Tuberculosis (TB), mainly caused by the bacterium Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death and the leading cause from a single infectious agent (above HIV/AIDS) . Approximately 5%-10% of the estimated 1.7 billion people infected with MTB will develop TB disease during their lifetime, and about 90% of cases occur among adults . The latest World Health Organization (WHO) global TB report has estimated that around 10 million people including 5.8 million men, 3.2 million women and 1.0 million children developed TB disease in 2017, and 1.3 million deaths occurred in the same year .
There are several reasons for the incremental prevalence of TB, and the widely spread of HIV/TB co-infection and drug-resistant TB (DR-TB, especially multi-drug resistant TB (MDR-TB)) are the two major reasons [3, 4]. TB is one of the most common opportunistic infection among HIV-positive people, and is the leading cause of death among HIV/TB co-infection patients, which resulted in 300, 000 deaths in 2017 . Globally in 2017, there were an estimated 558, 000 new cases of rifampicin (RIF)- resistant TB (RR-TB), and 82% of them were MDR-TB patients. The latest treatment outcome data show treatment success rates of 82% for TB, 77% for HIV-associated TB, 55% for MDR/RR-TB and 34% for extensively drug-resistant TB (XDR-TB) . Specific targets set in the End TB Strategy include a 95% reduction in TB deaths and a 90% reduction in TB incidence by 2035, compared with levels in 2015. The most immediate milestones, set for 2020, are a 35% reduction in TB deaths and a 20% reduction in the TB incidence rate, compared with levels in 2015 [5, 6]. Obviously, novel anti-TB agents effective against both drugsusceptible and DR-TB especially MDR-TB are needed to achieve this goal.
Benzofuran and isatin derivatives possess diverse biological properties [7-10], and some of them which are exemplified by TAM16 (Fig. 1) endow with promising in vitro and in vivo anti-TB potential [11, 12]. TAM16 not only showed broad-spectrum in vitro anti-TB activities, but also exhibited excellent in vivo physiochemical, toxicological and pharmacological properties . 1, 2, 3- Triazole tethered benzofuran-isatin-imine hybrids displayed excellent in vitro anti-TB activities against both drug-susceptible and MDR-TB strains, and the most active hybrid 1 (MIC: 0.25 and 0.5 mg/mL) was highly active against MTB H37Rv and MDR-TB strains , suggesting benzofuran-isatin-imine hybrids are potential anti-TB candidates. The structure-activity relationship (SAR) for isatin-containing hybrids indicated that the linker between the isatin and the other pharmacophores played a pivotal role in exertion of the anti-TB activities [14-19], and introduction of hydrogen bond donors such as hydroxylimine and thiosemicarbazide were favorable to the activity [12, 13].
|Fig. 1. Chemical structures of TAM16, isatin and 1, 2, 3-triazole tethered benzofuran-isatin-imine hybrid 1.|
Based on the above facts and as a continuous program to develop novel anti-TB candidates, a series of novel benzofuranisatin-hydroxylimine/-thiosemicarbazide hybrids 7a-l tethered via propylene, butylene and pentylene were designed, synthesized and evaluated for their anti-mycobacterial activity in vitro against both drug-susceptible and MDR-TB strains as well as cytotoxicity in VERO cells. The design strategy was depicted in Fig. 2.
|Fig. 2. Design strategy for benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids tethered via different linkers.|
All of the desired benzofuran-isatin-hydroxylamine/-thiosemicarbazide hybrids 7a-l can be obtained by the synthetic route depicted in Scheme 1. 5-Methoxyisatin/5-fluoroisatin/isatin 1a-c were alkylated with 1, 3-dibromopropane, 1, 4-dibromobutane, or 1, 5-dibromopentane 2a-c generated N-(3-bromopropyl/4-bromobutyl/5-bromopentyl)isatin derivatives 3a-f (yield: 63%-81%) [15, 16]. Cyclization of ethyl 3-(4-substituted phenyl)-3-oxopropanoate 4a-c and benzoquinone with copper(Ⅱ) triflate (Cu(OTf)2) as catalyst provided benzofuran intermediates 5a-c . Treatment of isatin derivatives 3a-f and benzofuran intermediate 5a-c with potassium carbonate as base yielded the key intermediates 6a-h (51%-77%). Finally, condensations of 6a-h with hydroxylamine hydrochloride or thiosemicarbazide hydrochloride in the presence of sodium bicarbonate provided benzofuran-isatin-hydroxylamine/-thiosemicarbazide hybrids 7a-l (19%-54%).
