesis of cofactor F₄₂₀ and Ddn function suggest that nitroaromatic compounds 6c and 8a act through a prodrug‐type activation mechanism likely mediated by Ddn. Meanwhile, nitrofuran compound 1 is also proposed to act through a prodrug‐type mechanism, although potentially activated by a different enzyme. Considering the observed resistance of anti‐TB compounds in clinical use, molecules with novel mechanism of action are urgently required. Furthermore, THP‐1 cytotoxicity and intracellular Mtb activity of selected compounds were promising. While, additional lead optimisation and target deconvolution studies are necessary to advance these compounds along the drug discovery pipeline, our initial hit elaboration study clearly demonstrates that resorufin‐based compounds, particularly non‐nitroaromatic analogues with the ability to circumvent resistance, have potential for further exploration as a promising class of anti‐tuberculosis treatments.

Experimental Methods

General Experimental

Chemicals and solvents were purchased from standard suppliers and no further purifications were required. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. ¹H NMR and ¹³C NMR spectra were recorded on a BRUKER Avance III Nanobay 400 MHz NMR spectrometer equipped with a BACS 60 automatic sample changer at 400 MHz and 101 MHz, respectively. Chemical shifts were reported in parts per million (ppm) and all peaks being referenced through the residual deuterated solvent peak. Multiplicity was indicated as followed: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); br s (broad singlet). The coupling constants were reported in Hz. Poor solubility of nitroaromatic resorufin compounds prohibited the acquisition of ¹³C NMR spectra for compounds 1, 6b–g, 8a. Thin‐layer chromatography analysis (TLC) was performed on precoated silica gel aluminium‐backed plates. Visualization was by using either stains such as ninhydrin or under UV light at 254 nm and 365 nm. Flash column chromatography was performed using P60 silica gel (40–63 μm).

Low resolution mass spectrometry was obtained by Agilent 1260 Infinity II LCMS SQ equipped with a 1260 Infinity G1312B Binary pump and a 1260 Infinity G1367E 1260 HiP ALS autosampler. Detection of UV reactive compounds was performed at wavelengths of 214 nm and 254 nm and were recorded by a 1290 Infinity G4212iA 1290 DAD variable wavelength detector. LCMS data was processed through the LC/MSD Chemstation Rev.B.04.03 SP2 coupled with MassHunter Easy Access Software. The LC component was run as a reverse phase HPLC using a Raptor C18 3.0 × 50 mm 2.7 ‐microncolumn at 35 °C. The following buffers were used: Buffer A: 0.1 % formic acid in water; buffer B: 0.1 % formic acid in MeCN. The following gradient was used with a Poroshell 120 EC−C18 3.0 × 50 mm 2.7‐micron column with a flow rate of 0.5 mL/min and a total run time of 5 min: 0–2 min 5 %‐100 % buffer B; 2–4.5 min 100 % buffer B; 4.5–5 min 100 %‐5 % buffer B. Mass spectra were recorded in positive and negative ion mode with a scan range of 100–1000 m/z. UV detection was run at 214 nm and 254 nm. High resolution mass spectrometry was obtained by Agilent 6224 TOF LC/MS Mass Spectrometer coupled to an Agilent 1290 Infinity (Agilent, Palo Alto, CA). All data were acquired and reference mass corrected via a dual‐spray electrospray ionisation (ESI) source. Each scan or data point on the Total Ion Chromatogram (TIC) is an average of 13,700 transients, producing a spectrum every second. Mass spectra were created by averaging the scans across each peak and background subtracted against the first 10 s of the TIC. Acquisition was performed using the Agilent Mass Hunter Data Acquisition software version B.05.00 Build 5.0.5042.2 and analysis was performed using Mass Hunter Qualitative Analysis version B.05.00 Build 5.0.519.13. The mass spectrometer drying gas flow was at 11 L/min at a temperature of 325 °C in electrospray ionization mode. The nebulizer was setup at 45 psi with a capillary voltage of 4000 V. The fragmentor, skimmer and OCT RFV voltage were 160 V, 65 V and 750 V, respectively. The scan range acquired were 100–1500 m/z. Analytical high‐performance liquid chromatography (HPLC) was performed on Agilent 1260 Analytical HPLC with a 1260 DAD: G4212B detector and a Zorbax Eclipse Plus C18 Rapid Resolution 4.6 × 100 mm 3.5‐micron column. The eluent system was made up of solvent A (H₂O with 0.1 % formic acid) and solvent B (MeCN with 0.1 % formic acid). Samples used the same method: gradient starts from 95 % solvent A and 5 % solvent B and reaches 100 % solvent B in 8 min, sustained at 100 % solvent B for 1 min, returned to 95 % solvent A and 5 % solvent B over 0.1 min and sustained at 95 % solvent A and 5 % solvent B for 0.9 min. The retention times (t _R) are recorded in min. Automated C18 column chromatography when used was performed using cartridge 40 g, C18 silica gel, 20–35 μm spherical particles, 100 Å, eluent: 5 % MeCN/H₂O (5 CV), gradient 5→100 % MeCN/H₂O (20 CV).

Synthesis

General Procedure A: Alkylation

A mixture of the nucleophile (1.0 equiv.) and Cs₂CO₃ (2.0 equiv.) were suspended in DMF (20 mL per 100 mg of nucleophile) and stirred for 5 min at rt. The corresponding alkyl halide was added to the mixture and stirred at the specified temperature until reaction completion (monitored by LCMS and TLC). The mixture was cooled to rt then concentrated until most of the DMF was evaporated.

7‐((5‐Nitrofuran‐2‐yl)methoxy)‐3H‐phenoxazin‐3‐one (1) ^[13]

A mixture of resorufin sodium salt (118 mg, 0.500 mmol, 1.0 equiv.) and 2‐(bromomethyl)‐5‐nitrofuran (103 mg, 0.500 mmol, 1.0 equiv.) were reacted at 40 °C for 20 h according to general procedure A. The reaction was diluted in CH₂Cl₂ and washed copiously with sat. NaHCO₃ solution and then brine. The organic layer was dried with MgSO₄ and concentrated to give product as an orange solid (80 mg, 47 %). ¹H NMR (400 MHz, DMSO‐d₆ ) δ 7.81 (d, J=8.9 Hz, 1H), 7.71 (d, J=3.7 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.30 (d, J=2.7 Hz, 1H), 7.17 (dd, J=8.9, 2.7 Hz, 1H), 7.09 (d, J=3.8 Hz, 1H), 6.80 (dd, J=9.8, 2.1 Hz, 1H), 6.29 (d, J=2.1 Hz, 1H), 5.42 (s, 2H). LCMS: m/z (ESI) 339.0 (M+H⁺, 100 %). HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₀N₂O₆ [M+H]⁺ 339.0612, found 339.0611. HPLC (254 nm): t_R 6.52 min, >95 %.

7‐((4‐Nitrobenzyl)oxy)‐3H‐phenoxazin‐3‐one (6b) ^[37]

A mixture of resorufin sodium salt (150 mg, 0.635 mmol, 1.0 equiv.) and 4‐nitrobenzyl bromide (165 mg, 0.765 mmol, 1.2 equiv.) were reacted at rt for 20 h according to general procedure A. The crude material was purified by FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the title compound as an orange solid (40 mg, 18 %). ¹H NMR (400 MHz, 10 % TFA‐d in CDCl₃) δ 8.38 – 8.31 (m, 3H), 8.25 (d, J=9.5 Hz, 1H), 7.70 (d, J=8.7 Hz, 2H), 7.65 – 7.59 (m, 2H), 7.45 (dd, J=7.3, 2.5 Hz, 2H), 5.52 (s, 2H). LCMS: m/z (ESI) 349.0 (M+H⁺, 100 %), t_R 3.07 min. HRMS (ESI‐TOF) m/z: calcd for C₁₉H₁₂N₂O₅ [M+H]⁺ 349.0819, found 349.0817. HPLC (254 nm): t_R 6.86 min, >95 %.

