Compounds for the treatment of tuberculosis

Abstract

The present invention relates to a compound having the formula (Ia) or (Ib) wherein R1 is hydrogen or methyl; R2 is unsubstituted or substituted alkyl; R3 is aryl or heteroaryl, which is optionally substituted by one or more groups selected from halogen, alkyl or alkoxy; and, in formula (Ia), X is CH or N and Y is NH, S or O, or in formula (Ib), X is NH, S or O and Y is CH or N. The present invention further relates to a method for synthesizing the compounds of the present invention, a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and bedaquiline (BDQ), an analogue of bedaquiline (BDQ) or a mixture thereof, and the use of said composition or compound for the treatment of tuberculosis.

Description

Technical Field

[0001] The present invention relates to compounds and compositions for the treatment of tuberculosis. Background Art

[0002] The following discussion of background art is intended to facilitate an understanding of the present invention. However, it should be understood that the discussion is not an admission or recognition that any of the materials mentioned was published, known, or part of common general knowledge in any jurisdiction as of the priority date of the present application.

[0003] Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. There is a need for new treatment strategies to address the widespread prevalence of TB and the spread of multi-drug resistant (MDR) and extensively drug-resistant (XDR) forms of TB, which remain a serious public health challenge worldwide.

[0004] Bedaquiline (BDQ; ) is an anti-tuberculosis compound belonging to the chemical class of diarylquinolines. However, despite the clinical success of BDQ, clinical resistance to BDQ has been reported in patients with extensively drug-resistant tuberculosis (XDR-TB).

[0005] WO 2018 / 151681 A2 relates to specific pyrimidine compounds for the treatment of tuberculosis and compositions containing them.

[0006] There is a continuing need to develop additional compounds or compositions for the treatment of tuberculosis. Summary of the Invention

[0007] In one aspect of the present invention, there is provided a compound having formula (Ia) or (Ib)

[0008]

[0009]

[0010] wherein

[0011] R1 is hydrogen or methyl;

[0012] R2 is unsubstituted or substituted alkyl;

[0013] R3 is aryl or heteroaryl, optionally substituted with one or more groups selected from halogen, alkyl, or alkoxy; and,

[0014] in formula (Ia), X is CH or N and Y is NH, S, or O, or,

[0015] In formula (Ib), X is NH, S or O and Y is CH or N.

[0016] In a preferred embodiment of the present invention, in formula (Ia), X is N and Y is NH, or in formula (Ib), X is NH and Y is N.

[0017] More preferably, R1 is a methyl group at the 6-position of the pyrimidine ring.

[0018] Preferably, R2 is an ethyl group or a -CH2COOCH2CH3 group.

[0019] In another preferred embodiment of the present invention, R3 is an aryl group.

[0020] Most preferably, the aryl group is substituted with one or more halogen atoms.

[0021] Specifically, the compound is selected from the group consisting of

[0022]

[0023]

[0024]

[0025] and its tautomers.

[0026] In another aspect, the present invention relates to a composition comprising a compound according to the present invention or a pharmaceutically acceptable salt thereof and bedaquiline (BDQ), an analogue of bedaquiline (BDQ) or a mixture thereof.

[0027] Preferably, the analogue of bedaquiline (BDQ) comprises a racemate of a compound having formula (V):

[0028] Or

[0029] a racemate of a compound having formula (VI)

[0030]

[0031] In another aspect, the present invention relates to a compound according to the present invention for use in therapy.

[0032] In another aspect, the present invention relates to a compound according to the present invention for the treatment of bacterial infections.

[0033] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0034] In another aspect, the present invention relates to the use of a compound according to the present invention in the manufacture of a medicament for the treatment of bacterial infections.

[0035] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0036] In another aspect, the present invention relates to a composition according to the present invention for use in therapy.

[0037] In another aspect, the present invention relates to a composition according to the present invention for the treatment of bacterial infections.

[0038] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0039] In another aspect, the present invention relates to the use of a composition according to the present invention in the manufacture of a medicament for the treatment of bacterial infections.

[0040] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0041] In another aspect, the present invention relates to a method of treating a subject suffering from a bacterial infection, the method comprising the step of administering to the subject a therapeutically effective amount of a compound or composition according to the present invention.

[0042] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0043] In another aspect, the present invention relates to a method for synthesizing a compound according to the present invention, wherein X is NH and Y is N, or X is N and Y is NH, the method comprising the following steps:

[0044] (a) Reacting 4-nitrobenzene-1,2-diamine with unsubstituted or R3-substituted benzoic acid in the presence of an acid;

[0045] (b) Reducing the nitro group to obtain the corresponding amine derivative; and

[0046] (c) Reacting the amine derivative of step (b) with 2-chloro-pyrimidin-4-amine substituted with R1 and R2. Description of the Drawings

[0047] Figure 1:(A) Growth inhibition of Mycobacterium tuberculosis H37Rv by the compound of formula (II) (named GaMF1.39). (B) Growth inhibition of Mycobacterium bovis BCG cells by the compound of formula (II) (named GaMF1.39). (C) Killing kinetics of BDQ and the compound of formula (II) against Mycobacterium smegmatis mc 2 155. In 10x MIC 50 of the compound of formula (II) and 200x MIC 50 of BDQ, bacteria were grown in liquid broth (LBT). Samples of the bacterial cultures were collected at different time points (from t = 0 days until t = 4 days) and plated onto Middlebrook 7H11 agar plates. The plates were incubated at 37 °C for 3 days until colonies appeared.

