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Enantiopure antituberculosis candidates synthesized from (−)-fenchone
European Journal of Medicinal Chemistry 77 (2014) 243e247
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enantiopure antituberculosis candidates synthesized from ()-fenchone Georgi M. Dobrikov a, *, Violeta Valcheva b, Yana Nikolova a, Iva Ugrinova c, Evdokia Pasheva c, Vladimir Dimitrov a, * a b c
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Bl. 9, Acad. G. Bonchev Str., Sofia 1113, Bulgaria Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Bl. 26, Acad. G. Bonchev Str., Sofia 1113, Bulgaria Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, Bl. 21, Acad. G. Bonchev Str., Sofia 1113, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2014 Received in revised form 6 March 2014 Accepted 8 March 2014 Available online 12 March 2014
The synthesis of new enantiopure N-acyl compounds derived from ()-fenchone has been performed. The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed for most of them moderate activity. The structures bearing sulfonamide functionality have comparable activity to ethambutol and possess low cytotoxicity. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Fenchone Chiral Amides Antimycobacterial Tuberculosis
1. Introduction Despite the availability of highly efficacious treatment for decades, tuberculosis (TB) remains a major global health challenge. In 1993, the World Health Organization (WHO) declared TB a global public health emergency, at a time when an estimated 7e8 million cases and 1.3e1.6 million deaths occurred each year. In 2010 there were an estimated 8.5e9.2 million cases and 1.2e1.5 million deaths (including deaths from TB among HIV-positive people). TB is the second leading cause of death from an infectious disease worldwide (after HIV, which caused an estimated 1.8 million deaths in 2008) [1]. An urgent need for highly potent, more effective drugs with fewer or no side effects and shorter treatment periods to combat the increasing TB pandemic is therefore apparent. Potential anti-TB drug candidates such as diarylquinolones (TMC207), nitroimidazoles (OPC67683 and PA824), pyrroles (LL3858), diamines [2] are in different stages of clinical trials [3,4]. The incorporation of lipophilic polycyclic aliphatic compounds into drug-structures with promising anti-TB applications has enjoyed much attention from
* Corresponding authors. E-mail address: [email protected] (G.M. Dobrikov). http://dx.doi.org/10.1016/j.ejmech.2014.03.025 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
researchers for several years starting with the discovery of SQ109 [5]. The recent studies of Onajole et al. [2,6] confirm the importance of further investigations of polycyclic compounds as potent anti-TB agents. In the course of our research for novel anti-tubercular compounds, we recently demonstrated the role of chirality and wide spectrum of substituted acyl groups, attached to nitrogen atom of (R)-2-aminobutanol [7,8]. The present study aims at investigating of new subclass diastereomerically pure bicyclic N-substituted compounds with fenchane skeletons and the possibility of further enhancing/improving their anti-TB activity. Based on this, 15 novel amidoalcohols and 2 other N-carbonyl derivatives bearing the lipophilic fenchane skeleton were synthesized and screened for activity against drug sensitive (H37Rv) strain of tuberculosis. 2. Results and discussion 2.1. Chemistry The starting aminoalcohol 3 (Scheme 1) was synthesized by using enantiomerically pure ()-fenchone as a natural source of chirality. Desired amides 13e21, 25e27 and 31e33 were synthesized through three main methods of N-acylation of 3, as shown below (Schemes 2e4). It was found that the N-acylation by using
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acylchlorides or activated acids (TBTU) is more efficient than the aminolysis of esters applied previously [7]. Compounds 36e37 were synthesized by other methods (Scheme 5). All of the synthesized compounds inherit the configuration of 3 and were isolated in high purity after column chromatography or crystallization. Scheme 1. Synthesis of compounds 2 and 3.
Scheme 2. Synthesis of compounds 13e21.
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245
Scheme 5. Synthesis of compounds 36 and 37. Scheme 3. Synthesis of compounds 25e27.
2.1.1. Synthesis of compounds 2 and 3 The synthesis of starting aminoalcohol 3 was performed in two steps through intermediate 2 (Scheme 1), by modification of described procedure [9]. The addition of in situ prepared LiCH2CN to ()-fenchone was performed at 85 C in THF without using of anhydrous CeCl3. Other key moment was the hydrolysis of the reaction mixture e the aqueous NH4Cl was added at 85 C and the reaction immediately allowed to r.t. This improved procedure leads to quantitative yield of 2 as pure diastereoisomer after column chromatography. Reduction of 2 to aminoalcohol 3 was performed quantitatively with LiAlH4 in Et2O, according to the literature [9]. The diastereomerical purity and configurations of 2 and 3 were unambiguously confirmed by 1D and 2D NMR experiments. 2.1.2. Synthesis of compounds 13e21 Series of amidoalcohols 13e21 were synthesized in good to excellent yields using standard conditions for acylation of 3 (0 C and Et3N in dry DCM) with acid chlorides 4e12 (Scheme 2). Sulfochloride 12 was prepared from cinnamic acid as described [10].
