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Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents
Bioorganic & Medicinal Chemistry Letters 22 (2012) 954–957
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents Yinghong Yang a,b, , Zhenling Wang a, , Jianzhong Yang a, Tao Yang a, Weiyi Pi a, Wei Ang c, Yanni Lin a, Yuanyuan Liu a, Zicheng Li b,⇑, Youfu Luo a,⇑, Yuquan Wei a a
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, PR China c Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China b
a r t i c l e
i n f o
Article history: Received 10 August 2011 Revised 29 November 2011 Accepted 3 December 2011 Available online 9 December 2011 Keywords: Diphenyl ether nucleus Antituberculosis H37Rv BCG murine model
a b s t r a c t A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated against Mycobacterium tuberculosis H37Rv by a microdilution method, with MIC values ranging from 4 to 64 lg/mL. Through structure–activity relationship studies, the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group were found to play a significant role in the antitubercular activity. The most potent compound 6c showed an MIC value of 4 lg/mL and a good safety profile in HepG2 cell line by the MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing a good lead for subsequent optimization. Ó 2011 Elsevier Ltd. All rights reserved.
Tuberculosis (TB), one of the leading causes of death in the world, poses a significant challenge to the public health. The World Health Organization (WHO) has estimated that one-third of the world’s population is currently infected with the TB bacillus.1 According to the annual report on global control of TB published by the WHO in 2010, there were about 9.4 million incident cases of TB globally in 2009, and approximately 1.7 million people died of TB, among these deaths 23% were HIV-positive.2 People who are HIV-positive are more likely to be infected with TB, so the co-infection with HIV has been responsible for this serious situation. On the other hand, the emergence of multi drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) also contributes to the increased morbidity and mortality. MDR-TB, describes strains of tuberculosis that are resistant to at least the two main first-line TB drugs-isoniazid (INH) and rifampicin. XDR-TB is defined as resistance not only to rifampicin and isoniazid but also fluoroquinolones and to at least one of the injectable second-line drugs. It is also said that there was an estimated 0.44 million cases of MDR-TB in 2008, and 58 countries and territories had reported at least one case of XDR-TB by July 2010.2 This worsening situation leads to an urgent necessity for development of new agents to fight against this disease. Diphenyl ether nucleus is an important unit found in several synthetic and natural agents possessing wide range of pharmaco⇑ Corresponding authors. Tel./fax: +86 28 85503817 (Y.L.).
E-mail addresses: [email protected] (Z. Li), [email protected] (Y. Luo). These authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.12.022
logical activities. A classic chemical with this nucleus is triclosan, which is launched in the early 1970s. It is widely used in a variety of cleaning and personal care products for its broad spectrum antimicrobial activity against a variety of Gram-positive and Gramnegative bacteria3 and continuous studies have been focused on triclosan and its derivatives in recent years. 4 FAB-001 (Fig. 1), a FabI inhibitor with the diphenyl ether nucleus developed by FAB Pharma (Paris, France), is currently being evaluated in phase-I trials for the treatment of drug-resistant staphylococci.5 Moreover, lots of novel diphenyl ether analogues were designed to exhibit enhanced antimycobacterial activity.6 Kini and co-workers synthesized a series of heterocycle substituted diphenyl ether derivatives and all the compounds showed good antitubercular activity against Mycobacterium tuberculosis strain H37Rv.7 Diaryl amide skeleton is also a class of privileged structure with broad range of biological activities such as antiviral,8 anti-inflammatory,9 analgesic,10 antibacterial,11 melanin synthesis inhibitor12 and S1P4 antagonist.13 Recently, in a high throughput screening for inhibitors of M. tuberculosis H37Rv, several diaryl amides14 (Fig. 1) were found to possess potent activity in combination with the relatively low toxicity. Encouraged by the prominent activity of diphenyl ether derivatives and diaryl amides, we proposed to synthesize some novel diphenyl ether analogues containing the diaryl amide moiety by molecular hybridization approach, aiming to search for potential candidates with better antitubercular activities and lower toxicities. In this Letter, we designed and synthesized 22 compounds with a diphenyl ether nucleus, and their antitubercular activity against M. tuberculosis H37Rv in vitro were evaluated by a microdilution
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
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Scheme 2. Synthetic route of compound 6v. Reagents and conditions: (i) CuI, N,Ndimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2, CHCl3, reflux; (vi) 3-chloroaniline (5a), CH2Cl2, Et3N, rt. Figure 1. Chemical structures of triclosan, FAB-001 and diaryl amides.