7‐((5‐Nitrothiophen‐2‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6c) ^[14]

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 2‐(bromomethyl)‐5‐nitrothiophene (114 mg, 0.515 mmol, 1.2 equiv.) was reacted at rt for 16 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as a dark orange solid (10 mg, 6 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.09 (d, J=4.2 Hz, 1H), 7.82 (d, J=8.9 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.38 (d, J=4.2 Hz, 1H), 7.27 (d, J=2.6 Hz, 1H), 7.16 (dd, J=8.9, 2.7 Hz, 1H), 6.80 (dd, J=9.8, 2.1 Hz, 1H), 6.29 (d, J=2.1 Hz, 1H), 5.59 (s, 2H). LCMS: m/z (ESI) 355.0 (M+H⁺, 100 %). t_R 3.70 min. HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₀N₂O₅SNa [M+Na]⁺ 377.0203, found 377.0221. HPLC (254 nm): t_R 6.68 min, 95 %.

7‐((1‐Methyl‐5‐nitro‐1H‐imidazol‐2‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6d)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and S9 (74.7 mg, 0.425 mmol, 1.0 equiv.) was reacted at 70 °C for 16 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (32 mg, 21 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.48 (s, 1H), 7.82 (d, J=8.9 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.30 (d, J=2.7 Hz, 1H), 7.18 (dd, J=8.9, 2.7 Hz, 1H), 6.80 (dd, J=9.8, 2.1 Hz, 1H), 6.30 (d, J=2.0 Hz, 1H), 5.42 (s, 2H), 3.82 (s, 3H). LCMS: m/z (ESI) 353.1 (M+H⁺, 100 %), t_R 3.24 min. HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₂N₄O₅ [M+H]⁺ 353.0880, found 353.0885 HPLC (254 nm): t_R 5.54 min, >95 %.

7‐((1‐Methyl‐2‐nitro‐1H‐imidazol‐5‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6e) ^[14]

A mixture of resorufin sodium salt (41.8 mg, 0.178 mmol, 1.0 equiv.) and S4 (37.5 mg, 0.213 mmol, 1.2 equiv.) was reacted at 50 °C for 3 h according to general procedure A. The crude material was extracted between CH₂Cl₂ and H₂O. The organic layer was washed with sat. NaHCO₃ solution, brine, dried over Na₂SO₄, filtered then concentrated. Purification by FCC (3 % MeOH/CH₂Cl₂) afforded the product as an orange solid (13 mg, 20 %). ¹H NMR (400 MHz, 10 % TFA‐d in CDCl₃) δ 8.35 (d, J=9.3 Hz, 1H), 8.25 (d, J=9.5 Hz, 1H), 7.67 – 7.50 (m, 3H), 7.45 (s, 1H), 7.31 – 7.27 (m, 1H), 5.49 (s, 2H), 4.21 (s, 1H). LCMS: m/z (ESI) 353.1 (M+H⁺, 100 %), t_R 3.24 min. HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₂N₄O₅ [M+2Na]²⁺ 199.0296, found 199.0290. HPLC (254 nm): t_R 5.38 min, >90 %. Spectral data is in agreement with the literature.

7‐((1‐Methyl‐4‐nitro‐1H‐imidazol‐2‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6f)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and S7 (74.7 mg, 0.425 mmol, 1.0 equiv.) was reacted at 70 °C for 16 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated under vacuum. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (21 mg, 14 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.48 (s, 1H), 7.82 (d, J=8.9 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.31 (d, J=2.7 Hz, 1H), 7.18 (dd, J=8.9, 2.7 Hz, 1H), 6.80 (dd, J=9.8, 2.1 Hz, 1H), 6.30 (d, J=2.1 Hz, 1H), 5.42 (s, 2H), 3.82 (s, 3H). LCMS: m/z (ESI) 353.2 (M+H⁺), t_R 3.29 min. HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₂N₄O₅ [M+H]⁺ 353.0880, found 353.0880. HPLC (254 nm): t_R 5.65 min, >95 %.

7‐(Benzyloxy)‐3H‐phenoxazin‐3‐one (6g) ^[38]

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and benzyl bromide (60.6 μL, 0.510 mmol, 1.2 equiv.) was reacted at rt for 16 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (32 mg, 25 %). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J=8.9 Hz, 1H), 7.47 – 7.35 (m, 6H), 7.02 (dd, J=8.9, 2.6 Hz, 1H), 6.89 (d, J=2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.1 Hz, 1H), 6.32 (d, J=2.1 Hz, 1H), 5.18 (s, 2H). LCMS: m/z (ESI) 304.1 (M+H⁺, 100 %), t _R 3.63 min. HRMS (ESI‐TOF) m/z: calcd for C₁₉H₁₃NO₃ [M+H]⁺ 304.0968, found 304.0983. HPLC (254 nm): t_R 7.03 min, 91 %. Spectral data is in agreement with the literature.

7‐(Methoxymethoxy)‐3H‐phenoxazin‐3‐one (6h) ^[22]

A mixture of resorufin sodium salt (500 mg, 2.13 mmol, 1.0 equiv.) and bromomethyl methyl ether (208 μL, 2.55 mmol, 1.2 equiv.) were reacted at rt for 20 h according to general procedure A. The crude material was purified by FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the product as an orange solid (253 mg, 46 %). ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J=8.8 Hz, 1H), 7.42 (d, J=9.9 Hz, 1H), 7.10 – 6.98 (m, 2H), 6.84 (dd, J=9.8, 2.0 Hz, 1H), 6.33 (d, J=2.0 Hz, 1H), 5.27 (s, 2H), 3.51 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.5, 161.2, 149.9, 146.2, 145.5, 134.9, 134.5, 131.7, 129.1, 114.9, 107.0, 102.9, 94.7, 56.7. LCMS: m/z (ESI) 258.1 (M+H⁺, 100 %), t_R 3.35 min. HRMS (ESI‐TOF) m/z: calcd for C₁₄H₁₁NO₄ [M+H]⁺ 258.0761, found 258.0759. HPLC (254 nm): t_R 5.98 min, >95 %. Spectral data is in agreement with the literature.

7‐(2‐Methoxyethoxy)‐3H‐phenoxazin‐3‐one (6i)

A mixture of resorufin sodium salt (50 mg, 0.213 mmol, 1 equiv.) and 2‐bromoethyl methyl ether (24.0 μL, 0.255 mmol, 1.2 equiv.) was reacted at rt for 16 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (36 mg, 62 %). ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 6.98 (dd, J=8.9, 2.7 Hz, 1H), 6.86 – 6.79 (m, 2H), 6.32 (d, J=2.0 Hz, 1H), 4.25 – 4.19 (m, 2H), 3.83 – 3.77 (m, 2H), 3.47 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.0, 150.0, 145.9, 145.7, 134.8, 134.4, 131.7, 128.6, 114.2, 106.9, 100.9, 70.8, 68.5, 59.5. LCMS: m/z (ESI) 272.1 (M+H⁺, 60 %), t _R 3.25 min. HRMS (ESI‐TOF) m/z: calcd for C₁₅H₁₃NO₄ [M+H]⁺ 272.0917, found 272.0921. HPLC (254 nm): t_R 5.66 min, >95 %.

7‐Methoxy‐3H‐phenoxazin‐3‐one (6j) ^[37]

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and methyl iodide (32 μL, 0.510 mmol, 1.2 equiv.) were reacted at rt for 20 h according to general procedure A. The crude material was purified by FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the product as a dark red solid (40 mg, 41 %). ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J=8.9 Hz, 1H), 7.42 (d, J=6.8 Hz, 1H), 7.02 – 6.76 (m, 3H), 6.34 (s, 1H), 3.93 (s, 3H). LCMS: m/z (ESI) 228.1 (M+H⁺, 100 %), t_R 3.31 min. HRMS (ESI‐TOF) m/z: calcd for C₁₃H₉NO₃ [M+H]⁺ 228.0655, found 228.0654. HPLC (254 nm): t_R 5.82 min, >95 %. Spectral data is in agreement with the literature.