2.1.3. Synthesis of compounds 25e27 The amidoalcohols 25e27 were synthesized using simple solvent-free aminolysis of esters 22e24 with 3 by heating at 90e 100 C (Scheme 3). Compounds 25e27 were isolated in moderate yields and high purities after column chromatography (or crystallization in case of the low-soluble 27). 2.1.4. Synthesis of compounds 31e33 Compounds 31e33 were prepared through the coupling reaction between 3 and acids 28e30, respectively, in presence of O(Benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluoroborate (TBTU) as activating agent (Scheme 4). TBTU was chosen as one of the most effective coupling reagent in peptide synthesis [11]. Amides 31e33 were isolated in high yields after column chromatography. In case of anthranilic acid (29) preliminary protection of the aromatic amino group was not necessary, due to its low reactivity in comparison to the high nucleophilic amino group of 3. To the best of our knowledge, there are no data published concerning acid 30 and its preparation was introduced in detail in the part named “Supplementary data”. 2.1.5. Synthesis of compounds 36e37 Urea 36 was synthesized through reaction between 3 and ethyl isocyanate (34) at 0 C in dry DCM (Scheme 5). The pure product was obtained in quantitative yield after crystallization from heptane/MTBE. The N-carbobenzyloxy (Cbz) protected guanidine derivative 37 was prepared in quantitative yield from 3 and commercially available 1,3-bis(benzyloxycarbonyl)-2-methyl-isothiourea (35) according analogous procedure described in literature [12]. 2.2. Biology There are no data regarding the antimycobacterial and cytotoxic activity of the synthesized compounds.
Scheme 4. Synthesis of compounds 31e33.
2.2.1. In vitro antimycobacterial activity The synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv (Table 1; results are presented in mM) using the method of Canetti (see Section 4.2). All compounds are in agreement with the formal Lipinski’s rule of five (except 18 and 37) and were designed in order to investigate their antimycobacterial activity in comparison with the activity of ethambutol (EMB), and the starting bicyclic aminoalcohol 3. Most
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Table 1 In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds 3, 13e21, 25e27, 31e33 and 36e37. Entry
Compound
Antimycobacterial activity toward reference strain of Mycobacterium tuberculosis H37Rv, MIC (mM)
Cytotoxicity toward human embryonal kidney cell line 293T, IC50 (mM)
Selectivity index, SIa
LogPb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
3 13 14 15 16 17 18 19 20 21 25 26 27 31 32 33 36 37 EMB$2HClc
15.20 13.92 10.30 15.27 22.15 18.22 13.00 17.62 7.02 6.14 19.87 18.92 11.44 19.52 18.96 14.17 14.91 9.85 7.22
42.3 18.9 62.3 23.8 72.5 178.6 21.6 64.8 97.9 335.6 18.8 83.9 106.7 70.3 81.5 62.1 121.2 82.4 Not tested
2.8 1.4 6.0 1.6 3.3 9.8 1.7 3.7 13.9 54.7 0.9 4.4 9.3 3.6 4.3 4.4 8.1 8.4 e
2.13 2.80 2.91 4.24 4.76 3.86 9.72 4.36 3.91 3.96 2.79 3.99 4.53 3.42 3.41 3.14 2.31 6.16 0.06d
a b c d
0.28 0.40 0.42 0.38 0.40 0.42 0.39 0.60 0.44 0.45 0.40 0.46 0.55 0.49 0.43 0.68 0.40 0.65
Selectivity index: SI ¼ IC50/MIC. LogP, octanolewater partitioning coefficient, was calculated using ACDLabs/ChemSketch 2012 (www.acdlabs.com). EMB$2HCl e ethambutol dihydrochloride (reference compound). LogP of EMB$2HCl is known in the literature: N.R. Budha, R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
of the amides (15, 17, 19, 25e27 and 36) contain pharmacophore groups known in our previous studies [7,8]. It is interesting to point out that the introduction of various substituents at N-atom of 3 doesn’t affect dramatically the activity of the derivatives. As a whole, the compounds in this study (including 3) showed moderate but stable level of antimycobacterial activity (in most cases 30e70% of the activity of reference EMB; 48% for 3). Compounds 20e21 and 37 demonstrated MIC near to EMB. The activity of sulfonamide 20 was expected since recent studies of Malwal et al. [13,14] revealed the importance of 2,4-dinitrosulfonamide group as in vivo source of sulfur dioxide that is a key agent for selective radical damaging of bacterial biomacromolecules. In this context, it is interesting to note that the activity of cinnamic sulfonamide 21 (MIC 6.14 mM) is comparable with that of 20 although the structures are rather different in respect of the aromatic moieties. Compound 37 is a rare example of antitubercular agent possessing in its structure the guanidine motif. This structural motif is common in antibiotics like streptomycin, capreomycin, viomycin [3,15] and some synthetic polyamines [16]. 2.2.2. In vitro cytotoxic activity The stable level of antimycobacterial activity gave us a good reason for further more detailed evaluation of the cytotoxic effect of those compounds and SI (selectivity index) calculation. The cytotoxic activity of the tested compounds was investigated against human embryonic kidney cell line (HEK293) using the MTT dye reduction assay. The corresponding IC50 values (in mM) of the tested compounds were calculated using nonlinear regression analysis and summarized in Table 1. As it could be seen the compounds demonstrated a wide range of cytotoxicity with IC50 between 18.8 and 335.6. Almost all of the substances have shown acceptable low to moderate cytotoxicity to the cells with only four exceptions (13, 15, 18 and 25) which showed high cytotoxicity. Compounds 20e21 and 37 demonstrated MIC near to EMB and low cytotoxic effect. It is important to note that the most potent antimycobacterial sample e the cinnamic sulfonamide 21 showed excellent SI (54.7) and therefore it is of particular interest due to its highly favorable features low cytotoxicity and significant antimycobacterial activity which makes it a good candidate for an efficient therapeutic agent.
