Patent Application
20250205234
The present invention generally relates to anti-tubercular drugs and more particularly relates to anti-tubercular compositions for enhanced whole ATP synthesis inhibition and anti-tuberculosis activity.
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Tuberculosis (TB) is one of the major bacteria-caused infectious diseases that resulted in 1.5 million deaths in 2020, which for the first time in over a decade has increased according to the World Health Organization's 2021 Global TB report (WHO. Global Tuberculosis Report 2011; ISBN 978-92-4-003702-1; World Health Organization: Geneva, 2021). Multidrug resistant-(MDR) and extensively drug-resistant (XDR) bacterial strains of Mycobacterium tuberculosis (Mtb) which causes TB are spreading worldwide, causing major global health issues. Drug tolerance of Mtb is proposed to be due to the ability of the pathogen to enter a metabolically quiescent state, in which it is phenotypically tolerant to drug challenge with conventional chemotherapeutics (Gomez, J. E. & Mckinney, J. D. M., Tuberculosis (Edinb) 2004, 84, 29-44). In 2012, the anti-tuberculosis diarylquinoline bedaquiline (BDQ) was approved. BDQ kills with delay metabolically quiescent cells by targeting the mycobacterial F1F0 ATP synthase, and brought oxidative phosphorylation (OXPHOS) and the electron transport chain (ETC) complexes (
The mycobacterial F1F0 ATP synthase which is a latent ATPase and essential for proper growth and colony formation, consists of the subunits α3:β3:γ:ε:b-δ:b′:a:c9. Its H+-translocating FO domain (subunits a:c9) uses the proton motive force (PMF), generated by the ETC complexes NADH dehydrogenase (in case of NDH-1) and cytochrome bcc:aa3 (cyt-bcc:aa3), to drive rotation of the central stalk subunits γ-ε. The latter causes sequential binding, entrapment and phosphorylation of ADP to ATP within the nucleotide-binding and catalytic α3:β3-headpiece. The peripheral stalk subunits b-δ:b′ smoothen transmission of power between the rotary c-ring and the α3:β3:γ:ε domain.
Mycobacterial specific modifications of the F-ATP synthase subunits α, δ and γ, including a C-terminal elongation, an inserted domain or a 12-14 amino acids extra loop, respectively, have been described as regulative or essential features for catalysis. For example, the mycobacterial extra loop of subunit γ, absent in the human homologue and other prokaryotes, is important for ATP synthesis as well as ATP hydrolysis and ATP-driven proton pumping regulation, and has been identified as a new drug target. The most recent M. smegmatis F-ATP synthase cryo-electron microscopy (EM) structure shows a conformation in which one of the polar γ-loop residues forms a salt bridge with an arginine residue of the peripheral stalk subunit b′ during rotation (Montgomerya, M. G. et al., Proc. Natl. Acad. Sci. U.S.A 2021, 118, e2111899118).
Therefore, there exists a need for new compositions for specific targeting of the mycobacterial F-ATP synthase, and anti-TB potency in macrophages.
Aspects and embodiments of the invention will be discussed by reference to the following numbered clauses.
It has been surprisingly found that a F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F1 domain of the F1F0-ATP synthase can synergistically be used in combination with one or more other active agents to treat a bacterial infection. For example, it has been found that the combination of a F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F1 domain of the F1F0-ATP synthase (e.g. GaMF1.39) with an NADH Dehydrogenase inhibitor (e.g. CFZ), a cyt-bcc:aa3 inhibitor (e.g. Q203), and/or a F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F0 domain of the F1F0-ATP synthase (e.g. TBAJ876) showed enhanced whole ATP synthesis inhibition and anti-tuberculosis activity.
Thus, in a first aspect of the invention, there is provided a combination comprising:
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.
The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions.
As will be appreciated, the compounds of (i) differ from the compounds of (iv) in respect of the domain of the F1F0-ATP synthase that they bind to.
Any suitable F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F1 domain of the F1F0-ATP synthase may be used herein.