method. Compound 6c was found to be the most potent antitubercular agents with MIC of 4 lg/mL, and it was then assessed in vivo using a BCG infection mice model. The target compounds were synthesized utilizing the reaction sequence as shown in Schemes 1 and 2. Iodobenzene or substituted iodobenzene (1) was employed as starting material, it was condensated with compound 2 to give compound 3, via the N,N-dimethyl glycine-promoted Ullmann coupling reaction.15 Compound 3 was further hydrolyzed to compound 4 in excellent yields under reflux conditions in a solution of sodium hydroxide. Compound 4 was then treated with thionyl chloride to obtain the corresponding acyl chloride, and it was reacted with various amines 5 in presence of triethylamine in anhydrous dichloromethane to give the target compounds 6a–v. The structures of 6a–v were fully characterized by 1H NMR, 13C NMR, and ESI-MS analysis and all the analytical data were documented in the Supplementary data. The purity of all compounds was above 97.0% determined by HPLC normalization method. All the synthesized compounds (6a–v) were evaluated for their antimycobacterial activity against M. tuberculosis H37Rv by a microdilution method in 96-wells plates.16 Briefly, M. tuberculosis was grown in Middlebrook 7H9 broth containing oleic acid-albumindextrose-catalase (OADC) enrichment, and was diluted with 7H9OADC medium to 104 CFU/mL. An amount of 100 lL of this bacterial culture was then added to a microtiter well containing serial dilutions of compounds 6a–v. The plates were then incubated at 37 °C in a moisturized incubator and visually examined for growth. INH was used as a positive control. The MIC values are shown in Table 1. As shown in Table 1, all the compounds of this series exhibited antitubercular activity with MIC ranging from 4 to 64 lg/mL, and compound 6c was found to be the most potent with MIC of 4 lg/mL. Compounds 6a (R2 = 3-Cl-Ph) and 6b (R2 = 4-Br-Ph) exhibited lower activity than compound 6c (R2 = 3,4-di Cl-Ph), and 3-Cl substituted compound 6a was slightly more effective compared with the 4-Br substituted compound 6b. When the two chlorine
atoms in 6c were replaced by two fluorine atoms in 6d (R2 = 3,4di F-Ph), the activity was decreased a lot, and so was 6e (R2 = 2Cl-4-Br-Ph) with a chlorine atom and a bromine atom. All these data suggested that the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group are very important in the antitubercular activity. Compounds 6f (R2 = 2-CN-Ph), 6g (R2 = 4-CF3-Ph) introduced with electron-withdrawing substituents and compounds 6h (R2 = 4-OCF3-Ph), 6i (R2 = 2,4-di OCH3-Ph) with electro-donating substituents all showed moderate activity (MIC = 32 lg/mL) without potency improvement. In particular, introduction of a nitro unit in the phenyl ring of R2 group (compound 6j, R2 = 3-NO2-Ph; 6k, R2 = 4-NO2-Ph) did not increase antitubercular activity, although lots of compounds with the NO2 substituent, such as nitrofunans and nitroimidazoles, possessed broad spectrum antimicrobial activities by DNA damage. Next, compounds 6m–6q with more complex aromatic or heterocyclic rings were synthesized, compound 6n (R2 = 6-methoxybenzo[d]thiazol-2-yl) exhibited relatively higher level of activity with MIC of 16 lg/mL compared with 6m and 6o–6q. Further modifications on the length of the aliphatic chain between the B ring and the amide bond showed that all the derivatives (6s–6v, n = 0, 2, 3) possess similar potency (MIC = 32 lg/mL) with compound 6a (n = 1). Compound 6s with amide bond at meta position of B ring and compound 6v at para position demonstrated similar activities. Neither molecule 6q with chlorine atom nor 6r with fluorine atom of R1 group in A ring exhibited increased antimycobacterial activity compared with molecule 6a. Further studies with modifications of the A and B rings are in progress in our lab. Thus, at this stage, all the modifications indicated that the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group play a significant role in the antitubercular activity. The safety profile of compounds 6a–v was tested on human cancer cell lines HepG2 at the concentration of 20 lM using the MTT method. Briefly, cells (2500/well) were seeded in 96-well plates and cultured for 24 h, followed by treatment with the target compounds 6a–v for another 48 h. Twenty microliter of 5 mg/ml MTT was added per well and incubated for another 2.5 h at 37 °C. Then the supernatant fluid was removed and MTT formazan
Scheme 1. Synthetic route of compounds 6a–u. Reagents and conditions: (i) CuI, N,N-dimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2, CHCl3, reflux; (vi) NH2R2 (5a–u), CH2Cl2, Et3N, rt.
