Carborane clusters increase the potency of bis-substituted cyclam derivatives against Mycobacterium tuberculosis

Abstract

Bis-substituted cyclam derivatives have recently emerged as a promising new class of antibacterial agents, displaying excellent activity against drug-resistant Mycobacterium tuberculosis (Mtb) and in vivo efficacy in a zebrafish assay. Herein we report the synthesis and biological activity of new carborane derivatives within this class of antitubercular compounds. The resulting carborane–cyclam conjugates incorporating either hydrophobic closo-1,2-carborane or anionic, hydrophilic nido-7,8-carborane clusters display promising activity in an antibacterial assay employing the virulent Mtb strain H37Rv. The most active of these carborane derivatives exhibit MIC₅₀ values of <1 μM, making them the most active compounds in this unique class of antibacterial cyclams reported to date.

Bis-substituted cyclam derivatives have recently emerged as a promising new class of antibacterial agents, displaying excellent activity against drug-resistant Mycobacterium tuberculosis (Mtb). Carborane pendants enhance this activity.

Tuberculosis (TB) is one of the top ten causes of death globally.^1–6 TB is a highly contagious disease, and the emergence of drug resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) has led to a resurgence in cases, particularly in underdeveloped countries. According to the World Health Organization (WHO), there were 1.3 million deaths caused by TB in 2020 including 230 000 people below the age of 14.^3,7–9 Current treatments for multi-drug resistant TB (MDR-TB) and extensively-drug resistant TB (XDR-TB) require a combination of the four frontline drugs isoniazid, ethambutol, pyrazinamide and rifampicin over an extended time frame.^1,2,10 Mild toxicity is associated with these multi-drug regimens, causing unwanted side effects such as nausea and vomiting. As a consequence, the strict adherence required of patients to prevent the development of resistance in surviving bacteria is often compromised, and patient compliance is thus difficult to achieve. To reduce the burden on patients there is a need for better, faster acting drugs, capable of inhibiting multi-drug resistant strains of bacteria.^4,5,11

We have recently reported a series of macrocyclic compounds with promising activity against Mtb, including compounds that are potent against drug-resistant strains and active in vivo in a zebrafish model (Danio rerio).^12,13 These compounds consist of hydrophobic aromatic pendant groups linked to a macrocyclic cyclam core (1–6, Fig. 1). A correlation was observed between bulkier, hydrophobic pendant aromatic groups and increased Mtb inhibition.

Fig. 1. Previously reported bis-substituted cyclam derivatives and their activity against Mtb strain H37Rv.^12,13.

Carboranes are a unique class of cluster compounds, consisting of boron and carbon atoms which form a robust polyhedral framework that is stabilized by σ-electron delocalization.^14,15 Dicarba-closo-dodecaborane (closo-carborane) is the most common type of carborane and it exists in three isomeric forms ortho (1,2-), meta (1,7-) and para (1,12-), which vary in the relative position of the two carbon atoms. These icosahedral closo-carborane cages possess excellent kinetic stability to hydrolysis and high hydrophobicity.^14,15 They are ca. 5.5 Å in diameter, which is slightly larger than a phenyl ring (approximately 4.7 Å),¹⁶ and can also occupy a greater volume than a rotating phenyl ring. As such, carboranes are sometimes used as bioisosteres for phenyl groups to tweak hydrophobicity and promote favourable intermolecular interactions, including unusual ‘dihydrogen’ bonding interactions, with a target biological receptor or enzyme. With this in mind, we set out to explore the potential of using bis-substituted cyclam derivatives conjugated to carborane cages as antitubercular agents.

Boron-containing moieties such as carboranes, boronic acids and esters, azaborines, and benzoxaboroles have previously been investigated as alternatives to traditional organic functional groups in order to improve the bioactivity of existing drugs or discover completely new agents in the treatment of disease.¹⁴ Several boron-containing compounds have been successfully introduced into the clinic, most notably the 26S proteasome inhibitor bortezomib (Velcade®), a well-established primary treatment for multiple myeloma,¹⁷ and more recently, tavaborole (Kerydin®), a topical antifungal agent for the treatment of onychomycosis, and crisaborole (Eucrisa®), used to treat mild to moderate atopic dermatitis. In contrast, there have been few studies to date that describe boron-containing antibacterial compounds. There are a small number of examples containing benzoxaboroles, diazaborines, carboranes, boronic acids and esters that exhibit inhibitory effects towards Escherichia coli,^18,19Staphylococcus aureus,^18–21Pseudomonas aeruginosa^18,21 and Enterococcus faecium.^21–23 There have been very few reports of boron-containing candidates that are active against Mtb,^24–26 with only three papers describing Mtb inhibitory compounds that contain boron clusters reported to date: a series of isoniazid–carborane derivatives,²⁵ dihydrofolate reductase inhibitors based upon pyrimidine antifolates,²⁷ and analogues of SQ109, an ethylenediamine derivative of the frontline drug ethambutol.²⁶

Herein we report a series of novel bis-substituted cyclam derivatives bearing carborane clusters which show very low micromolar activity against the highly-virulent Mtb strain H37Rv. High potency (MIC₅₀ 0.64–3.22 μM) was achieved for all new carborane compounds assayed, making these derivatives the most active of all bis-substituted cyclam derivatives assessed to date. Our work provides compelling evidence that incorporation of closo-1,2- and nido-7,8-carborane cages into bis-substituted cyclams can significantly enhance the bioactivity of this unique class of antibacterial agents.