In particular embodiments of the invention that may be mentioned herein, the F1F0-ATP synthase inhibitor that selectively binds to the F1 domain of the F1F0-ATP synthase may be a compound of formula Ia or Ib:
For the absolute avoidance of doubt, the F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F0 domain of the F1F0-ATP synthase is not a compound of formula Ia or Ib, nor is it diarylquinoline bedaquiline. Any other F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F0 domain of the F1F0-ATP synthase may be used herein. For example, the F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F0 domain of the F1F0-ATP synthase may be TBAJ876:
References herein (in any aspect or embodiment of the invention) to compounds of formula Ia and formula Ib include references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula Ia or Ib with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula Ia or Ib in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
Examples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric acids.
Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
As mentioned above, also encompassed by formula Ia or Ib are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.
For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
“Pharmaceutically functional derivatives” of compounds of formula Ia or Ib as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula Ia or Ib.
The term “prodrug” of a relevant compound of formula Ia or Ib includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
Prodrugs of compounds of formula Ia or Ib may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula Ia or Ib wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula Ia or Ib is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.
Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. I-92, Elsevier, New York-Oxford (1985).
Compounds of formula Ia or Ib, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula Ia or Ib”.
Compounds of formula Ia or Ib may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
Compounds of formula Ia or Ib may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
Compounds of formula Ia or Ib may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
The terms “patient” and “patients” include references to mammalian (e.g. human) patients. As used herein the terms “subject” or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
The term “halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.
Unless otherwise stated, the term “aryl” when used herein includes C6-14 (such as C6-10) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. C6-14 aryl groups include phenyl, naphthyl and the like, such as 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.
Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, acyclic or cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably C1-10 alkyl and, more preferably, C1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl.
The term “heteroaryl” when used herein refers to an aromatic group containing one or more heteroatom(s) (e.g. one to four heteroatoms) preferably selected from N, O and S (so forming, for example, a mono-, bi-, or tricyclic heteroaromatic group). Heteroaryl groups include those which have between 5 and 14 (e.g. 10) members and may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic. However, when heteroaryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Heterocyclic groups that may be mentioned include benzothiadiazolyl (including 2,1,3-benzothiadiazolyl), isothiochromanyl and, more preferably, acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzoxadiazolyl (including 2,1,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro-2H-1,4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1,3-benzoselenadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1,2-a]pyridyl, indazolyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isoxazolyl, naphthyridinyl (including 1,6-naphthyridinyl or, preferably, 1,5-naphthyridinyl and 1,8-naphthyridinyl), oxadiazolyl (including 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl and 1,3,4-oxadiazolyl), oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroisoquinolinyl (including 1,2,3,4-tetrahydroisoquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl), tetrahydroquinolinyl (including 1,2,3,4-tetrahydroquinolinyl and 5,6,7,8-tetrahydroquinolinyl), tetrazolyl, thiadiazolyl (including 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl and 1,3,4-thiadiazolyl), thiazolyl, thiochromanyl, thiophenetyl, thienyl, triazolyl (including 1,2,3-triazolyl, 1,2,4-triazolyl and 1,3,4-triazolyl) and the like. Substituents on heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. Heteroaryl groups may also be in the N- or S-oxidised form. Particularly preferred heteroaryl groups include pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, thiophenyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzoimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pterdinyl. Particularly preferred heteroaryl groups include monocylic heteroaryl groups.
For the avoidance of doubt, references herein to compounds of formula Ia or Ib include, where the context permits, references to any of compounds of formula Ia or Ib. Further, references to any of compounds of formula Ia or Ib include references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
Further embodiments of the invention that may be mentioned include those in which the compound of formula Ia or Ib is isotopically labelled. However, other particular embodiments of the invention that may be mentioned include those in which the compound of formula Ia or Ib is not isotopically labelled.
The term “isotopically labelled”, when used herein includes references to compounds of formula Ia or Ib in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to “one or more positions in the compound” will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula Ia or Ib. Thus, the term “isotopically labelled” includes references to compounds of formula Ia or Ib that are isotopically enriched at one or more positions in the compound.
The isotopic labelling or enrichment of the compound of formula Ia or Ib may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 35S, 18F, 37Cl, 77Br, 82Br and 125I).
When the compound of formula Ia or Ib is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula Ia or Ib that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.
In particular embodiments that may be mentioned herein, in formula Ia, X may be N and Y may be NH. In further embodiments that may be mentioned herein, in Formula Ib, X may be NH and Y may be N.