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Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
Table 1 Antimycobacterial activity and toxicity of compounds 6a–v
O R1
n
A
B
H N
R2
O
O A
6a-u Compound
R1
n
H N
B 6v
R2
O H37Rv (lg/ml) MIC90
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t 6u 6v INH a b c d
H H H H H H H H H H H H H H H H Cl F H H H —
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 3 —
Cl
3-Cl-Ph– 4-Br-Ph– 3,4-Di Cl-Ph– 3,4-Di F-Ph– 2-Cl-4-Br-Ph2-CN-Ph– 4-CF3-Ph– 4-OCF3-Ph– 2,4-Di OCH3-Ph– 3-NO2-Ph– 4-NO2-Ph– Naphthalen-1-yl– Diphenylmethyl– 6-Methoxybenzo[d]thiazol-2-yl– 5-Methylpyridin-2-yl– Thiazol-2-yl– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– —
HepG2 (%) b
a
16 16 NTd 8 16 NT 8 16 NT 16 8 32 8 8 8 16 16 16 16 16 16 16 0.0625
MIC
IRc@20 lM
32 64 4 32 32 32 32 32 32 32 32 64 32 16 32 32 32 32 32 32 32 32 0.0625
7.95 15.70 7.00 26.73 12.81 19.08 10.31 4.71 4.11 0.95 36.41 3.31 4.84 4.29 12.21 39.89 27.06 23.52 7.07 25.05 NT 29.20 3.51
MIC90 is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 100 CFU of bacteria inoculated merely. MIC is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 10 CFU of bacteria inoculated merely. IR is the mean inhibitory rate calculated from three independent experiments measured at 48 h after treatment with the test compound at the concentration of 20 lM. NT means not tested.
precipitate was dissolved in 150 lL of DMSO, shaken mechanically for 15–20 min. The optical density of each well was measured at 570 nm, using a SpectraMAX M5 microplate spectrophotometer. The inhibitory rates at 20 lM are given in Table 1, all the active compounds showed low toxicity in this assay and the most potent compound 6c exhibited good safety profile with inhibitory rates as low as 7%. The most potent derivative 6c was further tested for in vivo antitubercular activity using a murine model of BCG infection17 as described in detail in the Supplementary data. Female BALB/c mice were infected intravenously with bacterial culture containing approximately 1 105 CFU of M. bovis BCG, and compound 6c was orally administered at a dose of 25 mg/kg once daily. After six weeks of treatment, mice were sacrificed. Lung and spleen tissues were homogenized and plated on Modified Lowenstein-Jensen Medium Base. BCG colonies were counted after six weeks of incubation at 37 °C. Data were expressed as mean log10 CFU per organ
Table 2 Multiplication of M. bovis BCG Group
BCG + vehicle BCG + 6c BCG + INH
Na
6 8 8
Log10 CFU (mean ± SD) Lung
Spleen
4.64 ± 0.17 2 ± 0.89⁄ 2 ± 0.45⁄
4.79 ± 0.25 2 ± 0.74⁄ 3 ± 0.24⁄
Significantly different: ⁄P <0.01 versus vehicle-treated group. a N means the number of treated mice in each group.
and results of one several representative experiments were shown in Table 2. Compound 6c-treated mice had a significant reduction in bacterial numbers in the lungs, as well as in spleens, compared to vehicle controls. And there is no remarkable difference in bacterial numbers between compound 6c group and INH group, both in lungs and spleens.
Figure 2. Histopathological appearance of lungs of infected mice treated with vehicle (A), infected mice treated with compound 6c (B), and infected mice treated with INH (C). Magnification, 100.
