Bioorganic & Medicinal Chemistry Letters 24 (2014) 2530–2534
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Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin Zˇiga Hodnik a, Joanna M. Łos´ b, Aleš Zˇula a, Nace Zidar a, Zˇiga Jakopin a, Marcin Łos´ b, Marija Sollner Dolenc a, Janez Ilaš a, Grzegorz We˛grzyn b,⇑, Lucija Peterlin Mašicˇ a,⇑, Danijel Kikelj a a b
University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, 1000 Ljubljana, Slovenia ´ sk, Department of Molecular Biology, Wita Stwosza 59, 80-308 Gdan ´ sk, Poland University of Gdan
a r t i c l e
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Article history: Received 3 March 2014 Revised 28 March 2014 Accepted 29 March 2014 Available online 8 April 2014 Keywords: Biofilm Indole Inhibition Marine Oroidin
a b s t r a c t Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indolebased analogue 4b (MBIC50 = 20 lM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms. Ó 2014 Elsevier Ltd. All rights reserved.
The growth of bacteria is faster on surfaces that present a protective and nutrient-rich environment compared to growth in water and in liquids with low nutrient contents.1 The adhesion of bacterial cells to inert or living surfaces results in the formation of biofilms, that is, structured communities that are encased in a self-produced extracellular matrix of biomolecules.1,2 The extracellular matrix, which consists of polysaccharides, proteins and nucleic acids, is the primary reason for profound protection from the host’s immune system and for a nearly 1000-fold increase in biofilm resistance to conventional antibiotics when compared to planktonic bacteria.3,4 The robust resistance of biofilms to the hazards of their environment, for example, predators, antibiotics and host immune systems,2,5,6 results not only in biofouling in the food and marine industries but also in various nosocomial and chronic infections in patients, which result in perpetual inflammation and tissue damage.3,7 The formation of biofilms on inert surfaces includes indwelling medical devices, catheters and contact lenses, and infections of living surfaces most often include lung infections in cystic fibrosis patients, periodontitis, endocarditis and urinary tract
⇑ Corresponding authors. Tel.: +48 58 523 6024; fax: +48 58 523 5501 (G.W.); tel.: +386 1 4769 635; fax: +386 1 4258 031 (L.P.M.). E-mail addresses: [email protected] (G. We˛grzyn), lucija. [email protected] (L. Peterlin Mašicˇ). http://dx.doi.org/10.1016/j.bmcl.2014.03.094 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
infections.4,8 Furthermore, an estimate by the National Institutes of Health states that 80% of microbial infections are biofilm-based.9 Facultative anaerobic cocci Staphylococcus aureus and Streptococcus mutans are among common Gram-positive bacteria that form infectious biofilms.1,10 Nosocomial infections are frequently caused by omnipresent S. aureus bacteria, which are commonly found in the anterior nares in humans and can enter the circulatory system through an epithelial breach.11,12 Osteomyelitis, indwelling medical device infections, chronic wound infections, endocarditis and periodontitis exemplify the most prevalent biofilm-based infections caused by S. aureus. While periodontitis is sometimes caused by S. mutans, its most common biofilm-based infection remains dental caries. S. mutans is considered to be one of the earliest colonisers of human teeth after their eruption, and its fermentation of dietary carbohydrates and consequential production of acids poses one of the most important factors for the formation of dental caries.10 Ubiquitous biofouling presents a considerable threat to immobile marine organisms such as sponges and seaweeds, which do not possess an immune system that can protect them against microbes. Hence, they have evolved to biosynthesise various secondary metabolites that function as a defence system against bacterial infections.1,13 Among the most investigated anti-biofilm agents from marine sponges are the halogenated furanones from the seaweed Delisea pulchra,14,15 terpenoids16,17 and especially 2aminoimidazole-based alkaloids.18–20 The 2-aminoimidazole-based
Zˇiga Hodnik et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2530–2534
marine alkaloid oroidin (Fig. 1) and various series of its analogues were extensively studied as biofilm inhibitors, primarily by Melander and coworkers.18,20–27 Although numerous analogues of oroidin have been observed to control biofilm development,18,20–27 to our knowledge, indolebased analogues of oroidin have not yet been studied. In contrast, various studies outlined the ability of indole to regulate the growth of various bacterial biofilms and therefore represents an important quorum sensing molecule.28–30 In the present work, we first replaced the dibromopyrrole moiety of oroidin with the isosteric indole and 5-fluoroindole moieties, and evaluated the anti-biofilm activity of synthesised analogues 131 and 231 (Table 1) against two Gram-positive biofilm-forming strains of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus mutans and against a Gram-negative biofilm-forming strain of Pseudomonas aeruginosa (cf. Supplementary material). While indole-based analogue 1 did not exhibit any anti-biofilm activity against the tested strains, its 5-fluoroindole analogue 2 attenuated the formation of MRSA and S. mutans biofilms to 8.1% and 18.7%, respectively, at 100 lM (Table 1). Based on these encouraging anti-biofilm activities we further explored the structure–activity relationship of
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indole-based series of compounds by synthesising conformationally constrained analogues (Fig. 1) in which the central alkene region of oroidin was replaced with synthetically more favourable 1,3phenylene (compounds 3a–14) and 1,4-phenylene (compounds 15a–17b) moieties. To study the structure–activity relationship of our small library of conformationally constrained indole-based analogues of oroidin we (i) evaluated the effect of the rigidification of analogues 1 and 2 on anti-biofilm activity, (ii) compared the 1,3substituted series to the 1,4-substituted series of analogues and (iii) studied the influence of various substituents on indole and 2-aminoimidazole moieties on anti-biofilm activity (Fig. 1). Syntheses of oroidin and compounds 1–5, 7, 8, 9 and 11–15b were performed as previously described.31,32 Compounds 10a (cf. Supplementary material), 16a and 17a were synthesised using O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluorobo rate (TBTU)-promoted coupling of tert-butyl 2-amino-4-(3-aminophenyl)-1H-imidazole-1-carboxylate31 or tert-butyl 2-amino-4-(4aminophenyl)-1H-imidazole-1-carboxylate31 with the corresponding indole-2-carboxylic acids.33 Subsequent treatment of compounds 10a, 16a and 17a with gaseous hydrogen chloride gave compounds 10, 16b and 17b. Compound 6a was synthesised from 10a, using
Br HN H2 N
HN
H N
N
Br O
oroidin
HN H2 N
HN
H N
N
R1 R 1 = H or F
O 1-2 rigidification O N HN R3
N H
H N
HN H2 N
N R2
HN
H N
N
N NH
N
1,3-substitution
3a-14
H2 N
R1 1,4-substitution
O
R1
R2
H N R1
O 15a-17b
R 1 = H, F, Cl,... R 2 = H or Boc R 3 = H or CH3
R1 = H, F or Cl R 2 = H or Boc
Figure 1. Design of conformationally constrained indole-based analogues of oroidin with potential anti-biofilm activity.