|Scheme 1. Synthesis of benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids 7a-l|
The anti-mycobacterial activity of benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids 7a-l, precursor 6a along with the first-line anti-TB agents isoniazid (INH) and rifampicin (RIF) against MTB H37Rv strains and cytotoxicity towards VREO cells were investigated (Table 1).
All benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids 7a-l and precursor 6a with MIC values ranging from < 0.016 mg/mL to 0.156 mg/mL exhibited great anti-mycobacterial activities against MTB H37Rv, and all hybrids except 7i were more potent than the first-line anti-TB agents INH and RIF (MIC: 0.078mg/mL), and nine of them 7a, 7b, 7e-h and 7j-l and precursor 6a (MIC: < 0.06 mg/mL) were comparable to the parent compound TAM16 (MIC: < 0.06 mg/mL). The SAR revealed that for benzofuran-isatin-hydroxylimine hybrids, shorter linker between isatin and benzofuran was more favorable to the activity. In general, hybrids with thiosemicarbazide fragment were more potent than the corresponding hydroxylamine analoges, and introduction of -F at para-position of phenyl position (R3) was detrimental to the activity.
All hybrids also displayed acceptable toxicological profiles with CC50 ranging from 8 mg/mL to 128 mg/mL. The structure-cytotoxicity relationship study indicated that compared with ketone, introduction of either hydroxylimine or thiosemicarbazide at C-3 position of isatin motif could increase the cytotoxicity as evidenced by ketone 6a (CC50: 512 mg/mL) was less toxic than hybrids 7a (CC50: 128 mg/mL) and 7e (CC50: 128 mg/mL), and the relative contribution order was ketone > hydroxylimine > thiosemicarbazide. Introduction of -F at C-5 position of isatin and para-position of phenyl position resulted in the increment of cytotoxicity. In general, hybrids with longer linkers also showed higher cytotoxicity.
To evaluate the selectivity profiles of these hybrids, the selectivity index (SI) values from the ratio of CC50=MICMTB H37Rv were calculated (Table 1). All hybrids possessed high SI values (SI: ≥205), suggesting that this kind of hybrids hold excellent selectivity profiles.
Encouraged by their strong potency against the drug sensitive MTB H37Rv strain, the most active nine benzofuran-isatinhydroxylimine/thiosemicarbazide hybrids 7a, 7b, 7e–h and 7j–l and precursor 6a were selected for further evaluation for in vitro activity against two clinical MDR-TB isolates (resistant to INH and RIF). The in vitro activity of selected compounds against MDR-TB isolates was listed in Table 2.
All of the selected nine benzofuran-isatin-hydroxylimine/ thiosemicarbazide hybrids 7a, 7b, 7e-h and 7j-l and precursor 6a demonstrated considerable activity against the tested two clinical MDR-TB isolates with MIC values ranging from 0.22 mg/mL to 14.18 mg/mL. All hybrids were more potent than RIF and INH (MIC: >40 mg/mL), but were less active than TAM16 (MIC: < 0.016 mg/mL) against the two clinical MDR-TB isolates. The SAR indicated that benzofuran-isatin-thiosemicarbazide hybrids were more active than the corresponding hydroxylimine analogs. Hybrid 7f (MIC: 0.22 and 0.86 mg/mL, respectively) was found to be most active against the tested two clinical MDR-TB isolates, and it was >51-fold more potent than RIF and INH (MIC: >40 mg/mL).
