Design, Synthesis and Antimycobacterial Activity of Novel Imidazo[1,2-a]pyridine Amide-Cinnamamide Hybrids

We report herein the design and synthesis of a series of novel imidazo[1,2-a]pyridine amide-cinnamamide hybrids linked via an alkyl carbon chain. All 38 new hybrids were evaluated for their antimycobacterial activity against M. tuberculosis (MTB) H37Rv ATCC 27294 using the microplate Alamar Blue assay (MABA). Although the hybrids are less active than the two reference compounds, the promising activity (MICs: 4 μg/mL) of 2,6-dimethylimidazo[1,2-a]pyridine amide-cinnamamide hybrids 11e and 11k could be a good starting point to further find new lead compounds against multi-drug-resistant tuberculosis.


Introduction
Tuberculosis (TB), including multi-drug-resistant TB (MDR-TB) and extensively-drug-resistant TB (XDR-TB), as well as the lethal combination represented by HIV co-infection, constitutes an unacceptable burden of human suffering and loss [1,2]. For example, the current therapy requires at least 20 months of treatment for MDR-TB. For these infections, several novel candidates are currently in clinical trials [3][4][5], and one of them, Bedaquiline, was approved by the FDA in December 2012 for the treatment of MDR-TB. However, its wide application may be limited because of serious adverse effects, such as cardiac arrhythmias [6]. Therefore, there is still an urgent need for new anti-TB drugs that target novel biological pathways in M. tuberculosis (MTB), shorten therapy and reduce the burden of latent infection [7].
On the other hand, trans-cinnamic acid derivatives are an important class of molecules by reason of their wide spectrum of pharmacological profiles, including antioxidative [18], antitumor [19], antibacterial [20] and antitubercular [21] properties. It is of interest to note that cinnamic acid was used for TB even before the current therapy was discovered [22]. Additionally, cinnamic acid was found to act synergistically with isoniazid, rifamycin and other known anti-TB agents against MTB [23]. Additionally, rifamycin SV, a hybrid derivative of cinnamic acid and rifamycin, was observed to show higher activity against most of the tested MTB and MDR-MTB strains than its individual counterparts [24]. Recently, several natural products containing a cinnamic acid moiety were reported as anti-TB agents [25][26][27][28], such as pisoniamide (Figure 1), a natural cinnamamide isolated from Pisonia aculeate [29].
Molecules 2016, 21,49 2 of 14 ( Figure 1), were observed to have nanomolar potency against MTB H37Rv and MDR-MTB strains in our lab [17]. On the other hand, trans-cinnamic acid derivatives are an important class of molecules by reason of their wide spectrum of pharmacological profiles, including antioxidative [18], antitumor [19], antibacterial [20] and antitubercular [21] properties. It is of interest to note that cinnamic acid was used for TB even before the current therapy was discovered [22]. Additionally, cinnamic acid was found to act synergistically with isoniazid, rifamycin and other known anti-TB agents against MTB [23]. Additionally, rifamycin SV, a hybrid derivative of cinnamic acid and rifamycin, was observed to show higher activity against most of the tested MTB and MDR-MTB strains than its individual counterparts [24]. Recently, several natural products containing a cinnamic acid moiety were reported as anti-TB agents [25][26][27][28], such as pisoniamide (Figure 1), a natural cinnamamide isolated from Pisonia aculeate [29]. In our continuous program in the search of potent and safe IMB1502 derivatives, we intended to construct a new class of hybrids as attractive anti-TB agents by molecular hybridization between IMB1502 and pisoniamide. A detailed structural comparison revealed that both of them are composed of a delocalized aromatic pharmacophore (green) and a hydrophobic moiety (purple) connected via a same ethylidyne linkage (black, Figure 1). Therefore, a series of novel hybrid structures containing IPA and cinnamamide moieties linked via an alkyl carbon chain (ethylidyne or propylidyne) were designed and synthesized in this study (Figure 1), with the hope that these target compounds would exhibit improved anti-MTB activity.

Chemistry
Detailed synthetic pathways to cinnamamide derivatives 5-6 and novel hybrids 11-14 are depicted in Schemes 1 and 2, respectively. Commercially available cinnamic acids 1a-l were treated with thionyl chloride at reflux to give the corresponding acyl chlorides 2a-l. Condensation of the resulting 2a-l with tert-butyl (2-aminoethyl)carbamate or tert-butyl (3-aminopropyl)carbamate in the presence of triethylamine (NEt3) yielded 3a-l and 4a-g, respectively, which were hydrolyzed with trifluoroacetic acid (TFA) to afford the desired cinnamamide derivatives 5a-l and 6a-g as TFA salts (Scheme 1). In our continuous program in the search of potent and safe IMB1502 derivatives, we intended to construct a new class of hybrids as attractive anti-TB agents by molecular hybridization between IMB1502 and pisoniamide. A detailed structural comparison revealed that both of them are composed of a delocalized aromatic pharmacophore (green) and a hydrophobic moiety (purple) connected via a same ethylidyne linkage (black, Figure 1). Therefore, a series of novel hybrid structures containing IPA and cinnamamide moieties linked via an alkyl carbon chain (ethylidyne or propylidyne) were designed and synthesized in this study (Figure 1), with the hope that these target compounds would exhibit improved anti-MTB activity.