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
Meanwhile, lung tissues were harvested for histological evaluation. As shown in Figure 2, perivascular and peribronchiolar accumulations of resident epithelioid macrophages, granulocytes, and lymphocytes located in the parenchyma, close to major airways, and blood vessels were observed in lung tissues of BCG model mice, and these pathological changes were significantly attenuated by treatment with compound 6c. In conclusion, 22 compounds with a diphenyl ether nucleus were synthesized and submitted to antimycobacterial activity evaluation against M. tuberculosis H37Rv by microdilution method. All these compounds exhibited promising antitubercular activity with MIC ranging from 4 to 64 lg/mL, and N-(3,4-dichlorophenyl)-2-(3-phenoxyphenyl)acetamide (6c) was identified as the most potent inhibitor with MIC of 4 lg/mL. Safety profile studies in HepG2 cell line were performed by MTT assay, all the effective compounds possessed low inhibitory rates at 20 lM. Further evaluation of compound 6c in a murine model of BCG infection, indicating compound 6c could be a good lead for subsequent optimization to get better antitubercular agents. Acknowledgments This work was supported by the National Major Program of China during the 12th Five-Year Plan Period(2012ZX09103-101-068). The authors are grateful to Shanghai Pulmonary Hospital for in vitro antitubercular screening, and thank Dr. Ruiliang Jin for helpful discussions. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.12.022. References and notes 1. http://www.who.int/mediacentre/factsheets/fs104/en/. 2. Global Tuberculosis Control, 2010. http://www.who.int/tb/publications/ global_report/2010/en/index.html.
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3. (a) Jones, R. D.; Jampani, H. B.; Newman, J. L.; Lee, A. S. Am. J. Infect. Control 2000, 28, 184; (b) Scheweizer, H. P. FEMS Microbiol. Lett. 2001, 202, 1. 4. (a) Sharma, S.; Ramya, T. N. C.; Surolia, A.; Surolia, N. Antimicrob. Agents Chemother. 2003, 47, 3859; (b) Perozzo, R.; Kuo, M.; Sidhu, A. B. S.; Valiyaveetti, J. T.; Bittman, R.; Jacobs, W. R., Jr.; Fidock, D. A.; Sacchettini, J. C. J. Biol. Chem. 2002, 277, 13106; (c) Chhibber, M.; Kumar, G.; Parasuraman, P.; Ramya, T. N. C.; Surolia, N.; Surolia, A. Bioorg. Med. Chem. 2006, 14, 8086; (d) Mishra, S.; Karmodiya, K.; Parasuraman, P.; Surolia, A.; Surolia, N. Bioorg. Med. Chem. Lett. 2008, 16, 5536. 5. Butler, M. S.; Cooper, M. A. J. Antibiot. 2011, 64, 413. 6. (a) Tonge, P. J.; Kisker, C.; Slayden, R. A. Curr. Top. Med. Chem. 2007, 7, 489; (b) Ende, C. W. A.; Knudson, S. E.; Liu, N.; Childs, J.; Sullivan, T. J.; Boyne, M.; Xu, H.; Gegina, Y.; Knudson, D. L.; Johnson, F.; Peloquin, C. A.; Slayden, R. A.; Tonge, P. J. Bioorg. Med. Chem. Lett. 2008, 18, 3029. 7. (a) Kini, S. G.; Bhat, A. R.; Bryant, B.; Williamson, J. S.; Dayan, F. E. Eur. J. Med. Chem. 2009, 44, 492; (b) Kini, S. G.; Bhat, A. R.; Pan, Z.; Dayan, F. E. J. Enzyme Inhib. Med. Chem. 2010, 25, 730. 8. Derpoorten, K. V.; Balzarini, J.; Clercq, E. D.; Poupaert, J. H. Biomed. Pharmacother. 