7‐Ethoxy‐3H‐phenoxazin‐3‐one (6k) ^[38]

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and bromoethane (63.5 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 72 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (21 mg, 20 %). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=9.0 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 6.93 (dd, J=8.9, 2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.1 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.33 (d, J=2.1 Hz, 1H), 4.14 (q, J=7.0 Hz, 2H), 1.48 (t, J=7.0 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.2, 150.0, 145.9, 145.5, 134.8, 134.3, 131.7, 128.4, 114.2, 106.8, 100.6, 64.8, 14.7. LCMS: m/z (ESI) 242.1 (M+H⁺, 100 %), t_R 3.46 min. HRMS: (ESI‐TOF) m/z calcd for C₁₄H₁₁NO₃ [M+H]⁺ 242.0812, found 242.0810. HPLC (254 nm): t_R 6.26 min, >95 %. Spectral data is in agreement with the literature.

7‐Propoxy‐3H‐phenoxazin‐3‐one (6l)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 1‐iodopropane (82.6 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (33 mg, 30 %). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J=8.9 Hz, 1H), 7.39 (d, J=9.8 Hz, 1H), 6.92 (dd, J=8.9, 2.6 Hz, 1H), 6.81 (dd, J=9.8, 2.1 Hz, 1H), 6.78 (d, J=2.6 Hz, 1H), 6.30 (d, J=2.0 Hz, 1H), 4.01 (t, J=6.5 Hz, 2H), 1.86 (h, J=7.4 Hz, 2H), 1.06 (t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.4, 150.0, 145.8, 145.5, 134.8, 134.2, 131.7, 128.4, 114.2, 106.8, 100.6, 70.7, 22.5, 10.5. LCMS: m/z (ESI) 256.1 (M+H⁺, 100 %), t_R 3.77 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₃NO₃ [M+H]⁺ 256.0968, found 256.0963. HPLC (254 nm): t_R 6.92 min, >95 %.

7‐Butoxy‐3H‐phenoxazin‐3‐one (6m)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 1‐bromobutane (91.8 μL, 0.850 mmol, 2.0 equiv.) were reacted at 60 °C for 48 h according to general procedure A. The mixture was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution and brine, then dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by FCC (20 % EtOAc/CH₂Cl₂) to obtain the product as a red solid (45 mg, 39 %). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d, J=8.9 Hz, 1H), 7.41 (d, J=9.8 Hz, 1H), 6.93 (dd, J=8.9, 2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.1 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.32 (d, J=2.1 Hz, 1H), 4.06 (t, J=6.5 Hz, 2H), 1.88 – 1.78 (m, 2H), 1.58 – 1.46 (m, 2H), 1.00 (t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.5, 150.0, 145.9, 145.5, 134.8, 134.2, 131.7, 128.4, 114.3, 106.8, 100.6, 69.0, 31.1, 19.3, 13.9. LCMS: m/z (ESI) 270.1 (M+H⁺, 100 %), t _R 3.72 min. HRMS (ESI‐TOF) m/z: calcd for C₁₆H₁₅NO₃; [M+H]⁺ 270.1125, found 270.1120. HPLC (254 nm): t _R 7.50 min, >95 %.

7‐(4‐Fluorobutoxy)‐3H‐phenoxazin‐3‐one (6n)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 1‐bromo‐4‐fluorobutane (91.6 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic phase was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by FCC (20 % EtOAc/CH₂Cl₂) to obtain the product as an orange solid (20 mg, 16 %). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=8.9 Hz, 1H), 7.41 (d, J=9.8 Hz, 1H), 6.92 (dd, J=8.9, 2.6 Hz, 1H), 6.82 (dd, J=9.8, 2.1 Hz, 1H), 6.80 (d, J=2.6 Hz, 1H), 6.31 (d, J=2.1 Hz, 1H), 4.54 (dt, J=47.6, 5.7 Hz, 2H), 4.12 (t, J=6.0 Hz, 2H), 2.05 – 1.94 (m, 3H), 1.92 – 1.84 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.1, 150.0, 145.8, 145.7, 134.8, 135.3, 131.7, 128.5, 114.1, 106.9, 100.7, 83.7 (d, J=165.3 Hz), 68.6, 27.2 (d, J=20.1 Hz), 25.3 (d, J=5.05 Hz). LCMS: m/z (ESI) 288.1 (M+H⁺ _, 100 %). HRMS: (ESI‐TOF) m/z calcd for C₁₆H₁₄FNO₃ [M+H]⁺ 288.1030, found 288.1033. HPLC (254 nm): t_R 6.38 min, >95 %.

7‐Isopropoxy‐3H‐phenoxazin‐3‐one (6o)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 2‐bromopropane (79.9 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 24 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic phase was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by FCC (20 % EtOAc/CH₂Cl₂) to obtain the product as an orange solid (13 mg, 12 %). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 6.91 (dd, J=8.9, 2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.0 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.33 (d, J=2.1 Hz, 1H), 4.66 (hept, J=6.0 Hz, 1H), 1.41 (d, J=6.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 162.4, 150.1, 145.9, 145.4, 134.8, 134.2, 131.8, 128.3, 115.0, 106.8, 101.4, 71.5, 22.0. LCMS: m/z (ESI) 256.1 (M+H⁺, 100 %), t_R 3.53 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₃NO₃ (M+H⁺) 256.0968, found 256.0963. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₃NO₃ (M+Na⁺) 278.0788, found 278.0799. HPLC (254 nm): t_R 6.43 min, >95 %.

7‐Isobutoxy‐3H‐phenoxazin‐3‐one (6p)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 1‐bromo‐2‐methylpropane (93 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The resulting solid after the addition of H₂O was filtered under vacuum and washed with 1 M NaOH aq. solution (50 mL) and H₂O (50 mL) to afford the title compound as an orange solid (56 mg, 48 %). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d, J=8.9 Hz, 1H), 7.41 (d, J=9.8 Hz, 1H), 6.93 (dd, J=8.9, 2.6 Hz, 1H), 6.82 (dd, J=9.8, 2.1 Hz, 1H), 6.80 (d, J=2.6 Hz, 1H), 6.31 (d, J=2.1 Hz, 1H), 3.82 (d, J=6.5 Hz, 2H), 2.14 (hept, J=13.3, 6.7 Hz, 1H), 1.06 (d, J=6.7 Hz, 7H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.5, 150.0, 145.9, 145.5, 134.8, 134.3, 131.7, 128.4, 114.3, 106.8, 100.6, 75.5, 28.3, 19.3. LCMS: m/z (ESI) 270.1 (M+H⁺, 100 %), t_R 3.70 min. HRMS (ESI‐TOF) m/z: calcd for C₁₆H₁₅NO₃ [M+H]⁺ 270.1125, found 270.1117. HPLC (254 nm): t_R 7.59 min, >95 %.

7‐(Cyclopentyloxy)‐3H‐phenoxazin‐3‐one (6q)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and bromocyclopropane (91.2 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The mixture was extracted between EtOAc and H₂O. The organic layer was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered and concentrated. The crude was purified by FCC (20 % EtOAc/CH₂Cl₂) to afford the product as a red solid (33 mg, 28 %). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J=8.9 Hz, 1H), 7.39 (d, J=9.8 Hz, 1H), 6.88 (dd, J=8.9, 2.6 Hz, 1H), 6.80 (dd, J=9.8, 2.1 Hz, 1H), 6.75 (d, J=2.6 Hz, 1H), 6.30 (d, J=2.1 Hz, 1H), 4.83 (tt, J=5.8, 2.5 Hz, 1H), 2.04 – 1.60 (m, 8H). ¹³C NMR (101 MHz, CDCl₃) δ 186.3, 162.6, 150.0, 145.8, 145.3, 134.8, 134.1, 131.6, 128.2, 115.1, 106.7, 101.4, 81.0, 33.0, 24.2. LCMS: m/z (ESI) 282.1 (M+H⁺, 100 %), t_R 3.71 min. HRMS: (ESI‐TOF) m/z calcd for C₁₇H₁₅NO₃ [M+H]⁺ 282.1125, found 282.1119. HPLC (254 nm): t_R 7.56 min, >95 %.