Table 1 Anti-biofilm activities of oroidin and its indole-based analogues
HN H 2N
HN
H N
N
R1
O 1-2
Compounds
Oroidin 1 2
R1
— H F
Staphylococcus aureus
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. n.a. 8.1 ± 0.7
— — 60
18.0 ± 8.6 n.a. 18.7 ± 9.7
— — 60
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
Zˇiga Hodnik et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2530–2534
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standard catalytic hydrogenation procedure. Following treatment of 6a with gaseous hydrogen chloride gave compound 6b (cf. Supplementary material). The synthesised compounds were screened for their anti-biofilm activity in strains of MRSA, Streptococcus mutans and Pseudomonas aeruginosa using a modified biofilm formation assay (cf. Supplementary material), described by Christensen et al.34 The MBIC50 values of analogues 2, 4b, 12, 14, 15b, 16a and 16b were determined at multiple concentrations using a modified biofilm formation assay.35 The anti-biofilm activities against MRSA and Streptococcus mutans are presented in Tables 1–3. Oroidin and its indole-based analogues (1–17b) did not display any anti-biofilm activity against Pseudomonas aeruginosa (results not shown). 1,3-Substituted indole-based analogues 3b, 4b, 8, 11, 12 and 14 exhibited moderate to strong inhibition of MRSA biofilm formation,
while most of the 1,3-substituted indole-based analogues attenuated the formation of S. mutans biofilms (Table 2). The substitution at position 5 of the indole ring was also found to be important for anti-biofilm activity of 1,3-substituted analogues. In contrast to unsubstituted analogue 3b (MRSA BF: 13.9%, S. mutans BF: 14.2%), the hydroxy-(5b) and nitro-substituted (10) analogues displayed weaker activities against S. mutans biofilm (BF: 53.4% and 33.3%, respectively) and were inactive against MRSA biofilm formation. The amino-substituted analogues 6a and 6b did not display any anti-biofilm activities in both tested strains. The weak inhibitions of hydroxy-(5a and 5b) and amino-substituted analogues (6a and 6b) further suggest that polar 5-indole substituents negatively affect the activities against MRSA and S. mutans biofilms. The influence of the indole moiety substitution pattern on the anti-biofilm activity is further illustrated by 3-carboxamidoindole-based
Table 2 Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
O N
N H
H2 N
R3
H N
O
Compounds
3a 3b 4a 4b 5a 5b 6a 6b 7 8 9 10 11 12 13 14
N H
HN
H N
3a - 6a
R1
R1
3b - 6b 7 - 12
R3
H H OCF3 OCF3 OH OH NH2 NH+3Cl F Cl OCH3 NO2 OBn H — —
— H — H — H — H H H H H H CH3 — —
NH
HN
S
14
Staphylococcus aureus
H N
HN
13
R1
N H
H 2N
N H
H 2N
O
H +ClN
O
H +ClN
HN
N O
O
H+ClN
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. 13.9 ± 2.3 n.a. 34.1 ± 6.1 n.a. n.a. n.a. n.a. n.a. 44.0 ± 8.3 n.a. n.a. 31.4 ± 12.2 19.1 ± 6.4 n.a. 23.0 ± 10.6
— — — 20 — — — — — — — — — 70 — 90
30.6 ± 5.2 14.2 ± 2.0 n.a. 17.0 ± 3.9 57.6 ± 11.5 53.4 ± 7.0 n.a. n.a. 8.7 ± 3.5 6.8 ± 3.3 10.5 ± 1.8 33.3 ± 4.9 8.2 ± 2.4 8.8 ± 1.9 23.9 ± 2.9 10.1 ± 1.3
— — — 20 — — — — — — — — — 80 — 60
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
Table 3 Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
H2 N R
2
N NH
N
H N R1
O 15a-17b
Compounds
15a 15bc 16a 16bc 17a 17bc
R1
H H F F Cl Cl
R2
Boc H Boc H Boc H
Staphylococcus aureus
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. 11.1 ± 3.0 13.1 ± 4.7 10.9 ± 4.7 78.0 ± 20.3 n.a.
— 90 80 60 — —
22.6 ± 7.2 8.1 ± 3.5 56.0 ± 20.6 3.9 ± 3.0 n.a. 38.2 ± 13.3
— 60 120 50 — —
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments. c HCl salt.
