Design, Synthesis and Antitubercular Activity of Novel Isoniazid‐Cyclic‐Amine‐Azachalcones Hybrids

In this work, it is described the design of twenty-four heterocyclic amine-azachalcones compounds through molecular hybridization of chalcone scaffold and fragments of isoniazid, fluoroquinolones, and linezolid with antituberculosis potential. The new compounds were synthesized via Claisen-Schmidt condensation, providing yields of 36-95%. Fifteen compounds showed antituberculosis activity against Mycobacterium tuberculosis H37Rv strain. Two amine-azachalcones 15 and 17 showed relevant biological activity with minimum inhibitory concentration (MIC) values of 6.62 and 4.85 μM, respectively. Compound 12 showed the best profile of antitubercular activity with MIC = 9.54 μM and selectivity index (SI) = 9.33. It was found that morpholine group is important to increase potency of antimycobacterial activity but also to add some toxicity to the chalcone molecular framework. The results described herein would be a guide in the designing of novel and optimized antitubercular derivatives based on the chalcone scaffold.


Introduction
Tuberculosis (TB) disease is classified as one of the top ten leading causes of death in the world, due to its aggressive infection promoted by a unique agent. Moreover, one-fourth of the global population was detected with latent tuberculosis, occurring without any disease symptoms. 1 In 2017, ten million people manifested TB and the situation aggravated even more with 1. 3

million deaths. 1
The first-line treatment for TB involves the use of five different drugs such as isoniazid, pyrazinamide, streptomycin, ethambutol, and rifampicin, which can be used in association. 2 When the first-line drugs cannot be used, it is recommended the second-line treatment with levofloxacin or moxifloxacin, bedaquiline, and linezolid. 3 Failures in chemotherapy are related to several aspects such as high complexity (association of three or more drugs); long period of treatment (6 to 9 months or up to 24 months); 3,4 countless adverse effects (nausea, vomiting, and epigastric pain), 1,5 and bacterial resistance.
Bacterial resistance is a cause of alarm for the health systems throughout the world. 3,6 Bacterial strains of TB have been classified into two types, TB multidrug-resistant (resistance to the first-line drugs), and TB extremely resistant (resistance to first and second-line drugs). 6 In this context, it is reinforced the relevance to search into new antituberculosis drug candidates. 7 Natural products remain a fundamental source of inspiration in the discovery and development of new bioactive compounds. 8 Through secondary metabolism, natural products are able to create several types of substances, with multiple biological activities. 9 Among the secondary metabolites, our research group is especially interested in chalcone scaffold, as a synthetic target. Chalcones are considered a privileged structure and have been widely used as a template in medicinal chemistry studies. Due to its structural diversity, chalcones have shown great potential in the molecular modification process, leading to the synthesis of compounds containing different pharmacophore groups, which can enhance its pharmacological effect. 10 Recent reports 10 aimed at chalcones as compounds with a wide range of biological activities such as anticancer, cancer-preventative effects, anti-inflammatory, antibacterial, antidiabetic, antioxidant, antimicrobial, antiviral, antimalarial and neuroprotective effects. It is noteworthy that chalcone compounds are emerging as potential candidates for new antitubercular drugs ( Figure 1). [10][11][12][13][14][15] Structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) information has demonstrated that the nitrofuran scaffold as a substituent in chalcones 1-2 improved its antitubercular activities. 16,17 However, many studies showed other structural combinations, aiming for new antitubercular agents, as seen in chalcones 3-4, 18,19 by the use of creative and intuitive molecular hybridization approach (Figure 1). 20 In order to expand the chemical space of chalcones, this study aimed to synthesize and evaluate the antitubercular activity of twenty-four heterocyclic amine-azachalcone derivatives designed by molecular hybridization of chalcone scaffold 5 with structural fragments of isoniazid 6, fluoroquinolones 7 and linezolid 8 (Scheme 1). Isoniazid 6 is the best and the oldest drug employed to treat TB, fluoroquinolone 7 and linezolid 8 are modern broadspectrum antibiotics, which have shown antitubercular activity. [21][22][23] Results and Discussion Chemistry Firstly, it was necessary to obtain p-aminated acetophenones with the appropriate substitution patterns ( Table 1). The synthesis of acetophenones 35a-35h occurred via aromatic nucleophilic substitution of 4-fluoroacetophenone 33 in the presence of cyclic amines 34a-34h, using K 2 CO 3 and dimethyl sulfoxide (DMSO) as a solvent. Products were obtained in 71 to 99% yields; the reaction times were observed for 24 or 48 h until the complete consumption of starting materials (Scheme 2). 24 Amine-azachalcones 9-32 were obtained via Claisen-Schmidt condensation of p-aminated acetophenones 35a-35h and carbaldehydes 36a-36c using a solution of NaOH in ethanol as solvent, which was added dropwise, providing the compounds 9-32 with yields ranging from 36 to 95% (Table 1). 25 Structural modifications intended to increase the complexity of cyclic amines bonded to the aromatic ring, in order to generate a variation in the molecular volume in R'. Thus, compounds were obtained from simple N-heterocyclic amine templates, as pyrrolidine, to more complex molecules like 1-(pyrimidin-2-yl)piperazine.
When positional isomers were compared, pyridine compounds substituted with nitrogen at position 4 showed  Unknown synthetic compounds 9-32 were characterized by 1 H and 13 C nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS).