7‐(Cyclohexylmethoxy)‐3H‐phenoxazin‐3‐one (6r)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and (bromomethyl)cyclohexane (119 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 72 h according to general procedure A. The crude material was purified by FCC (20 % EtOAc/CH₂Cl₂) to afford the product as an orange solid (35 mg, 27 %). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d, J=8.9 Hz, 1H), 7.41 (d, J=9.8 Hz, 1H), 6.92 (dd, J=8.9, 2.6 Hz, 1H), 6.82 (dd, J=9.8, 2.0 Hz, 1H), 6.78 (d, J=2.6 Hz, 1H), 6.31 (d, J=2.0 Hz, 1H), 3.85 (d, J=6.0 Hz, 2H), 1.92 – 1.67 (m, 6H), 1.38 – 1.16 (m, 3H), 1.15 – 1.00 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.6, 150.0, 145.9, 145.4, 134.8, 134.2, 131.7, 128.4, 114.3, 106.8, 100.6, 74.6, 37.6, 29.9, 26.5, 25.8. LCMS: m/z (ESI) 310.2 (M+H⁺, 100 %), t_R 4.24 min. HRMS (ESI‐TOF) m/z: calcd for C₁₉H₁₉NO₃ [M+H]⁺ 310.1438, found 310.1444. HPLC (254 nm): t_R 8.73 min, >95 %.

7‐(2‐Hydroxyethoxy)‐3H‐phenoxazin‐3‐one (6s)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 2‐bromoethanol (90.4 μL, 1.28 mmol, 3.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. Purification by C18 reverse phase column chromatography afforded the product as a red solid (40 mg, 36 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.77 (d, J=8.9 Hz, 1H), 7.53 (d, J=9.8 Hz, 1H), 7.12 (d, J=2.6 Hz, 1H), 7.07 (dd, J=8.9, 2.6 Hz, 1H), 6.79 (dd, J=9.8, 2.1 Hz, 1H), 6.27 (d, J=2.1 Hz, 1H), 4.97 (t, J=5.4 Hz, 1H), 4.16 (t, J=4.8 Hz, 2H), 3.82 – 3.71 (m, 2H). ¹³C NMR (101 MHz, DMSO‐d₆ ) δ 185.3, 162.9, 149.7, 145.3, 145.1, 134.9, 133.7, 131.3, 127.8, 114.2, 105.6, 100.8, 70.9, 59.3. LCMS: m/z (ESI) 258.1 (M+H⁺, 100 %), t_R 3.03 min. HRMS (ESI‐TOF) m/z: calcd for C₁₄H₁₁NO₄ [M+H]⁺ 258.0761, found 258.0757. HPLC (254 nm): t_R 4.50 min, 94 %.

7‐((Methylthio)methoxy)‐3H‐phenoxazin‐3‐one (6t)

A mixture of resorufin sodium salt (200 mg, 0.850 mmol, 1.0 equiv.) and chloromethyl methyl sulfide (140 μL, 1.70 mmol, 2.0 equiv.) were reacted at rt for 2 h according to general procedure A and the mixture was concentrated under reduced pressure. The crude material was purified FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the product as a dark orange solid (36 mg, 15 %). ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J=8.9 Hz, 1H), 7.43 (d, J=9.8 Hz, 1H), 6.99 (dd, J=8.9, 2.6 Hz, 1H), 6.89 (d, J=2.6 Hz, 1H), 6.85 (dd, J=9.8, 2.0 Hz, 1H), 6.34 (d, J=2.0 Hz, 1H), 5.24 (s, 2H), 2.29 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.5, 161.0, 149.9, 146.2, 145.5, 134.9, 134.5, 131.7, 129.0, 114.8, 107.0, 102.4, 73.2, 14.9. LCMS: m/z (ESI) 274.1 (M+H⁺, 80 %), t_R 3.41 min. HRMS (ESI‐TOF) m/z: calcd for C₁₄H₁₁NO₃S [M+H]⁺ 274.0532, found 274.0540. HPLC (254 nm): t _R 6.72 min, 89 %.

7‐(3,3,3‐Trifluoro‐2‐hydroxypropoxy)‐3H‐phenoxazin‐3‐one (6u)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 3‐bromo‐1,1,1‐trifluoro‐2‐propanol (88.2 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was extracted between EtOAc and H₂O. The organic phase was washed with 1 M NaOH solution, brine, dried over MgSO₄, filtered then concentrated. Purification by FCC (20 % EtOAc/CH₂Cl₂) afforded the product as an orange solid (17 mg, 12 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.78 (d, J=8.9 Hz, 1H), 7.53 (d, J=9.8 Hz, 1H), 7.20 (d, J=2.7 Hz, 1H), 7.09 (dd, J=8.9, 2.7 Hz, 1H), 6.79 (dd, J=9.8, 2.1 Hz, 1H), 6.76 (d, J=5.9 Hz, 1H), 6.27 (d, J=2.1 Hz, 1H), 4.52 – 4.41 (m, 1H), 4.36 (dd, J=10.8, 3.9 Hz, 1H), 4.25 (dd, J=10.8, 6.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO‐d ₆) δ 185.3, 161.9, 149.7, 145.5, 145.2, 134.9, 133.8, 131.3, 128.1, 124.9 (q, J=283.4 Hz), 114.05, 105.7, 101.1, 67.5 (q, J=1.7 Hz), 67.5 (q, J=29.7 Hz). LCMS: m/z (ESI) 326.1 (M+H⁺, 100 %), t_R 3.30 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₀F₃NO₄ [M+H]⁺ 326.0635, found 326.0632. HPLC (254 nm): t_R 5.74 min, >95 %.

((3‐Oxo‐3H‐phenoxazin‐7‐yl)oxy)methyl acetate (6v)[ ³⁹ , ⁴⁰ ]

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and bromomethyl acetate (83.4 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 2 h according to general procedure A. Crude mixture was purified by FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the product as an orange solid (30 mg, 25 %). ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.9 Hz, 1H), 7.04 (dd, J=8.9, 2.6 Hz, 1H), 6.99 (d, J=2.6 Hz, 1H), 6.85 (dd, J=9.8, 2.0 Hz, 1H), 6.33 (d, J=2.0 Hz, 1H), 5.83 (s, 2H), 2.16 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 169.6, 160.3, 149.7, 146.9, 145.4, 134.9, 134.8, 131.9, 129.5, 114.5, 107.2, 102.7, 84.7, 21.0. LCMS: m/z (ESI) 286.1 (M+H⁺, 100 %), t _R 3.22 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₁NO₅ [M+H]⁺ 286.0710, found 286.0705. HPLC (254 nm): t_R 5.67 min, >95 %. Spectral data is in agreement with the literature.

7‐((Tetrahydro‐2H‐pyran‐3‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6w)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 3‐(bromomethyl)oxane (152 mg, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was purified by automated C18 reverse phase column chromatography to afford the product as an orange solid (13 mg, 10 %). ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 6.93 (dd, J=8.9, 2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.0 Hz, 1H), 6.80 (d, J=2.6 Hz, 1H), 6.32 (d, J=2.0 Hz, 1H), 4.05 – 3.98 (m, 1H), 3.97 – 3.94 (m, 2H), 3.90 – 3.82 (m, 1H), 3.55 – 3.47 (m, 1H), 3.45 – 3.38 (m, 1H), 2.26 – 2.13 (m, 1H), 1.99 – 1.87 (m, 1H), 1.75 – 1.62 (m, 2H), 1.55 – 1.44 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.1, 150.0, 145.81, 145.77, 134.9, 134.4, 131.7, 128.6, 114.1, 106.9, 100.7, 70.5, 70.4, 68.7, 35.8, 26.0, 24.9. LCMS: m/z (ESI) 312.1 (M+H⁺, 100 %), t_R 3.38 min. HRMS: (ESI‐TOF) m/z calcd for C₁₈H₁₇NO₄ [M+H]⁺ 312.1230, found 312.1232. HPLC (254 nm): t_R 6.22 min, >95 %.

