Design, Synthesis and biological evaluation of novel imidazo[1,2-a]pyridinecarboxamides as potent anti-tuberculosis agents

Abstract

Tuberculosis (TB) is a highly infectious disease that has been plaguing the human race for centuries. The emergence of multidrug resistant strains of TB has been detrimental to the fight against tuberculosis with very few safe therapeutic options available. As part of an ongoing effort to identify potent anti-tuberculosis agents, we synthesized and screened a series of novel imidazo[1,2-a]pyridinecarboxamide derivatives for their anti-tuberculosis properties. These compounds were designed based on reported anti-tuberculosis properties of the indolecarboxamides (I2Cs) and imidazo[1,2-a]pyridinecarboxamides (IPAs). In this series, we identified compounds 15 and 16 with excellent anti-TB activity against H37Rv strain of tuberculosis (MIC = 0.10 – 0.19 μM); these compounds were further screened against selected clinical isolates of Mtb. Compounds 15 and 16 showed excellent activities against multidrug resistant (MDR) and extensively drug resistant (XDR) strains of TB (MIC range: 0.05 – 1.5 μM) with excellent selectivity indices. In addition, preliminary ADME studies on compound 16 showed favorable pharmacokinetic properties.

INTRODUCTION

Tuberculosis (TB) is an insidious and air-borne infectious disease caused by Mycobacterium tuberculosis that mainly affects the lungs. TB is one of the top 10 leading cause of mortality worldwide from a single infectious disease (above HIV/AIDS). In 2018, the World Health Organization (WHO) estimated about 10 million people fell ill with TB worldwide while 1.5 million died as a result of TB infection (0.25 million among people living with HIV).(1) An estimated 0.9 million new cases of TB among people living with HIV was reported in 2018, 72% of whom lived in Africa.(1) In addition, eight counties namely: India, China, Indonesia, Philippines, Pakistan, Nigeria, Bangladesh and South Africa, accounted for two third of new TB cases reported globally.(1) The global health burden of TB infection is further aggravated by HIV co-infection, thus making TB one of the leading cause of death among people living with HIV.(1, 2) Active drug-susceptible TB is treatable and curable using a standard 6 month course of 4 antimicrobial drugs (first-line anti-TB drugs namely isoniazid, rifampin, pyrazinamide and ethambutol), however the long duration of treatment, poor drug quality, inability to provide the required drug regimens for the duration of treatment and/or inappropriate drug use has given rise to the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) TB.(2) The burden of TB is further exacerbated by the emergence of MDR-TB and XDR-TB and current TB drugs are inadequate in putting an end to this multidrug resistance epidemic.

In August 2019, the FDA approved PA-824 (pretomanid) in combination with TMC207 (bedaquiline) and Linezolid for the treatment of certain type of extremely treatment-resistant TB of the lungs.(3) Noteworthy, TMC207 (bedaquiline; approved 2012) was the first anti-TB drug approved in over four decades by the FDA for treatment of MDR-TB in adults when there are no other alternatives. However, bedaquiline (TMC207) is associated with side effect such as irregular heart rhythm,(4) which show that the search for newer, highly efficient, fast acting drug with low toxicity is far from over. Bedaquiline targets the membrane-bound adenosine triphosphate (ATP) synthase enzyme of M. tuberculosis,(5-7) while the exact target of pretomanid is not yet known. Pretomanid was also reported to show activity against both replicating and hypoxic nonreplicating strains of tuberculosis.(7-9)

Our TB drug discovery campaign led to the identification of indole-2-carboxamides which showed excellent activity against susceptible, MDR and XDR strains of Mtb.(10-12) The indole-2-carboxamides has been reported to inhibit Mtb growth by targeting the MmpL3 protein (which belongs to the resistance, nodulation and cell division (RND) family of membrane transporters), an essential membrane transporter.(10-14) In addition, we recently reported that indole-2-carboxamide (1) showed anti-M. abscessus activity by acting on the MmpL3 protein of this organism.(15, 16)

Another heterocyclic aromatic carboxamide, which has been extensively researched as a highly potent anti-TB scaffold, is the imidazopyridine-3-carboxamides (IPA).(17-24) Using genome sequencing technique, it was established that IPA inhibitor family exert its anti-TB properties by targeting M. tuberculosis QcrB, an essential component of the electron transport chain.(25)

Structural Activity Relationship (SAR) studies of the indole-2-carboxamides (I2C) showed the importance of cyclic aliphatic ring on the amide nitrogen(10, 12) in order to enhance and maintain activity, this motivated us to investigate the anti-TB activity of IPAs with varying size of cyclic aliphatic rings on the amide nitrogen at both positions 2 and 3 of the imidazopyridine ring. Herein, we report the design, synthesis, biological evaluation and in vitro pharmacokinetic (PK) of novel imidazo[1,2-a]pyridine carboxamide derivatives 3 – 19, our goal is to design and investigate small molecules with potent anti-TB activity.