[0048] Figure 2 :(A) Inhibition of intracellular ATP synthesis in Mycobacterium bovis BCG cells by the compound of formula (II) (named GaMF1.39) compared to BDQ and the compound of formula (VI) (named BDQ1). Inhibition of ATP synthesis by the compound of formula (II) in inside-out membrane vesicles (IMV), from Mycobacterium smegmatis having an IC for Mycobacterium smegmatis of 90 nM 50 (B) and an IC of 8.7 nM for Mycobacterium bovis BCG IMV 50 .

[0049] Figure 3 : 300 nM of the compound of formula (II) (named GaMF1.39) increased the potency of the racemate of the compound of formula (V) in ATP synthesis inhibition (Δ) when compared to the inhibition by this racemate alone (▲).

[0050] Figure 4 : Synergistic effect of the compound of formula (II) (named GaMF1.39) and the racemate of the compound of formula (VI) (named BDQ1) on ATP synthesis inhibition in IMV of Mycobacterium smegmatis. When the compound of formula (II) was added in a concentration-dependent manner to the racemate of the compound of formula (VI) (300 nM), ATP synthesis was significantly reduced to almost complete inhibition.

[0051] Figure 5: Synergistic effect of the compound of formula (II) (named GaMF1.39) and the compound of formula (VII) (named BDQ) on the inhibition of ATP synthesis in the IMV of Mycobacterium smegmatis. When the compound of formula (II) was added to the racemate (300 nM) of the compound of formula (VII) in a concentration-dependent manner, the ATP synthesis was significantly reduced to almost complete inhibition. Detailed Description

[0052] Specific embodiments of the present invention will now be described with reference to the accompanying drawings. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention. In addition, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0053] Throughout this specification, unless the context requires otherwise, the terms "comprising", "consisting of", etc. shall be construed as non-exhaustive, or in other words, meaning "including but not limited to".

[0054] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean including the stated features or groups of features, but not excluding any other features or groups of features.

[0055] Throughout this specification, unless the context requires otherwise, the word "include" or variations such as "includes" or "including" will be understood to mean including the stated features or groups of features, but not excluding any other features or groups of features.

[0056] As used herein, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0057] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid.

[0058] As used herein, the terms "treatment", "treat" and "therapy" and their synonyms refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down (mitigate) TB. Those in need of such treatment include those who have already been infected with TB and those who are susceptible to TB infection, or those to be protected from TB infection.

[0059] As used herein, the "therapeutically effective amount" of a compound is the amount of the active agent that is capable of preventing or at least slowing down (mitigating) TB. The dosage and administration of the compounds, compositions and formulations of the present invention can be determined by those of ordinary skill in the art of clinical pharmacology or pharmacokinetics. See, for example, Mordenti and Rescigno, (1992) Pharmaceutical Research. 9:17-25; Morenti et al., (1991) Pharmaceutical Research. 8:1351-1359; and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al. (eds) (Pergamon Press: New York, 1989), pp. 42-96. The effective amount of the compounds, compositions and formulations of the present invention to be used for treatment will depend, for example, on the treatment objective, the route of administration and the condition of the patient. As used in the specification herein, the term "patient" includes humans and animals. Thus, it will be necessary for the treating physician to determine (titer) the dosage and vary the route of administration as needed to obtain the best therapeutic effect. Typical daily doses can range from about 1 μg / kg / day to about 50 mg / kg / day or more of the patient's body weight, about 1 mg / kg / day to about 50 mg / kg / day, about 1 mg / kg / day to about 10 mg / kg / day, preferably about 1 μg / kg / day to about 10 mg / kg / day.

[0060] As used herein, the term "alkyl" means a branched or unbranched saturated hydrocarbon group having from 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl. The alkyl group can be cyclic or acyclic. The alkyl group can also be substituted or unsubstituted. As described herein, for example, the alkyl group can be substituted with one or more groups including but not limited to optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxy, nitro, silyl, sulfo-oxo, or mercapto. In a preferred embodiment, the term "alkyl" means a branched or unbranched alkyl group containing from 1 to 6 carbon atoms. In an even more preferred embodiment, the term "alkyl" means a branched or unbranched alkyl group having 1 to 4 carbon atoms.