7‐((Tetrahydro‐2H‐pyran‐4‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6x)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and 4‐bromomethyltetrahydropyran (152 mg, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was purified by automated C18 reverse phase column chromatography to afford the product as an orange solid (11 mg, 8 %). ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 6.93 (dd, J=8.9, 2.6 Hz, 1H), 6.83 (dd, J=9.8, 2.0 Hz, 1H), 6.80 (d, J=2.6 Hz, 1H), 6.32 (d, J=2.0 Hz, 1H), 4.08 – 4.00 (m, 2H), 3.91 (d, J=6.4 Hz, 2H), 3.46 (td, J=11.8, 2.1 Hz, 2H), 2.21 – 2.05 (m, 1H), 1.82 – 1.73 (m, 2H), 1.57 – 1.41 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 186.4, 163.2, 150.0, 145.8, 145.7, 134.8, 134.4, 131.7, 128.5, 114.1, 106.9, 100.7, 73.6, 67.6, 35.1, 29.7. LCMS: m/z (ESI) 312.2 (M+H⁺, 100 %), t_R 3.36 min. HRMS: (ESI‐TOF) m/z calcd for C₁₈H₁₇NO₄ [M+H]⁺ 312.1230, found 312.1236. HPLC (254 nm): t_R 6.09 min, >95 %.

7‐((3‐(Hydroxymethyl)oxetan‐3‐yl)methoxy)‐3H‐phenoxazin‐3‐one (6y)

A mixture of resorufin sodium salt (100 mg, 0.425 mmol, 1.0 equiv.) and (3‐(bromomethyl)oxetan‐3‐yl)methanol (96.0 μL, 0.850 mmol, 2.0 equiv.) were reacted at 70 °C for 48 h according to general procedure A. The crude material was purified by automated C18 reverse phase column chromatography to afford the product as an orange solid (34 mg, 25 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.78 (d, J=8.9 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.17 (d, J=2.6 Hz, 1H), 7.09 (dd, J=8.9, 2.7 Hz, 1H), 6.79 (dd, J=9.8, 2.1 Hz, 1H), 6.27 (d, J=2.0 Hz, 1H), 5.04 (t, J=5.4 Hz, 1H), 4.46 – 4.40 (m, 4H), 4.32 (s, 2H), 3.73 (d, J=5.5 Hz, 2H). ¹³C NMR (101 MHz, DMSO‐d₆ ) δ 185.2, 162.8, 149.7, 145.2, 134.9, 133.7, 131.3, 127.9, 115.6, 114.1, 105.6, 100.9, 74.5, 69.8, 62.0, 43.9. LCMS: m/z (ESI) 314.2 (M+H⁺, 100 %), t_R 3.49 min. HRMS (ESI‐TOF) m/z: calcd for C₁₇H₁₅NO₅ [M+H]⁺ 314.1023, found 314.1017. HPLC (254 nm): t_R 4.69 min, 94 %.

Methyl 2‐((3‐oxo‐3H‐phenoxazin‐7‐yl)oxy)acetate (6z)

A mixture of resorufin sodium salt (500 mg, 2.36 mmol, 1.0 equiv.) and methyl bromoacetate (448 μL, 4.71 mmol, 2.0 equiv.) were reacted at 70 °C for 7 h according to general procedure A. The resulting precipitate after the addition of H₂O was filtered and washed with H₂O (100 mL) to afford the title product as a brown solid (401 mg, 60 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.79 (d, J=8.9 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.15 (d, J=2.7 Hz, 1H), 7.09 (dd, J=8.9, 2.7 Hz, 1H), 6.79 (dd, J=9.8, 2.1 Hz, 1H), 6.27 (d, J=2.1 Hz, 1H), 5.02 (s, 2H), 3.72 (s, 3H). LCMS: m/z (ESI) 286.1 (M+H⁺, 100 %), t_R 3.22 min. HRMS (ESI‐TOF) m/z: calcd for C₁₅H₁₁NO₅ [M+H]⁺ 286.0710, found 286.0704. HPLC (254 nm): t_R 5.43 min, >95 %.

2‐((3‐Oxo‐3H‐phenoxazin‐7‐yl)oxy)acetic acid (7z)

Compound 6  z (50.0 mg, 0.175 mmol, 1.0 equiv.) and LiOH (12.6 mg, 0.526 mmol, 3.0 equiv.) were stirred in MeOH (10 mL) at 40 °C for 4 d. The mixture was evaporated, and then was purified automated C18 reverse phase column to afford the product as a dark orange solid (12 mg, 25 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.79 (d, J=8.8 Hz, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.10 (d, J=2.6 Hz, 1H), 7.06 (dd, J=8.8, 2.7 Hz, 1H), 6.79 (dd, J=9.8, 2.1 Hz, 1H), 6.27 (d, J=2.1 Hz, 1H), 4.89 (s, 2H). LCMS: m/z (ESI) 270.1 (M‐H⁻, 10 %). HRMS: (ESI‐TOF) m/z calcd for C₁₄H₉NO₅ [M+H]⁺ 272.0553, found 272.0547. HPLC (254 nm): t_R 4.62 min, >95 %.

4‐Iodo‐7‐((5‐nitrofuran‐2‐yl)methoxy)‐3H‐phenoxazin‐3‐one (8a)

To a mixture of 1 (20.0 mg, 0.059 mmol, 1.0 equiv.) in 1 mL DMSO at rt was added I₂ (22.5 mg, 0.089 mmol, 1.5 equiv.). After overnight stirring, the mixture was purified by automated C18 reverse phase column chromatography to obtain the product as a dark purple solid (5.1 mg, 19 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.86 (d, J=8.9 Hz, 1H), 7.72 (d, J=3.7 Hz, 1H), 7.57 (d, J=9.7 Hz, 1H), 7.38 (d, J=2.7 Hz, 1H), 7.20 (dd, J=8.9, 2.7 Hz, 1H), 7.08 (d, J=3.8 Hz, 1H), 6.99 (d, J=9.7 Hz, 1H), 5.49 (s, 2H). LCMS: m/z (ESI) 465.0 (M+H⁺, 5 %), t_R 3.49 min. HRMS: (ESI‐TOF) m/z calcd for C₁₇H₉IN₂O₆ [M+H]⁺ 464.9578, found 464.9581. HPLC (254 nm): t _R 7.02 min, >95 %.

7‐(Benzyloxy)‐4‐iodo‐3H‐phenoxazin‐3‐one (8g)

Compound 6g (20.0 mg, 0.066 mmol, 1.0 equiv.) and iodine (67.0 mg, 0.263 mmol, 4.0 equiv.) were stirred in DMSO (1 mL) for 16 h. The mixture was then extracted between EtOAc and H₂O. The organic layer was washed with brine, dried over MgSO₄, filtered, then concentrated to the crude product. The crude material was purified by FCC (eluent 20 % EtOAc/CH₂Cl₂) to afford the title compound as a red solid (12 mg, 42 %). ¹H NMR (400 MHz, CDCl₃) δ 7.80 – 7.73 (m, 1H), 7.52 – 7.35 (m, 6H), 7.10 – 7.04 (m, 2H), 7.02 (d, J=9.7 Hz, 1H), 5.21 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 180.8, 163.4, 151.2, 146.1, 144.4, 135.4, 134.8, 132.2, 131.6, 129.0, 128.8, 128.8, 127.7, 115.3, 101.4, 85.4, 71.2. LCMS: m/z (ESI) 429.9 (M+H⁺, 100 %), t_R 3.46 min. HRMS (ESI‐TOF) m/z: calcd for C₁₉ H₁₂INO₃ [2 M+K]⁺ 896.9355, found 896.9349. HPLC (254 nm): t_R 5.98 min, >95 %.