1997, 51, 464. 9. Angell, R. M.; Angell, T. D.; Bamborough, P.; Brown, D.; Brown, M.; Buckton, J. B.; Cockeril, S. G.; Edwards, C. D.; Jones, K. L.; Longstaff, T.; Smee, P. G.; Smith, K. J.; Somers, D. O.; Walker, A. L.; Willson, M. Bioorg. Med. Chem. Lett. 2008, 18, 324. 10. Fletcher, S. R.; McIver, E.; Lewis, S.; Burkamp, F.; Leech, C.; Mason, G.; Boyce, S.; Morrison, D. Bioorg. Med. Chem. Lett. 2006, 16, 2872. 11. Cheng, T. J. R.; Wu, Y. T.; Lo, K. H.; Chen, S. K.; Chen, Y. H.; Huang, W. I.; Yuan, C. H.; Guo, C. W.; Huang, L. Y.; Chen, K. T.; Shih, H. W.; Cheng, Y. S. E.; Cheng, W. C.; Wong, C. H. Bioorg. Med. Chem. 2010, 18, 8512. 12. Hwang, S.; Choi, S. Y.; Lee, J. H.; Kim, S.; In, J.; Ha, S. K.; Lee, E.; Kim, T. Y.; Kim, S. Y.; Choi, S.; Kim, S. Bioorg. Med. Chem. 2010, 18, 5602. 13. Urbano, M.; Guerrero, M.; Zhao, J.; Velaparthi, S.; Schaeffer, M. T.; Brown, S.; Rosen, H.; Roberts, E. Bioorg. Med. Chem. Lett. 2011, 21, 5470. 14. Ananthan, S.; Faaleolea, E. R.; Goldman, R. C.; Hobrath, J. V.; Kwong, C. D. K.; Laughon, B. E.; Maddry, J. A.; Mehta, A.; Rasmussen, L.; Reynolds, R. C.; Secrist, J. A.; Shindo, N.; Showe, D. N.; Sosa, M. I.; Suling, W. J.; White, E. L. Tuberculosis 2009, 89, 334. 15. Ma, D.; Cai, Q. Org. Lett. 2003, 5, 3799. 16. Wallace, R. J.; JR.; Nash, D. R.; Steele, L. C.; Steingrube, V. J. Clin. Microbiol. 1986, 24, 976. 17. (a) Gao, H.; Yue, Y.; Hu, L.; Xu, W.; Xiong, S. Vaccine 2009, 27, 5313; (b) Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova, O.; Joanis, B. S.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli, A. P.; Milano, A.; Rossi, E. D.; Belanova, M.; Bobovska, A.; Dianiskova, P.; Kordulakova, J.; Sala, C.; Fullam, E.; Schneider, P.; McKinney, J. D.; Brodin, P.; Christophe, T.; Waddell, S.; Butcher, P.; Bharath, S.; Gaonkar, S.; Shandil, R. K.; Balasubramanian, V.; Riccardi, G.; Cole, S. T. Science 2009, 324, 801.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents Yinghong Yang a,b, , Zhenling Wang a, , Jianzhong Yang a, Tao Yang a, Weiyi Pi a, Wei Ang c, Yanni Lin a, Yuanyuan Liu a, Zicheng Li b,⇑, Youfu Luo a,⇑, Yuquan Wei a a
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, PR China c Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China b
a r t i c l e
i n f o
Article history: Received 10 August 2011 Revised 29 November 2011 Accepted 3 December 2011 Available online 9 December 2011 Keywords: Diphenyl ether nucleus Antituberculosis H37Rv BCG murine model
a b s t r a c t A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated against Mycobacterium tuberculosis H37Rv by a microdilution method, with MIC values ranging from 4 to 64 lg/mL. Through structure–activity relationship studies, the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group were found to play a significant role in the antitubercular activity. The most potent compound 6c showed an MIC value of 4 lg/mL and a good safety profile in HepG2 cell line by the MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing a good lead for subsequent optimization. Ó 2011 Elsevier Ltd. All rights reserved.
