Bioorganic & Medicinal Chemistry Letters 27 (2017) 223–227

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/locate /bmcl

Antimycobacterial activity of novel hydrazide-hydrazone derivativeswith 2H-chromene and coumarin scaffold

http://dx.doi.org/10.1016/j.bmcl.2016.11.0710960-894X/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail addresses: violina@pharmfac.net, violina_stoyanova@abv.bg

(V.T. Angelova).

Violina T. Angelova a,⇑, Violeta Valcheva b, Nikolay G. Vassilev c, Rosen Buyukliev a, Georgi Momekov d,Ivan Dimitrov a, Luciano Saso e, Mirjana Djukic f, Boris Shivachev g

aDepartment of Chemistry, Faculty of Pharmacy, Medical University of Sofia, 2, Dunav St., 1000 Sofia, Bulgariab Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev St., 1113 Sofia, Bulgariac Laboratory of Nuclear Magnetic Resonance, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 9, Acad. G. Bonchev St.,1113 Sofia, Bulgariad Laboratory of Experimental Chemotherapy, Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University-Sofia, 2, Dunav St., 1000Sofia, BulgariaeDepartment of Physiology and Pharmacology ‘‘Vittorio Erspamer”, Sapienza University, P.le Aldo Moro 5, 00185 Rome, ItalyfDepartment for Toxicology, Faculty of Pharmacy, University of Belgrade, 450, Vojvode Stepe, St., 11221 Belgrade, SerbiagBulgarian Academy of Sciences, Institute of Mineralogy and Crystallography, 107, Acad. G. Bonchev St., 1113 Sofia, Bulgaria

a r t i c l e i n f o

Article history:Received 2 September 2016Revised 19 November 2016Accepted 23 November 2016Available online 25 November 2016

Keywords:Antitmycobacterial activity2H-ChromeneCoumarinCytotoxicityHydrazide-hydrazone

a b s t r a c t

This study reports the synthesis of new 2H-chromene or coumarin based acylhydrazones, which wereevaluated for their in vitro antimycobacterial activity against reference strain Mycobacterium tuberculosisH37Rv and compared to the first-line antituberculosis drugs, isoniazid (INH) and ethambutol (EMB). Themost active compounds 7m (MIC 0.13 lM), 7o (MIC 0.15 lM) and 7k (MIC 0.17 lM) demonstratedantimycobacterial activity at submicromolar concentration level and remarkably minimal associatedcytotoxicity in the human embryonic kidney cell line HEK-293T. Structure-activity relationship for thisclass of compounds has been established.

� 2016 Elsevier Ltd. All rights reserved.

9,10,12–16

Despite notable progress in the treatment of mycobacterialinfections, tuberculosis (TB) is still a public health concern world-wide.1,2 The dramatic increase in number of cases of multidrug-resistant (MDR-TB) and extensively drug-resistant tuberculosis(XDR-TB)3–5 require development of novel low toxic compoundswith improved efficacy6–8 Current treatment of TB with first andsecond line drugs require a minimum six months of therapy to pre-vent relapse. This long-term chemotherapy certainly increases therisk of drug resistance. Although isoniazid (INH) is still the mosteffective antituberculosis drug, recommended by WHO, it hastwo major drawbacks which need to be overcomed: hepatotoxicityand its deactivation via the genetically polymorphic N-ary-laminoacetyltransferases (NATs). Indeed hydrazones, isoniazidderivatives wherein N2 is blocked towards acetylation by NATs,appeared to be more effective and less hepatotoxic than isoni-azid.9–12 Various classes of pyridine, pyrimidine, coumarin, quino-line, benzoxazole, indole, purine, pyrrole, furan, benzofuran,

triazoles etc. based hydrazones have been evaluated fortheir antimycobacterial potential. Also, 2H-chromenes, coumarinsand their derivatives have gained great therapeutic importancefor the discovery of novel antimycobacterial compounds.17–24 Keriet al. (2015)25 have emphasized the strategic place of coumarinderivatives in medicinal chemistry particularly on their antituber-culosis effect.