3. Conclusions An efficient synthesis of new enantiopure chiral N-acyl compounds derived from natural ()-fenchone has been demonstrated. The obtained pure structures have been characterized by spectroscopic methods and the in vitro biological activity has been evaluated against M. tuberculosis H37Rv. The most potent compounds among this series were 20, 21 and 37 (MIC comparable to EMB). Almost all of the substances have shown acceptable low to moderate cytotoxicity. 4. Experimental 4.1. Chemistry For thin layer chromatography (TLC) aluminum sheets precoated with silica gel 60 F254 (Merck) were used. Flash column chromatography was carried out using silica gel 60 (0.040e 0.063 mm, 230e400 mesh ASTM, Merck). Commercially available solvents for reactions, TLC and column chromatography were used after distillation (and were dried when needed). Melting temperatures were determined in capillary tubes on an Electrothermal MEL-TEMP 1102D-230 VAC apparatus without corrections. The NMR spectra were recorded on a Bruker Avance IIþ 600 (600.13 for 1 H and 150.92 MHz for 13C NMR) spectrometer. In case of CDCl3 TMS was used as internal standard. For other deuterated solvents 1 H spectra were calibrated to the residual solvent peaks (DMSO-d6 d ¼ 2.50). 13C spectra were calibrated in all cases to the residual solvent peaks (CDCl3 d ¼ 77.00, DMSO-d6 d ¼ 39.52). 31P NMR spectra was recorded with full proton decoupling and using 85% H3PO4 as external standard. The calibration of the 31P NMR spectra was performed through changing of the spectrum reference frequency (specific for the used NMR probe). The following additional NMR techniques were used for all compounds: DEPT 135, COSY, HSQC and HMBC. For numbering of the atoms see Supplementary data. Mass spectra (MS) were recorded on a Thermo Scientific High Resolution Magnetic Sector MS DFS by chemical ionization (CI) or electrospray ionization (ESI), and are reported as fragmentation in m/z with relative intensities (%). Optical rotation [a]20 D measurements were obtained using a PerkineElmer 241 polarimeter.
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Elemental analyses were performed by the Microanalytical Laboratory for Elemental Analysis of the Institute of Organic Chemistry, Bulgarian Academy of Sciences. All fine chemicals were commercially available (from SigmaeAldrich, Merck, Fluka, Acros, Alfa Aesar). Dimethyl sulfoxide (DMSO) for testing of bioactivities was commercial (spectroscopic grade) and was used without distillation or purification. 4.2. Methodology for evaluation of antimycobacterial activity The antimycobacterial activity was determined through the proportional method of Canetti towards reference strain M. tuberculosis H37Rv. This method, recommended by the WHO, is the most commonly used one worldwide for exploration of sensitivity/ resistance of tuberculosis strains towards chemotherapeutics [17e 21]. It allows precise determination of the proportion of resistant mutants to a certain drug. A sterile suspension/solution of each tested compound was added to LöwensteineJensen egg based medium before its coagulation (30 min at 85 C). Each compound was tested at four concentrations e 5 mg/mL, 2 mg/mL, 0.2 mg/mL and 0.1 mg/mL (in DMSO). For some compounds, additional tests at concentrations 0.05, 1, 2 and 3 mg/mL were performed. Tubes with Löwensteine Jensen medium (5 mL) containing tested compounds and those without them (controls) were inoculated with a suspension of M. tuberculosis H37Rv (105 cells/mL) and incubated for 45 days at 37 C. The ratio between the number of colonies of M. tuberculosis grown in medium containing compounds and the number of colonies in control medium were calculated and expressed as percentage of inhibition. The MIC is defined as the minimum concentration of compound required to inhibit bacterial growth completely (0% growth). The MIC values are calculated and given as mM. 4.3. Methodology for evaluation of cytotoxicity The cell viability was examined as described previously [8]. Briefly a standard MTT-dye reduction based test have been used as described by Mosmann [22] with some modifications. The method is based on the biotransformation of the yellow tetrazolium salt MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a violet formazan via the mitochondrial succinate dehydrogenase in the viable cells. The exponentially growing cells (HEK e human embryonic kidney fibroblasts) were seeded in 96-well flat-bottomed micro-plates (100 mL/well) at a density of 105 cells per mL and after 24 h incubation at 37 C they were exposed to various concentrations of the tested compounds for 72 h. At least 8 wells were used for each concentration. After the incubation with the test compounds 10 mL MTT solution (5 mg/mL in PBS) aliquots were added to each well. The microplates were further incubated for 4 h at 37 C and the formazan crystals formed were dissolved through addition of 100 mL DMSO into each well. The absorbance was measured on an ELISA plate reader (Bio-Tek Instruments) with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain sample signal (OD570eOD630). The cell survival fractions
