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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights
Author's personal copy Bioorganic Medicinal Chemistry Letters 23(2013)4528-4531 Contents lists available at SciVerse Science Direct Bioorganic Medicinal Chemistry Letters LSEVIER journalhomepagewww.elsevier.com/locate/bmcl Tetrazole and triazole as bioisosteres of carboxylic acid: Discovery CrossMark of diketo tetrazoles and diketo triazoles as anti-HCV agents Wu-Hui Song Ming-Ming Liu C, Dong-Wei Zhong, Ye-lin Zhu, Mike Bosscher Lu Zhou De-Yong Ye, Zheng-Hong Yuan".* School of pharmacy, Fudan University, 826 Zhang-Heng road, Shanghai 201203, PR China Key Laboratory of Medical Molecular Virology y. 138 Yi-Xue-Yuan Road, Shanghai 200032, PR China a Dept of Chemistry, Trinity Christian College, 6601 W. College Dr. Palos Heights, IL 60463, Ug.270 Dong-An Road, Shanghai 200032, PR China Shanghai Cancer Center E Department of oncology, Shar ledical College, Fudan University, ARTICLE INFO ABSTRACT of diketo tetrazoles brary 2013 diketo acid, the active site inhibitor of HCV (Hepatitis C virus)polymerase NS5B. Among the synthesize 15June2013 compounds, 4-(4-fluorobenzyloxy ) phenyl diketo triazole(30)exhibited anti-HCV activity with an EC50 Available online 26 June 2013 value of 3. 9 HM and an SI value more than 128. The reduction of viral protein and mRNA levels were also validated, supporting the anti-HCV activity of compound 30. These results provide convincing evidence that the diketo tetrazoles and diketo triazoles can be developed as bioisosteres of a, y-diketo acid to exhi- bit potent inhibitory activity against HCV. e 2013 Elsevier Ltd. All rights reserved. Diketo triazoles Bioisosteres The world health organization estimates that 150 million peo- monoethyl ester of meconic acid(3, Fig. 2), dihydroxypyrimidine ple worldwide are chronically infected with Hepatitis c virus carboxylic acid (4, Fig. 2), 10 multihydroxyl flay (HCV) and 350,000 people each year die of HCv related diseases. Unfortunately, most of these compounds were Protective vaccination is not yet available and pegylated-interferon based antiviral assay because of the poor cellular permeability combined with ribavirin, the current standard therapy is often dif- Since the active site of NS5B is high conserved across all HCv geno- ficult for patients to tolerate and results in a sustained viral re- types'and the mutations at the active site(eg, S sponse (SvR)in only 50% of patients infected with the cantly reduce replication capacity the active site inhibitors predominant genotype 1.Although two new drugs boceprevir have the potential advantage to be active against all genotypes of and telaprevir as Ns3 protease inhibitors were approved by fDa the virus and the drug-resistant variants. 5 for the treatment of genotype 1 chronic hepatitis C recently, due To develop DKAs analogues with higher potential of active site to the potential of drug resistant strains and widespread infection, inhibitors of NS5B and better cellular permeability, we replaced the development of novel anti-HCV agents is still urgent. the free carboxylic acid of DKAs with their bioisosteres triazoles Aryl o, y-diketo acids(DKAs, 1, Fig. 1)were identified as specific, or tetrazoles and used the cell-based HCV replication system and reversible inhibitors of NS5B polymerase, a promising and val- test if a series of diketone triazoles and diketone tetrazoles could idated target for HCV therapies, in the low micromolar range. overcome the physiochemical and pharmacokinetic problems of Mechanistic studies showed that, as pyrophosphate(PPi, 2. DKAs Although a similar replacement of carboxylic acid with tria ig. 1)mimetic inhibitors, DKAs act as product-like analogues zole or tetrazole was successful in the research of integrase(IN) and chelate the two divalent cations(Mg ions)at the active site of NS5B. Due to the chemical and biological instability and poor membrane permeability of diketo acid group, several drug-like scaffolds were designed as analogues or mimics, such as the HO O-OH HO-P. du.cn(D -Y. Ye). 2 ontributed equally to this Letter. Figure 1. Structures of aryl a, y-diketo acids(DKAs, 1)and pyrophosphate(2)