4‐Iodo‐7‐(methoxymethoxy)‐3H‐phenoxazin‐3‐one (8h) ^[22]

Compound 6h (90.0 mg, 0.350 mmol, 1 equiv.) was dissolved in 10 mL CHCl₃ and stirred for 5 min. N‐Iodosuccinimide (118 mg, 0.525 mmol, 1.5 equiv.) was added to the orange solution and heated to reflux overnight. Once complete (TLC, eluent 20 % EtOAc/CH₂Cl₂), the red mixture was cooled down. The mixture was diluted with 20 mL CH₂Cl₂, washed with sat. sodium thiosulfate solution and concentrated. FCC (eluent 20 % EtOAc/CH₂Cl₂) afforded the title product as a red solid (32 mg, 24 %). ¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, J=8.8 Hz, 1H), 7.42 (d, J=9.7 Hz, 1H), 7.17 (d, J=2.6 Hz, 1H), 7.09 (dd, J=8.9, 2.5 Hz, 1H), 7.01 (d, J=9.7 Hz, 1H), 5.31 (s, 2H), 3.53 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 180.8, 161.7, 151.2, 145.8, 144.8, 134.8, 132.3, 131.5, 129.2, 115.8, 103.0, 94.7, 85.4, 56.7. LCMS: m/z (ESI) 384.0 (M+H⁺, 100 %), t_R 3.54 min. HRMS (ESI‐TOF) m/z: calcd for C₁₄H₁₀INO₄ [M+H]⁺ 383.9727, found 383.9733. HPLC (254 nm): t_R 7.04 min, >95 %.

4‐Iodo‐7‐(2‐methoxyethoxy)‐3H‐phenoxazin‐3‐one (8i)

Compound 6i (50.0 mg, 0.184 mmol, 1.0 equiv.) and iodine (93.5 mg, 0.369 mmol, 2.0 equiv.) were dissolved in DMSO (3 mL) and stirred at rt for 2 h. After 24 h (monitored by TLC, eluent 20 % EtOAc/CH₂Cl₂), the mixture was extracted between EtOAc (30 mL) and H₂O (30 mL). The organic layer was washed with brine (30 mL), dried over MgSO₄, filtered and concentrated under vacuum. FCC (eluent 20 % EtOAc/CH₂Cl₂) afforded the product as a red solid (17 mg, 23 %). ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J=8.9 Hz, 1H), 7.42 (d, J=9.8 Hz, 1H), 7.07 – 6.97 (m, 3H), 4.30 – 4.24 (m, 2H), 3.85 – 3.78 (m, 2H), 3.47 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 180.7, 163.5, 151.2, 146.0, 144.4, 134.8, 132.2, 131.6, 128.8, 115.3, 100.9, 85.3, 70.7, 68.7, 59.5. LCMS: m/z (ESI) 398.0 (M+H⁺, 100 %), t_R 3.46 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₂INO₄ [M+H]⁺ 397.9884, found 397.9884. HPLC (254 nm): t_R 6.44 min, >95 %.

4‐Bromo‐7‐(2‐methoxyethoxy)‐3H‐phenoxazin‐3‐one (9i)

Compound 6i (50.0 mg, 0.184 mmol, 1.0 equiv.) was dissolved in CHCl₃ (10.0 mL) and stirred for 5 min. N‐Bromosuccinimide (39.4 mg, 0.221 mmol, 1.2 equiv.) was added to the orange solution and heated to reflux overnight. After 24 h, (monitored by TLC, eluent 20 % EtOAc/CH₂Cl₂), the red mixture was cooled down. The mixture was diluted with CH₂Cl₂ (20.0 mL), washed with sat. sodium thiosulfate solution and concentrated under vacuum. FCC (eluent 20 % EtOAc/CH₂Cl₂) afforded the title product as a red solid (50 mg, 77 %). ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J=8.9 Hz, 1H), 7.43 (d, J=9.9 Hz, 1H), 7.03 (dd, J=8.9, 2.6 Hz, 1H), 7.00 (d, J=9.8 Hz, 1H), 6.98 (d, J=2.6 Hz, 1H), 4.28 – 4.24 (m, 2H), 3.84 – 3.79 (m, 2H), 3.47 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 179.3, 163.5, 147.4, 145.6, 144.6, 134.3, 133.2, 131.7, 128.5, 115.3, 105.3, 100.9, 70.7, 68.7, 59.5. LCMS: m/z (ESI) 350.0 (M⁷⁹Br+H⁺, 100 %), 352.0 (M⁸¹Br+H⁺, 100 %), t_R 3.52 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₂BrNO₄ [M⁷⁹Br+H]⁺ 350.0022, found 350.0038; C₁₅H₁₂ClNO₄ [M⁸¹Br+H]⁺ 352.0004, found 352.0020. HPLC (254 nm): t _R 6.24 min, >95 %.

4‐Chloro‐7‐(2‐methoxyethoxy)‐3H‐phenoxazin‐3‐one (10i)

Compound 6i (50.0 mg, 0.184 mmol, 1.0 equiv.) was dissolved in CHCl₃ (10.0 mL) and stirred for 5 min. N‐Chlorosuccinimide (29.5 mg, 0.221 mmol, 1.2 equiv.) was added to the orange solution and heated to reflux overnight. After 24 h, (monitored by TLC, eluent 20 % EtOAc/CH₂Cl₂), the red mixture was cooled down. The mixture was diluted with CH₂Cl₂ (20.0 mL), washed with sat. sodium thiosulfate solution and concentrated under vacuum. FCC (eluent 20 % EtOAc/CH₂Cl₂) afforded the title product as a red solid (224 mg, 40 %). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J=8.9 Hz, 1H), 7.41 (d, J=9.9 Hz, 1H), 7.01 (dd, J=8.9, 2.7 Hz, 1H), 6.96 (d, J=9.9 Hz, 1H), 6.95 (d, J=2.6 Hz, 1H), 4.28 – 4.21 (m, 2H), 3.82 – 3.78 (m, 2H), 3.46 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 179.1, 163.4, 145.44, 145.38, 144.5, 134.0, 133.2, 131.8, 128.3, 115.2, 113.3, 100.8, 70.6, 68.6, 59.4. LCMS: m/z (ESI) 306.0 (M³⁵Cl+H⁺, 100 %), 308.0 (M³⁷Cl+H⁺, 30 %), t_R 3.35 min. HRMS: (ESI‐TOF) m/z calcd for C₁₅H₁₂ClNO₄ [M³⁵Cl+H]⁺ 306.0528, found 306.0516; C₁₅H₁₂ClNO₄ [M³⁷Cl+H]⁺ 308.0504, found 308.0493. HPLC (254 nm): t _R 5.91 min, >95 %.

7‐Amino‐3H‐phenoxazin‐3‐one (13) ^[24]

Urea (2.93 g, 48.7 mmol, 3.0 equiv.), K₂CO₃ (2.47 g, 17.9 mmol, 1.1 equiv.), and KOH (2.73 g, 48.7 mmol, 3.0 equiv.) were dissolved in anhydrous DMSO (16 mL). Nitrobenzene (2.00 g, 1.67 mL, 16.3 mmol, 1.0 equiv.) was added dropwise to the solution. The mixture was then stirred at 90 °C for 5 h open to atmospheric air. The reaction was monitored by TLC (20 % EtOAc/CH₂Cl₂). After nitrobenzene was consumed (5 h), the reaction mixture was cooled to rt and poured into water (100 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, followed by filtration. The solvent was evaporated under vacuum and the crude residue was purified by FCC (eluent 10 % EtOAc/CH₂Cl₂) to obtain the product as a dark purple solid (701 mg, 35 %). Since the 4‐nitrosoaniline intermediate is unstable, it was immediately used in the following reaction.