In embodiments of the invention that may be mentioned herein:
In embodiments of the invention that may be mentioned herein, the F1F0-ATP synthase inhibitor or a pharmaceutically acceptable salt or solvate thereof that selectively binds to the F1 domain of the F1F0-ATP synthase is selected from the group consisting of:
As will be appreciated, any suitable combination of the active ingredients set out herein may be used. Thus, in embodiments of the invention the combination may comprise:
Any suitable NADH dehydrogenase inhibitor or a pharmaceutically acceptable salt or solvate thereof may be used herein. For example, the NADH dehydrogenase inhibitor or a pharmaceutically acceptable salt or solvate thereof may be clofazimine.
Any suitable cytochrome-bcc:aa3 inhibitor or a pharmaceutically acceptable salt or solvate thereof may be used herein. For example, the cytochrome-bcc:aa3 inhibitor or a pharmaceutically acceptable salt or solvate thereof may be Q203:
The combination disclosed herein may be presented as a single pharmaceutical formulation (containing all of the active ingredients in one formulation) or as a kit of parts comprising two or more pharmaceutical formulations (each formulation containing at least one active ingredient).
Thus, in aspects of the invention, there is provided:
The combinations, the pharmaceutical compositions and kits of parts described herein may be useful in the treatment of subjects who are suffering from a bacterial infection. Therefore, in a further aspect of the invention, there is disclosed a use of a combination as described herein, a composition as described herein or a kit of parts as described herein in medicine.
In further aspects of the invention, there is disclosed:
In particular embodiments of the uses, methods, combinations for use, compositions for use and kit of parts for use, the bacterial infection may be tuberculosis.
Further aspects and embodiments of the invention are listed in the following numbered Statements.
and tautomers thereof.
Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.
M. smegmatis mc2 155 (ATCC 35734) and M. bovis BCG (ATCC 700084) were gifted from from Prof. Thomas Dick lab. (Department of Microbiology and immunology, Yong Loo Lin School of medicine, National University of Singapore, Singapore). M. tuberculosis H37Rv was gifted from Prof. Thomas Dick lab. (Department of Microbiology and immunology, Yong Loo Lin School of medicine, National University of Singapore, Singapore). P. aeruginosa PAO1 WT, E. coli UTI 189 was gifted from Prof. Scott Rice, Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore. The cultures were maintained in complete Middlebrook 7H9 medium (Sigma-Aldrich) supplemented with 0.5% (vol/vol) glycerol (Promega), 0.05% Tween-80 (Sigma-Aldrich), and 10% Middlebrook albumin-dextrose-catalase (ADC) (Sigma-Aldrich). Corning T-25 mm2 tissue culture flasks, 96-well clear flat bottom polystyrene microplates and 96-well white half area microplate were used for the experiments. Bacterial colonies were grown on Middlebrook 7H10 agar (Sigma-Aldrich) supplemented with 0.5% (vol/vol) glycerol (Promega), 0.05% Tween-80 (Sigma-Aldrich), and 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC) (Sigma-Aldrich). Bac titer glo and Cell titer glo were purchased from Promega. The following reagents like MOPS ((N-Morpholino) propane sulfonic acid), ADP (Adenosine diphosphate), NADH (Nicotinamide adenine dinucleotide), Succinic acid, Potassium phosphate, Clofazamine, DMSO (dimethyl sulfoxide), phosphate-buffered saline (PBS), ACMA (9-Amino-6-chloro-2-methoxyacridine), Thiorodazine, Tyrphostin A9, M9 salts, MgSO4, CaCl2), 0.4% w/v glucose, Terfenadine/tolbutamide, ACN, MeOH, CFZ were obtained from Sigma-Aldrich. Ananerobic atmosphere generation packets for oxygen consumption assays were purchased from Sigma. Bedaquiline (BDQ) and Telacebec (Q203) were purchased from MedChem Express. Compound TBAJ-876 was synthesized by Bioduro LLC (Beijing, China).
M. smegmatis mc2 155 and M. bovis BCG were cultured in complete 7H9 medium and grown to mid-exponential phase. Cells were inoculated to an initial optical density of 0.005. For broth culture, M. smegmatis was incubated at 37° C. under shaking at 80 rpm for 2-3 days in T-25 mm2 tissue culture flasks. For M. bovis BCG, the culture was incubated at 37° C. under shaking at 80 rpm for 5 days in T-25 mm2 tissue culture flasks. To count colony forming units (CFU ml-1), each culture was serially diluted in phosphate-buffered saline (PBS) (pH 7.0) and spotted on to agar plates.