In conclusion, a series of novel benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids were designed, synthesized and evaluated for their in vitro anti-TB activities against drug-sensitive MTB H37Rv and MDR-TB isolates as well as cytotoxicity. All benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids exhibited considerable in vitro anti-mycobacterial activities against the tested three MTB strains, and all of them also showed acceptable cytotoxicity. The most active hybrid 7f was >4.8 and >51 folds more potent than the first line anti-TB agents RIF and INH against both drug-sensitive MTB H37Rv and MDR-TB isolates, respectively. The results demonstrated the potential utility of benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids as anti-TB agents.Appendix A. Supplementary data
Supplementarymaterial related to this article canbefound, in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.11.032.
World Health Organization, Global Tuberculosis Report, 2018. https://apps.who.int/iris/handle/10665/75938?locale=es
World Health Organization, Global Tuberculosis Report, 2017.
Y.L. Fan, J.B. Wu, X.W. Cheng, et al., Eur. J. Med. Chem. 146 (2018) 554-563. DOI:10.1016/j.ejmech.2018.01.080
S. Zhang, Z. Xu, C. Gao, et al., Eur. J. Med. Chem. 138 (2017) 501-513. DOI:10.1016/j.ejmech.2017.06.051
The Global Plan to End TB, 2016-2020, Geneva: Stop TB Partnership, 2015.
World Health Organization, The End TB Strategy-global Strategy and Targets for Tuberculosis Prevention, Care and Control After, 2015.
A. Baldisserotto, M. Demurtas, I. Lampronti, et al., Eur. J. Med. Chem. 156 (2018) 118-125. DOI:10.1016/j.ejmech.2018.07.001
R.J. Nevagi, S.N. Dighe, S.N. Dighe, Eur. J. Med. Chem. 97 (2015) 561-581. DOI:10.1016/j.ejmech.2014.10.085
Z. Xu, S. Zhang, C. Gao, et al., Chin. Chem. Lett. 28 (2017) 159-167. DOI:10.1016/j.cclet.2016.07.032
H.K. Shamsuzzaman, Eur. J. Med. Chem. 97 (2015) 483-504. DOI:10.1016/j.ejmech.2014.11.039
W. Zhang, S.C. Lun, S.H. Wang, et al., J. Med. Chem. 61 (2018) 791-803. DOI:10.1021/acs.jmedchem.7b01319
A. Aggarwal, M.K. Parai, N. Shetty, et al., Cell 170 (2017) 249-259. DOI:10.1016/j.cell.2017.06.025
F. Gao, H. Yang, T.Y. Lu, et al., Eur. J. Med. Chem. 159 (2018) 277-281. DOI:10.1016/j.ejmech.2018.09.049
Z. Xu, X.F. Song, Y.Q. Hu, et al., Eur. J. Med. Chem. 138 (2017) 66-71. DOI:10.1016/j.ejmech.2017.05.057
Z. Xu, S. Zhang, X.F. Song, et al., Bioorg. Med. Chem. Lett. 27 (2017) 3643-3646. DOI:10.1016/j.bmcl.2017.07.023
Z. Xu, X.F. Song, M. Qiang, et al., J. Heterocyclic Chem. 54 (2017) 3735-3741. DOI:10.1002/jhet.v54.6
Z. Xu, X.F. Song, Y.Q. Hu, et al., J. Heterocyclic Chem. 55 (2018) 77-82. DOI:10.1002/jhet.3004
L.S. Feng, M.L. Liu, S. Zhang, et al., Eur. J. Med. Chem. 46 (2011) 341-348. DOI:10.1016/j.ejmech.2010.11.023
L.S. Feng, M.L. Liu, B. Wang, et al., Eur. J. Med. Chem. 45 (2010) 3407-3412. DOI:10.1016/j.ejmech.2010.04.027