Author's personal copy Tetrazole and triazole as bioisosteres of carboxylic acid: Discovery of diketo tetrazoles and diketo triazoles as anti-HCV agents Wu-Hui Song b, , Ming-Ming Liu a,c, , Dong-Wei Zhong a , Ye-lin Zhu a , Mike Bosscher d , Lu Zhou a,⇑ , De-Yong Ye a,⇑ , Zheng-Hong Yuan b,⇑ a Key Laboratory of Smart Drug Delivery , Ministry of Education, School of Pharmacy, Fudan University, 826 Zhang-Heng Road, Shanghai 201203, PR China b Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, 138 Yi-Xue-Yuan Road, Shanghai 200032, PR China c Shanghai Cancer Center & Department of Oncology, Shanghai Medical College, Fudan University, 270 Dong-An Road, Shanghai 200032, PR China dDept. of Chemistry, Trinity Christian College, 6601 W. College Dr., Palos Heights, IL 60463, USA article info Article history: Received 5 February 2013 Revised 12 June 2013 Accepted 15 June 2013 Available online 26 June 2013 Keywords: Anti-HCV activity Diketo tetrazoles Diketo triazoles Bioisosteres abstract A series of diketo tetrazoles and diketo triazoles were designed and synthesized as bioisosteres of a,cdiketo acid, the active site inhibitor of HCV (Hepatitis C virus) polymerase NS5B. Among the synthesized compounds, 4-(4-fluorobenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activity with an EC50 value of 3.9 lM and an SI value more than 128. The reduction of viral protein and mRNA levels were also validated, supporting the anti-HCV activity of compound 30. These results provide convincing evidence that the diketo tetrazoles and diketo triazoles can be developed as bioisosteres of a,c-diketo acid to exhibit potent inhibitory activity against HCV. 2013 Elsevier Ltd. All rights reserved. The world health organization estimates that 150 million people worldwide are chronically infected with Hepatitis C virus (HCV) and 350,000 people each year die of HCV related diseases.1 Protective vaccination is not yet available and pegylated-interferon combined with ribavirin, the current standard therapy, is often dif- ficult for patients to tolerate and results in a sustained viral response (SVR) in only 50% of patients infected with the predominant genotype 1.2,3 Although two new drugs boceprevir and telaprevir as NS3 protease inhibitors were approved by FDA for the treatment of genotype 1 chronic hepatitis C recently, due to the potential of drug resistant strains4 and widespread infection, the development of novel anti-HCV agents is still urgent.5 Aryl a,c-diketo acids (DKAs, 1, Fig. 1) were identified as specific, and reversible inhibitors of NS5B polymerase, a promising and validated target for HCV therapies, in the low micromolar range.6 Mechanistic studies showed that, as pyrophosphate (PPi, 2, Fig. 1) mimetic inhibitors, DKAs act as product-like analogues and chelate the two divalent cations (Mg2+ ions) at the active site of NS5B.6,7 Due to the chemical and biological instability and poor membrane permeability of diketo acid group, several drug-like scaffolds were designed as analogues or mimics, such as the monoethyl ester of meconic acid (3, Fig. 2),8 dihydroxypyrimidine carboxylic acid (4, Fig. 2),9,10 multihydroxyl flavonoids,11,12 etc. Unfortunately, most of these compounds were inactive in cellbased antiviral assay because of the poor cellular permeability. Since the active site of NS5B is high conserved across all HCV genotypes13 and the mutations at the active site (e.g., S282T) signifi- cantly reduce replication capacity,14 the active site inhibitors have the potential advantage to be active against all genotypes of the virus and the drug-resistant variants.15 To develop DKAs analogues with higher potential of active site inhibitors of NS5B and better cellular permeability, we replaced the free carboxylic acid of DKAs with their bioisosteres triazoles or tetrazoles and used the cell-based HCV replication system to test if a series of diketone triazoles and diketone tetrazoles could overcome the physiochemical and pharmacokinetic problems of DKAs. Although a similar replacement of carboxylic acid with triazole or tetrazole was successful in the research of integrase (IN) 0960-894X/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.06.045 ⇑ Corresponding authors. Tel./fax: +86 21 51980125 (L.Z.). E-mail addresses: zhoulu@fudan.edu.cn (L. Zhou), dyye@shmu.edu.cn (D.