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analogue 13 (MRSA BF: not active, S. mutans BF: 23.9%), which in contrast to 2-carboxamidoindole-based analogue 3b, exhibited weaker activity against both tested strains. However, thieno [3,2-b]pyrrole-based analogue 14 (MRSA BF: 23.0%, S. mutans BF: 10.1%) displayed comparable potencies to those of analogue 3b. The influence on anti-biofilm activity was also evident in the case of substitution at the position 1 of the 2-aminoimidazole ring. Hence, the tert-butyloxycarbonyl (Boc)-substituted analogues 3a, 4a and 5a exhibited weaker anti-biofilm activities against S. mutans biofilm (BF: 30.6%, not active and 57.6%, respectively) compared to their unsubstituted analogues 3b (BF: 14.2%), 4b (BF: 17.0%) and 5b (BF: 53.4%) (Table 2). Similar effect was also evident in the case of MRSA biofilm, where in contrast to the unsubstituted analogues 3b (BF: 13.9%) and 4b (BF: 34.1%), analogues 3a and 4a did not display any activity. This suggests that introduction of larger substituents at position 1 of the 2-aminoimidazole moiety weakens the inhibition of biofilm formation. Comparison of the biofilm formation assay results for compounds 3b and 12 (MRSA BF: 19.1%, S. mutans BF: 8.8%) suggests that the substitution of 2amino group of the imidazole moiety does not contribute significantly to the inhibition of biofilm formation. In general, compared to the biofilm of MRSA strain, 1,3-substituted analogues displayed stronger inhibitions of S. mutans biofilm formation. Similar to 1,3-substituted analogues, 1,4-substituted indolebased analogues 15a–17b also inhibited the formation of MRSA or S. mutans biofilms (Table 3). Comparison of the potencies of indole (15b; MRSA BF: 11.1%, S. mutans BF: 8.1%) and 5-fluoroindole (16b; MRSA BF: 10.9%, S. mutans BF: 3.9%) analogues with the 5-chloroindole analogue 17b (MRSA BF: not active, S. mutans BF: 38.2%) suggests that in the case of 1,4-substituted analogues, introduction of larger substituents in position 5 of the indole moiety leads to the loss of anti-biofilm activity. As in the case of 1,3substituted analogues, 1,4-substituted analogues 15a–17b also indicated the relevance of substitution at position 1 of the 2aminoimidazole moiety for anti-biofilm activity. The Boc-substituted analogues 15a, 16a and 17a exhibited weaker inhibitions of MRSA and S. mutans biofilms, compared to unsubstituted analogues 15b, 16b and 17b (Table 3). With an aim to supplement the results of the biofilm formation assay, seven structurally diverse analogues (2, 4b, 12, 14, 15b, 16a, 16b) were selected for minimal biofilm inhibitory concentration (MBIC50) determination. The resulting MBIC50 values in the lower micromolar range were consistent with the screening results from the biofilm formation assay (Tables 1–3). Model compound 5-fluoroindole analogue 2 displayed an MBIC50 value of 60 lM against MRSA and S. mutans biofilms, while the most potent, 5-(trifluoromethoxy)indole-based analogue 4b exhibited an MBIC50 value of 20 lM against both tested biofilm-forming strains. Analogues 13 (MBIC50 = 60 lM) and 15b (MBIC50 = 60 lM) displayed better activities against S. mutans biofilms, while analogue 16a (MBIC50 = 80 lM) displayed stronger inhibition of MRSA biofilm formation. The other evaluated analogues displayed similar or even equal MBIC50 values for both biofilm-forming strains. Non-linear dose-response effects of the evaluated compounds were observed in the biofilm formation assay. Minor effects of the compounds on the biofilm formation were observed at low concentrations while the strong inhibitory effects, as well as linear dose–response effects, occurred after reaching a threshold concentration value. The latter suggests that the cellular machinery responsible for the synthesis of essential biofilm molecules and/or their incorporation into the biofilm structure is resistant to low concentrations of the oroidin analogues, which should be therefore applied in higher concentrations to either reach the cellular target and/or actively inhibit the biofilm-forming processes.
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To conclude, several novel synthetic indole-based analogues of the marine alkaloid oroidin exhibited antibiofilm activities in the low micromolar range against the Gram-positive biofilm-forming strains MRSA and S. mutans and therefore represent a novel class of 2-aminoimidazole-based inhibitors of S. aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of S. mutans biofilm formation. The anti-biofilm activities of the majority of the synthesized indolebased oroidin analogues illustrate the applicability of the indole moiety as a dibromopyrrole surrogate for generating inhibitors of MRSA and S. mutans biofilm formation. The latter suggests that indole-based oroidin analogues could act as inhibitors of bacterial biofilms by dispersing their structure as 2-aminoimidazol-based analogues18,23 and inhibit the quorum sensing mechanisms due to possession of the indole moiety.28–30 The SAR study revealed promising substituents at position 5 of the indole moiety for anti-biofilm activity against the tested bacterial strains. Finally, this study resulted in a potent, 5-(trifluoromethoxy)indole-based analogue 4b as a promising hit for further optimisation to generate novel inhibitors of S. aureus and S. mutans biofilms formation. Acknowledgments This work was supported by the European Union FP7 Integrated Project MAREX: Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications (Project No. FP7-KBBE-2009-3-245137), by the Slovenian Research Agency (Grant No. P1-0208 and Grant No. Z1-5458) and by the Ministry of Science and Higher Education, Poland (Grant No. 1544/7.PR UE/2010/7). M.L. acknowledges a support by the European Union, within the European Regional Development Fund, through the Grant Innovative Economy (POIG.01.01.02-00-008/08). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 094. References and notes 1. Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. Rev. Microbiol. 2004, 2, 95. 2. Hoiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O. Int. J. Antimicrob. Agents 2010, 35, 322. 3. Stowe, S. D.; Richards, J. J.; Tucker, A. T.; Thompson, R.; Melander, C.; Cavanagh, J. Mar. Drugs 2011, 9, 2010. 4. Taraszkiewicz, A.; Fila, G.; Grinholc, M.; Nakonieczna, J. Biomed Res. Int. 2013. 5. Matz, C.; McDougald, D.; Moreno, A. M.; Yung, P. Y.; Yildiz, F. H.; Kjelleberg, S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16819. 6. Hoiby, N.; Ciofu, O.; Bjarnsholt, T. Future Microbiol. 2010, 5, 1663. 7. Costerton, W.; Veeh, R.; Shirtliff, M.; Pasmore, M.; Post, C.; Ehrlich, G. J. Clin. Invest. 2003, 112, 1466. 8. Donlan, R. M.; Costerton, J. W. Clin. Microbiol. Rev. 2002, 15, 167. 9. Davies, D. Nat. Rev. Drug Disc. 2003, 2, 114. 10. Banas, J. A. Front. Biosci. 2004, 9, 1267. 11. Archer, N. K.; Mazaitis, M. J.; Costerton, J. W.; Leid, J. G.; Powers, M. E.; Shirtliff, M. E. Virulence 2011, 2, 445. 12. Fitzpatrick, F.; Humphreys, H.; O’Gara, J. P. Clin. Microbiol. Infect. 2005, 11, 967. 13. Haefner, B. Drug Discovery Today 2003, 8, 536. 14. Janssens, J. C. A.; Steenackers, H.; Robijns, S.; Gellens, E.; Levin, J.; Zhao, H.; Hermans, K.; De Coster, D.; Verhoeven, T. L.; Marchal, K.; Vanderleyden, J.; De Vos, D. E.; De Keersmaecker, S. C. J. Appl. Environ. Microbiol. 2008, 74, 6639. 15. Steenackers, H. P.; Levin, J.; Janssens, J. C.; De Weerdt, A.; Balzarini, J.; Vanderleyden, J.; De Vos, D. E.; De Keersmaecker, S. C. Bioorg. Med. Chem. 2010, 18, 5224. 16. Hertiani, T.; Edrada-Ebel, R.; Ortlepp, S.; van Soest, R. W. M.; de Voogd, N. J.; Wray, V.; Hentschel, U.; Kozytska, S.; Muller, W. E. G.; Proksch, P. Bioorg. Med. Chem. 2010, 18, 1297. 17. Skindersoe, M. E.; Ettinger-Epstein, P.; Rasmussen, T. B.; Bjarnsholt, T.; de Nys, R.; Givskov, M. Mar. Biotechnol. 2008, 10, 56. 18. Zˇula, A.; Kikelj, D.; Ilaš, J. Mini-Rev. Med. Chem. 1921, 2013, 13.
