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. 2020 Jan 29;12(3):207–222. doi: 10.4155/fmc-2019-0063

Bis-(imidazole/benzimidazole)-pyridine derivatives: synthesis, structure and antimycobacterial activity

Vasilichia Antoci 1, Dumitrela Cucu 1, Gheorghita Zbancioc 1, Costel Moldoveanu 1, Violeta Mangalagiu 2,*, Dorina Amariucai-Mantu 1, Aculina Aricu 3, Ionel I Mangalagiu 1,2,**
PMCID: PMC7421780  PMID: 31916456

Abstract

Aim:

Over the last decades, few significant achievements have been made in tuberculosis (TB) therapy. As a result, there is an urgent need for new anti-TB drugs.

Results:

Two new classes of bis-(imidazole/benzimidazole)-pyridine derivatives were designed, synthesized and evaluated for their antimycobacterial activity.

Conclusion:

The synthesis is efficient and straightforward, involving only two successive N-alkylations. The anti-TB assay reveal that our compounds have an excellent anti-TB activity against both replicating and nonreplicating Mtb, are not cytotoxic, exhibited a very good intracellular activity and are active against drug-resistant Mtb strains, some compounds have a bactericidal mechanism. The absorption, distribution, metabolism, excretion and toxicity studies performed for one compound are promising, indicating that it is a good candidate for a future drug.

Keywords: : ADMET, antimycobacterial, bis-(imidazole/benzimidazole)-pyridine, MBC, MIC

During the last two centuries, tuberculosis (TB) became an extremely aggressive disease responsible for millions of deaths around the world [1]. According to the WHO, TB now remains a leading infectious disease worldwide, almost a third of the world’s population (around 2 billion people) being infected with nonreplicating Mtb [1]. Drug-resistant TB, multidrug-resistant TB, extensively drug-resistant TB and totally drug-resistant TB are present in almost all countries all around the world, and making the situation even more challenging for global TB treatment. In 2017, there were around 700,000 new cases with resistance to rifampicin, the most effective first-line drug, of which 500,000 had multi-drug resistant-TB [1–3]. In addition, association of TB with HIV/AIDS complicates the situation for patients even more, with TB being the leading cause of death among the patients infected with HIV (in 2017, there were an estimated 400,000 TB deaths among HIV-positive people) [1,4,5]. As a result of these considerations, each year (in the last 5), there were an estimated two million TB deaths and about eight million new cases of TB reported [1].

The TB treatment is a challenging, complex and extremely difficult task. In the last 50 years, only two new drugs (bedaquiline [6] and delamanid [7]) have been developed in anti-TB therapies, for drug-resistant strains of Mtb. Therefore, the search for new anti-TB drugs active against Mtb remains one of the priority tasks of medicinal chemistry. There are currently several strategies used for the development of new anti-TB drugs [8–11], one of the most important and used strategy consist of searching for novel structures which the TB organism has never encountered with before. Nitrogen heterocyclic compounds, especially five and six member ring derivatives, represent the most effective and administered class of drugs in TB therapy. Among them, pyridine and imidazole/benzimidazole derivatives play a central role, the most effective and promising anti-TB drugs being related to these classes [8–18].

Encouraged by the above considerations and in continuation of our research in the area of new antimycobacterial compounds with azaheterocyclic skeleton [18–30], we report herein the design, synthesis and antimycobacterial activity of a new class of bis-(imidazole/benzimidazole)-pyridine (BIP) derivatives.

Experimental

Materials & measurements

Melting points were determined using an electrothermal Mel-Temp apparatus and were uncorrected (Barnstead International, IA, USA). The nuclear magnetic experiments (1H-NMR, 13C-NMR, 2D-COSY, 2D-HMQC, 2D-HMBC) were recorded using Bruker Avance III 500/Bruker Avance DRX 400 spectrometers (Bruker, Vienna, Austria) operating at 500/400 and 125/100 MHz for 1H and, respectively, 13C nuclei. The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, brs = broad singlet, ad = apparent doublet, at = apparent triplet. All chemical shifts are reported in δ units (p.p.m.) relative to the residual peak of solvent (ref: DMSO, 1H: 2.50 p.p.m.; 13C: 39.52 p.p.m.). Coupling constants (J) are given in Hz. The IR spectra were recorded on a Fourier transform IR (FTIR) Shimadzu Prestige 8400s spectrophotometer (Shimadzu, Kyoto, Japan). Thin layer chromatography (TLC) was carried out on Merck silica gel 60F254 plates. Visualization of the plates was achieved using a UV lamp (λmax = 254 or 365 nm). The ultrasonic bath Elma Transsonic T310 (power 34.5 W, frequency 35 kHz; Elma Schmidtbauer, Singen, Germany) was used for solubilizing the starting materials. The micro-analyses were in satisfactory agreement with the calculated values: C, ± 0.15; H, ± 0.10; N, ± 0.30.

Compounds 2a and 5 were previously reported [31].

General procedure for synthesis of starting materials 2a,b & 5

To a solution of imidazole/4-nitroimidazole (3 mmol, 3 equivalent, 0.20 g/0.34 g, dissolved in 15 ml tetrahydrofuran dry/35 ml dimethylformamide using US bath), or benzimidazole (2.4 mmol, 2.4 equivalent, 0.28 g, dissolved in 15 tetrahydrofuran dry), was added dropwise, at 0°C (ice), NaH (60% in mineral oil; 3.6 mmol, 3.6 equivalent, 0.14 g, suspended in 15 ml tetrahydrofuran dry). The sodium hydride was firstly washed three times with 5 ml n-hexane. Then, 2,6-bis(halogenomethyl)pyridine 1 (1 mmol, 1 equivalent, 0.18 g (X = -Cl for the solution of imidazole/4-nitroimidazole), 0.26 g (X = -Br for the solution of benzimidazole) dissolved in 15 ml tetrahydrofuran dry), was added dropwise. The resulting solutions were stirred for additional time (72, 24, 96 h) at room temperature, to give the corresponding starting materials 2a,b and 5. The solutions were filtered off, concentrated on rotary evaporator, than precipitated with diethyl ether for compounds 2a and 5, or dimethylformamide/methanol: 3/15 (v/v), for compound 2b. The formed precipitates were collected by filtration and dried in vacuo.

General procedure for synthesis of quaternary salts 4a–g

A solution of 2,6-bis((1H-imidazol-1-yl)methyl)pyridine 2a (1 mmol, 1 equivalent, 0.24 g, dissolved in 40 ml acetone using the ultrasound bath) and the α-halogeno ester and amide (2.4 mmol, 2.4 equivalent, 0.22 ml 3a, 0.44 g 3b) or the bromacetophenone derivatives (2.8 mmol, 2.8 equivalent, 0.56 g 3c, 0.78 g 3d, 0.65 g 3e, 0.68 g 3f, 0.64 g 3g) solubilized in acetone (15 ml), was magnetically stirred for 24 h, to give the corresponding quaternary salts 4a–g. The completion of the reactions was carried out using TLC. The obtained salts were filtered off, washed two times with the same solvent (10 ml) and dried in vacuo. No other purification required.