-Y. Ye), zhyuan@shmu.edu.cn (Z.-H. Yuan). These authors contributed equally to this Letter. Ar O O P O O P O HO OH HO OH OH O 1 2 Figure 1. Structures of aryl a,c-diketo acids (DKAs, 1) and pyrophosphate(2). Bioorganic & Medicinal Chemistry Letters 23 (2013) 4528–4531 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Author's personal copy W.-H. Song et aL/ Bioorg Med. Chem. Lett. 23(2013)4528-4531 O COOH N COOH 6 (5-CITEP) Figure 2. Structures of aryl diketo acid mimics such as monoethyl ester of meconic acid (3) dihydroxypyrimidine carboxylic acid (4), S-1360(5). 5-CITEP(6)and our most active compound 30. inhibitors as anti-HIV agents leading to the discovery of S-1360 (5. >50%)at 50 HM. The brief structure activities relationships (SArs Fig. 2)and 5-CITEP (6. Fig. 2), 6 related homologues of DKAs have were summarized as follows. The substitute of benzyloxy in the not been tested for anti-HCV activity to our knowledge. aryl ring is essential for the biological activity. The introduction As shown in scheme 1, the designed compounds tetrazole deriv- of chloride atom at the opposite position of benzyloxy can increase atives 10-22 and triazole derivatives 23-33 were synthesized via a the anti-HCV activities, when the benzyloxy was at 4-position of facile'one-pot' reaction as previously reported. 17,18 Reaction of the phenyl ring. in both tetrazole and triazole derivatives. but the ef- tarting material, 1H-tetrazol-5-ethyl formate(7), with 2-meth- fect of the fluoride atom on the Hcv activities was more compl xyproene in the presence p-TSA and followed by Claisen conden- cated. For the tetrazole derivatives, the introduce fluoride atom ition with various substituted aryl methyl ketone catalyzed by can increase the activities, when the benzyloxy was at 3-positie sodium ethoxide afford diketo tetrazole and triazole intermediate of phenyl ring. In comparison, the fluoride atom was more appro- 9 which was then deprotected by 4N hydrochloric acid to obtain priate when the benzyloxy was at 3-position of phenyl ring in the target compounds 10-33 triazole series. Moreover, HIV integrase inhibitors 5 and 6 were All synthesized diketo triazole and tetrazole derivatives were also synthesized according to the reference and their anti-HCV initially evaluated for their anti-HCV activities and cytotoxicity at activities evaluated. Unfortunately, both compounds possess the single concentration of 50 HM in an authentic HCV infection/ much weaker anti-HCV activities with less than 50% inhibition eplication system in the human hepatoma cell lines Huh-7, using 50 HM, which indicated that the substituents and sort of the aryl cell counting kit-8( CCK8)as previously reported. group played a critical role for the antiviral activities, even though The preliminary results of antiviral effect and cytotoxicity effect all the compounds contained aryl diketo tetrazole or aryl diketo are shown in Table 1, respectively. RG7128 was used as positive triazole groups control, which is a nucleoside clinical candidate in phase 2b. Ate Further, three of the nine active compounds( 14, 30, and 33) I of nine compounds exhibited more than 50% inhibition and all were selected to determine the ecso values of their anti-HCV activ- unds showed low cytotoxic activity(cell viability ratio ities and to test their cytotoxicity in higher concentration (500 HM). The results as shown in Table 2 suggested that the tria zole derivative 30 was the most potent molecule with an ECso va- lue of 3.9 HM. Additionally, compound 30 did not show clear "oneo -pot" synthesis cytotoxicity at 500 HM. Therefore, we further clarified the inhibi tory effect of compound 30 on the synthesis of viral protein and the replication of viral genome. Cell lysates were subjected to wes- tern blot analysis with the antibody of viral non-structural protein 7 X=N1022 X=C23-3 NS5A, in which the level of tubulin served as a loading control. As shown in Fig 3, the synthesis of HCV