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were calculated as percentage of the untreated control. In addition, IC50 values were derived from the concentration response curves. Acknowledgments This study was partially supported by the Bulgarian Science Fund e project DMU 02-1/2010. The financial support of the Bulgarian Science Fund for the purchase of Bruker Avance IIþ 600 NMR spectrometer in the framework of the Program “Promotion of the Research Potential through Unique Scientific Equipment” e project UNA-17/2005 is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.03.025. References [1] WHO Report 2011 e Global Tuberculosis Control; Webpage: http://www. who.int/tb/publications/global_report/2011/gtbr11_full.pdf. [2] O.K. Onajole, Y. Coovadia, H.G. Kruger, G.E.M. Maguire, M. Pillay, T. Govender, European Journal of Medicinal Chemistry 54 (2012) 1e9. [3] R.P. Tripathi, S.S. Bisht, A. Ajay, A. Sharma, M. Misra, M.Pd Gupt, Current Medicinal Chemistry 19 (2012) 488e517. [4] L.G. Dover, G.D. Coxon, Journal of Medicinal Chemistry 54 (2011) 6157e6165. [5] R.E. Lee, M. Protopopova, E. Crooks, R.A. Slayden, M. Terrot, C.E. Barry, Journal of Combinatorial Chemistry 5 (2003) 172e187. [6] O.K. Onajole, K. Govender, P. Govender, P.D. van Helden, H.G. Kruger, G.E.M. Maguire, K. Muthusamy, M. Pillay, I. Wiid, T. Govender, European Journal of Medicinal Chemistry 44 (2009) 4297e4305. [7] G.M. Dobrikov, V. Valcheva, M. Stoilova-Disheva, G. Momekov, P. Tzvetkova, A. Chimov, V. Dimitrov, European Journal of Medicinal Chemistry 48 (2012) 45e56. [8] G.M. Dobrikov, V. Valcheva, Y. Nikolova, I. Ugrinova, E. Pasheva, V. Dimitrov, European Journal of Medicinal Chemistry 63 (2013) 468e473. [9] V. Dimitrov, G. Dobrikov, M. Genov, Tetrahedron: Asymmetry 12 (2001) 1323e1329. [10] R.J.W. Cremlyn, Journal of the Chemical Society C (1968) 11e16. [11] E. Valeur, M. Bradley, Chemical Society Reviews 38 (2009) 606e631. [12] J. Quancard, A. Labonne, Y. Jacquot, G. Chassaing, S. Lavielle, Ph Karoyan, Journal of Organic Chemistry 69 (2004) 7940e7948. [13] S.R. Malwal, D. Sriram, P. Yogeeswari, H. Chakrapani, Bioorganic & Medicinal Chemistry Letters 22 (2012) 3603e3606. [14] S.R. Malwal, D. Sriram, P. Yogeeswari, V. Badireenath Konkimalla, H. Chakrapani, Journal of Medicinal Chemistry 55 (2012) 553e557. [15] Y.L. Janin, Bioorganic & Medicinal Chemistry 15 (2007) 2479e2513. [16] A. Nefzi, J. Appel, S. Arutyunyan, R.A. Houghten, Bioorganic & Medicinal Chemistry Letters 19 (2009) 5169e5175. [17] G. Canetti, N. Rist, J. Grosset, Revue de Tuberculose et de Pneumologie 27 (1963) 217e272. [18] G. Canetti, S. Froman, J. Grosset, P. Hauduroy, M. Langerova, H.T. Mahler, G. Meissner, D.A. Mitchison, L. Sula, Mycobacteria: laboratory methods for testing drug sensitivity and resistance, Bulletin of the World Health Organization 29 (1963) 565e578. [19] G. Canetti, W. Fox, A. Khomenko, H.T. Mahler, N.K. Menon, D.A. Mitchinson, N. Rist, N.A. Smelev, Bulletin de l’Organisation mondiale de la santé 41 (1969) 21e43. [20] L. Heifets, Conventional methods for antimicrobial susceptibility testing of Mycobacterium tuberculosis, in: I. Bastian, F. Portaels (Eds.), Multidrug-resistant Tuberculosis, Kluwer Academic Publishers, Dordrecht, The Netherlands, 2000. [21] CellTiter 96 Non-Radioactive Cell proliferation assay, Technical Bulletin #TB112, Promega Corporation USA, Revised 12/99. [22] T. Mosmann, Journal of Immunological Methods 65 (1983) 55e63.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enantiopure antituberculosis candidates synthesized from ()-fenchone Georgi M. Dobrikov a, *, Violeta Valcheva b, Yana Nikolova a, Iva Ugrinova c, Evdokia Pasheva c, Vladimir Dimitrov a, * a b c
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Bl. 9, Acad. G. Bonchev Str., Sofia 1113, Bulgaria Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Bl. 26, Acad. G. Bonchev Str., Sofia 1113, Bulgaria Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, Bl. 21, Acad. G. Bonchev Str., Sofia 1113, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2014 Received in revised form 6 March 2014 Accepted 8 March 2014 Available online 12 March 2014