GaMF1.39 was synthesized according to Hotra, A. et al., Angewandte Chemie Int. Ed. 2020, 59, 13295-13304.
To a suspension of 2,4-dichloro-6-methylpyrimidine (5.0 g, 30.7 mmol) in EtOH (50 mL) was added ethylamine hydrochloride (2.5 g, 30.7 mmol) and di-isopropylethylamine (2.5 eq.). The solution was heated at 50° C. for 24 hours before the reaction mixture was concentrated in vacuo to give a light yellow oil. The crude material was taken up in minimal amount of dichloromethane, adsorbed onto silica gel and purified by silica gel flash chromatography (0-100% EtOAc/Hexanes) to afford compound 2-chloro-N-ethyl-6-methylpyrimidin-4-amine as a light yellow solid (1.74 g, 33% yield). 1H NMR (400 MHZ, CDCl3) δ 6.06 (s, 1H), 5.06 (br. s, 1H), 3.32 (s, 2H), 2.33 (s, 3H), 1.24 (t, J=7.2 Hz, 3H); MS (ESI) m/z 172.1 [C7H1035ClN3+H]+, 174 (37Cl M++H+, 31); HRMS calcd for C7H11N335Cl (M++H+) 172.0642; found 172.0649 (4.1 PPM); FTIR (nujol, cm-1) vmax 3250, 1600, 968; mp: 74-75° C.
A mixture of 4-chlorobenzoic acid (292 mg, 1.87 mmol) and 4-nitrobenzene-1,2-diamine (300 mg, 1.96 mmol, 1.05 eq) in polyphosphoric acid (4 mL) was stirred at 140° C. for 4 hours. The reaction was quenched by pouring into water (5 mL) and adjusted to pH 7 with 10 N NaOH solution. The precipitate was filtered and dried in vacuo to give a black residue. The crude product was purified by silica gel flash chromatography (0-40% EtOAc/Hexanes) to afford compound 2-(4-chlorophenyl)-5-nitro-1H-benzo[d]imidazole as a light green solid (142 mg, 28% yield). 1H NMR (400 MHZ, DMSO-d6) δ 13.7 (br. s, 1H), 8.49 (s, 1H), 8.23 (d, J=6.8 Hz, 2H), 8.14 (dd, J=8.8, 2.4 Hz, 1H), 7.78 (d, J=8.4 Hz, 1H), 7.69 (d, J=8.4 Hz, 2H); MS (ESI) m/z 274.0 [C13H8ClN3O2+H]+.
A mixture of 2-(4-chlorophenyl)-5-nitro-1H-benzo[d]imidazole (132 mg, 0.482 mmol), iron powder (269 mg, 4.82 mmol, 10 eq.) and NH4Cl (258 mg, 4.82 mmol, 10 eq.) in 4:1 EtOH/water (5 mL) was heated at 80° C. for 5 hours. The mixture was filtered through a pad of celite, washing with MeOH. The filtrate was concentrated and the residue was taken up in water (10 mL) and extracted with EtOAc (3×5 mL). The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo give 2-(4-chlorophenyl)-1H-benzo[d]imidazol-5-amine as a brown oil (87.6 mg, 75% yield) which was used in the next step without further purification. 1H NMR (400 MHZ, CDCl3) δ 7.91 (d, J=8.4 Hz, 2H), 7.48-7.42 (m, 3H), 6.84 (s, 1H), 6.69 (dd, J=8.4, 2.0 Hz, 1H); MS (ESI) m/z 244.0 [C13H10ClN3+H]+.