Resorcinol (1.26 g, 11.5 mmol, 2.0 equiv.) and zinc chloride (859 mg, 6.30 mmol, 1.1 equiv.) were suspended in anhydrous EtOH (20 mL). Then, the mixture was stirred under reflux, and 4‐nitrosoaniline (700 mg, 5.73 mmol, 1.0 equiv.) was added portionwise to the solution over 1 h. The reaction was monitored by TLC. After the reaction was completed (2 h), the solvent was evaporated under vacuum and the residue was filtered through a silica plug (washing with CH₂Cl₂, followed by acetone) to obtain the crude product. Purification by C18 reverse phase column chromatography afforded the product as a black solid (201 mg, 17 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 7.48 (d, J=8.8 Hz, 1H), 7.41 (d, J=9.6 Hz, 1H), 7.01 (s, 2H), 6.71 (dd, J=8.8, 2.3 Hz, 1H), 6.62 (dd, J=9.7, 2.0 Hz, 1H), 6.49 (d, J=2.3 Hz, 1H), 6.15 (d, J=1.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO‐d ₆) δ 184.5*, 155.4, 150.0, 146.6, 139.4, 134.3, 132.0, 131.2, 126.1, 113.6, 104.8, 97.2. LCMS: m/z (ESI) 213.1 (M+H⁺, 100 %), t_R 2.83 min. HRMS (ESI‐TOF) m/z: calcd for C₁₂H₈N₂O₂ [M+H]⁺ 213.0659, found 213.0651. HPLC (254 nm): t_R 3.86 min, >95 %. *Carbon signal identified through HMBC experiment. Spectral data is in agreement with the literature.

3,6‐Dihydroxy‐9H‐xanthen‐9‐one (15) ^[41]

2,2′,4,4′‐Tetrahydroxybenzophenone (2.00 g, 8.12 mmol, 1.0 equiv.) and sodium acetate (13.3 mg, 0.162 mmol, 0.02 equiv.) were suspended in water (30.0 mL) in a thick‐walled glass sealed tube and heated to 200 °C overnight. The reaction was monitored by TLC (50 % EtOAc/PB). After 24 h, the mixture was allowed to cool down to rt. The mixture was filtered under vacuum and washed with water, MeCN and Et₂O, then dried to give the product as a pale pink solid (1.48 g, 80 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 10.81 (s, 2H), 7.98 (d, J=8.7 Hz, 2H), 6.86 (dd, J=8.7, 2.2 Hz, 2H), 6.82 (d, J=2.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO‐d₆ ) δ 173.9, 163.4, 157.5, 127.8, 114.0, 113.7, 102.1. LCMS: m/z (ESI) 228.9 (M+H⁺), t_R 2.90 min. Spectral data is in agreement with the literature.

3,6‐Bis((tert‐butyldimethylsilyl)oxy)‐9H‐xanthen‐9‐one (16) ^[41]

Compound 24 (1.48 g, 6.50 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (30.0 mL) under N₂. Imidazole (1.55 g, 22.8 mmol, 3.5 equiv.) was added to the reaction mixture, followed by portion‐wise addition of tert‐butyildimethylsilyl chloride (TBDMSCl) (2.94 g, 19.5 mmol, 3.0 equiv.). The reaction was stirred at rt under N₂. After reaction completion (4 h), the reaction mixture was extracted between Et₂O and water, then washed with brine. The organic layer was dried with MgSO₄, filtered and concentrated to give the crude product. The crude product was recrystallised in hot EtOH to give the product as white needles (2.47 g, 83 %). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (dd, J=8.0, 1.0 Hz, 2H), 7.01 – 6.68 (m, 4H), 1.01 (s, 18H), 0.29 (s,12H). ¹³C NMR (101 MHz, CDCl₃) δ 175.9, 161.5, 157.9, 128.4, 117.8, 116.6, 107.5, 25.7, 18.4, −4.19. LCMS: m/z (ESI) mass not observed, t_R 4.47 min. Spectral data is in agreement with the literature.

tert‐Butyl 4‐(6‐hydroxy‐3‐oxo‐3H‐xanthen‐9‐yl)‐3‐methylbenzoate (17) ^[42]

Compound 20 (408 mg, 1.28 mmol, 1.0 equiv.) was dissolved in anhydrous THF (5.00 mL) under N₂ gas and cooled to −40 °C. Isopropyl magnesium chloride (iPrMgCl, 2 M) (705 μL, 1.41 mmol, 1.1 equiv.) was then added dropwise to the reaction mixture. The mixture was stirred for 4 h between −10 °C and 0 °C. Compound 16 (644 mg, 1.41 mmol, 1.1 equiv.) was then added portion‐wise to the reaction mixture. The reaction mixture was warmed slowly to rt and stirred for 2 d. Monitored by TLC (5 % EtOAc/PB, and 5 % MeOH/CH₂Cl₂). To the mixture was added aq. 3 M HCl (8 mL) and the mixture and stirred for 2 h. Saturated aq. NH₄Cl solution (10 mL) was then added to the mixture, and then concentrated under vacuum. The residue was resuspended in 1 M HCl and EtOAc then filtered, washing with water, EtOAc and finally Et₂O to obtain an orange solid. The product was obtained after FCC (5 % MeOH/ CH₂Cl₂) as an orange solid (206 mg, 40 %). ¹H NMR (400 MHz, DMSO‐d₆ ) δ 8.03 (s, 1H), 7.99 – 7.94 (m, 1H), 7.47 (d, J=7.9 Hz, 1H), 7.32 – 7.25 (m, 4H), 7.11 (dd, J=9.2, 2.0 Hz, 2H), 2.06 (s, 3H), 1.60 (s, 9H). LCMS: m/z (ESI) 401.0 (M‐H⁻), t_R 2.86 min.

3‐Methyl‐4‐(6‐((5‐nitrofuran‐2‐yl)methoxy)‐3‐oxo‐3H‐xanthen‐9‐yl)benzoic acid (18)

A mixture of fluorescein 17 (50.0 mg, 0.635 mmol, 1.0 equiv.) and 4‐nitrobenzyl bromide (206 mg, 0.95 mmol, 1.2 equiv.) were reacted at rt for 20 h according to general procedure A. The resulting precipitate after the addition of H₂O was filtered and washed with water. The orange solid was stirred in 1 : 1 TFA/CH₂Cl₂ (4 mL) for 4 h. After completion, the volatiles were removed under reduced pressure. Purification by automated C18 reverse phase column chromatography afforded the product as an orange solid (23 mg, 20 % over 2 steps). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.04 (s, 1H), 7.97 (d, J=7.8 Hz, 1H), 7.70 (d, J=3.8 Hz, 1H), 7.46 – 7.39 (m, 2H), 7.08 (d, J=3.8 Hz, 1H), 7.02 (dd, J=9.0, 2.5 Hz, 1H), 6.92 (d, J=8.9 Hz, 1H), 6.86 (d, J=9.7 Hz, 1H), 6.45 (dd, J=9.7, 1.9 Hz, 1H), 6.27 (d, J=1.9 Hz, 1H), 5.43 (s, 2H), 2.08 (s, 3H). ¹³C NMR (101 MHz, DMSO‐d₆ ) δ 182.8, 166.9, 162.8, 158.4, 158.0, 154.3, 152.7, 151.9, 149.6, 136.5, 132.0, 131.3, 130.7, 129.6, 129.5, 129.1, 127.0, 117.6, 114.8, 114.7, 114.3, 113.5, 104.7, 101.7, 62.3, 19.1. LCMS: m/z (ESI) 472.1 (M+H⁺, 100 %), t_R 3.23 min. HRMS (ESI‐TOF) m/z: calcd for C₂₆H₁₇NO₈ [M+H]⁺ 472.1027, found 472.1026. HPLC (254 nm): t_R 5.98 min, >95 %.

tert‐Butyl 4‐iodo‐3‐methylbenzoate (20) ^[43]

4‐Iodo‐3‐methylbenzoic acid (1.10 g, 4.20 mmol, 1.0 equiv.), boc anhydride (2.71 g, 12.4 mmol, 3.0 equiv.) and DMAP (133 mg, 1.09 mmol, 0.26 equiv.) were dissolved in anhydrous THF (10.0 mL) and refluxed for 2 d, monitoring by TLC (15 % EtOAc/PB). When the reaction no longer progressed, it was cooled down to rt. Imidazole (1.01 g, 14.9 mmol, 3.5 equiv.) was added to remove unreacted boc anhydride and stirred at rt overnight. The mixture was then concentrated to an orange/pink residue and extracted between Et₂O and aq. 1 M HCl. The organic layer was washed with saturated NaHCO₃ and brine. The organic layer was dried with MgSO₄ and concentrated to give crude product as a pink liquid. The product was obtained by FCC (eluent 3 % EtOAc /PB) to obtain a pink liquid (838 mg, 63 %). ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J=8.2 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.47 – 7.42 (m, 1H), 2.47 (s, 3H), 1.58 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 165.6, 141.7, 139.1, 132.2, 130.4, 128.2, 106.9, 81.5, 28.3, 28.2. LCMS: m/z (ESI) mass not observed, t_R 4.80 min. Spectral data is in agreement with the literature.