The synthesis of new enantiopure N-acyl compounds derived from ()-fenchone has been performed. The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed for most of them moderate activity. The structures bearing sulfonamide functionality have comparable activity to ethambutol and possess low cytotoxicity. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Fenchone Chiral Amides Antimycobacterial Tuberculosis
1. Introduction Despite the availability of highly efficacious treatment for decades, tuberculosis (TB) remains a major global health challenge. In 1993, the World Health Organization (WHO) declared TB a global public health emergency, at a time when an estimated 7e8 million cases and 1.3e1.6 million deaths occurred each year. In 2010 there were an estimated 8.5e9.2 million cases and 1.2e1.5 million deaths (including deaths from TB among HIV-positive people). TB is the second leading cause of death from an infectious disease worldwide (after HIV, which caused an estimated 1.8 million deaths in 2008) [1]. An urgent need for highly potent, more effective drugs with fewer or no side effects and shorter treatment periods to combat the increasing TB pandemic is therefore apparent. Potential anti-TB drug candidates such as diarylquinolones (TMC207), nitroimidazoles (OPC67683 and PA824), pyrroles (LL3858), diamines [2] are in different stages of clinical trials [3,4]. The incorporation of lipophilic polycyclic aliphatic compounds into drug-structures with promising anti-TB applications has enjoyed much attention from
* Corresponding authors. E-mail address: [email protected] (G.M. Dobrikov). http://dx.doi.org/10.1016/j.ejmech.2014.03.025 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
researchers for several years starting with the discovery of SQ109 [5]. The recent studies of Onajole et al. [2,6] confirm the importance of further investigations of polycyclic compounds as potent anti-TB agents. In the course of our research for novel anti-tubercular compounds, we recently demonstrated the role of chirality and wide spectrum of substituted acyl groups, attached to nitrogen atom of (R)-2-aminobutanol [7,8]. The present study aims at investigating of new subclass diastereomerically pure bicyclic N-substituted compounds with fenchane skeletons and the possibility of further enhancing/improving their anti-TB activity. Based on this, 15 novel amidoalcohols and 2 other N-carbonyl derivatives bearing the lipophilic fenchane skeleton were synthesized and screened for activity against drug sensitive (H37Rv) strain of tuberculosis. 2. Results and discussion 2.1. Chemistry The starting aminoalcohol 3 (Scheme 1) was synthesized by using enantiomerically pure ()-fenchone as a natural source of chirality. Desired amides 13e21, 25e27 and 31e33 were synthesized through three main methods of N-acylation of 3, as shown below (Schemes 2e4). It was found that the N-acylation by using
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acylchlorides or activated acids (TBTU) is more efficient than the aminolysis of esters applied previously [7]. Compounds 36e37 were synthesized by other methods (Scheme 5). All of the synthesized compounds inherit the configuration of 3 and were isolated in high purity after column chromatography or crystallization. Scheme 1. Synthesis of compounds 2 and 3.
Scheme 2. Synthesis of compounds 13e21.
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 77 (2014) 243e247
245
Scheme 5. Synthesis of compounds 36 and 37. Scheme 3. Synthesis of compounds 25e27.
2.1.1. Synthesis of compounds 2 and 3 The synthesis of starting aminoalcohol 3 was performed in two steps through intermediate 2 (Scheme 1), by modification of described procedure [9]. The addition of in situ prepared LiCH2CN to ()-fenchone was performed at 85 C in THF without using of anhydrous CeCl3. Other key moment was the hydrolysis of the reaction mixture e the aqueous NH4Cl was added at 85 C and the reaction immediately allowed to r.t. This improved procedure leads to quantitative yield of 2 as pure diastereoisomer after column chromatography. Reduction of 2 to aminoalcohol 3 was performed quantitatively with LiAlH4 in Et2O, according to the literature [9]. The diastereomerical purity and configurations of 2 and 3 were unambiguously confirmed by 1D and 2D NMR experiments. 2.1.2. Synthesis of compounds 13e21 Series of amidoalcohols 13e21 were synthesized in good to excellent yields using standard conditions for acylation of 3 (0 C and Et3N in dry DCM) with acid chlorides 4e12 (Scheme 2). Sulfochloride 12 was prepared from cinnamic acid as described [10].