Tuberculosis (TB), one of the leading causes of death in the world, poses a significant challenge to the public health. The World Health Organization (WHO) has estimated that one-third of the world’s population is currently infected with the TB bacillus.1 According to the annual report on global control of TB published by the WHO in 2010, there were about 9.4 million incident cases of TB globally in 2009, and approximately 1.7 million people died of TB, among these deaths 23% were HIV-positive.2 People who are HIV-positive are more likely to be infected with TB, so the co-infection with HIV has been responsible for this serious situation. On the other hand, the emergence of multi drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) also contributes to the increased morbidity and mortality. MDR-TB, describes strains of tuberculosis that are resistant to at least the two main first-line TB drugs-isoniazid (INH) and rifampicin. XDR-TB is defined as resistance not only to rifampicin and isoniazid but also fluoroquinolones and to at least one of the injectable second-line drugs. It is also said that there was an estimated 0.44 million cases of MDR-TB in 2008, and 58 countries and territories had reported at least one case of XDR-TB by July 2010.2 This worsening situation leads to an urgent necessity for development of new agents to fight against this disease. Diphenyl ether nucleus is an important unit found in several synthetic and natural agents possessing wide range of pharmaco⇑ Corresponding authors. Tel./fax: +86 28 85503817 (Y.L.).
E-mail addresses: [email protected] (Z. Li), [email protected] (Y. Luo). These authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.12.022
logical activities. A classic chemical with this nucleus is triclosan, which is launched in the early 1970s. It is widely used in a variety of cleaning and personal care products for its broad spectrum antimicrobial activity against a variety of Gram-positive and Gramnegative bacteria3 and continuous studies have been focused on triclosan and its derivatives in recent years. 4 FAB-001 (Fig. 1), a FabI inhibitor with the diphenyl ether nucleus developed by FAB Pharma (Paris, France), is currently being evaluated in phase-I trials for the treatment of drug-resistant staphylococci.5 Moreover, lots of novel diphenyl ether analogues were designed to exhibit enhanced antimycobacterial activity.6 Kini and co-workers synthesized a series of heterocycle substituted diphenyl ether derivatives and all the compounds showed good antitubercular activity against Mycobacterium tuberculosis strain H37Rv.7 Diaryl amide skeleton is also a class of privileged structure with broad range of biological activities such as antiviral,8 anti-inflammatory,9 analgesic,10 antibacterial,11 melanin synthesis inhibitor12 and S1P4 antagonist.13 Recently, in a high throughput screening for inhibitors of M. tuberculosis H37Rv, several diaryl amides14 (Fig. 1) were found to possess potent activity in combination with the relatively low toxicity. Encouraged by the prominent activity of diphenyl ether derivatives and diaryl amides, we proposed to synthesize some novel diphenyl ether analogues containing the diaryl amide moiety by molecular hybridization approach, aiming to search for potential candidates with better antitubercular activities and lower toxicities. In this Letter, we designed and synthesized 22 compounds with a diphenyl ether nucleus, and their antitubercular activity against M. tuberculosis H37Rv in vitro were evaluated by a microdilution
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
955
Scheme 2. Synthetic route of compound 6v. Reagents and conditions: (i) CuI, N,Ndimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2, CHCl3, reflux; (vi) 3-chloroaniline (5a), CH2Cl2, Et3N, rt. Figure 1. Chemical structures of triclosan, FAB-001 and diaryl amides.
method. Compound 6c was found to be the most potent antitubercular agents with MIC of 4 lg/mL, and it was then assessed in vivo using a BCG infection mice model. The target compounds were synthesized utilizing the reaction sequence as shown in Schemes 1 and 2. Iodobenzene or substituted iodobenzene (1) was employed as starting material, it was condensated with compound 2 to give compound 3, via the N,N-dimethyl glycine-promoted Ullmann coupling reaction.15 Compound 3 was further hydrolyzed to compound 4 in excellent yields under reflux conditions in a solution of sodium hydroxide. Compound 4 was then treated with thionyl chloride to obtain the corresponding acyl chloride, and it was reacted with various amines 5 in presence of triethylamine in anhydrous dichloromethane to give the target compounds 6a–v. The structures of 6a–v were fully characterized by 1H NMR, 13C NMR, and ESI-MS analysis and all the analytical data were documented in the Supplementary data. The purity of all compounds was above 97.0% determined by HPLC normalization method. All the synthesized