NS5A proteins was inhibited by compound 30 in a dose-dependent manner. Moreover, quantita tive rT-PCR was also employed to examine the RNa level of HCv genome, which was normalized by cellular GAPDH mRNA. A OMe dose-dependent reduction of HCv RNa levels by compound 30 was also observed(Fig. 4), which confirmed compound 30 as a promising lead with anti-HCV activity. In conclusion, we designed and synthesized dike nd triazole derivatives to take advantage of known bioisosteres of carboxylic acids, starting from diketo acid, known active site inhibitors of NS5B. Among the synthesized compounds, 4-(4-fluo- robenzyloxy )phenyl diketo triazole(30)exhibited anti-HCV activ Scheme 1. Reagents and conditions: (a)2-methoxypropene, p-TSA, THF, rt, 1 h:(b) ity with an ECso of 3.9 HM and a selectivity index greater than aryl methyl ketone, NaoEt, 60C, 3 h: (C)4NHCI(aq)l rt too"C, h: 62-89]overalL. 128. Moreover, to confirm the antiviral activity of compound 30
Author's personal copy inhibitors as anti-HIV agents leading to the discovery of S-1360 (5, Fig. 2) and 5-CITEP (6, Fig. 2),16 related homologues of DKAs have not been tested for anti-HCV activity to our knowledge. As shown in scheme 1, the designed compounds tetrazole derivatives 10–22 and triazole derivatives 23–33 were synthesized via a facile ‘one-pot’ reaction as previously reported.17,18 Reaction of the starting material, 1H-tetrazol-5-ethyl formate (7), with 2-methoxyproene in the presence p-TSA and followed by Claisen condensation with various substituted aryl methyl ketone catalyzed by sodium ethoxide afford diketo tetrazole and triazole intermediate 9 which was then deprotected by 4N hydrochloric acid to obtain target compounds 10–33. All synthesized diketo triazole and tetrazole derivatives were initially evaluated for their anti-HCV activities and cytotoxicity at the single concentration of 50 lM in an authentic HCV infection/ replication system in the human hepatoma cell lines Huh-7, using cell counting kit-8 (CCK8) as previously reported.12 The preliminary results of antiviral effect and cytotoxicity effect are shown in Table 1, respectively. RG7128 was used as positive control, which is a nucleoside clinical candidate in phase 2b.19 A total of nine compounds exhibited more than 50% inhibition and all of the compounds showed low cytotoxic activity (cell viability ratio >50%) at 50 lM. The brief structure activities relationships (SARs) were summarized as follows. The substitute of benzyloxy in the aryl ring is essential for the biological activity. The introduction of chloride atom at the opposite position of benzyloxy can increase the anti-HCV activities, when the benzyloxy was at 4-position of phenyl ring, in both tetrazole and triazole derivatives. But the effect of the fluoride atom on the HCV activities was more complicated. For the tetrazole derivatives, the introduce fluoride atom can increase the activities, when the benzyloxy was at 3-position of phenyl ring. In comparison, the fluoride atom was more appropriate when the benzyloxy was at 3-position of phenyl ring in the triazole series. Moreover, HIV integrase inhibitors 5 and 6 were also synthesized according to the reference and their anti-HCV activities were evaluated. Unfortunately, both compounds possess much weaker anti-HCV activities with less than 50% inhibition at 50 lM, which indicated that the substituents and sort of the aryl group played a critical role for the antiviral activities, even though all the compounds contained aryl diketo tetrazole or aryl diketo triazole groups. Further, three of the nine active compounds (14, 30, and 33) were selected to determine the EC50 values of their anti-HCV activities and to test their cytotoxicity in higher concentration (500 lM). The results as shown in Table 2 suggested that the triazole derivative 30 was the most potent molecule with an EC50 value of 3.9 lM. Additionally, compound 30 did not show clear cytotoxicity at 500 lM. Therefore, we further clarified the inhibitory effect of compound 30 on the synthesis of viral protein and the replication of viral genome. Cell lysates were subjected to western blot analysis with the antibody