2.1.3. Synthesis of compounds 25e27 The amidoalcohols 25e27 were synthesized using simple solvent-free aminolysis of esters 22e24 with 3 by heating at 90e 100 C (Scheme 3). Compounds 25e27 were isolated in moderate yields and high purities after column chromatography (or crystallization in case of the low-soluble 27). 2.1.4. Synthesis of compounds 31e33 Compounds 31e33 were prepared through the coupling reaction between 3 and acids 28e30, respectively, in presence of O(Benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluoroborate (TBTU) as activating agent (Scheme 4). TBTU was chosen as one of the most effective coupling reagent in peptide synthesis [11]. Amides 31e33 were isolated in high yields after column chromatography. In case of anthranilic acid (29) preliminary protection of the aromatic amino group was not necessary, due to its low reactivity in comparison to the high nucleophilic amino group of 3. To the best of our knowledge, there are no data published concerning acid 30 and its preparation was introduced in detail in the part named “Supplementary data”. 2.1.5. Synthesis of compounds 36e37 Urea 36 was synthesized through reaction between 3 and ethyl isocyanate (34) at 0 C in dry DCM (Scheme 5). The pure product was obtained in quantitative yield after crystallization from heptane/MTBE. The N-carbobenzyloxy (Cbz) protected guanidine derivative 37 was prepared in quantitative yield from 3 and commercially available 1,3-bis(benzyloxycarbonyl)-2-methyl-isothiourea (35) according analogous procedure described in literature [12]. 2.2. Biology There are no data regarding the antimycobacterial and cytotoxic activity of the synthesized compounds.
Scheme 4. Synthesis of compounds 31e33.
2.2.1. In vitro antimycobacterial activity The synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv (Table 1; results are presented in mM) using the method of Canetti (see Section 4.2). All compounds are in agreement with the formal Lipinski’s rule of five (except 18 and 37) and were designed in order to investigate their antimycobacterial activity in comparison with the activity of ethambutol (EMB), and the starting bicyclic aminoalcohol 3. Most
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Table 1 In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds 3, 13e21, 25e27, 31e33 and 36e37. Entry
Compound
Antimycobacterial activity toward reference strain of Mycobacterium tuberculosis H37Rv, MIC (mM)
Cytotoxicity toward human embryonal kidney cell line 293T, IC50 (mM)
Selectivity index, SIa
LogPb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
3 13 14 15 16 17 18 19 20 21 25 26 27 31 32 33 36 37 EMB$2HClc
15.20 13.92 10.30 15.27 22.15 18.22 13.00 17.62 7.02 6.14 19.87 18.92 11.44 19.52 18.96 14.17 14.91 9.85 7.22
42.3 18.9 62.3 23.8 72.5 178.6 21.6 64.8 97.9 335.6 18.8 83.9 106.7 70.3 81.5 62.1 121.2 82.4 Not tested
2.8 1.4 6.0 1.6 3.3 9.8 1.7 3.7 13.9 54.7 0.9 4.4 9.3 3.6 4.3 4.4 8.1 8.4 e
2.13 2.80 2.91 4.24 4.76 3.86 9.72 4.36 3.91 3.96 2.79 3.99 4.53 3.42 3.41 3.14 2.31 6.16 0.06d
a b c d
0.28 0.40 0.42 0.38 0.40 0.42 0.39 0.60 0.44 0.45 0.40 0.46 0.55 0.49 0.43 0.68 0.40 0.65
Selectivity index: SI ¼ IC50/MIC. LogP, octanolewater partitioning coefficient, was calculated using ACDLabs/ChemSketch 2012 (www.acdlabs.com). EMB$2HCl e ethambutol dihydrochloride (reference compound). LogP of EMB$2HCl is known in the literature: N.R. Budha, R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
of the amides (15, 17, 19, 25e27 and 36) contain pharmacophore groups known in our previous studies [7,8]. It is interesting to point out that the introduction of various substituents at N-atom of 3 doesn’t affect dramatically the activity of the derivatives. As a whole, the compounds in this study (including 3) showed moderate but stable level of antimycobacterial activity (in most cases 30e70% of the activity of reference EMB; 48% for 3). Compounds 20e21 and 37 demonstrated MIC near to EMB. The activity of sulfonamide 20 was expected since recent studies of Malwal et al. [13,14] revealed the importance of 2,4-dinitrosulfonamide group as in vivo source of sulfur dioxide that is a key agent for selective radical damaging of bacterial biomacromolecules. In this context, it is interesting to note that the activity of cinnamic sulfonamide 21 (MIC 6.14 mM) is comparable with that of 20 although the structures are rather different in respect of the aromatic moieties. Compound 37 is a rare example of antitubercular agent possessing in its structure the guanidine motif. This structural motif is common in antibiotics like streptomycin, capreomycin, viomycin [3,15] and some synthetic polyamines [16]. 2.2.2. In vitro cytotoxic activity The stable level of antimycobacterial activity gave us a good reason for further more detailed evaluation of the cytotoxic effect of those compounds and SI (selectivity index) calculation. The cytotoxic activity of the tested compounds was investigated against human embryonic kidney cell line (HEK293) using the MTT dye reduction assay. The corresponding IC50 values (in mM) of the tested compounds were calculated using nonlinear regression analysis and summarized in Table 1. As it could be seen the compounds demonstrated a wide range of cytotoxicity with IC50 between 18.8 and 335.6. Almost all of the substances have shown acceptable low to moderate cytotoxicity to the cells with only four exceptions (13, 15, 18 and 25) which showed high cytotoxicity. Compounds 20e21 and 37 demonstrated MIC near to EMB and low cytotoxic effect. It is important to note that the most potent antimycobacterial sample e the cinnamic sulfonamide 21 showed excellent SI (54.7) and therefore it is of particular interest due to its highly favorable features low cytotoxicity and significant antimycobacterial activity which makes it a good candidate for an efficient therapeutic agent.