General procedure for synthesis of quaternary salts 6a–g

To a solution of 2,6-bis((1H-benzo[d]imidazol-1-yl)methyl)pyridine 5 (1 mmol, 1 equivalent, 0.34 g, dissolved in 40 ml acetone using the ultrasound bath) were added the α-halogeno ester and amide (4.8 mmol, 4.8 equivalent, 0.73 ml 3a, 0.89 g 3b) or the bromacetophenone derivatives (4.5 mmol, 4.5 equivalent, 0.89 g 3c, 1.25 g 3d, 1.05 g 3e, 1.1 g 3f, 1.03 g 3g) solubilized in acetone (20 ml). The solutions were refluxed for 12 h and magnetically stirred for another 12 h, resulting the desired salts 6a–g. TLC was used for follow the evolution of chemical reactions. The precipitates were collected by filtration, then washed with acetone (3 times with 10 ml) and dried in vacuum. No other purification required.

Spectral data of starting materials 2a,b & 5

2,6-bis((1H-imidazol-1-yl)methyl)pyridine (2a)

Appearance: white powder. Yield, 76%. melting point (mp) 98–106°C. FTIR (KBr) cm-1 = 3101, 2987, 1595, 1506, 1431, 1230. 1H-NMR (400 MHz, DMSO-d6) δ = 7.81–7.77 (t, J = 7.6 Hz, 1H, H4), 7.75 (s, 2H, 2xH2′), 7.20 (s, 2H, 2xH5′), 7.05–7.03 (d, J = 7.6 Hz, 2H, 2xH3), 6.93 (s, 2H, 2xH4′), 5.30 (s, 4H, 2xCH2). 13C-NMR (100 MHz, DMSO-d6) δ = 156.7 2xC2, 138.5 C4, 137.7 2xC2′, 128.6 2xC4′, 120.3 2xC3, 119.8 2xC5′, 51.1 2xCH2. Anal. Calcd. for C13H13N5: C, 65.25; H, 5.48; N, 29.27; found: C, 65.40; H, 5.38; N, 29.57.

2,6-bis((4-nitro-1H-imidazol-1-yl)methyl)pyridine (2b)

Appearance: white powder. Yield, 72%. mp 189–195°C. FTIR (KBr) cm-1 = 3140, 3119, 1595, 1525, 1485, 1330. 1H-NMR (400 MHz, DMSO-d6) δ = 8.36 (ad, 2H, 2xH2′), 7.91–7.87 (at, J = 8.0 Hz, 3H, 2xH5′, H4), 7.34–7.32 (d, J = 7.6 Hz, 2H, 2xH3), 5.42 (s, 4H, 2xCH2). 13C-NMR (100 MHz, DMSO-d6) δ = 154.9 2xC2, 146.9 2xC4′, 138.7 C4, 137.7 2xC5′, 121.8 2xC2′, 121.2 2xC3, 51.6 2xCH2. Anal. Calcd. for C13H11N7O4: C, 47.42; H, 3.37; N, 29.78; found: C, 47.32; H, 3.30; N, 29.98.

2,6-bis((1H-benzo[d]imidazol-1-yl)methyl)pyridine (5)

Appearance: white powder. Yield, 64%. mp 154–160°C. FTIR (KBr) cm-1 = 3120, 2990, 1590, 1526, 1451, 1270. 1H-NMR (500 MHz, DMSO-d6) δ = 8.35 (s, 2H, 2xH2′), 7.74–7.71 (t, J = 8.0 Hz, 1H, H4), 7.66–7.65 (d, J = 8.0 Hz, 2H, 2xH7′), 7.40–7.38 (d, J = 8.0 Hz, 2H, 2xH4′), 7.19–7.11 (m, 6H, 2xH6′, 2xH3, 2xH5′), 5.54 (s, 4H, 2xCH2). 13C- NMR (125 MHz, DMSO-d6) δ = 155.9 2xC2, 144.4 2xC2′, 143.4 2xC3′a, 138.4 C4, 133.7 2xC7′a, 122.4 2xC5′, 121.5 2xC6′, 120.9 2xC3, 119.4 2xC7′, 110.6 2xC4′, 49.4 2xCH2. Anal. Calcd. for C21H17N5: C, 74.32; H, 5.05; N, 20.63; found: C, 74.47; H, 5.15; N, 20.33.

Spectral data of quaternary salts 4a–g & 6a–g

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-methoxy-2-oxoethyl)-1H-imidazol-3-ium) bromide (4a)

Appearance: white powder. Yield, 50%. mp 83–85°C. FTIR (KBr) cm-1 = 3095, 2983, 1730, 1593, 1440, 1240, 1070. 1H-NMR (500 MHz, DMSO-d6) δ = 9.33 (s, 2H, 2xH2′), 8.00–7.97 (t, J = 7.5 Hz, 1H, H4), 7.80 (s, 2H, 2xH4′), 7.75 (s, 2H, 2xH5′), 7.53–7.52 (d, J = 8.0 Hz, 2H, 2xH3), 5.67 (s, 4H, 2xCH2-α), 5.39 (s, 4H, 2xCH2-β), 3.76 (s, 6H, 2xCH3). 13C-NMR (125 MHz, DMSO-d6) δ = 167.4 2xCO, 153.4 2xC2, 138.8 C4, 137.8 2xC2′, 123.6 2xC4′, 123.0 2xC5′, 122.2 2xC3, 52.8 2xCH3, 52.6 2xCH2-α, 49.7 2xCH2-β. Anal. Calcd. for C19H23Br2N5O4: C, 41.85; H, 4.25; N, 12.84; found: C, 41.95; H, 4.34; N, 12.65.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-amino-2-oxoethyl)-1H-imidazol-3-ium) iodide (4b)

Appearance: yellowish powder. Yield, 68%. mp 182–184°C. FTIR (KBr) cm-1 = 3348, 3140, 3079, 2940, 1679, 1584, 1520, 1432. 1H-NMR (500 MHz, DMSO-d6) δ = 9.15 (s, 2H, 2xH2′), 8.00–7.97 (t, J = 7.5 Hz, 1H, H4), 7.88 (brs, 2H, 2x(-NH-)), 7.68 (s, 4H, 2xH4′, 2xH5′), 7.55 (brs, 2H, 2x(-NH-)), 7.51–7.49 (d, J = 7.5 Hz, 2H, 2xH3), 5.59 (s, 4H, 2xCH2-α), 5.01 (s, 4H, 2xCH2-β). 13C-NMR (125 MHz, DMSO-d6) δ = 166.7 2xCO, 153.5 2xC2, 138.9 C4, 137.8 2xC2′, 123.8 2xC4′, 122.5 2xC5′, 122.1 2xC3, 52.6 2xCH2-α, 50.6 2xCH2-β. Anal. Calcd. for C17H21I2N7O2: C, 33.52; H, 3.47; N, 16.09; found: C, 33.67; H, 3.57; N, 15.84.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-oxo-2-phenylethyl)-1H-imidazol-3-ium) bromide (4c)