of viral non-structural protein NS5A, in which the level of tubulin served as a loading control. As shown in Fig. 3, the synthesis of HCV NS5A proteins was inhibited by compound 30 in a dose-dependent manner. Moreover, quantitative RT-PCR was also employed to examine the RNA level of HCV genome, which was normalized by cellular GAPDH mRNA. A dose-dependent reduction of HCV RNA levels by compound 30 was also observed (Fig. 4), which confirmed compound 30 as a promising lead with anti-HCV activity. In conclusion, we designed and synthesized diketo tetrazole and triazole derivatives to take advantage of known bioisosteres of carboxylic acids, starting from diketo acid, known active site inhibitors of NS5B. Among the synthesized compounds, 4-(4-fluorobenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activity with an EC50 of 3.9 lM and a selectivity index greater than 128. Moreover, to confirm the antiviral activity of compound 30, O O N N H N O F O O COOH OH O O 3 N N OH OH COOH HO 4 O O N N H O N F 5 (S-1360) O O N N N H N HN 6 (5-ClTEP) Cl 30 Figure 2. Structures of aryl diketo acid mimics such as monoethyl ester of meconic acid (3), dihydroxypyrimidine carboxylic acid (4), S-1360 (5), 5-CITEP (6) and our most active compound 30. N N X H N O EtO N N X N O EtO OMe N N X N O OMe O Ar Ar O O N N X H N "one-pot" synthesis a b c 7 9 X = N 10-22 X = C 23-33 8 Scheme 1. Reagents and conditions: (a) 2-methoxypropene, p-TSA, THF, rt, 1 h; (b) aryl methyl ketone, NaOEt, 60 C, 3 h; (c) 4 N HCl(aq), rt to 0 C, 2 h; 62–89% overall. W.-H. Song et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4528–4531 4529
Author's personal copy 4530 W.-H. Song ef al / Bioorg Med. Chem. Lett. 23(2013)4528-4531 Anti-HCV activities for the 24 synthesized compounds Compound Inhibition ratioe- 50 HM(%) 1234 xNNNNNNNNNNNNNcccccccc 28.70 62 20 12345 64 Furan-2-yl 14.5 34 3-(Benzyloxy )phenyl 3-(4-Fluorobenzyloxy )phenyl 29 azyloxy )phenyl C (4-Fluorobenzyl)furan-2-yl RG7128 75.3%@2pM The valuated in an authentic HCV infection/replication system by measured the EgFP autofluorescence in Huh-7 cell lines. The values represent The best three which were further selected to determine the ECso values. Table 2 25 CCso(uM) sid >500 >77 Cv assay was evaluated in an authentic HCv infection/ replication system by measured the EGFP autofluorescence in Huh-7 cell lines. The ECso value of each compound was the concentration required to inhibit HCV RNA replication by 50% and was estimated by lir ition of 5 conce ons. The values represent average of triplicate results. 005 The CCso value of each compound means concentration required to reduce cell proliferation by50‰. Selectivity index(Si): ratio of CCso to eC uM 1 HM 5 AM 10 AM RG7128 Compound 30 the reductionof the viral protein and mrNa levels we ompound 30 in ndent manner. RG7128(2 HM) served as positive control and relative by western blot and Real-time PCR. This study disc ne levels were calculated as a ratio of the hcv genome levels to gADPh lead compound to design more potent anti-HCV structural modification of compound 30 as an anti-HCV candidate RG7128 is currently in progress. Mock Acknowledge S5A This work was supported by grants from the National Natural lin Science Foundation of China(No. 20902013 and No 30973641) National Basic Research Program of China(No. 2009CB522504) ompound 30 in a dose-dependent manner. RG719 pression was inhibited by Shanghai Committee of Science and Technology of China(Grant Figure 3. Western blotting showed that HC No 13ZR1403900), China Postdoctoral Science Foundation(No. control and tubulin was loading controL 2013M531126), and"Zhuo Xue "Program of Fudan Universi