A solution of 2-chloro-N-ethyl-6-methylpyrimidin-4-amine (35.0 mg, 0.204 mmol) and 2-(4-chlorophenyl)-1H-benzo[d]imidazol-5-amine (74.5 mg, 0.306 mmol, 1.5 eq.) in n-BuOH (1 mL) was heated in a microwave reactor at 180° C. for 2 hours. The reaction mixture was azeotroped with toluene to give a white residue which was purified by preparative HPLC (20-50% MeCN/H2O; 0.1% formic acid) to afford desired compound (formate salt) as an off-white solid upon lyophilisation (12.0 mg, 15.5% yield). 1H NMR (400 MHZ, DMSO-d6) δ 12.6 (br. s, 1H), 9.00 (s, 1H), 8.28 (s, 1H), 8.14 (d, J=8.8 Hz, 2H), 7.59 (d, J=8.8 Hz, 2H), 7.45 (q, J=8.4 Hz, 2H), 6.98 (br. s, 1H), 5.78 (s, 1H), 3.37 (br. s, 2H), 2.14 (s, 3H), 1.18 (t, J=7.2 Hz, 3H); 13C NMR (100 MHZ, DMSO-d6) δ 163.2, 163.0, 159.6, 133.8, 129.3, 129.1, 128.9, 127.8, 127.7, 34.9, 23.3, 14.7; MS (ESI) m/z 379.1 [C20H19ClN6+H]+; HRMS (ESI) m/z calc'd for [C20H19ClN6+H]+ 379.1433, found 379.1428; Melting point=142-143° C.
The growth inhibition dose-response assay was carried out using the broth microdilution method as described previously (Moreira, W., Aziz, D. B. & Dick, T., Front. Microbiol. 2016, 7, 199). Plates with the M. bovis BCG strain were incubated at 37° C. on an orbital shaker set at 80 rpm for 5 days. At the end of the incubation period, the culture in all wells was manually resuspended and the OD600 was read using a TECAN Infinite Pro 200 plate reader. The MIC50 reported represents the concentration that inhibits 50% of growth compared to drug free control.
Preparation of M. smegmatis IMVs, ATP synthesis and ATP-driven proton translocation assays were performed according to Hotra, A. et al., FEBS J. 2016, 283, 1947-1961.
Recombinant mycobacterial F-ATP synthase was reconstituted according to Harikishore, A. et al., ACS Chemical Biol. 2022, 17, 529-535. For inhibitor studies with GaMF1.39 (90- and 180 nM) or BDQ (4 nM), proteoliposomes containing the reconstituted F-ATP synthase were additionally preincubated for 10 min at 4° C. with the respective inhibitor, before the ATP synthesis measurements were carried out.
Methylene blue assay Aliquots of 1.5 ml (OD600=0.3), log-phase cultures of M. bovis BCG were transferred into clear 2 ml screw-cap glass vials along with test compounds. Thiorodazine was used as a positive control. The cultures were incubated in the presence of the drugs for 96 h at 37° C. before the addition of methylene blue dye at a final concentration of 0.001%. Upon addition of methylene blue, all vials were tightly closed and incubated at 37° C. in a hypoxic jar. Oxygen in the jar was removed by Anaerobic atmosphere Generation (AnaeroGen) sachet. The dye decolorizes when the oxygen in the culture is used up. The color changes and the differences in the exponentially growing cultures and broth reflect the relative oxygen consumption, thus indirectly indicate the respiration of each culture.
ATP-driven proton translocation into IMV's of M. smegmatis was measured by a decrease of 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence using a Cary Eclipse
Fluorescence spectrophotometer (Varian Inc., Palo Alto) according to Haagsma, A. C. et al., FEMS microbiology letters, 2010, 313, 68-74. IMV's (0.18 mg per ml) were preincubated at 37° C. in 10 mM HEPES-KOH (pH 7.5), 100 mM KCl, 5 mM MgCl2 containing 2 μM ACMA and a baseline was monitored for 5 min. The reaction was started by adding 2 mM ATP, or 2 mM NADH. After about 8 min, any proton gradient was collapsed by the addition of 2 μM of the uncoupler SF6847. The excitation and emission wavelengths were 410 nm and 480 nm, respectively.
As shown by the minimal inhibitory concentration (MIC50) of 6.8 μM (
Since GaMF1.39 lacked the ability to induce uncoupled proton pumping (
Bacterial Killing Assay of M. smegmatis mc2 155
M. smegmatis mc2 155 cultures were grown to exponential phase and diluted to an OD600 Of 0.005 and aliquoted onto T-25 mm2 tissue culture flasks. Test compounds were dispensed into each flask and were incubated at 37° C. with shaking at 180 rpm for 5 days. Approximately 10 μl of culture was taken out from each flask, followed by the serial dilution with PBS. 25 μl of cultures of respective dilutions were plated on each quadrant of 7H10 agar plates. The agar plates were incubated at 37° C. for 3 days. Bacterial viability was determined by counting the colony-forming units (CFU).