[0061] Throughout this specification, the term "alkyl" is generally used to refer to both unsubstituted alkyl and substituted alkyl; where appropriate, substituted alkyl is also specifically referred to herein by identifying one or more specific substituents on the alkyl group. For example, the term "haloalkyl" specifically refers to an alkyl group substituted with one or more halogen atoms (e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups. The term "azaalkyl" specifically refers to an alkyl group in which at least one carbon is replaced by nitrogen. The term "oxaalkyl" specifically refers to an alkyl group in which at least one carbon is replaced by oxygen.

[0062] As used herein, the terms "alkoxy" and "alkoxyl" refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an "alkoxy" group can be defined as —OA1, where A1 is an alkyl or cycloalkyl group as defined above. "Alkoxy" also includes oligomers of alkoxy as just described; that is, alkoxy can be a polyether, such as -OA1-OA2 or -OA1—(OA2) a —OA3, where "a" is an integer from 1 to 200, and A1, A2, and A3 are alkyl and / or cycloalkyl groups.

[0063] As used herein, the terms "derivative" or "analogue" refer to a compound having a structure similar or related to the compound to which the term is applied.

[0064] As used herein, the bond of a substituent on a ring structure does not point to its specific position but to the center of the ring structure, meaning that the substituent can be attached to any possible position on the ring structure. As an example, R1 of formula (Ia) or formula (Ib) can be attached to any one of the possible positions on the pyrimidine ring, i.e., to its 5- or 6-position.

[0065] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as limiting the scope of the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as the individual values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. The range is not limited to integers and may include fractional measurements. This applies regardless of the breadth of the range.

[0066] For those of ordinary skill in the art, after reading the following description of the specific embodiments of the present invention in conjunction with the accompanying drawings, other aspects of the present invention will become apparent.

[0067] In one aspect of the present invention, there is provided a compound having the formula (Ia) or (Ib)

[0068]

[0069]

[0070] wherein

[0071] R1 is hydrogen or methyl;

[0072] R2 is unsubstituted or substituted alkyl;

[0073] R3 is aryl or heteroaryl, optionally substituted by one or more groups selected from halogen, alkyl, or alkoxy; and,

[0074] in formula (Ia), X is CH or N and Y is NH, S, or O, or,

[0075] in formula (Ib), X is NH, S, or O and Y is CH or N.

[0076] Advantageously, the compounds of the present invention target F-ATP synthase. F1F0 ATP synthase (F-ATP synthase) is one of the essential enzymes that meet the energy requirements of both the proliferative aerobic and hypoxic dormant phases of the mycobacterial life cycle. The enzyme is composed of nine stoichiometric subunits α3:β3:γ:δ:ε:a:b:b’:c9 and is divided into a membrane-embedded F0 domain (a:b:b’:c9) and a water-soluble F1 portion (α3:β3:γ:δ:ε). The F1 domain contains three catalytic αβ-pairs that form an α3:β3 hexamer, in which ATP synthesis or ATP hydrolysis occurs. The catalytic α3:β3-top is connected to the ion-pumping F0 portion via two central stalk subunits γ, ε and a peripheral stalk. The F0 domain contains subunits a, b and b’ and a ring structure composed of 9 c subunits. It is proposed that the rotational movement of the c-ring triggers the rotation of the central subunits γ and ε, resulting in consecutive conformational changes of the nucleotide-binding subunits α and β, followed by the synthesis of ADP + Pi into ATP.

[0077] It has been shown that F-ATP synthase is very important for the optimal growth of Mycobacterium smegmatis and Mycobacterium tuberculosis (Mtb) (the latter causes TB). This is different in other prokaryotes and eukaryotes (i.e., humans), where the enzyme is not necessary for growth on fermentable carbon sources and where increased glycolytic flux can compensate for the loss of oxidative phosphorylation. This difference is attributed to the very high amount of ATP required to synthesize mycobacterial cells. The uniqueness of the mycobacterial F-ATP synthase also lies in its lack of proton translocation ability and its low or latent ATPase activity in the fast or slow growth forms, respectively.

[0078] The clinical success of BDQ (an inhibitor of F1F0 ATP synthase) confirms the enzyme complex as a valid key target for anti-tuberculosis drug development.

[0079] In addition, F-ATP synthase belongs to the orchestra of enzymes that form the electron transport chain (ETC), to which cytochrome c oxidase (cyt-bc1-aa3) and the bacterium-specific cytochrome bd-type menaquinol oxidase (cyt-bd) belong, and F-ATP synthase contributes to ATP generation.

[0080] More advantageously, the compounds of the present invention target the soluble F1 portion of the mycobacterial F1F0-ATP synthase in drug-resistant MDR and XDR-TB. The basis for this concept lies in the inventors' novel insights into the natural paradigm of energy fixation within mycobacteria, the new drug target responsible for ATP synthesis within the key catalyst, and the development of new compounds. In addition, the compounds of the present invention have been found to contribute to the synergistic efficacy with new BDQ analogs in various drug combinations, thus addressing the challenges of MDR and XDR-TB.

[0081] The compounds of the present invention are benzamide analogs of the compounds described in WO 2018 / 151681 A2, which have not been described previously.