CHEMISTRY

Following efficient amide coupling protocol (Scheme 1), seventeen novel imidazopyridine carboxamide derivatives were generated. Briefly, ethyl acetoacetate or ethyl benzoylacetate was reacted with N-bromosuccinimide (NBS) to afford the corresponding ethyl-2-bromoacetoacetate or ethyl-2-bromobenzoylacetate, which was subsequently reacted with substituted 2-amino pyridine (21-23) to afford the various substituted imidazo[1,2-a]pyridine-3-carboxylates. Basic hydrolysis of the ester derivatives afforded the corresponding acids 24 - 27.

Scheme 1^a.

^aReagents and conditions: (a) (i) ethyl acetoacetate or ethyl benzoylacetate, NBS, H₂O, reflux, 1.5 h (ii) substituted 2-aminopyridine, reflux, 3 h; (b) LiOH, EtOH, reflux, 3 h; (c) EDC·HCl, HOBt, corresponding amine, Et₃N, CH₂Cl₂, rt, 12–16 h, yield: 33 - 77%.

The substituted imidazopyridine carboxylic acids (24 – 28) were reacted with their corresponding cycloalkyl amines in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and hydroxybenzotriazole (HOBt) as coupling agents and triethylamine as a base to obtain compounds 2–19 (Scheme 1).

DISCUSSION

Structure-Activity Relationships:

In general, the biological activity of the analogs was found to depend on the number of carbon spacers between the amide Nitrogen and the cyclic aliphatic ring. The reference compound 2 showed similar anti-TB activity (MIC = 0.2 μM) as reported in literature.(25) Replacement of the aromatic side chain on the amide nitrogen with cyclic aliphatic rings such as N-cyclohexyl, N-cycloheptyl and N-cyclooctyl groups (compounds 3 – 5) resulted in a loss of activity against susceptible strain of Mtb. Furthermore, replacing the methyl group at position 6 of the imidazo[1,2-a]pyridine ring with a chloro group at positions 6 or 7 (compounds 6 and 7) did not improve their activities against TB (MIC >200 μM). In addition, replacing the methyl group at 2-position of the imidazo[1,2-a]pyridine ring with a phenyl group (compound 8) did not show any improved activity (MIC > 160 μM).

Compounds 9 - 11 bearing one carbon spacer between the amide nitrogen and the cycloalkyl moieties (i.e. cyclohexyl, cycloheptyl and cyclooctyl groups) turned out to be 267-, 133- and 133-fold more potent against M.tb compared to their counterparts compounds 3 - 5 with no carbon spacers. On the other hand, compound 12 bearing one carbon spacer between the amide nitrogen and a polycyclic group (1-adamantyl derivative) showed poor anti-TB activity compared to its monocyclic counterparts 9 – 11. To complete the investigation of the effect of the carbon spacer between amide nitrogen and cycloalkyl group, a two and three carbon spacers were installed and investigated. Compounds 13 (bearing a cyclohexyl group) and 14 (bearing a cycloheptyl group) with two carbon spacers showed similar activities as their one carbon spacer counterparts 9 and 10. On the other hand, compound 15 (bearing a cyclohexyl group) with a three carbon spacers was 4-fold more active than compounds 9 (one carbon spacer) and 13 (two carbon spacers) respectively. Switching the methyl group at position 6 of compound 15 to a 6-chloro (compound 16) resulted in approximately 2-fold drop in activity, while switching the methyl group at position 2 on the imidazopyridine ring to a phenyl group (compound 17, with chloro substituent at position 7) resulted in a complete loss in activity, suggesting that a methyl or small group at the 2-position is essential for activity. Noteworthy, compounds 15 and 16 were approximately 1.5 - 3-fold more active than isoniazid against H37Rv strain of TB. Next, we examined the effect of the position of the amide group on the imidazopyridine ring. The amide group at position 2 of the imidazo[1,2-a]pyridine ring (18 and 19) proved to be less active than the amide group at position 3, implying that amide substituent at the position 3 is essential for anti-TB activity.