Therefore, we focused on the synthesis of 2H-chromene or cou-marin compounds with various substituted hydrazide-hydrazonepharmacophore attached to the 3rd position in the chromene ring.The antimycobacterial activity of the synthesized compoundsagainst Mycobacterium tuberculosis H37Rv strain and the toxicityagainst human embryonal kidney cell line HEK-293T wereevaluated.

The synthesis of 2H-chromane and coumarin based hydrazonesis presented in Scheme 1. Compounds 3a–c were prepared fromthe condensation of salicylaldehyde 1 and a,b-unsaturated aldehy-des 2a–c under base-promoted conditions.26 For the preparation of2H-chromene-3-carbaldehyde 3a and 2-methyl-2H-chromene-3-carbaldehyde 3b we applied the synthetic strategy of Azizmoham-madi et al.,27 while the synthesis of 2-phenyl-2H-chromene-3-car-

Scheme 1. Synthetic route for 2H-chromene based hydrazones 7a–q and coumarin based hydrazone 8a–e; *hydrazone derivatives 7l–k and 8a–c have been describedelsewhere.29 Conditions: (a) K2CO3, 1,4-dioxane, reflux 2 h; (b) pyridine, benzoic acid, toluene, 25 �C, 24 h, (c) POCl3, DMF, 60 �C; (d) abs. EtOH, r.t., 15–30 min.

Fig. 1. ORTEP view of the molecular structure of (a) 7i and (b) 7k, atomicdisplacement parameters are at 50 % probability, H atoms are shown as sphereswith arbitrary radii.

224 V.T. Angelova et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 223–227

baldehyde 3c was performed according to Córdova et al.,28,29 withpyrrolidine as a catalyst and benzoic acid as an additive in toluene.The synthesis of the hydrazide-hydrazones 7a–q and 8a–f involvedequimolar condensation of 2H-chromene-3-carbaldehyde 3a–c or4-chlorocoumarin-3-carbaldehyde 4 with appropriate hydrazides6a–f in abs. ethanol, at room temperature (Scheme 1).

The reaction progress was monitored by thin layer chromatog-raphy (TLC). The proposed structures are confirmed from FTIR, 1HNMR, 13C NMR, 2D (1H–1H COSY, 1H–13C COSY, HMBC andNOESY) and HR-MS spectral techniques. The novel hydrazonederivatives 7a–q and 8a–e were obtained as a single E geometricalisomer and the observed duplication pattern in DMSO was due tothe presence of syn/anti amide conformers.30–34 Configuration (E)and a synperiplanar conformation of the compounds was provedby NOESY spectrum, measured in DMSO-d6 (Supplementary,Figs. S1 and S2). Additionally, the configuration of the C@N doublebond in structures 7i (with low activity) and 7k (with high activity)was confirmed by X-ray diffraction crystallography ((Fig. 1), Sup-plementary, Figs. S1–S4, Tables S2, S3).

The synthesized compounds 7a–q and 8a–e were evaluatedin vitro against M. tuberculosis H37Rv, using the Canetti method.35

Isoniazid and ethambutol were used as controls. The results of thein vitro antimycobacterial screening are summarized in Table 1.The established antimycobacterial activity of the tested seriesprompted us to evaluate the cytotoxic potential of the promisingleads (i.e. compounds that show activity at submicromolar concen-trations), thus determining the selectivity (SI) of the antiprolifera-tive effects (Table 1.). The cytotoxicity of the selected compoundswas assessed against the human embryonal kidney cell line 293-T, after 72 h of continuous exposure.