Appearance: white powder. Yield, 85%. mp 165–167°C. FTIR (KBr) cm-1 = 3080, 2920, 1687, 1583, 1525, 1430. 1H-NMR (500 MHz, DMSO-d6) δ = 9.30 (s, 2H, 2xH2′), 8.06–8.01 (m, 5H, 4xH2′′, H4), 7.82–7.81 (ad, 4H, 2xH4′, 2xH5′), 7.77–7.74 (t, J = 7.5 Hz, 2H, 2xH4′′), 7.62–7.57 (m, 6H, 4xH3′′, 2xH3), 6.21 (s, 4H, 2xCH2-β), 5.74 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 191.4 2xCO, 153.6 2xC2, 138.8 C4, 137.8 2xC2′, 134.5 2xC4′′, 133.6 2xC1′′, 129.0 4xC3′′, 128.1 4xC2′′, 123.9 2xC4′, 122.9 2xC5′, 122.1 2xC3, 55.6 2xCH2-β, 52.6 2xCH2-α. Anal. Calcd. for C29H27Br2N5O2: C, 54.65; H, 4.27; N, 10.99; found: C, 54.55; H, 4.36; N, 10.79.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-bromophenyl)-2-oxoethyl)-1H-imidazol-3-ium) bromide (4d)

Appearance: white powder. Yield, 92%. mp 238–240°C. FTIR (KBr) cm-1 = 3115, 3070, 2960, 1695, 1583, 1560, 610. 1H-NMR (500 MHz, DMSO-d6) δ = 9.26 (s, 2H, 2xH2′), 8.04–8.01 (t, J = 8.0 Hz, 1H, H4), 7.97–7.95 (d, J = 9.0 Hz, 4H, 4xH2′′), 7.83–7.82 (d, J = 8.5 Hz, 4H, 4xH3′′), 7.80 (s, 2H, 2xH4′), 7.78 (s, 2H, 2xH5′), 7.57–7.55 (d, J = 8.0 Hz, 2H, 2xH3), 6.16 (s, 4H, 2xCH2-β), 5.72 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 190.8 2xCO, 153.6 2xC2, 138.9 C4, 137.8 2xC2′, 132.6 2xC1′′, 132.1 4xC3′′, 130.1 4xC2′′, 128.7 2xC4′′, 123.9 2xC5′, 122.9 2xC4′, 122.2 2xC3, 55.6 2xCH2-β, 52.6 2xCH2-α. Anal. Calcd. for C29H25Br4N5O2: C, 43.8; H, 3.17; N, 8.81; found: C, 43.7; H, 3.26; N, 9.01.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-chlorophenyl)-2-oxoethyl)-1H-imidazol-3-ium) bromide (4e)

Appearance: yellowish powder. Yield, 77%. mp 209–212°C. FTIR (KBr) cm-1 = 3126, 3086, 2972, 1697, 1589, 1558, 758. 1H-NMR (500 MHz, DMSO-d6) δ = 9.23 (s, 2H, 2xH2′), 8.05–8.02 (m, 5H, 4xH2′′, H4), 7.78 (s, 2H, 2xH4′), 7.76 (s, 2H, 2xH5′), 7.69–7.67 (d, J = 8.5 Hz, 4H, 4xH3′′), 7.56–7.55 (d, J = 8.0 Hz, 2H, 2xH3), 6.13 (s, 4H, 2xCH2-β), 5.71 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 190.6 2xCO, 153.6 2xC2, 139.4 2xC4′′, 138.9 C4, 137.9 2xC2′, 132.3 2xC1′′, 130.0 4xC2′′, 129.2 4xC3′′, 123.9 2xC5′, 122.9 2xC4′, 122.2 2xC3, 55.6 2xCH2-β, 52.7 2xCH2-α. Anal. Calcd. for C29H25Br2Cl2N5O2: C, 49.32; H, 3.57; N, 9.92; found: C, 49.20; H, 3.67; N, 9.74.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-nitrophenyl)-2-oxoethyl)-1H-imidazol-3-ium) bromide (4f)

Appearance: pink powder. Yield, 81%. mp 233–236°C. FTIR (KBr) cm-1 = 3120, 2935, 1698, 1580, 1523, 1473, 1348. 1H-NMR (500 MHz, DMSO-d6) δ = 9.31 (s, 2H, 2xH2′), 8.40–8.39 (d, J = 9.0 Hz, 4H, 4xH3′′), 8.29–8.28 (d, J = 9.0 Hz, 4H, 4xH2′′), 8.05–8.02 (t, J = 8.0 Hz, 1H, H4), 7.83–7.82 (add, 4H, 2xH4′, 2xH5′), 7.58–7.57 (d, J = 8.0 Hz, 2H, 2xH3), 6.28 (s, 4H, 2xCH2-β), 5.75 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 190.9 2xCO, 153.6 2xC2, 150.4 2xC4′′, 138.9 C4, 138.2 2xC1′′, 137.8 2xC2′, 129.7 4xC2′′, 124.0 4xC3′′, 123.8 2xC4′, 123.0 2xC5′, 122.2 2xC3, 56.0 2xCH2-β, 52.7 2xCH2-α. Anal. Calcd. for C29H25Br2N7O6: C, 47.89; H, 3.46; N, 13.48; found: C, 47.99; H, 3.37; N, 13.65.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-imidazol-3-ium) bromide (4g)

Appearance: white powder. Yield, 92%. mp 210–213°C. FTIR (KBr) cm-1 = 3076, 2933, 1683, 1600, 1244, 1168. 1H-NMR (500 MHz, DMSO-d6) δ = 9.26 (s, 2H, 2xH2′), 8.07–7.98 (m, 5H, H4, 4xH2′′), 7.79–7.78 (ad, 4H, 2xH4′, 2xH5′), 7.57–7.56 (d, J = 7.5 Hz, 2H, 2xH3), 7.11–7.09 (d, J = 8.5 Hz, 4H, 4xH3′′), 6.10 (s, 4H, 2xCH2-β), 5.72 (s, 4H, 2xCH2-α), 3.87 (s, 6H, 2xCH3). 13C-NMR (125 MHz, DMSO-d6) δ = 189.7 2xCO, 164.1 2xC4′′, 153.6 2xC2, 138.9 C4, 137.9 2xC2′, 130.6 4xC2′′, 126.4 2xC1′′, 123.9 2xC4′, 122.8 2xC5′, 122.2 2xC3, 114.3 4xC3′′, 55.7 2xCH3, 55.2 2xCH2-β, 52.6 2xCH2-α. Anal. Calcd. for C31H31Br2N5O4: C, 53.39; H, 4.48; N, 10.04; found: C, 53.26; H, 4.58; N, 9.86.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-methoxy-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) bromide (6a)

Appearance: white powder. Yield, 39%. mp 184–188°C. FTIR (KBr) cm-1 = 3135, 3080, 2933, 1729, 1600, 1431, 1234, 1069. 1H-NMR (500 MHz, DMSO-d6) δ = 9.93 (s, 2H, 2xH2′), 8.05–8.03 (m, 3H, 2xH4′, H4), 7.70–7.68 (d, J = 8.0 Hz, 2H, 2xH3), 7.65–7.61 (t, J = 8.0 Hz, 4H, 2xH7′, 2xH5′), 7.44–7.41 (t, J = 8.0 Hz, 2H, 2xH6′), 5.96 (s, 4H, 2xCH2-α), 5.68 (s, 4H, 2xCH2-β), 3.77 (s, 6H, 2xCH3). 13C-NMR (125 MHz, DMSO-d6) δ = 167.1 2xCO, 152.9 2xC2, 143.7 2xC2′, 139.0 C4, 131.1 2xC3′a, 130.5 2xC7′a, 126.8 2xC5′, 126.6 2xC6′, 122.7 2xC3, 113.9 2xC4′, 113.5 2xC7′, 52.9 2xCH3, 50.7 2xCH2-α, 47.5 2xCH2-β. Anal. Calcd. for C27H27Br2N5O4: C, 50.25; H, 4.22; N, 10.85; found: C, 50.10; H, 4.13; N, 11.04.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-amino-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) iodide (6b)