Based on our preliminary results, the most active compounds (15 and 16) were selected for further screening against a panel of clinical isolates(26) of Mtb originally obtained from pulmonary TB patients (Table 2). Among those strains are one drug-sensitive (DS) (V4207), two MDR Mtb (KZN494 and V2475), two XDR Mtb (TF274, R506) respectively. In addition, the toxicity of these compounds against mammalian cells was evaluated on Vero cells, and their respective selectivity indices (SI) were calculated (Table 2). Compounds 15 and 16 maintained their excellent activity or showed only a moderate drop in activity (up to 2- fold for 15 and up to 8-fold for 16) against the tested clinical isolates of Mtb. Noteworthy is the fact that both compounds (15 and 16) showed a 2-fold increase in activity against the MDR Mtb strain KZN494 while compounds 15 maintained its excellent activity against XDR Mtb strain R506. The high activity of these compounds against MDR and XDR strains of TB indicates their potential use to treat drug-resistant Mtb strains. In addition, compounds 15 and 16 displayed high IC₅₀ values against Vero cells resulting in high SI indices suggesting their potential lack of toxicity towards mammalian cells.

Table 2.

Activity of imidazo[1,2-a]pyridine-3-carboxamides against Selected Clinical Isolates of Mtb [MIC (μM)] and Vero Cells [IC₅₀ (μM)].

Mtb MIC (μM)>
Compd	H37Rv/DS	V4207/DS	KZN494/MDRa	V2475/MDRa	R506/XDRb	TF274/XDRb	Vero cells IC50 (μM)	SIc
15	0.10	0.10	0.05	0.20	0.10	0.20	102.07	1024
16	0.19	0.37	0.09	0.37	0.75	1.5	≥191.67	≥1024

^aResistance to isoniazid and rifampin.

^bResistance to isoniazid, rifampin, levofloxacin, and kanamycin.

^cSelectivity index (SI) = IC₅₀ (Vero)/MIC.

As for the mode of action, the imidazo[1,2-a]pyridinecarboxamides (IPAs) are well known QcrB inhibitors of M. tuberculosis,(18, 22, 25, 27) thus we propose that these novel imidazo[1,2-a]pyridinecarboxamide compounds could potentially target and bind to QcrB, which encodes the beta subunit of the cytochrome bc1 complex of Mycobacterium tuberculosis. The cytochrome bc1 facilitates electron transfer and proton [H⁺] translocation, which is the driving force for ATP synthesis and essential for Mycobacterium tuberculosis.

Preliminary ADME Studies on Compound 16.

Based on the encouraging biological results described above, we conducted some selected ADME studies on compound 16 to assess its drug-like properties (Table 3). The compound exhibited high plasma protein binding in human (99.89%) and mouse (99.64%). As shown in Table 3, in the presence of compound 16 (10 μM) none of the tested CYP isoforms (with the exception of CYP3A4) showed greater than 48% inhibition.

Table 3.

ADME data for imidazo[1,2-a]pyridine-3-carboxamide 16.

Assay	16
Plasma protein binding (%), human/mouse	99.89/99.64
CYP inhibition (% inhibition at 10 μM)
CYP1A2	26.3
CYP2C9	32.2
CYP2C19	45.2
CYP2D6	16.4
CY3A4a	52.2
hERG IC50 (μM)	>10
Hepatocytes, % remaining at 2 hrs, starting concentration 1 μM	0.19 (human)
Liver microsomes, % remaining after 1 hr, starting concentration 1 μM	1.51 (human, 60 mins), 0.15 (mouse, 60 mins)

^aMidazolam is used as substrate

The potential inhibitory effect of compound 16 on human Ether-à-go-go related gene (hERG) channel was evaluated using manual patch-clamp system. Based on the result, compound 16 is suggested to be ranked as a weak inhibitor on hERG channel (low inhibition of hERG (IC₅₀ > 10 μM); thus implying low potential risk for blocking the cardiac calcium channel. Noteworthy, in the Ether-hERG analysis we found that compound 16 may encounter solubility problem in hERG assay buffer at the highest concentration, 30 μM. The IC₅₀ is therefore only estimated to be > 10 μM, as percentage hERG inhibition tested at 10 μM was found less than 50% (29.10% inhibition at 10 μM).

When incubated with human hepatocyte, over 95% of compound 16 degraded after 2 hours incubation using a starting concentration of 1 μM and the human hepatocytes. Compound 16 was unstable in liver microsomes with approximately 1.5% (human) and 0.15% (mouse) remaining unchanged after 1 hour using a starting concentration of 1 μM.