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19. a) Huigens, R. W.; Richards, J. J.; Parise, G.; Ballard, T. E.; Zeng, W.; Deora, R.; Melander, C. J. Am. Chem. Soc. 2007, 129, 6966; b) Melander, C.; Moeller, P. D. R.; Ballard, T. E.; Richards, J. J.; Huigens, R. W.; Cavanagh, J. Int. Biodeterior. Biodegrad. 2009, 63, 529. 20. Rogers, S. A.; Huigens, R. W.; Cavanagh, J.; Melander, C. Antimicrob. Agents Chemother. 2010, 54, 2112. 21. Bunders, C. A.; Richards, J. J.; Melander, C. Bioorg. Med. Chem. Lett. 2010, 20, 3797. 22. Peng, L. L.; DeSousa, J.; Su, Z. M.; Novak, B. M.; Nevzorov, A. A.; Garland, E. R.; Melander, C. Chem. Commun. 2011, 47, 4896. 23. Richards, J. J.; Ballard, T. E.; Huigens, R. W.; Melander, C. ChemBioChem 2008, 9, 1267. 24. Richards, J. J.; Reed, C. S.; Melander, C. Bioorg. Med. Chem. Lett. 2008, 18, 4325. 25. Rogers, S. A.; Melander, C. Angew. Chem., Int. Ed. 2008, 47, 5229. 26. Thompson, R. J.; Bobay, B. G.; Stowe, S. D.; Olson, A. L.; Peng, L. L.; Su, Z. M.; Actis, L. A.; Melander, C.; Cavanagh, J. Biochemistry 2012, 51, 9776. 27. Yeagley, A. A.; Su, Z. M.; McCullough, K. D.; Worthington, R. J.; Melander, C. Org. Biomol. Chem. 2013, 11, 130. 28. Hu, M. X.; Zhang, C.; Mu, Y. F.; Shen, Q. W.; Feng, Y. J. Indian J. Microbiol. 2010, 50, 362. 29. Lee, J.-H.; Lee, J. FEMS Microbiol. Rev. 2010, 34, 426. 30. Raut, J. S.; Shinde, R. B.; Karuppayil, M. S. Afr. J. Microbiol. Res. 2012, 6, 6005. 31. Zidar, N.; Montalvão, S.; Hodnik, Zˇ.; Nawrot, D. A.; Zˇula, A.; Ilaš, J.; Kikelj, D.; Tammela, P.; Peterlin Mašicˇ, L. Mar. Drugs 2014, 12, 940. 32. Zidar, N.; Jakopin, Zˇ.; Madge, D. J.; Chan, F.; Tytgat, J.; Peigneur, S.; Dolenc, M. S.; Tomašic´, T.; Ilaš, J.; Peterlin Mašicˇ, L.; Kikelj, D. Eur. J. Med. Chem. 2014, 74, 23. 33. General procedure for the syntheses of compounds 16a and 17a (with 16a as an example): To a suspension of 5-fluoroindole-2-carboxylic acid (122 mg, 1.09 mmol) and TBTU (380 mg, 1.18 mmol) in dichloromethane (5 mL) Nmethylmorpholine (0.501 mL, 4.56 mmol) was added, and the mixture stirred at rt for 0.5 h upon which a clear solution formed. tert-Butyl 2-amino-4-(4aminophenyl)-1H-imidazole-1-carboxylate (250 mg, 0.91 mmol) was added
and the mixture stirred at 35 °C for 24 h. The solvent was evaporated in vacuo, the residue dissolved in ethyl acetate (30 mL), and washed successively with water (2 10 mL), saturated aqueous NaHCO3 solution (2 10 mL), and brine (1 10 mL). The organic phase was dried over Na2SO4, filtered and the solvent evaporated under reduced pressure. The crude product was purified by flash column chromatography using ethyl acetate/petroleum ether or dichloromethane/methanol as an eluent, to afford 16a (178 mg, 45% yield) as a white solid; mp 191–193 °C; IR (KBr) m = 3291, 3179, 1679, 1664, 1590, 1514, 1488, 1448, 1409, 1335, 1315, 1257, 1237, 1204, 1143, 1114, 833, 755 cm1. 1H NMR (DMSO-d6) d 1.60 (s, 9H, t-Bu), 6.61 (s, 2H, NH2), 7.10 (dt, 1H, 3J = 8.8 Hz, 4 J = 2.4 Hz, Ar-H), 7.31 (s, 1H, Ar-H), 7.43–7.50 (m, 3H, 3Ar-H), 7.74 (d, 2H, 3 J = 8.4 Hz, 2Ar-H), 7.80 (d, 2H, 3J = 8.4 Hz, 2Ar-H), 10.29 (s, 1H, NH), 11.86 (s, 1H, NH); 13C NMR (DMSO-d6) d 27.85 (CCH3), 84.37 (CCH3), 104.09 (d, 1C, JC– F = 6 Hz), 105.90 (d, 1C, JC–F = 21 Hz), 108.84, 113.61 (d, 1C, JC–F = 10 Hz), 120.25, 122.96, 124.73, 126.42, 127.01 (d, 1C, JC–F = 11 Hz), 132.95, 133.59, 135.75, 136.33, 138.61, 147.48, 156.23 (d, 1C, JC–F = 203 Hz), 159.35; MS (ESI) m/z (%) = 436.2 (MH+, 100). HRMS for C23H23N5O3F: calculated 436.1785; found 436.1780. Anal. Calcd for C23H22N5O3F 0.35 H2O (%): C, 62.53; H, 5.18; N, 15.85. Found: C, 62.83; H, 5.21; N, 15.44. 34. Christensen, G. D.; Simpson, W. A.; Younger, J. J.; Baddour, L. M.; Barrett, F. F.; Melton, D. M.; Beachey, E. H. J. Clin. Microbiol. 1985, 22, 996. 35. MBIC50 determination: The MBIC50 (minimal biofilm inhibitory concentration) value was determined by measurement of effects of the tested compound at various concentrations. Several different concentrations between 0 and 120 lM were used and the minimal concentration giving at least 50% inhibition was determined. A series of dilutions changing by 10 lM was employed rather than the doubling dilution technique, as in our preliminary experiments we found that effects of particular oroidin derivatives were not linearly proportional to their concentrations. Instead, a more or less sharp threshold concentration was noted, below which relatively small changes were determined with a concentration increase, and above which the inhibition was significantly more pronounced with a relatively small increase in the tested compound level.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin Zˇiga Hodnik a, Joanna M. Łos´ b, Aleš Zˇula a, Nace Zidar a, Zˇiga Jakopin a, Marcin Łos´ b, Marija Sollner Dolenc a, Janez Ilaš a, Grzegorz We˛grzyn b,⇑, Lucija Peterlin Mašicˇ a,⇑, Danijel Kikelj a a b
University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, 1000 Ljubljana, Slovenia ´ sk, Department of Molecular Biology, Wita Stwosza 59, 80-308 Gdan ´ sk, Poland University of Gdan
a r t i c l e
i n f o
Article history: Received 3 March 2014 Revised 28 March 2014 Accepted 29 March 2014 Available online 8 April 2014 Keywords: Biofilm Indole Inhibition Marine Oroidin
a b s t r a c t Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indolebased analogue 4b (MBIC50 = 20 lM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms. Ó 2014 Elsevier Ltd. All rights reserved.