Appearance: yellow powder. Yield, 74%. mp 123–126°C. FTIR (KBr) cm-1 = 3349, 3168, 3040, 2915, 1680, 1598, 1420. 1H-NMR (500 MHz, DMSO-d6) δ = 9.70 (s, 2H, 2xH2′), 8.05–8.02 (m, 3H, H4, 2x(-NH-)), 7.90–7.88 (d, J = 8.5 Hz, 2H, 2xH4′), 7.72 (brs, 2H, 2x(-NH-)), 7.69–7.68 (d, J = 7.5 Hz, 2H, 2xH3), 7.64–7.61 (t, J = 8.0 Hz, 2H, 2xH5′), 7.60–7.59 (d, J = 8.0 Hz, 2H, 2xH7′), 7.42–7.38 (t, J = 8.0 Hz, 2H, 2xH6′), 5.89 (s, 4H, 2xCH2-α), 5.26 (s, 4H, 2xCH2-β). 13C-NMR (125 MHz, DMSO-d6) δ = 166.4 2xCO, 152.9 2xC2, 143.8 2xC2′, 138.9 C4, 131.2 2xC3′a, 130.5 2xC7′a, 126.6 2xC5′, 126.4 2xC6′, 122.6 2xC3, 113.4 2xC4′, 113.4 2xC7′, 50.5 2xCH2-α, 48.4 2xCH2-β. Anal. Calcd. for C25H25I2N7O2: C, 42.33; H, 3.55; N, 13.82; found: C, 42.43; H, 3.50; N, 13.97.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-oxo-2-phenylethyl)-1H-benzo[d]imidazol-3-ium) bromide (6c)

Appearance: white powder. Yield, 50%. mp 203–205°C. FTIR (KBr) cm-1 = 3139, 2973, 1697, 1586, 1543, 1480. 1H-NMR (500 MHz, DMSO-d6) δ = 9.88 (s, 2H, 2xH2′), 8.12–8.11 (d, J = 8.0 Hz, 4H, 4xH2′′), 8.09–8.06 (m, 3H, H4, 2xH4′), 7.81–7.78 (t, J = 7.5 Hz, 2H, 2xH4′′), 7.75–7.73 (d, J = 8.0 Hz, 2H, 2xH3), 7.68–7.59 (m, 8H, 2xH7′, 4xH3′′, 2xH5′), 7.45–7.42 (t, J = 8.0 Hz, 2H, 2xH6′), 6.49 (s, 4H, 2xCH2-β), 6.02 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 191.3 2xCO, 153.0 2xC2, 143.8 2xC2′, 139.0 C4, 134.6 2xC4′′, 133.6 2xC1′′, 131.6 2xC3′a, 130.5 2xC7′a, 129.0 4xC3′′, 128.5 4xC2′′, 126.7 2xC5′, 126.5 2xC6′, 122.7 2xC3, 113.9 2xC4′, 113.4 2xC7′, 53.4 2xCH2-β, 50.7 2xCH2-α. Anal. Calcd. for C37H31Br2N5O2: C, 60.26; H, 4.24; N, 9.50; found: C, 60.16; H, 4.33; N, 9.35.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-bromophenyl)-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) bromide (6d)

Appearance: white powder. Yield, 77%. mp 193–195°C. FTIR (KBr) cm-1 = 3140, 3020, 2943, 1694, 1585, 1550, 1470, 620. 1H-NMR (500 MHz, DMSO-d6) δ = 9.83 (s, 2H, 2xH2′), 8.08–8.05 (m, 3H, H4, 2xH4′), 8.02–8.01 (d, J = 9.0 Hz, 4H, 4xH2′′), 7.87–7.85 (d, J = 8.5 Hz, 4H, 4xH3′′), 7.74–7.72 (d, J = 7.5 Hz, 2H, 2xH3), 7.68–7.66 (d, J = 8.5 Hz, 2H, 2xH7′), 7.62–7.59 (t, J = 7.5 Hz, 2H, 2xH5′), 7.46–7.43 (t, J = 7.5 Hz, J = 8.0 Hz, 2H, 2xH6′), 6.43 (s, 4H, 2xCH2-β), 6.00 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 190.7 2xCO, 153.0 2xC2, 143.8 2xC2′, 139.0 C4, 132.6 2xC1′′, 132.1 4xC3′′, 131.6 2xC3′a, 130.5 2xC7′a, 130.4 4xC2′′, 128.8 2xC4′′, 126.7 2xC5′, 126.5 2xC6′, 122.7 2xC3, 114.0 2xC4′, 113.4 2xC7′, 53.3 2xCH2-β, 50.7 2xCH2-α. Anal. Calcd. for C37H29Br4N5O2: C, 49.64; H, 3.26; N, 7.82; found: C, 49.74; H, 3.17; N, 7.98.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-chlorophenyl)-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) bromide (6e)

Appearance: white powder. Yield, 83%. mp 175–178°C. FTIR (KBr) cm-1 = 3136, 3068, 2970, 1695, 1589, 1564, 1483, 765. 1H-NMR (400 MHz, DMSO-d6) δ = 9.84 (2H, s, 2xH2′), 8.11–8.09 (d, J = 8.4 Hz, 4H, 4xH2′′), 8.07–8.05 (d, J = 8.0 Hz, 3H, 2xH4′, H4), 7.74–7.67 (m, 8H, 2xH3, 4xH3′′, 2xH7′), 7.63–7.59 (t, J = 7.6 Hz, 2H, 2xH5′), 7.46–7.42 (t, J = 7.6 Hz, 2H, 2xH6′), 6.44 (s, 4H, 2xCH2-β), 6.00 (s, 4H, 2xCH2-α). 13C-NMR (100 MHz, DMSO-d6) δ = 190.4 2xCO, 153.0 2xC2, 143.8 2xC2′, 139.5 2xC4′′, 139.0 C4, 132.3 2xC1′′, 131.6 2xC3′a, 130.5 2xC7′a, 130.3 4xC2′′, 129.1 4xC3′′, 126.7 2xC5′, 126.5 2xC6′, 122.7 2xC3, 113.9 2xC4′, 113.4 2xC7′, 53.4 2xCH2-β, 50.7 2xCH2-α. Anal. Calcd. for C37H29Br2Cl2N5O2: C, 55.11; H, 3.62; N, 8.69; found: C, 54.96; H, 3.57; N, 8.85.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-nitrophenyl)-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) bromide (6f)