In summary, we describe the synthesis, structure-activity relationships and preliminary ADME analysis of novel imidazo[1,2-a]pyridinecarboxamide derivatives as anti-TB agents. Modifications to the N-methylthiophen-2-yl group at position 3 of the imidazo[1,2-a]pyridine ring in the lead compound 2 have been explored to create compounds with improved biological activities. Compounds 15 and 16 were identified as the most potent compounds in the series against susceptible strain of tuberculosis, and further screening of these compounds (15 and 16) showed excellent activity against MDR and XDR clinical isolates of TB. In addition, compound 16 showed acceptable PK properties, further study is currently underway to improve its stability in liver microsome and hepatocyte. These compounds adds to the growing list of imidazopyridine carboxamide derivatives that have been reported to possess anti-TB properties in literature(17, 18, 20, 22-24, 27-30).

Experimental details

1. Chemistry method

General information.

7-chloroimidazo[1,2-a]pyridine-2-carboxylic acid was purchased from Combi-blocks. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker spectrometer (300 and 75 MHz), with TMS as an internal standard. Standard abbreviation indicating multiplicity was used as follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet, m = multiplet and br = broad. HRMS experiments were performed on micOTOF-Q (Bruker) instrument. TLC was performed with Merck 60 F254 silica gel plates. Flash chromatography was performed using YamazenFlash® Rf system with Yamazen pre-packed columns or alternatively using Sigma-Aldrich silica gel (230–400 mesh). Purities of final compounds were established by analytical HPLC, which was carried out using the Agilent 1100 HPLC system with a Synergi 4 μm Hydro-RP 80A column, on a variable wavelength detector G1314A. Method: flow rate = 1.0 mL/min; gradient elution over 20 minutes, from 30% MeOH-H₂O to 100% MeOH with 0.05% TFA, and 100% MeOH with 0.05% TFA for an additional 5 minutes. The purity of all tested compounds was >95% (unless otherwise stated) as determined by the method described above.

General procedure for the synthesis of 2-19

To a solution of the appropriate carboxylic acid (1 equiv) in anhydrous dichloromethane (CH₂Cl₂) (4 mL/mmol) at room temperature were added anhydrous hydroxybenzotriazole (HOBt, 1 equiv) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 1 equiv) under an argon atmosphere. After stirring for 10 min, the appropriate substituted amine (1 equiv) and triethylamine (1.5 equiv) were added, and the reaction mixture was stirred at room temperature until disappearance of the starting material (usually 12 to 16 h). After this time water (2 mL) was added, and the mixture was extracted with EtOAc (3×10 mL), the organic layers were separated, washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (dichloromethane–methanol) gradient, 0-10% methanol in dichloromethane over 20-30 minutes (unless specified differently) to obtain the imidazopyridinamides in yields ranging from 33 to 77%.

Experimental details

2,6-dimethyl-N-(thiophen-2-ylmethyl)imidazo[1,2-a]pyridine-3-carboxamide (2).

Yield 65% (off-white solid). HPLC: t_R 8.0 min, purity 98.6%. ¹H NMR (300 MHz, CDCl₃) δ 9.21 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.23 (d, J = 3.0 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H), 7.05 (d, J = 3.0 Hz, 1H), 6.98-6.95 (t, J = 3.0, 6.0 Hz, 1H), 6.19 (s, 1H), 4.86 (d, J = 6.0 Hz, 2H), 2.65 (s, 3H), 2.35 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 161.4, 145.5, 145.1, 141.1, 130.1, 127.0, 126.0, 135.3, 123.0, 115.7, 114.9, 38.3, 18.4, 16.8. HRMS (ESI) calcd for C₁₅H₁₆N₃OS ([M+H]⁺) 286.1009; found: 286.0986.

N-cyclohexyl-2, 6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (3).

Yield 70 % (white solid). HPLC: t_R 9.0 min, purity 97.9%. ¹H NMR (300 MHz, CDCl₃) δ 9.16 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 5.67 (d, J = 6.0 Hz, 1H), 4.08-3.93 (m, 1H), 2.61 (s, 3H), 2.33 (s, 3H), 2.11-1.99 (m, 2H), 1.81-1.67 (m, 2H), 1.55-1.16 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 160.9, 144.9, 144.5, 129.7, 125.9, 122.7, 115.6, 115.4, 48.1, 33.4, 25.6, 24.8, 18.3, 16.6. HRMS (ESI) calcd for C₁₆H₂₂N₃O ([M+H]⁺) 272.1757; found: 272.1744.