7‐((5‐Nitrofuran‐2‐yl)methoxy)‐2H‐chromen‐2‐one (22)

A mixture of umbelliferone (78.0 mg, 0.481 mmol, 1.0 equiv.) and 2‐(bromomethyl)‐5‐nitrofuran (119 mg, 0.578 mmol, 1.2 equiv.) were reacted at rt according to general procedure A. The mixture was extracted between EtOAc and H₂O. The organic phase was washed brine, then dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by FCC (20 % EtOAc/CH₂Cl₂) to afford the product as a brown solid (52 mg, 38 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.01 (d, J=9.5 Hz, 1H), 7.72 – 7.65 (m, 2H), 7.18 (d, J=2.4 Hz, 1H), 7.10 – 7.04 (m, 2H), 6.33 (d, J=9.5 Hz, 1H), 5.36 (s, 2H). ¹³C NMR (101 MHz, DMSO‐d₆ ) δ 160.4, 160.1, 155.2, 153.2, 151.8, 144.2, 129.6, 114.4, 113.5, 113.06, 113.04, 112.8, 101.7, 61.8. LCMS: m/z (ESI) 288.1 (M+H⁺, 100 %), t _R 3.42 min. HRMS (ESI‐TOF) m/z: calcd for C₁₄H₉NO₆ [M+H]⁺ 288.0503, found 288.0502. HPLC (254 nm): t_R 6.08 min, >95 %.

Biology

Bacterial Strains and Media

Mycobacterium tuberculosis strain mc²6206 used in this study was obtained from Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine. Mycobacterial strains were grown on Middlebrook 7H9 medium (Difco, Sparks, MD) supplemented with 10 % (v/v) OADC enrichment (Difco), 0.2 % (v/v) glycerol, 0.05 % (v/v) tyloxapol, Pantothenate (50 mg/L) and L‐leucine 50 mg/L. Cultures were grown at 37 °C.

Minimal Inhibitory Concentration Assay (MIC₉₀)

Cultures of Mycobacterium tuberculosis mc²6206 were harvested by centrifugation at 4,000 rpm for 10 min. The supernatant removed, and the cell pellet resuspended in phosphate buffered saline (PBS) to an optical density at 600 nm (OD₆₀₀) of 0.01. The central 80 wells of a 96 well plate were set up to contain in triplicate, a 2‐fold dilution series of test compound in 50 μL 7H9 medium. 50 μL of culture suspension were added to the central 80 wells of the 96 well plates to give a final assay volume of 100 μL. Rows A and H contained 200 μL of sterile distilled water to minimise evaporation from culture wells during incubation. Wells B−F of column 1 contained 100 μL of PBS. Wells B−F of column 12 contained 50 μL of media and 50 μL of resuspended culture with no additional compound as positive controls. The plates were then incubated for 7 days at 37 °C after which the minimal inhibitory concentrations (MIC₉₀) were determined using light scattering measurements by reading plates in a microplate reader (Varioskan Lux, Thermo Scientific) at OD₆₀₀ and MIC₉₀ were calculated in Graph pad.

THP‐1 Cytotoxicity Assay

THP‐1 were seeded and differentiated with 100 ng/mL (PMA) at 1×10⁵ cell well. Microtitre plates were incubated at 37 °C with 5 % CO₂ to adhere and proliferate for 24 h. Following incubation, media was discarded, and 100 μL of prepared antimicrobial solutions were added, in triplicate, to the cells growing in the 96 well microtitire plates and further incubated for 24 h at 37 °C with 5 % CO₂. Following the 24 hours incubation period, microtitre plates were removed from the incubator, and the media and antimicrobial solutions were discarded into disinfectant. The wells were washed twice gently in pre‐warmed sterile Phosphate Buffered Saline (PBS) and discarded. 50 μL of pre‐warmed complete RPMI was added to each well, except for the negative control wells which received 50 μL of SDS at 10 %, then 10 μL of tetrazolium dye 3‐(4,5‐dimethylthiazol‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) at a concentration of 5 mg/mL in PBS was added to all wells. MTT was mixed gently by tapping, and the plate was re‐incubated at 37 °C with 5 % CO₂ for a further 4 h. Following incubation, contents of the well were discarded, 100 μL of DMSO was added to each well to dissolve the MTT dye, and the plate was re‐incubated for 10 min. The absorbance was measured by spectrophotometer (Varioskan™ Flash Multimode Reader, Thermo Scientific, USA) at 570 nm with dual‐band mode.

THP‐1 Macrophage Infection Studies

The antibiotic sensitivity of M. tuberculosis within THP‐1 infected macrophages were determined using previously described protocols, ^[44] with modifications. Briefly, the human monocytic cell line THP‐1 (ATCC Cat# TIB‐202) was cultured in standard RPMI 1640 macrophage medium supplemented with 10 % inactivated fetal bovine serum and 1 mM sodium pyruvate at 37 °C with 5 % CO₂. THP‐1 monocytes (5×10⁵ cells/well) were differentiated overnight using 100 ng/mL phorbol myristate acetate (PMA) and seeded in a 24 well‐plate. The next day differentiated macrophages were infected with a mid‐logarithmic phase culture of M. tuberculosis mc²6206 WT cells (OD 0.4–0.8) at a multiplicity of infection (MOI) of 10 : 1 (10 bacteria/1 cell). Infection was allowed to proceed for 1 h. Cells were then washed 3 times with pre‐warmed complete RPMI to remove extracellular bacilli. RPMI media containing supplements (pantothenic acid 25 μg/mL and leucine 50 μg/mL), 0.1 % BSA and compounds at varying concentrations were added to the infected cells and incubated at 37 °C with 5 % CO₂. After 24 h, infected cells were lysed in distilled water containing 0.1 % tyloxapol for 5 min at rt to determine the number of CFU/mL on Middlebrook 7H11 OADC agar supplemented with (pantothenic acid 25 μg/mL and leucine 50 μg/mL). The percentage of M. tuberculosis (mc²6206) cell viability was determined by normalizing CFU/mL counts at day 3 following compound treatment relative to the DMSO control.

Supporting Information

The authors have cited additional references within the Supporting information.[ ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷ , ⁴⁸ , ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² , ⁵³ ] The Supporting Information, including experimental sections (materials and methods), compound characterization, ¹H NMR, ¹³C NMR, LCMS, HRMS, HPLC purity analysis of final compounds, and X‐ray crystallographic data of compounds 8g, 8h and 9i is available free of charge at: www.ccdc.cam.ac.uk/data_request/cif

Abbreviations

DMAP

4‐dimethylaminopyridine

d

day(s)

DMF

N,N‐dimethylformamide

DMSO

dimethyl sulfoxide

EtOAc

ethyl acetate

FCC

flash column chromatography

HPLC

high performance liquid chromatography

IC₅₀

concentration that causes inhibition by 50 %

LCMS

liquid chromatograph mass spectrometry

M

molar

MIC

minimum inhibitory concentration;

Min

minute(s)

Mtb

Mycobacterium tuberculosis

NMR

nuclear magnetic resonance

OD₆₀₀

optical density at 600 nm

PB

petroleum benzine

rt

room temperature

SAR

structure‐activity relationship

TB

tuberculosis

TFA

trifluoroacetic acid

THF

tetrahydrofura

TLC

thin layer chromatography.

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

Tran E., Cheung C.-Y., Li L., Carter G. P., Gable R. W., West N. P., Kaur A., Gee Y. S., Cook G. M., Baell J. B., Jörg M., ChemMedChem 2024, 19, e202400482. 10.1002/cmdc.202400482

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.