Antitubercular activity
The antitubercular activity of compounds 9-32 was evaluated against Mycobacterium tuberculosis H37Rv strain ( Table 2). Minimum inhibitory concentrations (MICs) were defined as the lowest concentration that resulted in 90% of M. tuberculosis growth inhibition. Fifteen compounds of the series demonstrated some level of activity against M. tuberculosis.
The presence of a six-membered hetero-aromatic ring bonding directly to the cyclic amine group did not contribute to the antituberculosis activity (MIC > 250 µM) since the bulkiest azachalcones 27-32 were inactive.

Conclusions
In conclusion, twenty-four heterocyclic amineazachalcones 9-32 were obtained from moderate to good yields. Antituberculosis activity was evaluated against the M. tuberculosis H37Rv strain. Although the higher potency of compounds 15 and 17 (MIC = 6.62 and 4.85 µM, respectively) towards antituberculosis activity, both analogues were considered more cytotoxic (SI = 1.39 and 3.49, respectively). However, azachalcone 12 showed the best profile of activity and selectivity index (MIC = 9.54 µM; SI = 9.33), which provide important information about the SAR.
Morpholine group as a substituent in molecular template was related to higher potency and relevant biological activity, but on the other hand it also increased the cytotoxicity of azachalcones. Further research might explore new structural modifications, in order to provide more information about SAR of azachalcones, and also, optimize the hit-like compounds of this series.

General remarks
All solvents were purchased from Synth ® , São Paulo, Brazil, and distilled before use according to the standard procedure. All reactions were performed under an atmosphere of dry nitrogen and monitored by thin-layer chromatography (TLC) using prepared plates (silica gel 60 F254 on aluminum purchased from Sigma-Aldrich ® , St. Louis, MO, USA). The chromatograms were examined under both 254 and 360 nm UV light or with the developing agent ethanolic vanillin and heat. Flash column chromatography was performed on silica gel 60 (particle size 200-400 mesh ASTM, purchased from Sigma-Aldrich ® , St. Louis, MO, USA) and eluted with hexane or hexane/ethyl acetate in different ratios. Melting points were determined using Fisatom 430D equipment. The 13 C and 1 H NMR spectra were recorded in CDCl 3 solutions using a Bruker 75 or 300 MHz spectrometer. Chemical shifts (d) were expressed as parts per million (ppm) downfield from tetramethylsilane as the internal standard. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) measurements were carried out on a quadrupole time-of-flight instrument (UltrOTOF-Q, BrukerDaltonics, Billerica, MA, USA). General reagents as 4-fluoro-acetophenone 33, compound 35b, cyclic amines 34a, 34c-34h, carbaldehydes 36a-36c, bases, were purchased from Sigma-Aldrich ® , St. Louis, MO, USA.

Cytotoxicity assay
Fibroblasts (NIH/3T3) obtained from Rio de Janeiro Cell Bank (Brazil) were seeded in 96-well plates (1 × 10 4 cells mL −1 ) and incubated with synthetic compounds at 37 °C, 5% CO 2 for 48 h at the concentrations of 0.25-250 µg mL −1 . 29,30 Doxorubicin (Sigma-Aldrich ® , St. Louis, MO, USA) was used as the reference drug at concentrations of 0.025-25 µg mL −1 . Cell growth was estimated by the sulforhodamine B colorimetric method (SRB). 31 DMSO (Vetec ® , Rio de Janeiro, RJ, Brazil) was used as a negative control at the concentration necessary to solubilize the highest concentration of the test samples and did not interfere with cell viability. The percentage of growth was calculated as described by Monks et al. 32 IC 50 (half maximal inhibitory concentration on fibroblast cells) was determined by nonlinear regression analysis (Microcal Origin 6.0 ®33 and Microsoft Office Excel 2007 ® ). Selectivity index (SI) was calculated according to de Medeiros et al. 34

Supplementary Information
Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.