N-cycloheptyl-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (4).

Yield 74 % (white powder). HPLC: t_R 10.3 min, purity 97.9%. ¹H NMR (300 MHz, CDCl₃) δ 9.18 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 5.74 (d, J = 6.0 Hz, 1H), 4.29-4.10 (m, 1H), 2.66 (s, 3H), 2.33 (s, 3H), 2.15-1.98 (m, 2H), 1.76-1.44 (m, 10H); ¹³C NMR (75 MHz, CDCl₃) δ 160.7, 144.9, 144.5, 129.7, 125.9, 122.7, 115.7, 115.4, 50.4, 35.4, 28.0, 24.2, 18.3, 16.7. HRMS (ESI) calcd for C₁₇H₂₄N₃O ([M+H]⁺) 286.1914; found: 286.1911.

N-cyclooctyl-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (5).

Yield 61 % (white powder). HPLC: t_R 12.5 min, purity 98.7%. ¹H NMR (300 MHz, CDCl₃) δ 9.16 (s, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.16 (d, J = 9.0 Hz, 1H), 5.82 (d, J = 9.0 Hz, 1H), 4.28-4.20 (m, 1H), 2.66 (s, 3H), 2.32 (s, 3H), 1.95 (m, 2H), 2.02-1.60 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 160.5, 146.0, 144.8, 144.1, 129.9, 125.9, 122.9, 115.5, 49.4, 32.5, 27.2, 25.5, 23.7, 18.3, 16.5. HRMS (ESI) calcd for C₁₈H₂₆N₃O ([M+H]⁺) 300.2070; found: 300.2072.

6-chloro-N-cyclooctyl-2-methylimidazo[1,2-a]pyridine-3-carboxamide (6).

Yield 50 % (white powder). HPLC: t_R 15.3 min, purity 97.8%. ¹H NMR (300 MHz, CDCl₃) δ 9.49 (s, 1H), 7.49 (d, J = 9.0 Hz, 1H), 7.27 (d, J = 3.0 Hz, 1H), 5.78 (d, J = 6.0 Hz, 1H), 4.32-4.15 (m, 1H), 2.68 (s, 3H), 2.03-1.99 (m, 2H), 1.71-1.61 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 160.1, 145.3, 144.1, 128.0, 126.1, 121.4, 116.6, 116.2, 49.5, 32.5, 27.2, 25.5, 23.7, 16.6. HRMS (ESI) calcd for C₁₇H₂₃ClN₃O ([M+H]⁺) 320.1524; found: 320.1531.

7-chloro-N-cyclooctyl-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7).

Yield 51 % (white powder). HPLC: t_R 13.9 min, purity 97.9%. ¹H NMR (300 MHz, CDCl₃) δ 9.17 (d, J = 6.0 Hz, 1H), 7.41 (s, 1H), 6.76 (d, J = 6.0 Hz, 1H), 5.85 (d, J = 6.0 Hz, 1H), 4.19-4.13 (m, 1H), 2.57 (s, 3H), 1.91-1.86 (m, 2H), 1.65-1.48 (m, 12H).; ¹³C NMR (75 MHz, CDCl₃) δ 160.0, 145.5, 145.4, 133.2, 128.1, 115.9, 115.2, 114.3, 49.4, 32.4, 27.1, 25.4, 23.6, 16.4. HRMS (ESI) calcd for C₁₇H₂₃ClN₃O ([M+H]⁺) 320.1524; found: 320.1537.

7-chloro-N-cyclooctyl-2-phenylimidazo[1,2-a]pyridine-3-carboxamide (8).

Yield 56 % (white powder). HPLC: t_R 19.7 min, purity 97.7%. ¹H NMR (300 MHz, CDCl₃) δ 9.62 (s, 1H), 7.67-7.64 (m, 2H), 7.61 (d, J = 9.0 Hz, 1H), 7.51-7.49 (m, 3H), 7.34 (d, J = 9.0 Hz, 1H), 5.82 (d, J = 9.0 Hz, 1H), 4.12-4.07 (m, 1H), 1.77-1.71 (m, 2H), 1.51-1.27 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 159.5, 148.3, 144.4, 133.5, 129.7, 129.6, 129.0, 128.5, 126.4, 121.9, 117.3, 115.4, 49.2, 31.4, 27.2, 25.3, 23.3. HRMS (ESI) calcd for C₂₂H₂₅ClN₃O ([M+H]⁺) 382.1681; found: 382.1701.