The growth of bacteria is faster on surfaces that present a protective and nutrient-rich environment compared to growth in water and in liquids with low nutrient contents.1 The adhesion of bacterial cells to inert or living surfaces results in the formation of biofilms, that is, structured communities that are encased in a self-produced extracellular matrix of biomolecules.1,2 The extracellular matrix, which consists of polysaccharides, proteins and nucleic acids, is the primary reason for profound protection from the host’s immune system and for a nearly 1000-fold increase in biofilm resistance to conventional antibiotics when compared to planktonic bacteria.3,4 The robust resistance of biofilms to the hazards of their environment, for example, predators, antibiotics and host immune systems,2,5,6 results not only in biofouling in the food and marine industries but also in various nosocomial and chronic infections in patients, which result in perpetual inflammation and tissue damage.3,7 The formation of biofilms on inert surfaces includes indwelling medical devices, catheters and contact lenses, and infections of living surfaces most often include lung infections in cystic fibrosis patients, periodontitis, endocarditis and urinary tract
⇑ Corresponding authors. Tel.: +48 58 523 6024; fax: +48 58 523 5501 (G.W.); tel.: +386 1 4769 635; fax: +386 1 4258 031 (L.P.M.). E-mail addresses: [email protected] (G. We˛grzyn), lucija. [email protected] (L. Peterlin Mašicˇ). http://dx.doi.org/10.1016/j.bmcl.2014.03.094 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
infections.4,8 Furthermore, an estimate by the National Institutes of Health states that 80% of microbial infections are biofilm-based.9 Facultative anaerobic cocci Staphylococcus aureus and Streptococcus mutans are among common Gram-positive bacteria that form infectious biofilms.1,10 Nosocomial infections are frequently caused by omnipresent S. aureus bacteria, which are commonly found in the anterior nares in humans and can enter the circulatory system through an epithelial breach.11,12 Osteomyelitis, indwelling medical device infections, chronic wound infections, endocarditis and periodontitis exemplify the most prevalent biofilm-based infections caused by S. aureus. While periodontitis is sometimes caused by S. mutans, its most common biofilm-based infection remains dental caries. S. mutans is considered to be one of the earliest colonisers of human teeth after their eruption, and its fermentation of dietary carbohydrates and consequential production of acids poses one of the most important factors for the formation of dental caries.10 Ubiquitous biofouling presents a considerable threat to immobile marine organisms such as sponges and seaweeds, which do not possess an immune system that can protect them against microbes. Hence, they have evolved to biosynthesise various secondary metabolites that function as a defence system against bacterial infections.1,13 Among the most investigated anti-biofilm agents from marine sponges are the halogenated furanones from the seaweed Delisea pulchra,14,15 terpenoids16,17 and especially 2aminoimidazole-based alkaloids.18–20 The 2-aminoimidazole-based
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marine alkaloid oroidin (Fig. 1) and various series of its analogues were extensively studied as biofilm inhibitors, primarily by Melander and coworkers.18,20–27 Although numerous analogues of oroidin have been observed to control biofilm development,18,20–27 to our knowledge, indolebased analogues of oroidin have not yet been studied. In contrast, various studies outlined the ability of indole to regulate the growth of various bacterial biofilms and therefore represents an important quorum sensing molecule.28–30 In the present work, we first replaced the dibromopyrrole moiety of oroidin with the isosteric indole and 5-fluoroindole moieties, and evaluated the anti-biofilm activity of synthesised analogues 131 and 231 (Table 1) against two Gram-positive biofilm-forming strains of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus mutans and against a Gram-negative biofilm-forming strain of Pseudomonas aeruginosa (cf. Supplementary material). While indole-based analogue 1 did not exhibit any anti-biofilm activity against the tested strains, its 5-fluoroindole analogue 2 attenuated the formation of MRSA and S. mutans biofilms to 8.1% and 18.7%, respectively, at 100 lM (Table 1). Based on these encouraging anti-biofilm activities we further explored the structure–activity relationship of
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indole-based series of compounds by synthesising conformationally constrained analogues (Fig. 1) in which the central alkene region of oroidin was replaced with synthetically more favourable 1,3phenylene (compounds 3a–14) and 1,4-phenylene (compounds 15a–17b) moieties. To study the structure–activity relationship of our small library of conformationally constrained indole-based analogues of oroidin we (i) evaluated the effect of the rigidification of analogues 1 and 2 on anti-biofilm activity, (ii) compared the 1,3substituted series to the 1,4-substituted series of analogues and (iii) studied the influence of various substituents on indole and 2-aminoimidazole moieties on anti-biofilm activity (Fig. 1). Syntheses of oroidin and compounds 1–5, 7, 8, 9 and 11–15b were performed as previously described.31,32 Compounds 10a (cf. Supplementary material), 16a and 17a were synthesised using O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluorobo rate (TBTU)-promoted coupling of tert-butyl 2-amino-4-(3-aminophenyl)-1H-imidazole-1-carboxylate31 or tert-butyl 2-amino-4-(4aminophenyl)-1H-imidazole-1-carboxylate31 with the corresponding indole-2-carboxylic acids.33 Subsequent treatment of compounds 10a, 16a and 17a with gaseous hydrogen chloride gave compounds 10, 16b and 17b. Compound 6a was synthesised from 10a, using
Br HN H2 N
HN
H N
N
Br O
oroidin
HN H2 N
HN
H N
N
R1 R 1 = H or F
O 1-2 rigidification O N HN R3
N H
H N
HN H2 N
N R2
HN
H N
N
N NH
N
1,3-substitution
3a-14
H2 N
R1 1,4-substitution
O
R1
R2
H N R1
O 15a-17b
R 1 = H, F, Cl,... R 2 = H or Boc R 3 = H or CH3
R1 = H, F or Cl R 2 = H or Boc
Figure 1. Design of conformationally constrained indole-based analogues of oroidin with potential anti-biofilm activity.