3. Conclusions An efficient synthesis of new enantiopure chiral N-acyl compounds derived from natural ()-fenchone has been demonstrated. The obtained pure structures have been characterized by spectroscopic methods and the in vitro biological activity has been evaluated against M. tuberculosis H37Rv. The most potent compounds among this series were 20, 21 and 37 (MIC comparable to EMB). Almost all of the substances have shown acceptable low to moderate cytotoxicity. 4. Experimental 4.1. Chemistry For thin layer chromatography (TLC) aluminum sheets precoated with silica gel 60 F254 (Merck) were used. Flash column chromatography was carried out using silica gel 60 (0.040e 0.063 mm, 230e400 mesh ASTM, Merck). Commercially available solvents for reactions, TLC and column chromatography were used after distillation (and were dried when needed). Melting temperatures were determined in capillary tubes on an Electrothermal MEL-TEMP 1102D-230 VAC apparatus without corrections. The NMR spectra were recorded on a Bruker Avance IIþ 600 (600.13 for 1 H and 150.92 MHz for 13C NMR) spectrometer. In case of CDCl3 TMS was used as internal standard. For other deuterated solvents 1 H spectra were calibrated to the residual solvent peaks (DMSO-d6 d ¼ 2.50). 13C spectra were calibrated in all cases to the residual solvent peaks (CDCl3 d ¼ 77.00, DMSO-d6 d ¼ 39.52). 31P NMR spectra was recorded with full proton decoupling and using 85% H3PO4 as external standard. The calibration of the 31P NMR spectra was performed through changing of the spectrum reference frequency (specific for the used NMR probe). The following additional NMR techniques were used for all compounds: DEPT 135, COSY, HSQC and HMBC. For numbering of the atoms see Supplementary data. Mass spectra (MS) were recorded on a Thermo Scientific High Resolution Magnetic Sector MS DFS by chemical ionization (CI) or electrospray ionization (ESI), and are reported as fragmentation in m/z with relative intensities (%). Optical rotation [a]20 D measurements were obtained using a PerkineElmer 241 polarimeter.
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Elemental analyses were performed by the Microanalytical Laboratory for Elemental Analysis of the Institute of Organic Chemistry, Bulgarian Academy of Sciences. All fine chemicals were commercially available (from SigmaeAldrich, Merck, Fluka, Acros, Alfa Aesar). Dimethyl sulfoxide (DMSO) for testing of bioactivities was commercial (spectroscopic grade) and was used without distillation or purification. 4.2. Methodology for evaluation of antimycobacterial activity The antimycobacterial activity was determined through the proportional method of Canetti towards reference strain M. tuberculosis H37Rv. This method, recommended by the WHO, is the most commonly used one worldwide for exploration of sensitivity/ resistance of tuberculosis strains towards chemotherapeutics [17e 21]. It allows precise determination of the proportion of resistant mutants to a certain drug. A sterile suspension/solution of each tested compound was added to LöwensteineJensen egg based medium before its coagulation (30 min at 85 C). Each compound was tested at four concentrations e 5 mg/mL, 2 mg/mL, 0.2 mg/mL and 0.1 mg/mL (in DMSO). For some compounds, additional tests at concentrations 0.05, 1, 2 and 3 mg/mL were performed. Tubes with Löwensteine Jensen medium (5 mL) containing tested compounds and those without them (controls) were inoculated with a suspension of M. tuberculosis H37Rv (105 cells/mL) and incubated for 45 days at 37 C. The ratio between the number of colonies of M. tuberculosis grown in medium containing compounds and the number of colonies in control medium were calculated and expressed as percentage of inhibition. The MIC is defined as the minimum concentration of compound required to inhibit bacterial growth completely (0% growth). The MIC values are calculated and given as mM. 4.3. Methodology for evaluation of cytotoxicity The cell viability was examined as described previously [8]. Briefly a standard MTT-dye reduction based test have been used as described by Mosmann [22] with some modifications. The method is based on the biotransformation of the yellow tetrazolium salt MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a violet formazan via the