N-(cyclohexylmethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (9).

Yield 58 % (off-white solid). HPLC: t_R 10.8 min, purity 99%. ¹H NMR (300 MHz, CDCl₃) δ 9.19 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 5.86 (s, 1H), 3.35-3.31 (t, J = 6.0, 6.0 Hz, 2H), 2.67 (s, 3H), 2.33 (s, 3H), 1.81-1.60 (m, 6H), 1.32-1.14 (m, 3H), 1.06-0.96 (m, 2H).; ¹³C NMR (75 MHz, CDCl₃) δ 161.8, 145.0, 144.6, 129.8, 126.0, 122.8, 115.6, 115.3, 45.6, 38.1, 31.0, 26.4, 25.8, 18.3, 16.7. HRMS (ESI) calcd for C₁₇H₂₄N₃O ([M+H]⁺) 286.1914; found: 286.1895.

N-(cycloheptylmethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (10).

Yield 47 % (off-white powder). HPLC: t_R 12.4 min, purity 98.3%. ¹H NMR (300 MHz, CDCl₃) δ 9.20 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 5.85 (s, 1H), 3.37-3.33 (t, J = 6.0, 6.0 Hz, 2H), 2.69 (s, 3H), 2.34 (s, 3H), 1.91-1.71 (m, 4H), 1.72-1.68 (m, 2H), 1.58-1.45 (m, 5H), 1.34-1.24 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 161.8, 145.0, 144.6, 129.8, 126.0, 122.8, 115.7, 115.4, 45.9, 39.8, 32.3, 28.3, 26.4, 18.4, 16.7. HRMS (ESI) calcd for C₁₈H₂₆N₃O ([M+H]⁺) 300.2070; found: 300.2058.

N-(cyclooctylmethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (11).

Yield 72 % (off-white powder). HPLC: t_R 11.9 min, purity 99%. ¹H NMR (300 MHz, CDCl₃) δ 9.12 (s, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 5.91 (s, 1H), 3.30-3.26 (t, J = 6.0, 6.0 Hz, 2H), 2.63 (s, 3H), 2.29 (s, 3H), 1.79-1.27 (m, 15H); ¹³C NMR (75 MHz, CDCl₃) δ 161.7, 144.9, 144.6, 129.7, 125.9, 122.7, 115.6, 115.4, 46.1, 37.9, 30.4, 26.9, 26.2, 25.4, 18.3, 16.6. HRMS (ESI) calcd for C₁₉H₂₈N₃O ([M+H]⁺) 314.2227; found: 314.2213.

N-(adamantan-1-ylmethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (12).

Yield 73 % (tan solid). HPLC: t_R 12.5 min, purity 99%. ¹H NMR (300 MHz, CDCl₃) δ 9.17 (s, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 5.87-5.82 (t, J = 6.0, 9.0 Hz, 1H), 3.19 (d, J = 6.0 Hz, 2H), 2.70 (s, 3H), 2.31 (s, 3H), 2.05-1.96 (m, 3H), 1.73-1.56 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 161.9, 145.0, 144.5, 129.7, 126.0, 122.8, 115.6, 115.4, 50.7, 40.4, 36.9, 33.9, 28.2, 18.3, 16.9. HRMS (ESI) calcd for C₂₁H₂₈N₃O ([M+H]⁺) 338.2227; found: 338.2212.

N-(2-cyclohexylethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (13).

Yield 51 % (off-white powder). HPLC: t_R 12.6 min, purity 98.5%. ¹H NMR (300 MHz, CDCl₃) δ 9.12 (s, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 5.82-5.79 (t, J = 3.0, 6.0 Hz, 1H), 3.50-3.43 (q, J = 6.0, 6.0, 9.0 Hz, 2H), 2.65 (s, 3H), 2.32 (s, 3H), 1.74-1.60 (m, 5H), 1.53-1.46 (q, J = 6.0, 21.0, 12.0 Hz, 2H), 1.33 (m, 1H), 1.22-1.15 (m, 3H), 0.98-0.90 (t, J = 12.0, 12.0 Hz, 2H).; ¹³C NMR (75 MHz, CDCl₃) δ 160.6, 144.9, 144.6, 129.7, 125.9, 122.7, 115.6, 115.3, 37.3, 35.6, 33.2, 26.4, 26.2, 18.3, 16.6. HRMS (ESI) calcd for C₁₈H₂₆N₃O ([M+H]⁺) 300.2070; found: 300.2073.

N-(2-cycloheptylethyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (14).