Table 1 Anti-biofilm activities of oroidin and its indole-based analogues
HN H 2N
HN
H N
N
R1
O 1-2
Compounds
Oroidin 1 2
R1
— H F
Staphylococcus aureus
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. n.a. 8.1 ± 0.7
— — 60
18.0 ± 8.6 n.a. 18.7 ± 9.7
— — 60
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
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standard catalytic hydrogenation procedure. Following treatment of 6a with gaseous hydrogen chloride gave compound 6b (cf. Supplementary material). The synthesised compounds were screened for their anti-biofilm activity in strains of MRSA, Streptococcus mutans and Pseudomonas aeruginosa using a modified biofilm formation assay (cf. Supplementary material), described by Christensen et al.34 The MBIC50 values of analogues 2, 4b, 12, 14, 15b, 16a and 16b were determined at multiple concentrations using a modified biofilm formation assay.35 The anti-biofilm activities against MRSA and Streptococcus mutans are presented in Tables 1–3. Oroidin and its indole-based analogues (1–17b) did not display any anti-biofilm activity against Pseudomonas aeruginosa (results not shown). 1,3-Substituted indole-based analogues 3b, 4b, 8, 11, 12 and 14 exhibited moderate to strong inhibition of MRSA biofilm formation,
while most of the 1,3-substituted indole-based analogues attenuated the formation of S. mutans biofilms (Table 2). The substitution at position 5 of the indole ring was also found to be important for anti-biofilm activity of 1,3-substituted analogues. In contrast to unsubstituted analogue 3b (MRSA BF: 13.9%, S. mutans BF: 14.2%), the hydroxy-(5b) and nitro-substituted (10) analogues displayed weaker activities against S. mutans biofilm (BF: 53.4% and 33.3%, respectively) and were inactive against MRSA biofilm formation. The amino-substituted analogues 6a and 6b did not display any anti-biofilm activities in both tested strains. The weak inhibitions of hydroxy-(5a and 5b) and amino-substituted analogues (6a and 6b) further suggest that polar 5-indole substituents negatively affect the activities against MRSA and S. mutans biofilms. The influence of the indole moiety substitution pattern on the anti-biofilm activity is further illustrated by 3-carboxamidoindole-based
Table 2 Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
O N
N H
H2 N
R3
H N
O
Compounds
3a 3b 4a 4b 5a 5b 6a 6b 7 8 9 10 11 12 13 14
N H
HN
H N
3a - 6a
R1
R1
3b - 6b 7 - 12
R3
H H OCF3 OCF3 OH OH NH2 NH+3Cl F Cl OCH3 NO2 OBn H — —
— H — H — H — H H H H H H CH3 — —
NH
HN
S
14
Staphylococcus aureus
H N
HN
13
R1
N H
H 2N
N H
H 2N
O
H +ClN
O
H +ClN
HN
N O
O
H+ClN
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. 13.9 ± 2.3 n.a. 34.1 ± 6.1 n.a. n.a. n.a. n.a. n.a. 44.0 ± 8.3 n.a. n.a. 31.4 ± 12.2 19.1 ± 6.4 n.a. 23.0 ± 10.6
— — — 20 — — — — — — — — — 70 — 90
30.6 ± 5.2 14.2 ± 2.0 n.a. 17.0 ± 3.9 57.6 ± 11.5 53.4 ± 7.0 n.a. n.a. 8.7 ± 3.5 6.8 ± 3.3 10.5 ± 1.8 33.3 ± 4.9 8.2 ± 2.4 8.8 ± 1.9 23.9 ± 2.9 10.1 ± 1.3
— — — 20 — — — — — — — — — 80 — 60
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
Table 3 Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
H2 N R
2
N NH
N
H N R1
O 15a-17b
Compounds
15a 15bc 16a 16bc 17a 17bc
R1
H H F F Cl Cl
R2
Boc H Boc H Boc H
Staphylococcus aureus
Streptococcus mutans
% Of biofilm formationa
MBIC50b (lM)
% Of biofilm formationa
MBIC50b (lM)
n.a. 11.1 ± 3.0 13.1 ± 4.7 10.9 ± 4.7 78.0 ± 20.3 n.a.
— 90 80 60 — —
22.6 ± 7.2 8.1 ± 3.5 56.0 ± 20.6 3.9 ± 3.0 n.a. 38.2 ± 13.3
— 60 120 50 — —
a Percentage of biofilm formation compared to the negative control. Compounds were tested at 100 lM. Each value is the mean of three independent experiments. n.a. = not active. b The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments. c HCl salt.