[0082] In various embodiments, in formula (Ia), X is N and Y is NH, or, in formula (Ib), X is NH and Y is N.

[0083] In various embodiments, R1 is a methyl group at the 6-position of the pyrimidine ring.

[0084] In various embodiments, R2 is an ethyl group or a -CH2COOCH2CH3 group.

[0085] In various embodiments, R3 is an aryl group.

[0086] In various embodiments, the aryl group is substituted with one or more halogen atoms.

[0087] In various specific embodiments, the compound is selected from the group consisting of

[0088]

[0089]

[0090] and its tautomers.

[0091] The test results for compounds (II), (III), and (IV) are shown in Table 1.

[0092] Table 1: Test results of benzamide analog compounds

[0093]

[0094] Very unexpectedly, all of these analogs showed good IC 50 and MIC 50 values, where one of these compounds (the compound having formula (II)) was even significantly superior to the main compound of WO 2018 / 151681 A2 (named "cpd6" there).

[0095] Compounds having formula (II) exhibit a 10-fold improvement with respect to the minimum inhibitory concentration (MIC 50 ), being 3 μM in MtbH37Rv (see Table 1 above; Figure 1 A), and 6.8 μM in Mycobacterium bovis BCG ( Figure 1 B). Compounds having formula (II) are bactericidal against Mycobacterium smegmatis mc2 155 at 20-fold their MIC 50 , as shown by the observed inhibition of cell growth as in Figure 1 C. In contrast, BDQ has a delayed bactericidal activity, as previously reported (A. Koul, L. Vranckx, N. Dhar, H. W. Gohlmann, E. Ozdemir, J. M. Neefs, M. Schulz, P. Lu, E. Mortz, J. D. McKinney, K. Andries, D. Bald, Nat Commun[Nature Communications] 2014, 5, 3369.).

[0096] To determine whether the antimycobacterial activity is attributed to oxidative phosphorylation inhibition, an intracellular ATP synthesis assay on Mycobacterium bovis BCG was performed ( Figure 2 A). Compounds having formula (II) have an effect on ATP levels at an IC 50 of 3.3 μM, indicating its ability to inhibit ATP synthesis intracellularly, and the compound having formula (VI) (named BDQ-1) reveals an IC 50 of 3.4 nM for the inhibition of intracellular ATP synthesis, when compared to BDQ (IC 50 = 11.5 nM; Figure 2 A) with a slight improvement. The IC 50 of BDQ as a control was found to be 11.5 nM, which is similar to the reported IC 50 (Preiss L, Langer JD, Yildiz Eckhardt-Strelau L, Guillemont JEG, Koul A & Meier T (2015) Structure of the mycobacterial ATP synthase F O rotor ring in complex with anti-TB drug bedaquiline[The structure of the rotor ring of the mycobacterial ATP synthase F in complex with the anti-TB drug bedaquiline OStructure of the rotor ring. Sci. Adv. [Science Advances] 1: e1500106.). Interestingly, the compounds with formula (II) provide an 18-fold enhancement in ATP synthesis inhibition, about 90 nM for Mycobacterium smegmatis ( Figure 2 B) and even 8.7 nM for Mycobacterium bovis BCG IMV ( Figure 2 C).

[0097] The compounds with formula (II) (with a clogP value of 6.51) have lower lipophilicity compared to BDQ (clogP = 7.25) and have good metabolic stability in mouse liver microsomes (T 1 / 2 is 29.6 min, CL hep is 60.5 ml / min / kg, and Cl int is 46.8 ml / min / mg protein).

[0098] To explore the anti-TB potential of the compounds with formula (II), THP1 cells (monocytes) were infected with Mycobacterium bovis BCG, which has a genome similar to that of Mycobacterium tuberculosis.

[0099] In a second aspect, the present invention relates to a composition comprising a compound according to the invention or a pharmaceutically acceptable salt thereof and bedaquiline (BDQ), an analogue of bedaquiline (BDQ), or a mixture thereof.

[0100] In various embodiments, the bedaquiline (BDQ) comprises a compound having formula (VII):

[0101]

[0102] The synergistic effect of the racemate of the compound with formula (VII) (BDQ) and different concentrations of the compound with formula (II) was tested on Mycobacterium smegmatis IMV. As Figure 5 shown, the addition of 3x IC 50 (270 nM) of the compound with formula (II) completely inhibited ATP synthesis.

[0103] In various embodiments, the analogue of bedaquiline (BDQ) comprises the racemate of a compound having formula (V):

[0104] Or

[0105] the racemate of a compound having formula (VI):

[0106]

[0107] The asterisks in formulas (V) and (VI) denote chiral carbon atoms.