Bacterial Killing Assay of M. bovis BCG
M. bovis BCG cultures were grown to exponential phase and diluted to an OD600 of 0.005 and aliquoted onto T-25 mm2 tissue culture flasks. Test compounds were dispensed into each flask and were incubated at 37° C. with shaking at 110 rpm for 6 days. Approximately 10 μl of culture was taken out from each flask, followed by the serial dilution with PBS. 25 μl of cultures of respective dilutions were plated on each quadrant of 7H10 agar plates. The agar plates were incubated at 37° C. for 10 days. Bacterial viability was determined by counting the colony-forming units (CFU).
Zebrafish care and ethics statement: All Zebrafish experiments were approved by the NTU Institutional Animal Care and Use Committee under reference no #A20038. Experiments were done using wild-type AB zebrafish. The ages of the embryos are shown in hours post fertilization (hpf).
Dechorionated zebrafish wild-type embryos at 48 hpf (n=120) were used. The embryos were immersed respectively in 5 ml of E3 medium containing GaMF1.39 in 6-well plate for 5 days. DMSO was used as vehicle control. The embryos were observed under a microscope. Abnormal phenotypes and survival rate from each treatment group were observed for 5 sequential days. The experiment was repeated twice with three replicates each.
Biofilm assay was performed in a 24-well plate to evaluate the activity of BDQ and GaMF1.39 on P. aeruginosa PAO1 WT and E. coli UTI 189 biofilms. Overnight cultures of P. aeruginosa PAO1 WT and E. coli UTI 189 were diluted in M9 glucose media (1×M9 salts, 2 mM MgSO4, 0.1 mM CaCl2), 0.4% w/v glucose) to a final OD600 of 0.05. 1 ml of the diluted culture was then added into each well of the 24-well plate and incubated at 37° C. with 100 rpm shaking. Following 3 h of incubation, each well was washed once, and the culture media was replaced with 1 ml of 1×PBS (pH 7.4) or fresh M9 glucose medium containing 1-3 μM of BDQ or 3-9 μM of GaMF1.39. The treated samples were further incubated under the same conditions for 3 h. At t=6 h, the samples were collected. The 1×PBS (pH 7.4) buffer containing suspended bacteria cells with treatment was collected into 1.5 ml Eppendorf tubes and considered to be “planktonic samples”. Subsequently, each well was washed once with 1 ml 1×PBS (pH 7.4) before resuspending biofilm cells in the same volume of 1×PBS (pH 7.4). Biofilm cells were dislodged into the buffer by means of a cell scraper and 1 ml of the sample was collected into 1.5 ml Eppendorf tubes and labelled “biofilm samples”. The samples contained in Eppendorf tubes were sonicated in a water bath using the following settings: 5 min degas mode, 37 Hz, 100%, followed by 5 min pulse mode, 37 Hz, 100%. Subsequently, the samples were serially diluted and used for CFU counts. The experiment was repeated independently at least two times, with two technical replicate per independent experiment. CFU counts were analyzed using Graphpad Prism V9.3.0 using 2-way ANOVA and multiple comparison of column effect (concentration of compound against untreated control) within each row (planktonic vs biofilm samples).
Microsomal stability assay were outsourced and performed by Bioduro-Sundia Ltd, USA based on the following protocol as done in Obach, R. S., Drug Metab. Dispos., 1999, 27, 1350; McGinnity, D. F. & Riley, R. J., Biochem. Soc. Trans., 2001, 29, 135. Working solutions of each compound are prepared from 10 mM stock solution in DMSO diluted to a final concentration of 100 μM in 0.05 M phosphate buffer (pH 7.4). Aliquots of Liver Microsome working solution are transferred into 1.1 mL tubes using a multichannel pipette. Positive control (5 mixed) and test compound working solutions are transferred into the tubes. The mixtures are vortexed gently and then pre-incubated at 37° C. 5 mM NADPH were aliquoted into the tubes using a multichannel pipette and vortexed gently. At each time point of 0 min, 5 min, 15 min, 30 min and 60 min with NADPH or 0, 30 min and 60 min without NADPH, an aliquot is removed from each tube. Terfenadine/tolbutamide in ACN/MeOH (1:1, v/v) is added to quench and precipitate the microsomal incubations. Samples are capped and vigorously vortexed and then centrifuged at 4° C. An aliquot of each supernatant is transferred for LC-MS/MSC analysis. The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100 Å column (3.0 mm×30 mm, Phenomenex). Mobile phase—Solvent A: water with 0.1% formic acid, solvent B: ACN with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area) for each time point. The estimation of Clint (in μL/min/mg protein) is calculated using the following equation: CLint (μLmin−1 mg−1)=ln(2)*1000/t1/2/protein concentration
The cLogP values were calculated using ChemDraw software Version 22.