mitochondrial succinate dehydrogenase in the viable cells. The exponentially growing cells (HEK e human embryonic kidney fibroblasts) were seeded in 96-well flat-bottomed micro-plates (100 mL/well) at a density of 105 cells per mL and after 24 h incubation at 37 C they were exposed to various concentrations of the tested compounds for 72 h. At least 8 wells were used for each concentration. After the incubation with the test compounds 10 mL MTT solution (5 mg/mL in PBS) aliquots were added to each well. The microplates were further incubated for 4 h at 37 C and the formazan crystals formed were dissolved through addition of 100 mL DMSO into each well. The absorbance was measured on an ELISA plate reader (Bio-Tek Instruments) with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain sample signal (OD570eOD630). The cell survival fractions
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were calculated as percentage of the untreated control. In addition, IC50 values were derived from the concentration response curves. Acknowledgments This study was partially supported by the Bulgarian Science Fund e project DMU 02-1/2010. The financial support of the Bulgarian Science Fund for the purchase of Bruker Avance IIþ 600 NMR spectrometer in the framework of the Program “Promotion of the Research Potential through Unique Scientific Equipment” e project UNA-17/2005 is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.03.025. References [1] WHO Report 2011 e Global Tuberculosis Control; Webpage: http://www. who.int/tb/publications/global_report/2011/gtbr11_full.pdf. [2] O.K. Onajole, Y. Coovadia, H.G. Kruger, G.E.M. Maguire, M. Pillay, T. Govender, European Journal of Medicinal Chemistry 54 (2012) 1e9. [3] R.P. Tripathi, S.S. Bisht, A. Ajay, A. Sharma, M. Misra, M.Pd Gupt, Current Medicinal Chemistry 19 (2012) 488e517. [4] L.G. Dover, G.D. Coxon, Journal of Medicinal Chemistry 54 (2011) 6157e6165. [5] R.E. Lee, M. Protopopova, E. Crooks, R.A. Slayden, M. Terrot, C.E. Barry, Journal of Combinatorial Chemistry 5 (2003) 172e187. [6] O.K. Onajole, K. Govender, P. Govender, P.D. van Helden, H.G. Kruger, G.E.M. Maguire, K. Muthusamy, M. Pillay, I. Wiid, T. Govender, European Journal of Medicinal Chemistry 44 (2009) 4297e4305. [7] G.M. Dobrikov, V. Valcheva, M. Stoilova-Disheva, G. Momekov, P. Tzvetkova, A. Chimov, V. Dimitrov, European Journal of Medicinal Chemistry 48 (2012) 45e56. [8] G.M. Dobrikov, V. Valcheva, Y. Nikolova, I. Ugrinova, E. Pasheva, V. Dimitrov, European Journal of Medicinal Chemistry 63 (2013) 468e473. [9] V. Dimitrov, G. Dobrikov, M. Genov, Tetrahedron: Asymmetry 12 (2001) 1323e1329. [10] R.J.W. Cremlyn, Journal of the Chemical Society C (1968) 11e16. [11] E. Valeur, M. Bradley, Chemical Society Reviews 38 (2009) 606e631. [12] J. Quancard, A. Labonne, Y. Jacquot, G. Chassaing, S. Lavielle, Ph Karoyan, Journal of Organic Chemistry 69 (2004) 7940e7948. [13] S.R. Malwal, D. Sriram, P. Yogeeswari, H. Chakrapani, Bioorganic & Medicinal Chemistry Letters 22 (2012) 3603e3606. [14] S.R. Malwal, D. Sriram, P. Yogeeswari, V. Badireenath Konkimalla, H. Chakrapani, Journal of Medicinal Chemistry 55 (2012) 553e557. [15] Y.L. Janin, Bioorganic & Medicinal Chemistry 15 (2007) 2479e2513. [16] A. Nefzi, J. Appel, S. Arutyunyan, R.A. Houghten, Bioorganic & Medicinal Chemistry Letters 19 (2009) 5169e5175. [17] G. Canetti, N. Rist, J. Grosset, Revue de Tuberculose et de Pneumologie 27 (1963) 217e272. [18] G. Canetti, S. Froman, J. Grosset, P. Hauduroy, M. Langerova, H.T. Mahler, G. Meissner, D.A. Mitchison, L. Sula, Mycobacteria: laboratory methods for testing drug sensitivity and resistance, Bulletin of the World Health Organization 29 (1963) 565e578. [19] G. Canetti, W. Fox, A. Khomenko, H.T. Mahler, N.K. Menon, D.A. Mitchinson, N. Rist, N.A. Smelev, Bulletin de l’Organisation mondiale de la santé 41 (1969) 21e43. [20] L. Heifets, Conventional methods for antimicrobial susceptibility testing of Mycobacterium tuberculosis, in: I. Bastian, F. Portaels (Eds.), Multidrug-resistant Tuberculosis, Kluwer Academic Publishers, Dordrecht, The Netherlands, 2000. [21] CellTiter 96 Non-Radioactive Cell proliferation assay, Technical Bulletin #TB112, Promega Corporation USA, Revised 12/99. [22] T. Mosmann, Journal of Immunological Methods 65 (1983) 55e63.