The majority of the tested compounds with high antimycobac-terial activity (7d, 7h, 7j, 7k, 7m, 7o, 7p, and 8e) caused a 50% cellgrowth inhibition within the tested concentration range (12.5–100 lM); 7h and 7o displayed high cytotoxicity; the other com-pounds (7j,7k, 7m, 7p, 7q and 8e) were less toxic to human cells;

and 7m showed marginal cytotoxicity. Thus, regarding the selec-tive inhibition (Selectivity index, SI) of mycobacteria, compounds7m (SI = 697.38), 7k (SI = 448.94) and 7h (SI = 117.83) could beconsidered as promising hybrid antituberculosis agents.

Table 1Antimycobacterial activity and cytotoxicity of the compounds 7a–q and 8a–f.

No. Compd. Structure MICb (lM) IC50c (lM) SId LogPa

1 7a 6.39 ND – 3.55

2 7b 6.48 36.04 – 3.24

3 7c 7.03 22.10 – 3.35

4 7d 0.74 47.32 63.94 2.76

5 7e >6.12 ND – 3.58

6 7f 6.20 ND – 3.24

7 7g >6.70 ND – 3.31

8 7h 0.18 21.21 117.83 2.84

9 7ie 6.84 23.33 – 3.23

10 7je 0.64 48.77 76.20 2.76

11 7ke 0.17 76.32 448.94 2.49

12 7l >5.14 13.18 – 4.11

13 7m 0.13 90.66 697.38 3.94

14 7n >5.54 ND – 4.23

15 7o 0.15 11.91 79.4 3.85

16 7p 1.34 30.87 – 3.81

(continued on next page)

V.T. Angelova et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 223–227 225

Table 1 (continued)

No. Compd. Structure MICb (lM) IC50c (lM) SId LogPa

17 7q 0.53 30.18 56.94 2.97

18 8ae 6.12 28.56 3.56

19 8be 5.83 33.07 – 3.15

20 8ce 6.10 12.56 – 3.05

21 8d 1.45 26.15 – 3.42

22 8e 0.67 30.18 45.04 2.85

Isoniazidf 1.45 – –EMB_2HClf 7.64 – –

ND – not determined.a LogP was calculated by MDL QSAR software.b Antimycobacterial activity towards a reference strain of Mycobacterium tuberculosis H37Rv, MIC (lM) was defined as the lowest concentration resulting in a complete

inhibition of the bacterial growth and reproduction.c In vitro cytotoxicity towards human embryonal kidney cell line 293T, IC50 (lM).d Selectivity index, SI ratio = IC50/MIC) by the MTT assay.e Prepared by a procedure previously reported.f Isoniazid (reference compound) and EMB_2HCl (ethambutol dihydrochloride) – reference compounds.

226 V.T. Angelova et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 223–227

The structure-activity relationship of compounds 7a–q and 8a–e with respect to their antimycobacterial activity could be summa-rized as follows:

a) Chromene derivatives with strong charge donation (electrondonating group) and delocalization in the hydrazide moietyin 2-furyl 7d, 7h, 7o, isonicotinoyl 7k and 5-(4-methyl-1,2,3-tidiazinyl) 7q, or 4-methoxyphenyl 7m and 4-hydrox-yphenyl 7j fragments exhibited excellent activity againstM. tuberculosis H37Rv with MICs in the submicromolarrange. 2-Phenyl-2H-chromene derivatives 7m and 7o withMIC of 0.13 lM and 0.15 lM, respectively, differ only in abenzene ring (p-methoxyphenyl, 2-furyl substituents),demonstrated the highest activity. Among the tested hydra-zones, 7m showed the highest antimycobacterial effect andexhibited low cytotoxicity, indicating a favorable selectivityindex, SI = 697;

b) The substitution of the H-atom at the 2nd position in 2H-chromene moiety of hydrazones 7b and 7d, with a methylsubstituent in compounds 7f and 7h and with an phenylgroup in the case of 7m and 7o led to a significant increasein the antimycobacterial activity, which was not observed inhydrazones with a 4-chlorophenyl moiety 7a, 7e, 7l and 2-thienyl-substituted hydrazones 7c, 7g and 7n. Notably, thesubstitution of the 2-furyl and methoxyphenyl moiety inthe hydrazides with 2-thienyl or 4-chlorophenyl moietyresulted in a lower activity; fluorophenyl substitution 7pand 8e led to activity improvement compared to chlorophe-nyl substitution 7l.