Appearance: yellowish powder. Yield, 48%. mp 235–238°C. FTIR (KBr) cm-1 = 3135, 3018, 2955, 1685, 1583, 1525, 1488, 1348. 1H-NMR (500 MHz, DMSO-d6) δ = 9.88 (s, 2H, 2xH2′), 8.43–8.42 (d, J = 8.0 Hz, 4H, 4xH3′′), 8.35–8.33 (d, J = 8.5 Hz, 4H, 4xH2′′), 8.12–8.06 (m, 3H, 2xH4′, H4), 7.75–7.73 (d, J = 7.5 Hz, 2H, 2xH3), 7.71–7.70 (d, J = 8.5 Hz, 2H, 2xH7′), 7.64–7.61 (t, J = 7.5 Hz, 2H, 2xH5′), 7.48–7.45 (t, J = 7.5 Hz, 2H, 2xH6′), 6.55 (s, 4H, 2xCH2-β), 6.03 (s, 4H, 2xCH2-α). 13C-NMR (125 MHz, DMSO-d6) δ = 190.7 2xCO, 153.0 2xC2, 150.5 2xC4′′, 143.7 2xC2′, 139.0 C4, 138.3 2xC1′′, 131.6 2xC3′a, 130.5 2xC7′a, 130.0 4xC2′′, 126.7 2xC5′, 126.6 2xC6′, 123.9 4xC3′′, 122.7 2xC3, 114.1 2xC4′, 113.5 2xC7′, 53.9 2xCH2-β, 50.7 2xCH2-α. Anal. Calcd. for C37H29Br2N7O6: C, 53.70; H, 3.53; N, 11.85; found: C, 53.80; H, 3.62; N, 11.70.

1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) bromide (6g)

Appearance: white powder. Yield, 71%. mp 183–186°C. FTIR (KBr) cm-1 = 3138, 3016, 2931, 1683, 1599, 1566, 1242, 1172. 1H-NMR (500 MHz, DMSO-d6) δ = 9.89 (s, 2H, 2xH2′), 8.08–8.04 (m, 7H, 4xH2′′, 2xH4′, H4), 7.76–7.75 (d, J = 7.5 Hz, 2H, 2xH3), 7.70–7.68 (d, J = 8.0 Hz, 2H, 2xH7′), 7.62–7.58 (t, J = 8.0 Hz, 2H, 2xH5′), 7.45–7.42 (t, J = 8.0 Hz, 2H, 2xH6′), 7.14–7.12 (d, J = 8.5 Hz, 4H, 4xH3′′), 6.42 (s, 4H, 2xCH2-β), 6.01 (s, 4H, 2xCH2-α), 3.90 (6H, s, 2xCH3). 13C-NMR (125 MHz, DMSO-d6) δ = 189.5 2xCO, 164.2 2xC4′′, 153.0 2xC2, 143.8 2xC2′, 139.0 C4, 131.6 2xC3′a, 130.9 4xC2′′, 130.5 2xC7′a, 126.6 2xC5′, 126.5 2xC6′, 126.4 C1′′, 122.7 2xC3, 114.2 4xC3′′, 113.9 2xC4′, 113.4 2xC7′, 55.8 2xCH3, 53.0 2xCH2-β, 50.6 2xCH2-α. Anal. Calcd. for C39H35Br2N5O4: C, 58.73; H, 4.42; N, 8.78; found: C, 58.63; H, 4.47; N, 8.63.

Microbiology

Compounds were evaluated for antimycobacterial activity against Mycobacterium tuberculosis, as a part of the Center of Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) TB screening program under direction of the US National Institute of Health, the NIAID division. Antimycobacterial activities of the compounds were performed by TAACF at Southern Research Institute. All protocols concerning the antimycobacterial evaluation of tested compounds can be found in the Supplementary Figure 1.

Figure 1. . Atom identification in the spectral data of quaternary salts 4a–g and 6a–g.

Figure 1. 

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Results & discussion

Design & synthesis

One of the most useful strategies in drug discovery is the pharmacophore combination of well defined scaffolds. Bearing in mind this strategy, we decided to bring new contributions in the anti-TB fighting and, pursuing to achieve products with better activity, better pharmacological properties and alternative mechanisms of action, the present study is aimed to combine three pharmacophores: pyridine, imidazole/benzimidazole and p-chlorobenzoyl moieties. Structural necessities around pyridine and imidazole/benzimidazole moieties, for an anti-TB activity, are depicted in Figure 2.

Figure 2. . Design in the class of bis-(imidazole/benzimidazole)-pyridine derivatives.

Figure 2. 

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The first two pharmacophores units, pyridine and imidazole/benzimidazole moieties, are connected via a methylene (-CH2-) linker in 2,6- position of pyridine, respectively 1-position of imidazole/benzimidazole, the BIP derivatives type I being obtained. The p-chlorobenzoyl moiety was found to be a potent pharmacophoric unit for the antimycobacterial activity and also a useful antibacterial pharmacophore (due to its high lipophilicity) [19,24,25]. Consequently, we decide to anchor the p-chlorobenzoyl moiety in the 3-position of imidazole/benzimidazole, a second class of BIP derivatives, type II being obtained. We also decided to study the influence of other p-substituted (-H, -NO2, -OMe)-benzoyl moieties and nonbenzoyl moiety (-carbalkoxy, -acetamide) anchored to imidazole/benzimidazole, in order to allow structure–activity relationship (SAR) comparisons with p-chlorobenzoyl moiety.

In order to synthesize the BIP derivatives a direct approach was used, involving only two successive N-alkylations (Figure 3). A first N-alkylation of NH- imidazolic unit (from imidazole, 4-nitroimidazole and benzimidazole) is leading to the corresponding BIP derivatives type I, 2a, b and 5. The second alkylation consists in a quaternization reaction of N3-nitrogen atom from the imidazolic moiety with variously activated halogenated derivatives type 3 (2–bromo/iodo-alkyl esters/amide 3a, b or ϖ–bromo p-Z-substituted-acetophenones 3c–g), the BIP derivatives type II, 4a–g and 6a–g, being obtained.

Figure 3. . Reaction pathway to obtain bis-imidazole/benzimidazole-pyridine derivatives.

Figure 3. 

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For the BIP compound 2b, the quaternization reaction of N3-nitrogen atom from imidazole does not occur, the most probable because the imidazole ring is too deactivated (Figure 4).

Figure 4. . Structure–activity relationship correlations in the class of bis-(imidazole/benzimidazole)-pyridine derivatives.

Figure 4. 

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Using the modern tools of structural organic analysis (IR, 1H-NMR, 13C-NMR, 2D-COSY, 2D-HMQC, 2D-HMBC, and elemental analysis: C, H, N), we proved equivocally the structure of our compounds. In our structural organic analysis, we considered compound 6e as representative for the BIP derivatives (is the most active and promising anti-TB compound). The most informative signals furnished by 1H-NMR spectrum of 6e are those one of the aromatic protons H2′ (from imidazole ring) and H2′′ (from benzoyl), respectively the methylene protons: 2H (CH2-α pyridine) and 2H (CH2-β benzoyl). The most deshielded protons are H2′ (9.84 p.p.m., singlet) from benzimidazole moiety, due to the powerful deshielding effect of the positive nitrogen atom N3′ and N1′ nitrogen. The next deshielded protons are H2′′ from benzoyl moiety (8.11–8.09 p.p.m., doublet, J = 8.4 Hz), being deshielded by the electron withdrawing effect of the adjacent carbonyl ketone group and p-chlorophenyl moiety. The signals of the two methylene groups appear as singlet, at a very low field, unusual for this type of protons: 6.44 p.p.m. (CH2-β benzoyl) and 6.00 p.p.m. (CH2-α pyridine). This situation could be explained by the powerful electron withdrawing effect exerted by the CO carbonyl group and positive nitrogen atom N3′ from imidazole moiety (for CH2-β) from proximity, respectively nitrogen atom N1′ from imidazole and α-pyridine ring (for CH2-α).