[0108] Recently, BDQ analogs with potential for improvement in M. tuberculosis strains as well as pharmacological properties have been described (Tong AST, Choi PJ, Blaser A, Sutherland HS, Tsang SKY, Guillemont J, Motte M, Cooper CB, Andries K, Van den Broeck W, Franzblau SG, Upton AM, Denny WA, Palmer BD, Conole D. 2017. 6-Cyano Analogues of Bedaquiline as Less Lipophilic and Potentially Safer Diarylquinolines for Tuberculosis. ACS Med Chem Lett 8:1019-1024; Choi PJ, Sutherland HS, Tong AST, Blaser A, Franzblau SG, Cooper CB, Lotlikar J. 2017. 6-Cyano Analogues of Bedaquiline as Less Lipophilic and Potentially Safer Diarylquinolines for Tuberculosis. ACS Med Chem Lett 8:1019-1024; Choi PJ, Sutherland HS, Tong AST, Blaser A, Franzblau SG, Cooper CB, Lotlikar J. 2017. MU,Upton AM,Guillemont J,Motte M,Queguiner L,Andries K,Van den Broeck W,Denny WA,Palmer BD. 2017. Synthesis and evaluation of analogues of the tuberculosis drug bedaquiline containing heterocyclic B-ring units. Bioorg Med Chem 27:5190-5196; Sutherland HS,Tong AST,Choi PJ,Conole D,Blaser A,Franzblau SG,Cooper CB,Upton AM,Lotlikar MU,Denny WA,Palmer BD. 2018.Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles. Bioorg Med Chem 26:1797-1809; Sarathy, J., Ragunathan, P., Joon, S., Cooper, C., Upton, A., Grüber, G., and Dick, T. (2019) TBAJ-876 retains Bedaquiline’s activity against subunit c and ε of Mycobacterium tuberculosis F-ATP synthase. Antimicrob. Agents Chemother. 63(10). pii: e01191-19), including racemates of compounds of formula (V), which appear to interact with the subunit c-ring and subunit ε of Mycobacterium F-ATP synthase. Using an altered synthetic protocol, racemates of compounds of formula (V) were synthesized (see Examples section below).

[0109] Figure 3 It is shown that 300 nM of the compound of formula (II) increases the potential of the racemate of the compound of formula (V) in the inhibition of ATP synthesis (Δ) in Mycobacterium smegmatis IMV when compared to the inhibition (▲) of this racemate alone.

[0110] The synergy of the racemate of the compound of formula (VI) (named BDQ1) with different concentrations of the compound of formula (II) was tested on Mycobacterium smegmatis IMV. As Figure 4 shown, the addition of 3x IC 50 (270 nM) of the compound of formula (II) completely inhibited ATP synthesis.

[0111] In addition, human embryonic stem cell (hESC) line (E3) was used to examine potential drug-induced genotoxicity and perturbations to the hESC transcriptional program that lead to hESC differentiation. Assays revealed that the racemate (100 nM) of the tested compound of formula (VI) in combination with 300 nM of the compound of formula (II) showed neither substantial genotoxic effects nor induced major alterations in the overall transcriptional program.

[0112] In another aspect, the invention relates to a compound according to the invention for use in therapy.

[0113] In another aspect, the invention relates to a compound according to the invention for the treatment of bacterial infections, preferably for the treatment of tuberculosis, particularly for the treatment of multi-drug resistant or extensively drug-resistant tuberculosis.

[0114] In another aspect, the invention relates to the use of a compound according to the invention in the manufacture of a medicament for the treatment of bacterial infections.

[0115] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0116] In another aspect, the invention relates to a composition according to the invention for use in therapy.

[0117] In another aspect, the invention relates to a composition according to the invention for the treatment of bacterial infections, preferably for

[0118] the treatment of tuberculosis, particularly for the treatment of multi-drug resistant or extensively drug-resistant tuberculosis.

[0119] In another aspect, the invention relates to the use of a composition according to the invention in the manufacture of a medicament for the treatment of bacterial infections.

[0120] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0121] In another aspect, the invention relates to a method of treating a subject suffering from a bacterial infection, the method comprising the step of administering to the subject a therapeutically effective amount of a compound or composition according to the invention.

[0122] Preferably, the bacterial infection is tuberculosis, particularly multi-drug resistant or extensively drug-resistant tuberculosis.

[0123] In another aspect, the invention relates to a method of synthesizing a compound according to the invention, wherein X is NH and Y is N, or X is N and Y is NH, the method comprising the following steps:

[0124] (a) In the presence of an acid, react 4-nitrobenzene-1,2-diamine with unsubstituted or R3-substituted benzoic acid;

[0125] (b) Reduce the nitro group to obtain the corresponding amine derivative; and

[0126] (c) React the amine derivative of step (b) with 2-chloro-pyrimidin-4-amine substituted with R1 and R2.