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analogue 13 (MRSA BF: not active, S. mutans BF: 23.9%), which in contrast to 2-carboxamidoindole-based analogue 3b, exhibited weaker activity against both tested strains. However, thieno [3,2-b]pyrrole-based analogue 14 (MRSA BF: 23.0%, S. mutans BF: 10.1%) displayed comparable potencies to those of analogue 3b. The influence on anti-biofilm activity was also evident in the case of substitution at the position 1 of the 2-aminoimidazole ring. Hence, the tert-butyloxycarbonyl (Boc)-substituted analogues 3a, 4a and 5a exhibited weaker anti-biofilm activities against S. mutans biofilm (BF: 30.6%, not active and 57.6%, respectively) compared to their unsubstituted analogues 3b (BF: 14.2%), 4b (BF: 17.0%) and 5b (BF: 53.4%) (Table 2). Similar effect was also evident in the case of MRSA biofilm, where in contrast to the unsubstituted analogues 3b (BF: 13.9%) and 4b (BF: 34.1%), analogues 3a and 4a did not display any activity. This suggests that introduction of larger substituents at position 1 of the 2-aminoimidazole moiety weakens the inhibition of biofilm formation. Comparison of the biofilm formation assay results for compounds 3b and 12 (MRSA BF: 19.1%, S. mutans BF: 8.8%) suggests that the substitution of 2amino group of the imidazole moiety does not contribute significantly to the inhibition of biofilm formation. In general, compared to the biofilm of MRSA strain, 1,3-substituted analogues displayed stronger inhibitions of S. mutans biofilm formation. Similar to 1,3-substituted analogues, 1,4-substituted indolebased analogues 15a–17b also inhibited the formation of MRSA or S. mutans biofilms (Table 3). Comparison of the potencies of indole (15b; MRSA BF: 11.1%, S. mutans BF: 8.1%) and 5-fluoroindole (16b; MRSA BF: 10.9%, S. mutans BF: 3.9%) analogues with the 5-chloroindole analogue 17b (MRSA BF: not active, S. mutans BF: 38.2%) suggests that in the case of 1,4-substituted analogues, introduction of larger substituents in position 5 of the indole moiety leads to the loss of anti-biofilm activity. As in the case of 1,3substituted analogues, 1,4-substituted analogues 15a–17b also indicated the relevance of substitution at position 1 of the 2aminoimidazole moiety for anti-biofilm activity. The Boc-substituted analogues 15a, 16a and 17a exhibited weaker inhibitions of MRSA and S. mutans biofilms, compared to unsubstituted analogues 15b, 16b and 17b (Table 3). With an aim to supplement the results of the biofilm formation assay, seven structurally diverse analogues (2, 4b, 12, 14, 15b, 16a, 16b) were selected for minimal biofilm inhibitory concentration (MBIC50) determination. The resulting MBIC50 values in the lower micromolar range were consistent with the screening results from the biofilm formation assay (Tables 1–3). Model compound 5-fluoroindole analogue 2 displayed an MBIC50 value of 60 lM against MRSA and S. mutans biofilms, while the most potent, 5-(trifluoromethoxy)indole-based analogue 4b exhibited an MBIC50 value of 20 lM against both tested biofilm-forming strains. Analogues 13 (MBIC50 = 60 lM) and 15b (MBIC50 = 60 lM) displayed better activities against S. mutans biofilms, while analogue 16a (MBIC50 = 80 lM) displayed stronger inhibition of MRSA biofilm formation. The other evaluated analogues displayed similar or even equal MBIC50 values for both biofilm-forming strains. Non-linear dose-response effects of the evaluated compounds were observed in the biofilm formation assay. Minor effects of the compounds on the biofilm formation were observed at low concentrations while the strong inhibitory effects, as well as linear dose–response effects, occurred after reaching a threshold concentration value. The latter suggests that the cellular machinery responsible for the synthesis of essential biofilm molecules and/or their incorporation into the biofilm structure is resistant to low concentrations of the oroidin analogues, which should be therefore applied in higher concentrations to either reach the cellular target and/or actively inhibit the biofilm-forming processes.
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To conclude, several novel synthetic indole-based analogues of the marine alkaloid oroidin exhibited antibiofilm activities in the low micromolar range against the Gram-positive biofilm-forming strains MRSA and S. mutans and therefore represent a novel class of 2-aminoimidazole-based inhibitors of S. aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of S. mutans biofilm formation. The anti-biofilm activities of the majority of the synthesized indolebased oroidin analogues illustrate the applicability of the indole moiety as a dibromopyrrole surrogate for generating inhibitors of MRSA and S. mutans biofilm formation. The latter suggests that indole-based oroidin analogues could act as inhibitors of bacterial biofilms by dispersing their structure as 2-aminoimidazol-based analogues18,23 and inhibit the quorum sensing mechanisms due to possession of the indole moiety.28–30 The SAR study revealed promising substituents at position 5 of the indole moiety for anti-biofilm activity against the tested bacterial strains. Finally, this study resulted in a potent, 5-(trifluoromethoxy)indole-based analogue 4b as a promising hit for further optimisation to generate novel inhibitors of S. aureus and S. mutans biofilms formation. Acknowledgments This work was supported by the European Union FP7 Integrated Project MAREX: Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications (Project No. FP7-KBBE-2009-3-245137), by the Slovenian Research Agency (Grant No. P1-0208 and Grant No. Z1-5458) and by the Ministry of Science and Higher Education, Poland (Grant No. 1544/7.PR UE/2010/7). M.L. acknowledges a support by the European Union, within the European Regional Development Fund, through the Grant Innovative Economy (POIG.01.01.02-00-008/08). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 094. References and notes 1. Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. Rev. Microbiol. 2004, 2, 95. 2. Hoiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O. Int. J. Antimicrob. Agents 2010, 35, 322. 3. Stowe, S. D.; Richards, J. J.; Tucker, A. T.; Thompson, R.; Melander, C.; Cavanagh, J. Mar. Drugs 2011, 9, 2010. 4. Taraszkiewicz, A.; Fila, G.; Grinholc, M.; Nakonieczna, J. Biomed Res. Int. 2013. 5. Matz, C.; McDougald, D.; Moreno, A. M.; Yung, P. Y.; Yildiz, F. H.; Kjelleberg, S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16819. 6. Hoiby, N.; Ciofu, O.; Bjarnsholt, T. Future Microbiol. 2010, 5, 1663. 7. Costerton, W.; Veeh, R.; Shirtliff, M.; Pasmore, M.; Post, C.; Ehrlich, G. J. Clin. Invest. 2003, 112, 1466. 8. Donlan, R. M.; Costerton, J. W. Clin. Microbiol. Rev. 2002, 15, 167. 9. Davies, D. Nat. Rev. Drug Disc. 2003, 2, 114. 10. Banas, J. A. Front. Biosci. 2004, 9, 1267. 11. Archer, N. K.; Mazaitis, M. J.; Costerton, J. W.; Leid, J. G.; Powers, M. E.; Shirtliff, M. E. Virulence 2011, 2, 445. 12. Fitzpatrick, F.; Humphreys, H.; O’Gara, J. P. Clin. Microbiol. Infect. 2005, 11, 967. 13. Haefner, B. Drug Discovery Today 2003, 8, 536. 14. Janssens, J. C. A.; Steenackers, H.; Robijns, S.; Gellens, E.; Levin, J.; Zhao, H.; Hermans, K.; De Coster, D.; Verhoeven, T. L.; Marchal, K.; Vanderleyden, J.; De Vos, D. E.; De Keersmaecker, S. C. J. Appl. Environ. Microbiol. 2008, 74, 6639. 15. Steenackers, H. P.; Levin, J.; Janssens, J. C.; De Weerdt, A.; Balzarini, J.; Vanderleyden, J.; De Vos, D. E.; De Keersmaecker, S. C. Bioorg. Med. Chem. 2010, 18, 5224. 16. Hertiani, T.; Edrada-Ebel, R.; Ortlepp, S.; van Soest, R. W. M.; de Voogd, N. J.; Wray, V.; Hentschel, U.; Kozytska, S.; Muller, W. E. G.; Proksch, P. Bioorg. Med. Chem. 2010, 18, 1297. 17. Skindersoe, M. E.; Ettinger-Epstein, P.; Rasmussen, T. B.; Bjarnsholt, T.; de Nys, R.; Givskov, M. Mar. Biotechnol. 2008, 10, 56. 18. Zˇula, A.; Kikelj, D.; Ilaš, J. Mini-Rev. Med. Chem. 1921, 2013, 13.