compounds (6a–v) were evaluated for their antimycobacterial activity against M. tuberculosis H37Rv by a microdilution method in 96-wells plates.16 Briefly, M. tuberculosis was grown in Middlebrook 7H9 broth containing oleic acid-albumindextrose-catalase (OADC) enrichment, and was diluted with 7H9OADC medium to 104 CFU/mL. An amount of 100 lL of this bacterial culture was then added to a microtiter well containing serial dilutions of compounds 6a–v. The plates were then incubated at 37 °C in a moisturized incubator and visually examined for growth. INH was used as a positive control. The MIC values are shown in Table 1. As shown in Table 1, all the compounds of this series exhibited antitubercular activity with MIC ranging from 4 to 64 lg/mL, and compound 6c was found to be the most potent with MIC of 4 lg/mL. Compounds 6a (R2 = 3-Cl-Ph) and 6b (R2 = 4-Br-Ph) exhibited lower activity than compound 6c (R2 = 3,4-di Cl-Ph), and 3-Cl substituted compound 6a was slightly more effective compared with the 4-Br substituted compound 6b. When the two chlorine
atoms in 6c were replaced by two fluorine atoms in 6d (R2 = 3,4di F-Ph), the activity was decreased a lot, and so was 6e (R2 = 2Cl-4-Br-Ph) with a chlorine atom and a bromine atom. All these data suggested that the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group are very important in the antitubercular activity. Compounds 6f (R2 = 2-CN-Ph), 6g (R2 = 4-CF3-Ph) introduced with electron-withdrawing substituents and compounds 6h (R2 = 4-OCF3-Ph), 6i (R2 = 2,4-di OCH3-Ph) with electro-donating substituents all showed moderate activity (MIC = 32 lg/mL) without potency improvement. In particular, introduction of a nitro unit in the phenyl ring of R2 group (compound 6j, R2 = 3-NO2-Ph; 6k, R2 = 4-NO2-Ph) did not increase antitubercular activity, although lots of compounds with the NO2 substituent, such as nitrofunans and nitroimidazoles, possessed broad spectrum antimicrobial activities by DNA damage. Next, compounds 6m–6q with more complex aromatic or heterocyclic rings were synthesized, compound 6n (R2 = 6-methoxybenzo[d]thiazol-2-yl) exhibited relatively higher level of activity with MIC of 16 lg/mL compared with 6m and 6o–6q. Further modifications on the length of the aliphatic chain between the B ring and the amide bond showed that all the derivatives (6s–6v, n = 0, 2, 3) possess similar potency (MIC = 32 lg/mL) with compound 6a (n = 1). Compound 6s with amide bond at meta position of B ring and compound 6v at para position demonstrated similar activities. Neither molecule 6q with chlorine atom nor 6r with fluorine atom of R1 group in A ring exhibited increased antimycobacterial activity compared with molecule 6a. Further studies with modifications of the A and B rings are in progress in our lab. Thus, at this stage, all the modifications indicated that the two chlorine atoms at 3 and 4 positions in the phenyl ring of R2 group play a significant role in the antitubercular activity. The safety profile of compounds 6a–v was tested on human cancer cell lines HepG2 at the concentration of 20 lM using the MTT method. Briefly, cells (2500/well) were seeded in 96-well plates and cultured for 24 h, followed by treatment with the target compounds 6a–v for another 48 h. Twenty microliter of 5 mg/ml MTT was added per well and incubated for another 2.5 h at 37 °C. Then the supernatant fluid was removed and MTT formazan
Scheme 1. Synthetic route of compounds 6a–u. Reagents and conditions: (i) CuI, N,N-dimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2, CHCl3, reflux; (vi) NH2R2 (5a–u), CH2Cl2, Et3N, rt.
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Table 1 Antimycobacterial activity and toxicity of compounds 6a–v
O R1
n
A
B
H N
R2
O
O A
6a-u Compound
R1
n
H N
B 6v
R2
O H37Rv (lg/ml) MIC90
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t 6u 6v INH a b c d
H H H H H H H H H H H H H H H H Cl F H H H —
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 3 —
Cl
3-Cl-Ph– 4-Br-Ph– 3,4-Di Cl-Ph– 3,4-Di F-Ph– 2-Cl-4-Br-Ph2-CN-Ph– 4-CF3-Ph– 4-OCF3-Ph– 2,4-Di OCH3-Ph– 3-NO2-Ph– 4-NO2-Ph– Naphthalen-1-yl– Diphenylmethyl– 6-Methoxybenzo[d]thiazol-2-yl– 5-Methylpyridin-2-yl– Thiazol-2-yl– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– 3-Cl-Ph– —
HepG2 (%) b
a
16 16 NTd 8 16 NT 8 16 NT 16 8 32 8 8 8 16 16 16 16 16 16 16 0.0625
MIC
IRc@20 lM
32 64 4 32 32 32 32 32 32 32 32 64 32 16 32 32 32 32 32 32 32 32 0.0625
7.95 15.70 7.00 26.73 12.81 19.08 10.31 4.71 4.11 0.95 36.41 3.31 4.84 4.29 12.21 39.89 27.06 23.52 7.07 25.05 NT 29.20 3.51
MIC90 is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 100 CFU of bacteria inoculated merely. MIC is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 10 CFU of bacteria inoculated merely. IR is the mean inhibitory rate calculated from three independent experiments measured at 48 h after treatment with the test compound at the concentration of 20 lM. NT means not tested.