[0127] Examples

[0128] Materials and Methods

[0129] Preparation of inverted membrane vesicles from Mycobacterium smegmatis

[0130] Mycobacterium smegmatis was selected as an alternative model for Mycobacterium tuberculosis because there are several advantages to using Mycobacterium smegmatis. Mycobacterium smegmatis is saprophytic and, unlike Mycobacterium tuberculosis, it is not pathogenic and can be safely handled under biosafety level 2 (BSL2) conditions without the requirements of biosafety level 3 (BSL3). In addition, compared to the growth of Mycobacterium tuberculosis (generation time: approximately 24 hours), the growth of Mycobacterium smegmatis is much faster (generation time: approximately 3 hours). Moreover, while it takes almost 3 to 4 weeks for Mycobacterium tuberculosis to produce colonies on an agar plate, it only takes 2 to 3 days for Mycobacterium smegmatis to produce colonies on an agar plate, thus reducing the experimental duration. Importantly, Mycobacterium smegmatis inverted membrane vesicles (IMV) exhibit detectable ATP hydrolysis activity, which is crucial for enzyme assays to be used or conducted.

[0131] To purify the IMV of Mycobacterium smegmatis for ATP synthesis and hydrolysis assays, cells were grown overnight at 37 °C in 7H9 supplemented with 10% ADC, 0.5% glycerol, and 0.05% Tween 80 until they reached an OD600 value of 0.4. The culture was amplified in 200 ml of supplemented 7H9 and grown in a roller bottle (2 rpm) until OD 600= 0.4. This culture was used to inoculate a 5 l culture and allowed to grow overnight in a roller bottle until OD600 = 0.4. Approximately 5 g (wet weight) of Mycobacterium smegmatis was resuspended in 20 ml of membrane preparation buffer (50 mM MOPS, 2 mM MgCl2 pH 7.5) containing a protease inhibitor mixture without EDTA (1 tablet per 20 ml of buffer, Roche-USA) and 1.2 mg / ml lysozyme. The suspension was stirred at room temperature for 45 minutes and additionally supplemented with 300 μl of 1 M MgCl2 and 50 μl of DNase I (Thermo Fischer, USA) and stirring was continued for an additional 15 minutes at room temperature. All subsequent steps were carried out on ice. The cells were disrupted by passing through a microfluidizer model M-110L (M-110L) at 18,000 psi through three passes. The suspension containing the lysed cells was centrifuged at 4,200 x g for 20 min at 4 °C. The supernatant containing the membrane fraction was further subjected to ultracentrifugation at 45,000 x g for 1 h at 4 °C. The supernatant was discarded and the precipitated membrane fraction was resuspended in membrane preparation buffer containing 15% glycerol, aliquoted, snap-frozen and stored at -80 °C. The protein concentration in the vesicles was determined by the BCA method. The reconstituted membrane vesicles were stored at -80 °C.

[0132] ATP synthesis assay

[0133] ATP synthesis was measured in a flat-bottom white microtiter 96-well plate (Corning USA). The reaction mixture was prepared in assay buffer (50 mM MOPS, pH 7.5, 10 mM MgCl2) containing 10 μM ADP, 250 μM Pi and 1 mM NADH. The concentration of Pi was adjusted by adding 100 mM KH2PO4 salt dissolved in the assay buffer. ATP synthesis was initiated by adding reconstituted membrane vesicles of Mycobacterium smegmatis to a final protein concentration of 5 μg / ml. The reaction mixture was incubated at room temperature for 30 min and then 50 μl of CellTiter-glow reagent was added and the mixture was incubated for an additional 10 min at room temperature in the dark. The luminescence associated with the synthesized ATP generated was measured using a Tecan plate reader Infinite 200Pro (Tecan USA) with the following parameters: luminescence, integration time 500 ms, no attenuation.

[0134] Anti-mycobacterial activity

[0135] Screen test compounds and control drugs against Mycobacterium smegmatis mc2 155, Mycobacterium tuberculosis H37Rv, and Mycobacterium bovis BCG. Prepare an initial stock solution of the test compound at a concentration of 10 mM in 90% DMSO. Use ciprofloxacin as a positive control and the vehicle DMSO as a negative control. In the first method, test the compounds on microbial cultures at a fixed concentration of 50 μM. Each of the above strains was cultured at 37 °C in Middlebrook 7H9 liquid medium supplemented with 0.2% glycerol and 10% ADC (Albumin Dextrose Catalase) until logarithmic growth was achieved (OD 600 0.4 - 0.6). Obtain the test inoculum by diluting the suspension to an OD 600 0.1 in a test tube to a final volume of 1 ml and incubate it at 37 °C for 24 hours (Mycobacterium smegmatis) and 7 days (Mtb). Select test compounds that do not show visible bacillary growth compared to the positive and negative controls as hits.

[0136] General procedure for the synthesis of compounds having formula (Ia) or (Ib)

[0137] Compounds having formula (Ia) or (Ib) are obtained according to the following scheme (Scheme S1) by heating the corresponding aryl or heteroaryl acid (such as, for example, unsubstituted or substituted benzoic acid) with 4-nitrobenzene-1,2-diamine in the presence of polyphosphoric acid. Nitro reduction using Fe powder in the presence of ammonium chloride provides the benzimidazoleamine derivative, which is reacted with the corresponding substituted 2-chloropyrimidin-4-amine (named S2 in the scheme) under microwave heating to give the final product.