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19. a) Huigens, R. W.; Richards, J. J.; Parise, G.; Ballard, T. E.; Zeng, W.; Deora, R.; Melander, C. J. Am. Chem. Soc. 2007, 129, 6966; b) Melander, C.; Moeller, P. D. R.; Ballard, T. E.; Richards, J. J.; Huigens, R. W.; Cavanagh, J. Int. Biodeterior. Biodegrad. 2009, 63, 529. 20. Rogers, S. A.; Huigens, R. W.; Cavanagh, J.; Melander, C. Antimicrob. Agents Chemother. 2010, 54, 2112. 21. Bunders, C. A.; Richards, J. J.; Melander, C. Bioorg. Med. Chem. Lett. 2010, 20, 3797. 22. Peng, L. L.; DeSousa, J.; Su, Z. M.; Novak, B. M.; Nevzorov, A. A.; Garland, E. R.; Melander, C. Chem. Commun. 2011, 47, 4896. 23. Richards, J. J.; Ballard, T. E.; Huigens, R. W.; Melander, C. ChemBioChem 2008, 9, 1267. 24. Richards, J. J.; Reed, C. S.; Melander, C. Bioorg. Med. Chem. Lett. 2008, 18, 4325. 25. Rogers, S. A.; Melander, C. Angew. Chem., Int. Ed. 2008, 47, 5229. 26. Thompson, R. J.; Bobay, B. G.; Stowe, S. D.; Olson, A. L.; Peng, L. L.; Su, Z. M.; Actis, L. A.; Melander, C.; Cavanagh, J. Biochemistry 2012, 51, 9776. 27. Yeagley, A. A.; Su, Z. M.; McCullough, K. D.; Worthington, R. J.; Melander, C. Org. Biomol. Chem. 2013, 11, 130. 28. Hu, M. X.; Zhang, C.; Mu, Y. F.; Shen, Q. W.; Feng, Y. J. Indian J. Microbiol. 2010, 50, 362. 29. Lee, J.-H.; Lee, J. FEMS Microbiol. Rev. 2010, 34, 426. 30. Raut, J. S.; Shinde, R. B.; Karuppayil, M. S. Afr. J. Microbiol. Res. 2012, 6, 6005. 31. Zidar, N.; Montalvão, S.; Hodnik, Zˇ.; Nawrot, D. A.; Zˇula, A.; Ilaš, J.; Kikelj, D.; Tammela, P.; Peterlin Mašicˇ, L. Mar. Drugs 2014, 12, 940. 32. Zidar, N.; Jakopin, Zˇ.; Madge, D. J.; Chan, F.; Tytgat, J.; Peigneur, S.; Dolenc, M. S.; Tomašic´, T.; Ilaš, J.; Peterlin Mašicˇ, L.; Kikelj, D. Eur. J. Med. Chem. 2014, 74, 23. 33. General procedure for the syntheses of compounds 16a and 17a (with 16a as an example): To a suspension of 5-fluoroindole-2-carboxylic acid (122 mg, 1.09 mmol) and TBTU (380 mg, 1.18 mmol) in dichloromethane (5 mL) Nmethylmorpholine (0.501 mL, 4.56 mmol) was added, and the mixture stirred at rt for 0.5 h upon which a clear solution formed. tert-Butyl 2-amino-4-(4aminophenyl)-1H-imidazole-1-carboxylate (250 mg, 0.91 mmol) was added
and the mixture stirred at 35 °C for 24 h. The solvent was evaporated in vacuo, the residue dissolved in ethyl acetate (30 mL), and washed successively with water (2 10 mL), saturated aqueous NaHCO3 solution (2 10 mL), and brine (1 10 mL). The organic phase was dried over Na2SO4, filtered and the solvent evaporated under reduced pressure. The crude product was purified by flash column chromatography using ethyl acetate/petroleum ether or dichloromethane/methanol as an eluent, to afford 16a (178 mg, 45% yield) as a white solid; mp 191–193 °C; IR (KBr) m = 3291, 3179, 1679, 1664, 1590, 1514, 1488, 1448, 1409, 1335, 1315, 1257, 1237, 1204, 1143, 1114, 833, 755 cm1. 1H NMR (DMSO-d6) d 1.60 (s, 9H, t-Bu), 6.61 (s, 2H, NH2), 7.10 (dt, 1H, 3J = 8.8 Hz, 4 J = 2.4 Hz, Ar-H), 7.31 (s, 1H, Ar-H), 7.43–7.50 (m, 3H, 3Ar-H), 7.74 (d, 2H, 3 J = 8.4 Hz, 2Ar-H), 7.80 (d, 2H, 3J = 8.4 Hz, 2Ar-H), 10.29 (s, 1H, NH), 11.86 (s, 1H, NH); 13C NMR (DMSO-d6) d 27.85 (CCH3), 84.37 (CCH3), 104.09 (d, 1C, JC– F = 6 Hz), 105.90 (d, 1C, JC–F = 21 Hz), 108.84, 113.61 (d, 1C, JC–F = 10 Hz), 120.25, 122.96, 124.73, 126.42, 127.01 (d, 1C, JC–F = 11 Hz), 132.95, 133.59, 135.75, 136.33, 138.61, 147.48, 156.23 (d, 1C, JC–F = 203 Hz), 159.35; MS (ESI) m/z (%) = 436.2 (MH+, 100). HRMS for C23H23N5O3F: calculated 436.1785; found 436.1780. Anal. Calcd for C23H22N5O3F 0.35 H2O (%): C, 62.53; H, 5.18; N, 15.85. Found: C, 62.83; H, 5.21; N, 15.44. 34. Christensen, G. D.; Simpson, W. A.; Younger, J. J.; Baddour, L. M.; Barrett, F. F.; Melton, D. M.; Beachey, E. H. J. Clin. Microbiol. 1985, 22, 996. 35. MBIC50 determination: The MBIC50 (minimal biofilm inhibitory concentration) value was determined by measurement of effects of the tested compound at various concentrations. Several different concentrations between 0 and 120 lM were used and the minimal concentration giving at least 50% inhibition was determined. A series of dilutions changing by 10 lM was employed rather than the doubling dilution technique, as in our preliminary experiments we found that effects of particular oroidin derivatives were not linearly proportional to their concentrations. Instead, a more or less sharp threshold concentration was noted, below which relatively small changes were determined with a concentration increase, and above which the inhibition was significantly more pronounced with a relatively small increase in the tested compound level.