In the 13C-NMR spectrum, the most important data are furnished by the signals corresponding to the carbonyl group, C2 (from pyridine), C2′ (from imidazole ring) and the two carbons from the methylene groups (CH2-α pyridine and CH2-β benzoyl). The signals for carbonyl group appear at 190.4 p.p.m., typical for a C = O carbonyl ketone group. The C2 carbon is strongly deshielded (appears at 153.0 p.p.m.), being a α-endocyclic carbon from pyridine moiety. The C2′ carbon is also strongly deshielded (appears at 143.8 p.p.m.), in accordance with the powerful deshielding effect of the two adjacent nitrogen atoms from imidazole ring. The carbons from methylene groups appear at unusual low field: 53.4 p.p.m. (carbon from CH2-β benzoyl) and 50.7 p.p.m. (carbon from CH2-α pyridine). The same explications as far for methylene protons remain valid. The other remaining peaks form NMR spectra fully confirm the structure of compounds. Additional information concerning the 1H- and 13C-NMR spectra of compound 6e is shown in the Supplementary Information.

Antimycobacterial assay against Mycobacterium tuberculosis

The obtained compounds have been evaluated for their in vitro antimycobacterial activity against Mtb H37Rv (grown under aerobic conditions), on TAACF, US NIH, the NIAID division, at Southern Research Institute. The first step of assay consisted in the determination of relative solubility of our products in microbiological medium using turbidity as a standard parameter [32]. The results listed in Table 1 show that all the BIP compounds are soluble at the highest concentration (200 μM). For a future drug candidate, this excellent solubility microbiological medium is an excellent strength.

Table 1. . Solubility in microbiological medium and antimycobacterial activity of bis-(imidazole/benzimidazole)-pyridine compounds against Mtb H37Rv under aerobic conditions.

Compound MIC (μM) IC50 (μM) IC90 (μM) Compound solubility
        Starting concentration (μM) Lowest insoluble concentration (μM)
2a >200 >200 >200 200 N/A
2b† 92 77 100 200 N/A
4a >200 >200 >200 200 N/A
4b >200 >200 >200 200 N/A
4c >200 >200 >200 200 N/A
4e† 51 34 63 200 N/A
4f† 58 38 71 200 N/A
4g >200 160 >200 200 N/A
5† >50 43 >50 200 N/A
6a >200 >200 >200 200 N/A
6b >200 180 >200 200 N/A
6c† 56 25 60 200 N/A
6e† 17 7.9 18 200 N/A
6f† 24 14 21 200 N/A
6g† 58 28 6 200 N/A
Rifam picin 0.0060 0.0036 0.070 0.04 N/A
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†

Values indicate that compounds have a very good activity.

IC50: 50% inhibition of growth; IC90: 90% inhibition of growth; N/A: Compound was soluble at the highest concentration; MIC: Minimum inhibitory concentration.

Further, the in vitro screening assay allowed to determine the minimum inhibitory concentration (MIC), IC50 and IC90 (the concentrations that resulted in 50% and 90% inhibition of growth; Table 1) [33–37].

As can be seen, eight (2b, 5, 4e, 4f, 6c, 6e, 6f and 6g) from the 15 tested compounds had activity against Mtb H37Rv under aerobic conditions, with a MIC in the range of 17– 92 μM, and an IC50 in the range of 7.9 – 77 μM. From SAR point of view, the following remarks could be done: the BIP II compounds are more active than BIP I, which means that anchoring of a p-substituted-benzoyl moiety in the 3-position of imidazole/benzimidazole moiety is favorable for activity; the compounds bearing a benzimidazole moiety are more active than those one bearing an imidazole moiety. Compound 6e (with a p-chlorobenzoyl moiety anchored to benzimidazole) have shown the most pronounced antimycobacterial activity, which confirms our initial design considerations; and the p-substituted-benzoyl compounds are more much active than nonbenzoyl substituted (-carbalkoxy, -acetamide). Also, the active compounds are those one having in the para position of benzoyl moiety a chloride or nitro group, while replacing them with other atoms or group of atoms is leading to a dropping of antimycobacterial activity.

Five BIP derivatives, namely 2b, 5, 4f, 6c and 6f, with showed the best anti-TB profile in the primary assay, was passed into the secondary assays for evaluation of antimcyobacterial activity in a low throughput format. These assays include a revaluation of MIC, IC50 and IC90 against Mtb H37Rv grown under aerobic conditions, MIC under low oxygen, minimal bactericidal concentration (MBC), testing on drug-resistant Mtb, intracellular activity and cytotoxicity.

Initially, MIC, IC50 and IC90 were repeated at lower starting concentrations (Table 2). As we may notice from Table 2, the results of assay are reproducible, the obtained values being comparable with the previous ones listed in Table 1.

Table 2. . Activity against Mtb H37Rv at lower starting concentrations for bis-(imidazole/benzimidazole)-pyridine derivatives 2b, 5, 4f, 6c and 6f.

Compound MIC aerobic condition (μM) IC50 (μM) IC90 (μM)
2b† 101 93 100
5 >50 >50 >50
4f† 104 45 140
6c† 87 34 110
6f† 16 11 20
Rifampicin 0.0063 0.0040 0.0077
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Values indicate that compounds have a very good activity.

IC50: 50% inhibition of growth; IC90: 90% inhibition of growth; MIC: Minimum inhibitory concentration.

The bactericidal activity of BIP derivatives was determined subsequent to MIC assay. The MBC assay determines whether a drug's mechanism of action is bacteriostatic or bactericidal. A bactericidal mechanism of action is a truly advantage of drug candidates, because could shorten the standard treatment of TB with a minimum of 6 months. The MBC was determined against Mtb H37Rv grown in aerobic conditions, and indicate that three of the tested compounds (4f, 6c, 6f) have a bactericidal mechanism of action, Table 3. We may also notice from Table 3 that the activity of compounds 6c and 6f, are concentration dependent, which is associated with an optimal free drug maximum concentration to MIC ratio [38]. In the case of 4f molecule, a time dependent effect is observed, which is usually linked with a free drug concentrations existing above the MIC for a certain section of the dosing interval [38].

Table 3. . Minimal bactericidal concentration assay for compounds 2b, 5, 4f, 6c and 6f.

Compound MIC (μM) MBC (μM) Concentration dependent Time dependent
2b† 92 >200 ND ND
4f† 58 100 N Y
5 >50 >50 ND ND
6c† 56 100 Y N
6f† 18 50 Y N
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Values indicate that compounds have a very good activity.

MBC: Minimal bactericidal concentration; MIC: Minimum inhibitory concentration; N: No; ND: Not determined; Y: Yes.

Low oxygen recovery assay are furnishing essential information concerning activity against Mtb in a state of nonreplicating persistence (NRP). Nonreplicating state of Mtb is responsible for antimicrobial tolerance found in more and more cases of TB infections. As a result, the antimycobacterial activity against NRP Mtb H37Rv for compounds 2b, 5, 4f, 6c and 6f, were determined [39–41]. Results for the low oxygen recovery assay in terms of MIC, IC50 and IC90, are listed in Table 4.