[0138]

[0139] Scheme S1. Synthesis of compounds having formula (II), (III), and (IV)

[0140] 2-(4-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole S6a

[0141]

[0142] 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 h. 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 flash column chromatography on silica gel (0%-40% EtOAc / hexane) to afford compound S6a (142 mg, 28% yield) as a pale green solid; 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 [C 13 H8ClN3O2+H] + 。

[0143] 2-(3-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole S6b

[0144]

[0145] A mixture of 3-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 2 h. 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 flash column chromatography on silica gel (0%-40% EtOAc / hexane) to afford compound S6b (82.6 mg, 16% yield) as an orange solid; 1H NMR (400 MHz, DMSO-d6) δ 13.7 (br.s, 1H), 8.51 (s, 1H), 8.26 (s, 1H), 8.22 - 8.14 (m, 2H), 7.80 (d, J = 8.4 Hz, 1H), 7.65 (s, 2H); MS (ESI) m / z 274.0 [C 13 H8ClN3O2+H] + 。

[0146] 2-(4-Chlorophenyl)-1H-benzo[d]imidazol-5-amine S7a

[0147]

[0148] A mixture of S6a (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 h. The mixture was filtered through a Celite pad and washed with MeOH. The filtrate was concentrated and the residue was taken up in water (10 mL) and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give S7a (87.6 mg, 75% yield) as a brown oil, 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 [C 13 H 10 ClN3+H] + 。

[0149] 2-(3-Chlorophenyl)-1H-benzo[d]imidazol-5-amine S7b

[0150]

[0151] A mixture of S6b (82.6 mg, 0.302 mmol), iron powder (169 mg, 3.02 mmol, 10 eq.), and NH4Cl (161 mg, 3.02 mmol, 10 eq.) in 4:1 EtOH / water (5 mL) was heated at 80 °C for 3 h. The mixture was filtered through a Celite pad and washed with MeOH. The filtrate was concentrated and the residue was taken up in water (10 mL) and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give S7b (72.4 mg, 98% yield) as a brown oil, which was used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.88 - 7.85 (m, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 3.2 Hz, 2H), 6.82 (br.s, 1H), 6.68 (dd, J = 8.4, 2.0 Hz, 1H); MS (ESI) m / z 244.0 [C 13 H 10 ClN3+H] + 。

[0152] N 2-(2-(4-Chlorophenyl)-1H-benzo[d]imidazol-5-yl)-N 4 -ethyl-6-methylpyrimidine-2,4-diamine (Formula (II))

[0153]

[0154] A solution of 2-chloro-N-ethyl-6-methylpyrimidin-4-diamine S2 (35.0 mg, 0.204 mmol) and aniline S7a (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 h. 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 the compound (formate) of Formula (II), which was lyophilized to a grayish-white solid (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 [C 20 H 19 ClN6+H] + ; [C 20 H 19 ClN6+H] + The HRMS (ESI) m / z calculated value of [C

[0155] N 2 -(2-(3-Chlorophenyl)-1H-benzo[d]imidazol-5-yl)-N 4 -ethyl-6-methylpyrimidine-2,4-diamine (Formula (III))

[0156]

[0157] A solution of 2-chloro-N-ethyl-6-methylpyrimidine-4-diamine S2 (34.0 mg, 0.198 mmol) and aniline S7b (72.4 mg, 0.297 mmol, 1.5 eq.) in n-BuOH (1 mL) was heated in a microwave reactor at 180 °C for 2 h. The reaction mixture was azeotroped with toluene to give a white residue, which was purified by preparative HPLC (20%-60% MeCN / H2O; 0.1% formic acid) to afford the compound (formate) of formula (III), which was lyophilized to a white solid (38.4 mg, 51.2% yield); 1H NMR (400 MHz, DMSO-d6) δ 12.7 (br.s, 1H), 9.14 (s, 1H), 8.30 (s, 1H), 8.18 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.57-7.44 (m, 4H), 7.04 (br.s, 1H), 5.79 (s, 1H), 3.37 (br.s, 2H), 2.15 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.5, 163.0, 159.3, 148.7, 137.2, 133.7, 132.5, 130.8, 128.9, 125.7, 125.6, 124.6, 115.5, 35.0, 23.1, 14.7; MS (ESI) m / z 379.1 [C 20 H 19 ClN6+H] + ; [C 20 H 19 ClN6+H] + The HRMS (ESI) m / z calculated for [C

[0158] N 4 -ethyl-6-methyl-N 2 -(2-phenyl-1H-benzo[d]imidazol-5-yl)pyrimidine-2,4-diamine (Formula (IV))

[0159]