precipitate was dissolved in 150 lL of DMSO, shaken mechanically for 15–20 min. The optical density of each well was measured at 570 nm, using a SpectraMAX M5 microplate spectrophotometer. The inhibitory rates at 20 lM are given in Table 1, all the active compounds showed low toxicity in this assay and the most potent compound 6c exhibited good safety profile with inhibitory rates as low as 7%. The most potent derivative 6c was further tested for in vivo antitubercular activity using a murine model of BCG infection17 as described in detail in the Supplementary data. Female BALB/c mice were infected intravenously with bacterial culture containing approximately 1 105 CFU of M. bovis BCG, and compound 6c was orally administered at a dose of 25 mg/kg once daily. After six weeks of treatment, mice were sacrificed. Lung and spleen tissues were homogenized and plated on Modified Lowenstein-Jensen Medium Base. BCG colonies were counted after six weeks of incubation at 37 °C. Data were expressed as mean log10 CFU per organ
Table 2 Multiplication of M. bovis BCG Group
BCG + vehicle BCG + 6c BCG + INH
Na
6 8 8
Log10 CFU (mean ± SD) Lung
Spleen
4.64 ± 0.17 2 ± 0.89⁄ 2 ± 0.45⁄
4.79 ± 0.25 2 ± 0.74⁄ 3 ± 0.24⁄
Significantly different: ⁄P <0.01 versus vehicle-treated group. a N means the number of treated mice in each group.
and results of one several representative experiments were shown in Table 2. Compound 6c-treated mice had a significant reduction in bacterial numbers in the lungs, as well as in spleens, compared to vehicle controls. And there is no remarkable difference in bacterial numbers between compound 6c group and INH group, both in lungs and spleens.
Figure 2. Histopathological appearance of lungs of infected mice treated with vehicle (A), infected mice treated with compound 6c (B), and infected mice treated with INH (C). Magnification, 100.
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
Meanwhile, lung tissues were harvested for histological evaluation. As shown in Figure 2, perivascular and peribronchiolar accumulations of resident epithelioid macrophages, granulocytes, and lymphocytes located in the parenchyma, close to major airways, and blood vessels were observed in lung tissues of BCG model mice, and these pathological changes were significantly attenuated by treatment with compound 6c. In conclusion, 22 compounds with a diphenyl ether nucleus were synthesized and submitted to antimycobacterial activity evaluation against M. tuberculosis H37Rv by microdilution method. All these compounds exhibited promising antitubercular activity with MIC ranging from 4 to 64 lg/mL, and N-(3,4-dichlorophenyl)-2-(3-phenoxyphenyl)acetamide (6c) was identified as the most potent inhibitor with MIC of 4 lg/mL. Safety profile studies in HepG2 cell line were performed by MTT assay, all the effective compounds possessed low inhibitory rates at 20 lM. Further evaluation of compound 6c in a murine model of BCG infection, indicating compound 6c could be a good lead for subsequent optimization to get better antitubercular agents. Acknowledgments This work was supported by the National Major Program of China during the 12th Five-Year Plan Period(2012ZX09103-101-068). The authors are grateful to Shanghai Pulmonary Hospital for in vitro antitubercular screening, and thank Dr. Ruiliang Jin for helpful discussions. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.12.022. References and notes 1. http://www.who.int/mediacentre/factsheets/fs104/en/. 2. Global Tuberculosis Control, 2010. http://www.who.int/tb/publications/ global_report/2010/en/index.html.
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