Author's personal copy the reductionof the viral protein and mRNA levels were also tested by western blot and Real-time PCR. This study discovered a new lead compound to design more potent anti-HCV agents. Further structural modification of compound 30 as an anti-HCV candidate is currently in progress. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 20902013 and No. 30973641), National Basic Research Program of China (No. 2009CB522504), Shanghai Committee of Science and Technology of China (Grant No.13ZR1403900), China Postdoctoral Science Foundation (No. 2013M531126), and "Zhuo Xue" Program of Fudan University. Table 1 Anti-HCV activities for the 24 synthesized compounds Ar O O N N X H N Compound Ar X Inhibition ratio@a 50 lM (%) 10 Phenyl N 1.3 11 Furan-2-yl N 0.9 12 4-Nitrophenyl N 14.74 13 4-Methoxyphenyl N 28.70 14 2-(Benzyloxy)phenyl N 67.6c 15 3-(Benzyloxy)phenyl N 11.1 16 4-(Benzyloxy)phenyl N 54.9 17 2-(4-Fluorobenzyloxy)phenyl N 6.8 18 3-(4-Fluorobenzyloxy)phenyl N 62.7 19 4-(4-Fluorobenzyloxy)phenyl N 10.7 20 2-(4-Chlorobenzyloxy)phenyl N 36.2 21 3-(4-Chlorobenzyloxy)phenyl N 43.6 22 4-(4-Chlorobenzyloxy)phenyl N 64.9 23 Phenyl C 24.8 24 Furan-2-yl C 14.5 25 2-(Benzyloxy)phenyl C 34.4 26 3-(Benzyloxy)phenyl C 48.2 27 4-(Benzyloxy)phenyl C 12.1 28 2-(4-Fluorobenzyloxy)phenyl C 55.0 29 3-(4-Fluorobenzyloxy)phenyl C 43.4 30 4-(4-Fluorobenzyloxy)phenyl C 69.7c 31 2-(4-Chlorobenzyloxy)phenyl C 53.8 32 3-(4-Chlorobenzyloxy)phenyl C 29.2 33 4-(4-Chlorobenzyloxy)phenyl C 70.2c 5 (5-Chloroindol)-3-yl N 22.4 6 (4-Fluorobenzyl)furan-2-yl C 24.3 RG7128b 75.3% @ 2 lM a The anti-HCV assay was evaluated in an authentic HCV infection/replication system by measured the EGFP autofluorescence in Huh-7 cell lines. The values represent a means of triplicate results. b RG7128 was used as a reference positive control. c The best three compounds which were further selected to determine the EC50 values. Table 2 Anti-HCV antivities (EC50) and cytotoxicities (CC50) for selected 3 compounds Compound EC50a,b (lM) CC50c (lM) SId 14 9.2 >500 >54 30 3.9 >500 >128 33 6.5 >500 >77 a The anti-HCV assay was evaluated in an authentic HCV infection/replication system by measured the EGFP autofluorescence in Huh-7 cell lines. b The EC50 value of each compound was the concentration required to inhibit HCV RNA replication by 50% and was estimated by linear interpolation from inhibition of 5 concentrations. The values represent average of triplicate results. c The CC50 value of each compound means concentration required to reduce cell proliferation by 50%. d Selectivity index(SI): ratio of CC50 to EC50. Figure 3. Western blotting showed that HCV protein expression was inhibited by compound 30 in a dose-dependent manner. RG7128 (2 lM) served as positive control and tubulin was loading control. Figure 4. QRT-PCR showed that HCV replication was inhibited by compound 30 in a dose-dependent manner. RG7128 (2 lM) served as positive control and relative HCV genome levels were calculated as a ratio of the HCV genome levels to GADPH mRNA levels. 4530 W.-H. Song et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4528–4531
Author's personal copy W.-H. Song et aL/ Bioorg Med. Chem. Lett. 23(2013)4528-4531 References and notes 10. Koch, U: Attenni. B. Malancona, S: Colarusso, S: Conte, 1: Di 1.WhoFactSheet2012,http://www.who.intmediacentre/fae 1 Lee, H.S., Park, K, Lee, B: Kim, D. E. Chong, Y. Bioorg Med. Chem. 20 2. Fried, M. W: Shiffman, M. L Reddy, K. R. Smith, C: Marinos Liu, M. M.; Zhou, L; He, P. L; Zhang, Y N. Zhou, Y, Shen, Q: Chen, Jr. Haussinger, D; Diago, M: Carosi, G: Dhumeaux, D N. d2002 ano, V: Peters, M. G. Stefan, Z. Clin. Infect. Dis. 2009. 48, 313. 4. Hiraga,N: Imamura, M: Abe, H:: Hayes, c N5> 4. Hang, J.Q.; Yang. Y: Harris, S F Leveque, V. Whittington, HJ-; Rajyaguru, S. T: Onishi, M. Tsuge, M, leong, G. MCCown, M. F Wong, A: Giannetti, A M.J. Biol Chem. 2009, 284. 5. Se mma etao. hi d pga ee: at:s: 10. C De Francesco. R- Altamura. S: 16. Luo, Z G:Tan J: Zeng Y:Wang Hu. L M. Mini-Rev. Med. Chem. 2010. 10 omei, L; Koch, U: Neuner, P. I. Med. Chem. 2004, 47, 14. 17. Shimizu, S: Endo, T Izumi, K. Mikamiyama, H. Org Process Res Dev. 2007. 11 8. Pace. P: Nizi. E: Pacini. B: Pesci. S: Matassa. V: De francesco, R: Altamura. S 18. Liu, M, Zhong D: Zhou, J; Zhou, L: Ye. D. Chin /. Org Che m. 2012, 32, 1543. Summa, v. Bioorg Med. Chem. Lett. 2004. 14. 325 9. Summa V. Petrocchi A: Matassa. V G: Taliani. M. Laufer R: De francesco achakonda, S: Ross, B S; Zhang, H.R.: Bansal, S. Bioorg. Med. Chem Lett. 2010. R: Altamura. S: Pace, P. Med. Chem. 2004. 47, 5336 20.7376
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