[0160] A solution of 2-chloro-N-ethyl-6-methylpyrimidin-4-diamine S2 (30.0 mg, 0.175 mmol) and commercially available aniline S7c (54.8 mg, 0.262 mmol, 1.5 eq.) in n-BuOH (1 mL) was heated in a microwave reactor at 180 °C for 2 h. The reaction mixture was azeotroped with toluene to give a white residue, which was purified by preparative HPLC (5%-60% MeCN / H2O; 0.1% formic acid) to afford the compound (formate) of formula (IV), which was lyophilized to a white solid (46.2 mg, 76.7% yield); 1H NMR (400 MHz, DMSO-d6) δ 12.6 (br.s, 1H), 9.28 (s, 1H), 8.25 (s, 1H), 8.13 (d, J = 7.2 Hz, 2H), 7.52 (t, J = 7.2 Hz, 2H), 7.48 - 7.43 (m, 3H), 7.13 (br.s, 1H), 5.79 (s, 1H), 3.37 (br.s, 2H), 2.15 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.5, 163.0, 159.0, 150.4, 136.6, 130.4, 129.3, 128.8, 126.1, 115.3, 35.0, 22.8, 14.6; MS (ESI) m / z 345.2 [C 20 H 20 N6+H] + ; [C 20 H 20 N6+H] + The HRMS (ESI) m / z calculated for [C

[0161] General procedure for the synthesis of the racemate of the compound of formula (V)

[0162]

[0163] Reagents and conditions: (i) LiTMP, THF, -78 °C, 1.5 h then appropriate aldehyde, -78 °C, 4 h (4, 37%), (5, 55%); (ii) InCl3, Ph2SiHCl, DCE, 80 °C, 12 h, 44%; (iii) Et3SiH, TFA, CH2Cl2, 50 °C, 60%; (iv) lithium diisopropylamide (LDA), THF, -78 °C, 1.5 h then 8, then HOAc, (5307 (formed from the reaction of 11 with 8), 9%), (5366, 32%), (5316, 33%); (v) Pd(PPh3)4, Cs2CO3, PhMe / DMF, 110 °C, 5 h, 55%.

[0164] The synthesis of the racemate of the compound of formula (V) was carried out as follows:

[0165] Addition of lithium tetramethylpiperidide (LiTMP) to methoxyquinoline (1) and 5-isopropoxy-2-methoxynicotine aldehyde (2) and 2,3-dihydrobenzo[b][1,4]dioxine-5-carbaldehyde (3) provided the intermediate benzyl alcohols (4) and (5), respectively. Subsequent deoxygenation using InCl3, Ph2SiHCl, DCE and Et3SiH / TFA under acidic conditions gave the corresponding dihydro adducts (6) and (7), respectively. Unfortunately, due to the inhibition of the reaction by the basicity of the pyridine moiety, 6-bromo-3-((2,3-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (11) was not formed using either deoxygenation condition. Alternatively, a longer synthetic route was employed which used a Suzuki reaction between methoxyquinoline boronic acid (9) and 4-(bromomethyl)-2,3-dimethoxypyridine (10) to afford 6-bromo-3-((2,3-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (11).

[0166] The racemate of the compound of formula (V) was then synthesized by the addition of the appropriate benzylquinoline (11) and 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (8) mediated by LDA, as reported by Sutherland et al. (Sutherland HS, Tong AST, Choi PJ, Conole D, Blaser A, Franzblau SG, Cooper CB, Upton AM, Lotlikar MU, Denny WA, Palmer BD. 2018. Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles. Bioorg Med Chem 26:1797-1809). The resulting diarylquinoline was formed as a racemic mixture of two diastereoisomers, which was separated by column chromatography. The structure of the desired diastereoisomer was further determined by X-ray crystallography and characterized in ATP synthesis and cell growth assays.

[0167] General procedure for the synthesis of the compound of formula (VI)

[0168] The compound of formula (VI) was synthesized as shown in the following scheme:

[0169]

[0170] Those skilled in the art should further recognize that the variations and combinations of the features described above are not substitutes or replacements, but can be combined to form other embodiments that fall within the scope contemplated by the present invention.

Claims

1. A compound having the formula (Ia) wherein, The compound having the formula (Ia) is any one selected from the group consisting of:

2. A composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof and bedaquiline (BDQ), an analogue of bedaquiline (BDQ) or a mixture thereof; wherein, The analogue of bedaquiline BDQ is a racemate of the compound having the formula (V): or The racemate of the compound having the formula (VI):

3. Use of the compound according to claim 1 in the manufacture of a medicament for the treatment of tuberculosis.

4. The use according to claim 3, wherein, The tuberculosis is multi-drug resistant or extensively drug-resistant tuberculosis.

5. Use of the composition according to claim 2 in the manufacture of a medicament for the treatment of tuberculosis.

6. The use according to claim 5, wherein, The tuberculosis is multi-drug resistant or extensively drug-resistant tuberculosis.

7. A method for synthesizing the compound according to claim 1, wherein, The method comprises the following steps: (a) Reacting 4-nitrobenzene-1,2-diamine with unsubstituted or R3-substituted benzoic acid in the presence of an acid; (b) Reducing the nitro group to obtain the corresponding amine derivative; and (c) Reacting the amine derivative of step (b) with 2-chloro-pyrimidin-4-amine substituted with R1 and R2.

Images