Yield 33 % (tan solid). HPLC: t_R 14.2 min, purity 96.6%. ¹H NMR (300 MHz, CDCl₃) δ 9.07 (s, 1H), 7.37 (d, J = 9.0 Hz, 1H), 7.10 (s, J = 9.0 Hz, 1H), 5.89-5.85 (t, J = 6.0, 6.0 Hz, 1H), 3.46-3.40 (q, J = 6.0, 6.0 Hz, 2H), 2.59 (s, 3H), 2.26 (s, 3H), 1.71-1.32 (m, 13H), 1.22-1.13 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 161.6, 144.8, 144.6, 129.6, 125.8, 122.7, 115.5, 115.3, 37.9, 37.7, 37.0, 34.4, 28.4, 26.3, 18.3, 16.5. HRMS (ESI) calcd for C₁₉H₂₈N₃O ([M+H]⁺) 314.2227; found: 314.2220.

N-(3-cyclohexylpropyl)-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxamide (15).

Yield 77 % (tan solid). HPLC: t_R 14.0 min, purity 98%. ¹H NMR (300 MHz, CDCl₃) δ 9.17 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 5.81-5.77 (t, J = 6.0, 6.0 Hz, 1H), 3.49-3.42 (q, J = 6.0, 9.0, 6.0 Hz, 2H), 2.66 (s, 3H), 2.33 (s, 3H), 1.74-1.62 (m, 7H), 1.31-1.12 (m, 6H), 0.93-0.83 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 161.7, 144.9, 144.7, 129.8, 125.9, 122.8, 115.7, 115.4, 39.8, 37.4, 34.8, 33.3, 27.3, 26.6, 26.3, 18.3, 16.7. HRMS (ESI) calcd for C₁₉H₂₈N₃O ([M+H]⁺) 314.2227; found: 314.2224.

6-chloro-N-(3-cyclohexylpropyl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (16).

Yield 55 % (off-white powder). HPLC: t_R 17.7 min, purity 97.8%. ¹H NMR (300 MHz, CDCl₃) δ 9.50 (s, 1H), 7.52 (d, J = 12.0 Hz, 1H), 7.30 (d, J = 12.0 Hz, 1H), 5.84-5.83 (t, J = 3.0, 3.0 Hz, 1H), 3.51-3.44 (q, J = 6.0, 6.0, 6.0 Hz, 2H), 2.69 (s, 3H), 1.70-1.68 (m, 7H), 1.28-1.18 (m, 6H), 0.95-0.88 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 161.1, 145.3, 144.1, 128.2, 126.1, 121.5, 116.6, 116.1, 39.9, 37.3, 34.7, 33.3, 27.2, 26.6, 26.3, 16.5. HRMS (ESI) calcd for C₁₈H₂₅ClN₃O ([M+H]⁺) 334.1681; found: 334.1672.

7-chloro-N-(3-cyclohexylpropyl)-2-phenylimidazo[1,2-a]pyridine-3-carboxamide (17).

Yield 70 % (tan powder). HPLC: t_R 20.5 min, purity 93.8% ¹H NMR (300 MHz, CDCl₃) δ 9.65 (s, 1H), 7.68-7.61 (m, 3H), 7.52-7.51 (m, 3H), 7.37-7.33 (m, 1H), 5.81 (s, 1H), 3.31-3,24 (q, J = 6.0, 9.0, 6.0 Hz, 2H), 1.69-1.61 (m, 7H), 1.43-1.33 (m, 2H), 1.22-1.10 (m, 4H), 1.05-0.97 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 160.6, 148.4, 144.5, 133.5, 128.7, 129.1, 128.7, 126.8, 126.4, 122.0, 117.4, 115.2, 39.6, 37.3, 34.5, 33.2, 26.6, 26.5, 26.3. HRMS (ESI) calcd for C₂₃H₂₇ClN₃O ([M+H]⁺) 396.1837; found: 396.1837.

7-chloro-N-cyclooctylimidazo[1,2-a]pyridine-2-carboxamide (18).

Yield 46 % (white powder). HPLC: tR 17.5 min, purity 96.4%. ¹H NMR (300 MHz, CDCl₃) δ 8.21 (s, 1H), 8.11 (s, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.28 (d, J = 6.0 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 4.20-4.17 (m, 1H), 1.97-1.90 (m, 2H), 1.71-1.57 (m, 12H); ¹³C NMR (75 MHz, CDCl3) δ 161.0, 142.8, 141.4, 127.4, 124.2, 121.5, 118.4, 114.4, 49.2, 32.2, 27.3, 25.4, 23.7. HRMS (ESI) calcd for C₁₆H₂₁ClN₃O ([M+H]+) 306.1368; found: 306.1358.