c) 2-Methylchromene derivatives containing 4-hydroxyphenyland 4-pyridyl moieties 7j and 7k exhibited MICs of 0.64 lMand 0.17 lM, respectively, but the activity of coumarin con-taining hydrazones 8b and 8c with the same substituents inthe aromatic ring were less affected (MIC = 5.83, MIC = 6.10,respectively);

d) Except for the 5-(4-methyl-1,2,3-thiadiazole)-substitutedcoumarin derivative 8e and the 4-fluorophenyl substitutedcoumarin 8d which exhibited good activity with potencyequal to that of isoniazid, all other hydrazones with cou-marin scaffold 8a–c demonstrated antimycobacterialpotency lower than that of INH and MIC values ranging from5.83 to 6.12 lM.

e) All tested hydrazones appeared more lipophilic with logPvalues considerably higher than INH itself (logP = 0.06).We observed correlation between the antimycobacterialpotency and LogP values within each set of compounds(with the same chromene ring, but different substituentsin the aromatic rings I, II, III and IV, Table 1). The hydroxyland methoxy groups at para position in the aromatic ring,as well as 2-furyl- and 4-pyridyl-moieties were able to actas a hydrogen bond acceptor and decrease logP values ofthe compounds, which may be is important for their inhibi-tory activity against M. tuberculosis H37Rv.

SAR analysis revealed that the antimycobacterial activity of thesynthesized compounds does not depend only on their lipophiliccharacteristics. Activation of the hydrazide moiety by electrondo-nating groups seems to be also essential in the pharmacodynamics

V.T. Angelova et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 223–227 227

of these compounds. Evidently, coumarin and/or 2H-chromenemoieties acts as a scaffold and that the substituents at 2nd, and3rd positions in chromene ring and principally substituents inthe hydrazide moiety provide target specificity in each of thetested series (I, II, III, IV).

In summary, a series of new hybrid hydrazones bearing 2H-chromene or coumarin moieties were synthesized by means of aclassic procedure with satisfactory yields and their structures werefully supported by spectral data (IR, NMR, MS) and X-ray analisis.Twenty-two hydrazones were tested in vitro for antimycobacterialactivity (against M. tuberculosis H37Rv) and compared to referencefirst-line drugs isoniazid and ethambutol. Significantly improvedactivity with MICs the in micro- to submicromolar concentrationrange was observed by varying the substituents in hydrazone moi-ety and in the chromene nucleus. Some of the new compounds, 7d,7h, 7j, 7k, 7m, 7o, and 7q with logP values ranging from 2.49 to3.94 demonstrated considerably higher antimycobacterial potencythan INH. Among the tested hydrazones 7m has proven to bethe most effective compound against M. Tuberculosis H37Rv(MIC = 0.13 lM). Its antimycobacterial and cytotoxicity equieffec-tive concentrations vary 700-fold, indicating a favorable selectivityindex. Thus, this hydrazide-hydrazone class potentially defines apromising new candidate series for further investigations.

Acknowledgments

This study was supported by the National Research Fund ofBulgaria (projects UNA-17/2005 and DRNF-02/13/2009). Wegratefully appreciate the help of Yulian Voynikov with massspectrometry.

A. Supplementary data

Supplementary data (experimental procedures, characteriza-tion of final compounds and biological assays protocols) associatedwith this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.071.