Several methods were explored to determine the most efficient and highest yielding routes to carborane–cyclam conjugation. Direct carborane cage incorporation by the addition of nido-decaborane(14) to a terminal alkyne (Scheme 1), and C-vertex functionalization of carborane cages to facilitate common coupling reactions yielded three new closo-1,2-carborane conjugates with varying linkers. Regioselective deboronation of the closo-1,2-carborane cage using CsF in ethanol afforded three new nido-7,8-carborane derivatives with similar linkers.

Scheme 1. Synthesis of closo-1,2- (8, 9) and nido-7,8- (Cs₂·10) carboranyl–cyclam derivatives.

The direct reaction of nido-decaborane(14) with a terminal alkyne in the presence of CH₃CN is one effective route to substituted closo-1,2-carborane derivatives^28,29 and thus the reaction of a cyclam-appended alkyne with nido-decaborane(14) enabled cage formation with a minimal linker length (Scheme 1). The cyclam ring was appropriately functionalised prior to closo-carborane cage formation by means of bis-propargylation and Boc protection of the other macrocycle amines to afford compound 7, using conditions previously reported by Yu et al.³⁰ Standard reaction times for alkyne addition to nido-decaborane(14) typically require 4–5 hours, with yields ranging anywhere between 5–75%.^29,31 For the reaction of 7 with nido-decaborane, after heating at reflux for 24 hours to ensure conversion of both alkynes, compound 8 was isolated in 60% yield. A small quantity of the Lewis base CH₃CN is included in this reaction to afford the stabilised arachno adduct intermediate which is formed in situ after the loss of H₂ from nido-decaborane(14). Addition of the terminal alkyne and subsequent dissociation of CH₃CN resulted in the formation of a closo-1,2-carboranyl derivative 8, which was readily deprotected to afford hydrochloride salt 9. The products were characterised by ESI-MS and ¹¹B{¹H} NMR spectroscopy.

Closo-1,2-carborane cages (as seen in compounds 8 and 9) are stable under acidic conditions and are not susceptible to aerial oxidation. However, selective deboronation of a closo-1,2-carborane cage can be achieved in the presence of alkoxide or fluoride ions, leading to the expulsion of the most electron deficient BH unit (either B3 or the equivalent B6 in ortho isomers); this gives a nido-7,8-carborane bearing a formal negative charge which is delocalized across the entire cage. Closo-cage degradation of this type is often utilized to improve aqueous solubility, while maintaining many of the desired bulky characteristics of the cage. Thus, conjugate 9 was converted to the nido-7,8-carborane derivative by treatment with CsF in ethanol, yielding the ionic salt Cs₂·10. Cage conversion from closo to nido was confirmed spectroscopically in the ¹H NMR spectrum by the emergence of a broad peak at approximately −2 ppm, which corresponds to the two B–H–B hydrogen atoms located at the newly-opened face of the two nido-carborane cages.³² Deboronation of conjugate 9 to afford Cs₂·10 also corresponded with a significant and expected upfield shift of peaks in the ¹¹B{¹H} NMR spectrum, and the emergence of two new peaks at −33 and −37 ppm due to B10 and B1, respectively, consistent with data previously reported for other nido-7,8-carborane derivatives.³³ The formation of zwitterionic products (involving di-protonated macrocycles and anionic nido-carboranes) was ruled out by the detection of Cs⁺ counterions in the ICP-MS for all nido compounds Cs₂·10, Cs₂·15 and Cs₂·20.

Propargyl carborane 11 (ref. 34) was coupled to the bis-azido-cyclam derivative 12 (prepared using previously-reported methods³⁵) by a copper-catalyzed azide–alkyne cycloaddition using the Cu(ii)/sodium ascorbate conditions previously reported to be highly effective with this macrocyclic system.^30,35,36 Formation of the triazole linker was monitored by ¹H NMR spectroscopy with the emergence of the triazole C–H signal at 5.30 ppm, and disappearance of the terminal alkyne proton peak at 2.35 ppm. Coupled product 13 was deprotected using HCl in dioxane to afford the salt 14. Coupling of cages at both positions was confirmed by high resolution ESI-MS which showed a molecular ion peak [M + H]⁺ at m/z 703.69320 for 14 (m/z calculated for C₂₄H₅₉B₂₀N₁₀⁺ 703.69252). Closo-derivative 14 was converted in high yield to nido-7-8-analogue Cs₂·15 by treatment with CsF in ethanol (Scheme 2).

Scheme 2. Synthesis of ‘click’ coupled carborane–cyclam conjugates 14 and Cs₂·15.