7-chloro-N-(3-cyclohexylpropyl)imidazo[1,2-a]pyridine-2-carboxamide (19).

Yield 61 % (white powder). HPLC: t_R 18.7 min, purity 97.8%. ¹H NMR (300 MHz, CDCl₃) δ 8.21 (s, 1H), 8.11 (s, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.35-7.34 (t, 1H), 7.23 (d, J = 9.0 Hz, 1H), 3.47 (q, J = 6.0, 6.0, 6.0 Hz, 2H), 1.68-1.61 (m, 7H), 1.29-1.16 (m, 6H), 0.93-0.86 (m, 2H).; ¹³C NMR (75 MHz, CDCl₃) δ 162.1, 142.8, 141.2, 127.5, 124,2, 121.7, 118.4, 114.4, 39.6, 37.4, 34.6, 33.3, 27.1, 26.7, 26.3. HRMS (ESI) calcd for C₁₇H₂₃ClN₃O ([M+H]⁺) 320.1524; found: 320.1532.

2. Biological method

Minimum Inhibitory Concentration (MIC) determination.

MIC was determined by using MABA assay.

Mtb H37Rv and five clinical isolates were cultured to mid-log phase (OD600 = 0.5) in 7H9 broth at 37°C. The culture was diluted to OD600 of 0.001 using 7H9 without Tween 80 targeting 10⁵ colony forming units (CFU) per mL. Two-fold serial dilution was made for compounds from 64 to 0.0039 μg/mL using 100 μL of 7H9 without Tween 80 on 96-well plates. One hundred μL of the above-mentioned diluted culture was added to each testing wells. Test plates were incubated at 37 °C for 7 days, then 32.5 μL of alamarBlue reagent was added to all wells. Plates were further incubated for 16-18 h at 37° and read at Ex544/Em590 using a BMG Optima fluorescence plate reader. Data were analyzed and the lowest concentration that inhibited 90% of growth was defined as MIC.

Vero Cytotoxicity (IC₅₀) determination.

Cytotoxicity of selected compounds, expressed as IC₅₀, was determined using Vero cells and MABA assay. Briefly, Vero cell linage (ATCC CCL-81) was grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Flat-bottomed 96-well plate was seeded with 1 × 10⁴ cells per well. The plate was incubated at 37°C with 5% CO₂ for 16 h. For compound preparation, 2-fold serial dilution was made using a deep-well block using DMEM containing 5% FBS with a volume of 200 μL. Culture media was replaced with 160 μL of the compound-containing media, with 100% DMSO as positive (100% kill) control and media-only as blank (100% viability) control. The plate was incubated for 72 h and then washed twice with PBS before adding 100 μL of DMEM with 5% FBS medium freshly mixed with 10% alamarBlue. The plate was incubated for 2 h and then immediately read with a fluorescence microplate reader at 544Ex/590Em. The minimum concentration that killed at least 50% of the cells was identified as IC₅₀.

Figure 1.

Indole-2-carboxamide derivative 1; Imidazo[1,2-a]pyridine-3-carboxamide derivative 2 and hybrid structure.

Table 1.

MIC Values of Imidazo[1,2-a]pyridine carboxamide analogs

Compd	X	Y	R	MICa (μM)	Compd	X	Y	R	MICa (μM)
2	6-methyl	methyl		0.22	12	6-methyl	methyl		47.41
3	6-methyl	methyl		117.91	13	6-methyl	methyl		0.42
4	6-methyl	methyl		56.06	14	6-methyl	methyl		0.40
5	6-methyl	methyl		106.88	15	6-methyl	methyl		0.10
6	6-chloro	methyl		>200.13	16	6-chloro	methyl		0.19
7	7-chloro	methyl		≥200	17	7-chloro	phenyl		>161.66
8	7-chloro	phenyl		≥167.58	18	7-chloro	-		≥209.29
9	6-methyl	methyl		0.44	19	7-chloro	-		>200.13
10	6-methyl	methyl		0.42	INH b				0.29
11	6-methyl	methyl		0.80

^aThe lowest concentration of drug leading to at least a 90% reduction of bacterial growth signal by the microplate AlamarBlue assay (MABA). MIC values are reported as an average of three individual measurements

^bINH = Isoniazid.

Data availability statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.