References

1. Ködmön C, Zucs P, van der Werf M. Euro Surveill. 2016;21:12.2. European Centre for Disease P, Control WHOROfE. Stockholm, European Centre

for Disease Prevention and Control, 2015.3. Wood AJ, Iseman MD. N Engl J Med. 1993;329:784.4. Tiberi S, Payen M-C, Sotgiu G, et al. Eur Respir J. 2016;47:1235.5. Pang Y, Dong H, Tan Y, et al. Sci Rep. 2016;6:25330.6. Wong EB, Cohen KA, Bishai WR. Trends Microbiol. 2013;21:493.7. Dhanda SK, Vir P, Singla D, Gupta S, Kumar S, Raghava GP. PLoS One. 2016;11:

e0153771.8. P Barot K, Nikolova S, Ivanov I, et al. Mini Rev Med Chem. 2013;13:1664.9. Mathew B, Suresh J, Ahsan MJ, Mathew GE, Usman D, Subramanyan PN, Safna

S, Maddela S. Infectious Disorders-Drug Targets. 2015;15:76. Formerly CurrentDrug Targets-Infectious Disorders.

10. Negi VJ, Sharma A, Negi J, Ram V. Int J Pharm Chem. 2012;2:100.11. Eldehna WM, Fares M, Abdel-Aziz MM, Abdel-Aziz HA. Molecules.

2015;20:8800.12. Bijev A, Georgieva M. J Univ Chem Technol Metall. 2010;45:111.13. Martins F, Ventura C, Santos S, Viveiros M. Curr Pharm Des. 2014;20:4427.14. Lessigiarska I, Pajeva I, Prodanova P, Georgieva M, Bijev A. Med Chem.

2012;8:462.15. Koçyigit-Kaymakçıoglu B, Oruç-Emre EE, Unsalan S, Rollas S. Med Chem Res.

2009;18:277.16. Joshi SD, Kumar D, Dixit SR, et al. Eur J Med Chem. 2016.17. Cardoso SH, Barreto MB, Lourenço M, et al. Chem Biol Drug Des. 2011;77:489.18. Zheng P, Somersan-Karakaya S, Lu S, et al. J Med Chem. 2014;57:3755.19. Mahantesha B, Jambagi VB, Barigidad NN, Laxmeshwar S, Devaru V. Eur J Med

Chem. 2014;74:225.20. Patel RV, Kumari P, Rajani DP, Chikhalia KH. Med Chem Res. 2013;22:195.21. Alvey L, Prado S, Huteau V, et al. Bioorg Med Chem. 2008;16:8264.22. Alvey L, Prado S, Saint-Joanis B, et al. Eur J Med Chem. 2009;44:2497.23. Stec J, Abourashed EA. Pharm Pat Anal. 2015;4:317.24. Kawate T, Iwase N, Shimizu M, et al. Bioorg Med Chem Lett. 2013;23:6052.25. Keri RS, Sasidhar B, Nagaraja BM, Santos MA. Eur J Med Chem. 2015;100:257.26. Sheldrick GM. Acta Crystallogr A. 2008;64:112.27. Azizmohammadi M, Khoobi M, Ramazani A, et al. Eur J Med Chem. 2013;59:15.28. Ibrahem I, Sundén H, Rios R, Zhao G-L, Córdova A. CHIMIA Int J Chem.

2007;61:219.29. Sundén H, Ibrahem I, Zhao GL, et al. Chem Eur J. 2007;13:574.30. Angelova VT, Vassilev NG, Nikolova-Mladenova B, et al. Med Chem Res.

2016;2082.31. Palla G, Predieri G, Domiano P, Vignali C, Turner W. Tetrahedron. 1986;42:3649.32. Wyrzykiewicz E, Blaszczyk A. J Heterocycl Chem. 2000;37:975.33. Lopes AB, Miguez E, Kümmerle AE, Rumjanek VM, Fraga CAM, Barreiro EJ.

Molecules. 2013;18:11683.34. Martins F, Santos S, Ventura C, et al. Eur J Med Chem. 2014;81:119.35. Canetti G, Fox W, Khomenko A, et al. Bull World Health Organ. 1969;41:21.