Anti-MTB Activity
The target Compounds 11-14 were evaluated for their in vitro activity against MTB H37Rv ATCC 27294 using the microplate Alamar Blue assay (MABA) [32,33]. The minimum inhibitory concentration (MIC) is defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate bacterium-only controls, and MICs of 11-14 along with IMB1502 and isoniazid (INH) for comparison are presented in Table 1. The data reveal that all of the new synthesized hybrids 11-14 (MICs: 4->32 μg/mL) are much less active than the reference drug INH (MIC: 0.05 μg/mL) and the parent compound IMB1502 (MIC: 0.015 μg/mL), but fourteen of them have potential activity against this strain (MICs: 4-32 μg/mL). Among them, Compounds 11e and 11k display the highest activity (MICs: 4 μg/mL), and Compounds 11k and 11e have also promising activity with MICs of 8 and 16 μg/mL, respectively, against MTB H37Rv ATCC 27294.

Anti-MTB Activity
The target Compounds 11-14 were evaluated for their in vitro activity against MTB H37Rv ATCC 27294 using the microplate Alamar Blue assay (MABA) [32,33]. The minimum inhibitory concentration (MIC) is defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate bacterium-only controls, and MICs of 11-14 along with IMB1502 and isoniazid (INH) for comparison are presented in Table 1. The data reveal that all of the new synthesized hybrids 11-14 (MICs: 4->32 μg/mL) are much less active than the reference drug INH (MIC: 0.05 μg/mL) and the parent compound IMB1502 (MIC: 0.015 μg/mL), but fourteen of them have potential activity against this strain (MICs: 4-32 μg/mL). Among them, Compounds 11e and 11k display the highest activity (MICs: 4 μg/mL), and Compounds 11k and 11e have also promising activity with MICs of 8 and 16 μg/mL, respectively, against MTB H37Rv ATCC 27294.

Anti-MTB Activity
The target Compounds 11-14 were evaluated for their in vitro activity against MTB H37Rv ATCC 27294 using the microplate Alamar Blue assay (MABA) [32,33]. The minimum inhibitory concentration (MIC) is defined as the lowest concentration effecting a reduction in fluorescence of ě90% relative to the mean of replicate bacterium-only controls, and MICs of 11-14 along with IMB1502 and isoniazid (INH) for comparison are presented in Table 1. The data reveal that all of the new synthesized hybrids 11-14 (MICs: 4->32 µg/mL) are much less active than the reference drug INH (MIC: 0.05 µg/mL) and the parent compound IMB1502 (MIC: 0.015 µg/mL), but fourteen of them have potential activity against this strain (MICs: 4-32 µg/mL). Among them, Compounds 11e and 11k display the highest activity (MICs: 4 µg/mL), and Compounds 11k and 11e have also promising activity with MICs of 8 and 16 µg/mL, respectively, against MTB H37Rv ATCC 27294.

Chemistry
Melting points were determined in open capillaries and are uncorrected. 1 H-NMR spectra were determined on a Varian Mercury-400 spectrometer in DMSO-d6, D2O or CDCl3 using tetramethylsilane as an internal standard (see Supplementary Materials). Electrospray ionization (ESI) mass spectra and high resolution mass spectra (HRMS) were obtained on an MDSSCIEX Q-Tap mass spectrometer. The reagents were all of analytical grade or chemically pure. TLC was performed on silica gel plates (Merck, ART5554 60F254, Kenilworth, NJ, USA).

Chemistry
Melting points were determined in open capillaries and are uncorrected. 1 H-NMR spectra were determined on a Varian Mercury-400 spectrometer in DMSO-d 6 , D 2 O or CDCl 3 using tetramethylsilane as an internal standard (see Supplementary Materials). Electrospray ionization (ESI) mass spectra and high resolution mass spectra (HRMS) were obtained on an MDSSCIEX Q-Tap mass spectrometer. The reagents were all of analytical grade or chemically pure. TLC was performed on silica gel plates (Merck, ART5554 60F254, Kenilworth, NJ, USA).

General Procedure for the Synthesis of Imidazo[1,2-a]pyridine-3-carboxylic Acids 9, 10
To a solution of 7, 8 (4.0 mmol) in EtOH (30 mL) was added an aqueous solution of lithium hydroxide (12.0 mmol in 10 mL of water), and the mixture was stirred at room temperature overnight. The organic solvent was evaporated, and 1N HCl was added until the pH = 6. The residual was collected by filtration, washed with water and dried to give 9, 10. To a solution of 1.2 equiv of substituted cinnamic acid 1a-l (5 mmol) in 5 equiv of thionyl chloride (3.6 mL), a catalytic amount of DMF was added. The reaction mixture was refluxed for 4 h, and then, solvent was evaporated under vacuum to get the product 2a-l in the form of a solid residue in quantitative yield. The solid residue was directly added partially to an ice-cold stirred solution of 1.0 equiv of tert-butyl (2-aminoethyl)carbamate or tert-butyl (3-aminopropyl)carbamate and 2.0 equiv triethylamine in DCM (20 mL). After the addition, the mixture was warmed to room temperature and stirred for 2 h. Then, DCM (20 mL) was added and washed with 0.2 M HCl (40 mL), H 2 O (40 mL), 5% saturated. NaHCO3 (40 mL) and brine (40 mL), then dried over anhydrous magnesium sulfate. The solvent was removed in vacuo to give the corresponding cinnamamide derivatives 3a-l (65%-75%, from 1a-l) and 4a-g (59%-70%, from 1a-g) as a white solid. 3a-l, 4a-g (4 mmol) in DCM/TFA (9:1, 40 mL) were stirred at room temperature for 1 h. Solvents were removed in vacuo to yield 5a-l (100%) and 6a-g (100%) as a colorless oil.