The killing efficiency of GaMF1.39 against M. bovis BCG was tested at 8-fold its MIC50, which is shown clearly in flasks and on 7H10 agar plates (
To explore the anti-TB potency of the compound in macrophages, a THP-1 infection model was used. As shown in
Here, we present a detailed antimycobacterial activity study of the bactericidal GaMF1.39 analogue, evidence for its specific targeting of the mycobacterial F-ATP synthase, and anti-TB potency in macrophages, without altering biofilm formation or being toxic to zebrafish larvae.
A checkerboard titration assay was carried out as described previously (Hsieh, M. H. et al., Diagn. Microbiol. Infect. Dis. 1993, 16, 343-349; and Kaushik, A. et al., Antimicrob. Agents Chemother. 2015, 59, 6561-6567). Briefly, GaMF1.39 which was synthesized according to Hotra, A. et al., Angewandte Chemie Int. Ed. 2020, 59, 13295-13304, Q203, and CFZ, respectively, were added to complete 7H9 medium-containing 96-well flat-bottom Costar cell culture plates. Two-fold serial dilutions were done to allow 10 different concentrations of GaMF1.39 (0.1 μM to 60 μM) to be tested for interaction with 14 different concentrations Q203 (0.4 pM to 8 nM). Hence, a total of 140 different concentration combinations were tested between GaMF1.39 and Q203. In combination studies with CFZ, 10 different concentrations of GaMF1.39 (0.1 μM to 60 μM) were tested with 7 different concentrations of CFZ (0.03 μM to 2 μM). Each 96-well plate had a 7H9 medium-only control well and a drug-free bacterial culture control well. M. bovis (BCG) was cultured in complete 7H9 medium and grown to mid-exponential phase. Subsequently, the culture was diluted to an OD600 of 0.01 using complete 7H9 medium and added to each well in the 96-well plate to create a final OD600 value of 0.005. The plates were incubated for 6 days at 37° C. After the incubation period, the culture in each 96-well plate was manually resuspended, and the OD600 of each well was read using a Tecan Infinite Pro 200 plate reader. Calculation of the fractional inhibitory concentration index (FICI) was done to analyze the results. The FICI is calculated as (MIC of drug A in combination/MIC of drug A alone)+ (MIC of DARQ B in combination/MIC of DARQ B alone). This calculation was done only for wells which showed 50% inhibition of bacterial culture growth compared to drug-free bacterial culture wells. A FICI of ≤0.5 indicates synergy, a FICI of >0.5 to 4 indicates additivity (no interaction), and a FICI of >4 indicates antagonism (Odds, F. C., J. Antimicrob. Chemother. 2003, 52, 1).
Here, we investigated the pharmacodynamic influence of GaMF1.39 with CFZ, effecting the first complex of the ETC. As revealed in
Growth inhibition of the regimen comprising GaMF1.39+Q203 or GaMF1.39+TBAJ-876 correlated well with the depletion of intracellular ATP formed in the presence of the combinations (
Because of the lower FIC index (0.55) of the GaMF1.39+Q203 compared to the GaMF1.39+CFZ combination, we tested whether the increase in growth reduction of the GaMF1.39+Q203 regimen (
Taken together, the enzymes of the ETC are responsible for recycling of the reduced electron carriers NADH and FADH2 from central carbon metabolism, thereby facilitating redox balance, generating a PMF essential for maintaining transmembrane electrochemical gradients to regulate PMF-driven pumps and to drive ATP synthesis via OXPHOS (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202203339W | Apr 2022 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2023/050218 | 3/31/2023 | WO |