To access alkene-linked derivatives 19 and Cs₂·20, the homoallyl carborane derivative 16 (prepared by reacting closo-1,2-carborane with n-butyllithium and then 4-bromo-1-butene)³⁷ was first coupled with allyl bromide by cross metathesis using Grubbs 2nd generation catalyst to afford compound 17. Alkylation of the bridged cyclam 18 with compound 17 and subsequent hydrolysis of the cyclam bridge resulted in the free-base conjugate 19 as a colourless powder. The alternative reaction sequence employing alkylation of 18 with allyl bromide, followed by cross metathesis, resulted in an intractable mixture of products, purification of which proved difficult. Compound 19 was observed exclusively as the (E) stereoisomer by ¹H NMR spectroscopy, with ³J_HH = 15.3 Hz indicative of the (E) alkene geometry. As described above for 9 and 14, treatment of 19 with CsF in ethanol afforded the nido-carborane derivative Cs₂·20 in good yield (Scheme 3).

Scheme 3. Synthesis of (E)-alkenyl-linked closo-1,2- (19) and nido-7,8- (Cs₂·20) carborane–cyclam conjugates.

Minimum inhibitory concentrations (MICs) were determined for bis-substituted cyclam–carborane conjugates (Table 1) using a modified resazurin viability assay.^12,13,38 Inclusion of the carboranyl moiety in place of the phenyl ring of compounds 3 and 6 leads to a significant enhancement of MICs from >50 μM for 6 to 3.22 μM for 14. All three closo-1,2-carborane conjugates displayed low micromolar activity (<4 μM) against the Mtb strain H37Rv, even with the variation in nature and length of the linker between macrocycle and carborane. Importantly, the high biological activity was preserved irrespective of whether a hydrophobic closo-carborane or anionic nido-carborane cage was used in the case of 9 and 10. This suggests that the interaction of the carboranes with the biomolecular target is primarily governed by the steric bulk of the carborane, and potentially the formation of dihydrogen bonds between one or more cage B–H groups and H–O/N groups of amino acid residues, and not solely the hydrophobicity of the closo-carborane cage. Examples of both closo-1,2- and nido-7,8 carborane derivatives adopting similar conformations within receptor sites have been reported previously with carbonic anhydrase IX and human dihydrofolate reductase,^39,40 and thus similar inhibitory effects, whilst unusual, are indeed possible. The Mtb form of the latter enzyme is a known isoniazid target and, despite their ability to engage the protein active site in a single rotational orientation, nido- and closo-carborane pyrimidine antifolate derivatives were found to display only moderate activity against the attenuated Mtb strain H37Ra (MIC > 46 μM).⁴⁰ In contrast, our cyclam derivatives 9, Cs₂·10, and 19 are highly potent against the more virulent Mtb strain H37Rv and, in the case of 9 and Cs₂·10, there was little difference found in H37Rv inhibition between the closo-1,2- and nido-7,8-carboranes. Intriguingly, the nido-7,8-carborane derivatives Cs₂·15 and Cs₂·20 were found to be less water soluble than their closo-carborane analogues under the assay conditions, likely attributable to their formation of insoluble salts with one or more anions found in the 7H9 growth medium (e.g. phosphate, citrate, l-glutamate, or sulfate), so an accurate assessment of their biological activities could not be made in this assay.

Antibacterial activity of selected carborane–cyclam conjugates against Mtb strain H37Rv.

Compound	MIC (μM)	Ref.
1	3.13	13
9	0.80	This work
Cs2·10	0.78
14	3.22
Cs2·15	nda
19	0.64
Cs2·20	nda
Rifampicin	0.06	Controls
Isoniazid	0.16

^and = not determined.

In this work, we have developed new synthetic routes to a range of closo-1,2- and nido-7,8-carborane–cyclam conjugates and identified a series of novel compounds with improved bioactivity against the virulent Mtb strain H37Rv. The question of how these compounds inhibit Mtb remains to be answered. We have previously reported that copper(ii) and other metal complexes of related cyclam ligands show similar levels of bioactivity against Mtb to the parent cyclam ligands.^12,13 This raises the possibility that these compounds may operate by perturbing cellular copper concentration as reported for other antimycobacterial agents,^41,42 although further investigation is required to test this hypothesis. Work continues to address these questions and to expand on the initial findings to develop new boron-based therapeutic agents with potent activity against a wide range of pathogenic bacteria. The results of this work will be reported in due course.

This work was supported by the National Health and Medical Research Council (NHMRC) Project APP1084266. NS was supported by a Research Training Program Scholarship from the Australian Government (Department of Education, Skills and Employment). The authors acknowledge the facilities and the scientific and technical assistance of staff within the Sydney Analytical Core Research Facility and the School of Chemistry at the University of Sydney. We acknowledge and pay respect to the Gadigal people of the Eora Nation, the traditional owners of the land on which we research, teach, and collaborate at the University of Sydney.

Conflicts of interest

There are no conflicts to declare.