Table 4. . Low oxygen recovery assay for compounds 2b, 5, 4f, 6c and 6f.

Compound Low oxygen Normal oxygen
  MIC (μM) IC50 (μM) IC90 (μM) MIC (μM) IC50 (μM) IC90 (μM)
2b† 200 26 91 >200 >200 >200
5 >50 >50 >50 >50 >50 >50
4f† 183 25 65 129 49 78
6c† 200 16 130 >200 17 60
6f† 120 9.7 33 71 9.9 25
Rifampicin 0.13 0.0041 0.0065 0.096 0.0072 0.0025
Metronidazole 200 29 110 >200 >200 >200
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Values indicate that compounds have a very good activity.

IC50: 50% inhibition of growth; IC90: 90% inhibition of growth; MIC: Minimum inhibitory concentration.

As can be seen in Table 4, our compounds (except 5) had activity against NRP Mtb H37Rv superior to control metronidazole, with an IC50 in the range of 9.7– 26 μM, and a MIC in the range of 120–200 μM.

Next, MIC, IC50 and IC90 of compounds 2b, 5, 4f, 6c and 6f against five resistant isolates of Mtb strains [34–37] and nontuberculous mycobacteria (NTM) [34,37,42] under aerobic conditions were determined, Table 5. The NTM were Mycobacterium avium and Mycobacterium abscessus, while the resistant isolates were INH-R1 and INH-R2 (strains resistant to isoniazid), RIF-R1 and RIF-R2 (strains resistant to rifampicin), and FQ-R1 (strain resistant to fluoroquinolone).

Table 5. . MIC, IC50 and IC90 of compounds 2b, 5, 4f, 6c and 6f against Mtb resistant at different treatment and nontuberculous mycobacteria.

Compound INH-R1 INH-R2 RIF-R1 Mycobacterium avium
  MIC (μM) IC50 (μM) IC90 (μM) MIC (μM) IC50 (μM) IC90 (μM) MIC (μM) IC50 (μM) IC90 (μM) MIC (μM)
2b† 98 68 90 28 24 28 111 60 160 –
5 >50 >50 >50 >50 >50 >50 >50 >50 >50 –
4f† 55 46 52 65 39 75 67 35 100 >200
6c† 120 42 170 110 42 150 87 24 110 >200
6f† 24 13 21 25 13 22 25 15 24 200
C1 0.018 0.0084 0.022 0.065 0.0047 0.012 2 1.2 2.3 >200
C2 >200 >200 >200 >200 >200 >200 0.17 0.15 0.21 –
C3 1.2 0.64 1.4 1.4 0.84 1.4 0.76 0.59 0.91 –
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Values indicate that compounds have a very good activity.

C1: Rifampicin control; C2: Isoniazid control; C3: Levofloxacin control; FQ: Fluoroquinolone resistant strain; IC50: 50% inhibition of growth; IC90: 90% inhibition of growth; INH: Isoniazid resistant strain; MIC: Minimum inhibitory concentration; RIF: Rifampicin resistant strain.

Table 6. . MIC, IC50 and IC90 of compounds 2b, 5, 4f, 6c and 6f against Mtb resistant at different treatment and nontuberculous mycobacteria.

Compound RIF-R2 FQ-R1 Mycobacterium abscessus
  MIC (μM) IC50 (μM) IC90 (μM) MIC (μM) IC50 (μM) IC90 (μM) MIC (μM) IC50 (μM) IC90 (μM)
2b 68 38 61 102 66 92 – – –
5 >50 >50 >50 >50 >50 >50 – – –
4f† >200 40 120 87 49 91 110 89 97
6c† 100 44 120 200 40 >200 >200 120 200
6f† 30 17 31 38 18 38 88 56 68
C1 >50 >50 >50 0.027 0.013 0.039 3.3 2.1 3.1
C2 0.62 0.54 0.60 0.35 0.36 0.47 – – –
C3 1.1 0.60 1.2 20 12 22 – – –
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Values indicate that compounds have a very good activity.

C1: Rifampicin control; C2: Isoniazid control; C3: Levofloxacin control; FQ: Fluoroquinolone resistant strain; INH: Isoniazid resistant strain; MIC: Minimum inhibitory concentration; RIF: Rifampicin resistant strain.

The data from Table 5 indicate that Mtb monoresistant isolates were not resistant to our compounds (except 5) and, compounds 4f, 6c and 6f showed activity against M. abscessus, while against M. avium only compound 6f showed a moderate activity.

The cytotoxicity of BIP compounds were established by using the THP-1 human monocytic cell line [40], and measuring the IC50 values. Similar, the activity of compounds against intracellular Mtb H37Rv were determined by measuring viability in infected THP-1 cell line [40], and expressed in terms of IC50 and IC90, values. The obtained results are listed in Table 7.

Table 7. . Intracellular activity and cytotoxicity evaluation.

Compound Cytotoxicity IC50 (μM) IC50 intracell (μM) IC90 intracell (μM)
2b† >50 >200 >200
5† >50 >200 >200
4f† >50 26 48
6c† >50 37 >50
6f† >50 14 26
Control compound‡,§ 0.018‡ 0.23§ 0.29§
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Bold and italic values indicate that compounds have a very good activity.

‡

Staurosporine control.

§

Isoniazid control.

IC50: 50% inhibition of growth; IC90: 90% inhibition of growth.

As can be seen in Table 7, all our compounds are not cytotoxic and all tested compounds exhibited a very good intracellular activity, those one of compound 6f being remarkable. The data presented before indicate that compound 6f have a promising anti-TB profile and, as a result, a study concerning absorption, distribution, metabolism, excretion and toxicity (ADMET) have been done. The ADMET study begin with plasma–protein binding assay of compound 6f, using equilibrium dialysis method with a semipermeable membrane, which separates two compartments containing protein (human plasma) and buffer [43,44]. The obtained results are listed in Table 8.

Table 8. . Results of plasma–protein binding for compound 6f.

Compound Test species Mean plasma fraction unbound (%) Mean plasma fraction bound (%) Recovery
6f Human 7.8 92.2 24.5
Propranolol† Human 20.7 79.3 93
Warfarin† Human 0.51 99.5 96.7
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†

Control compounds: propranolol (normal binding) and warfarin (high-binding).

As it could be seen in Table 8, compound 6f have a value of plasma fraction bound lesser than 95% (92.2%), which means a lower clearance rate and a greater half-time in vivo and, consequently a more efficacy because of higher free drug concentration. The absorption properties of compound 6f through the intestinal epithelium were studied using the Caco-2 cell monolayer permeability assay, measuring in both directions (apical to basal, respectively basal to apical) the permeability of compound 6f [45–49]. The obtained results are listed in Table 9.

Table 9. . Results of Caco-2 assay of compound 6f.

Compound Mean A→B Papp† (10-6cm/s) Mean B→A Papp† (10-6cm/s) Efflux ratio‡
6f 0.47 0.095 0.20
Atenolol§ 0.13 0.37 2.8
Propranolol§ 12.7 25 2
Talinolol§ 0.077 3.3 43
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Papp is the apparent permeability;

‡

Efflux ratio (Re) is Papp (B→A)/Papp (A→B);

§

Control compounds.

The low active efflux (Re = 0.20) and low permeability (A→B Papp <2), indicate that compound 6f is poorly permeable and suggest a mechanism of absorption almost paracellular, with basically no involvement of transporter proteins (P-glycoprotein or others).

The early discovery of the potential drug–drug interactions is a necessary clue to increase the safety of pharmacotherapy, the drug–drug interactions being associated with the increasing of toxicity, decrease of pharmacological effect and adverse drug reactions [50]. One of the best methods used for detection of drug–drug interactions consist in drug metabolism determination via the cytochrome P450 system. In this respect, compound 6f was tested on while the resistant isolates were INH-R1 and INH-R2 (strains resistant to isoniazid), RIF-R1 and RIF-R2 (strains resistant to rifampicin), and FQ-R1 (strain resistant to fluoroquinolone; Table 10) [50–52 ].

Table 10. . Results of cytochrome P450 inhibition for compound 6f.

Compound IC50(μM)
  CYP3A4 – midazolam CYP3A4 – testosterone CYP2C9 CYP2D6 CYP2C8 CYP2B6 CYP2C19
6f >20 6.6 >20 0.92 >20 >20 8.8
Ketoconazole† 0.033 0.022 – – – – –
Sulfaphenazole† – – 0.16 – – – –
Quinidine† – – – 0.032 – – –
Montelukast† – – – – 0.14 – –
Tranylcypromine† – – – – – – 7.5
Ticlopidine† – – – – – 0.72 –
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†

Control compounds.

IC50: 50% inhibition of growth.

Compound 6f did not show inhibition (IC50 >20) on CYP3A4 – midazolam, CYP2C9, CYP2C8, CYP2B6 and have a low to moderate inhibition (IC50: 6.6 and 8.8) on CYP3A4 – testosterone and CYP2C19, these results indicating a low potential for drug–drug interactions. The microsomal binding assay is another important parameter in the prediction of in vivo pharmacokinetics from in vitro drug metabolism data. In order to determine microsomal stability of compounds, the assay is using pooled human liver S9 microsomes in the presence of the co-factor NADPH [53–55]. The obtained results are listed in Table 11.

Table 11. . In vitro microsomal stability assay of compound 6f.

Compound C (μM) Test species NADPH-dependent CLint† (μl/min/mg) NADPH-dependent T1/2‡ (min) NADPH-free CLint† (μl/min/mg) NADPH-free T1/2‡ (min)
6f 1 Human 21.7 106 <12.8 >180
Verapamil 1 Human 123 18.7 <12.8 >180
Dextromethorphan 1 Human 24.3 94.9 <12.8 >180
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Microsomal intrinsic clearance = ln(2)/(T1/2[microsomal protein]).

‡

Half-life = 0.693/-k, where k is the rate constant.

The data from Table 11 indicate that compound 6f is a low cleared compound, having a CLint<50 μl/min/mg. These indicate that compound 6f is likely to be slowly cleared in vivo, resulting in a higher duration of action. Finally, the cytotoxicity of compound 6f was determined on human liver cells (HepG2), using staurosporine as control (IC50 = 0.0086 μM) [56–59]. Compound 6f showed an IC50 >100 μM, having no cytotoxicity.

Conclusion

In summary, we report herein the design, synthesis and antimycobacterial activity of two new classes of BIP derivatives. The reaction pathway is advantageous and straight applicable, with two steps only: a N1-alkylations followed by a quaternization reaction of N3-nitrogen atom from the imidazolic moiety. 15 of the newly synthesized compounds were subject to the primary antimycobacterial assay against M. tuberculosis H37Rv under aerobic conditions, and reveal that eight (2b, 5, 4e, 4f, 6c, 6e, 6f and 6g) from the tested compounds had activity against Mtb H37Rv. SAR correlations indicate that the most valuable compounds are belong to the BIP II class, which prove that anchoring of a p-substituted-benzoyl moiety in the 3-position of imidazole/benzimidazole moiety is favorable for activity. From these, the most active compounds are those one having in the para position of benzoyl moiety a chloride or nitro group. All BIP compounds proved to be with excellent solubility in microbiological medium, which is an excellent strength for a future drug candidate. The best anti-TB five compounds (2b, 5, 4f, 6c and 6f) was subject to the secondary antimycobacterial screening, these assay indicating that our compounds are potent against both replicating and nonreplicating Mtb (superior to control metronidazole), posses a very good intracellular activity (those one of compound 6f being the highest), are active against drug-resistant Mtb strains, and have no cytotoxicity (on eukaryotic THP-1 human monocytic cells). Three of the tested compounds (4f, 6c, 6f) have a bactericidal mechanism of action and present a moderate to good activity against NTM. The pharmacokinetic ADMET screening of 6f, indicate a lower clearance rate, a great half-time in vivo, a low potential for drug–drug interactions with a high duration of action and no cytotoxicity (on HepG2 cells). We believe that, all these excellent assets make from compound 6f a good candidate for a future drug.

Future perspective

Considering the high versatility and aggressively of Mtb bacillus, the great and urgent demand of pharmaceutical industry, as well as enormous amount of effort for discovery of new anti-TB entities, we believe that substantially improvements in drug discovery of new antitubercular agents will be performed in the next decade. A special attention will be paid further to pyridine and imidazole scaffolds, which have an enormous potential of growth.

Summary points.

  • Design, synthesis and antimycobacterial activity of two new classes of bis-(imidazole/benzimidazole)-pyridine derivatives.

  • The reaction pathway is advantageous and straight applicable and, the newly 15 synthesized compounds was subject to antimycobacterial assay.

  • Interesting structure–activity relationship correlations have been done. Anchoring of a p-chloro-benzoyl moiety in the 3-position of imidazole/benzimidazole moiety is favorable for activity.

  • The anti-TB assay reveal that our compounds possess an excellent solubility in microbiological medium, have an excellent anti-TB activity against both replicating and nonreplicating Mtb, are not cytotoxic, exhibited a very good intracellular activity and are active against drug-resistant Mtb strains, some compounds have a bactericidal mechanism.

  • The pharmacokinetic absorption, distribution, metabolism, excretion and toxicity screening performed for one compound, indicating a lower clearance rate, a great half-time in vivo, a low potential for drug–drug interactions with a high duration of action and no cytotoxicity. We believe that, all these excellent assets make from this compound a good candidate for a future drug.

Supplementary Material

Click here for additional data file. (885KB, doc)

Acknowledgments

The authors are thankful to JP Boyce, the Senior Officer to Division of Microbiology and Infectious Diseases, NIAID. The authors also thank to CERNESIM Research Centre from Alexandru Ioan Cuza University of Iasi, for the NMR experiments.

Footnotes

Supplementary data

Supplementary data related to this article can be found in the online version, and contain NMR spectra of compound 6e and all protocols related to the antimycobacterial evaluation of tested compounds. To view the supplementary data that accompany this paper please visit the journal website at: www.future-science/doi/suppl/10.4155/fmc-2019-0063

Financial & competing interests disclosure

The authors are thankful for financial support to Romanian Ministry of Research and Innovation, Program 1 – Development of the national R&D system, Subprogram 1.2 – Institutional performance – RDI excellence financing projects, grant number 34PFE. Part of this work (biological tests) was supported by National Institutes of Health and the National Institute of Allergy and Infectious Diseases, contract no. HHSN27220110009I/HHSN27200004A19. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Open access

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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