Effects of N-pyrrole substitution on the anti-biofilm activities of oroidin derivatives against Acinetobacter baumannii

Article abstract of DOI:10.1016/j.bmcl.2008.06.089

Bacteria of the genus Acinetobacter spp. are rapidly emerging as problematic pathogens in healthcare settings. This is exacerbated by the bacteria's ability to form robust biofilms. Marine natural products incorporating a 2-aminoimidazole (2-AI) motif, namely from the oroidin class of marine alkaloids, have served as a unique scaffold for developing molecules that have the ability to inhibit and disperse bacterial biofilms. Herein we present the anti-biofilm activity of a small library of second generation oroidin analogues against the bacterium Acinetobacter baumannii.

Full text of DOI:10.1016/j.bmcl.2008.06.089

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4325–4327
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effects of N-pyrrole substitution on the anti-biofilm activities of oroidin
derivatives against Acinetobacter baumannii
*
Justin J. Richards, Catherine S. Reed, Christian Melander
North Carolina State University, Department of Chemistry, 2620 Yarborough Drive, Raleigh, NC 27695-8204, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacteria of the genus Acinetobacter spp. are rapidly emerging as problematic pathogens in healthcare set-
tings. This is exacerbated by the bacteria’s ability to form robust biofilms. Marine natural products incor-
porating a 2-aminoimidazole (2-AI) motif, namely from the oroidin class of marine alkaloids, have served
as a unique scaffold for developing molecules that have the ability to inhibit and disperse bacterial bio-
films. Herein we present the anti-biofilm activity of a small library of second generation oroidin ana-
logues against the bacterium Acinetobacter baumannii.
Received 1 May 2008
Revised 23 June 2008
Accepted 25 June 2008
Available online 28 June 2008
Keywords:
Biofilm
Ó 2008 Elsevier Ltd. All rights reserved.
Inhibition
Dispersion
Marine natural products
Oroidin
A bacterial biofilm is defined as a highly organized community
of bacteria whose growth is generally thought of as a developmen-
tal process.1–3 Industrially, biofilms are responsible for the microbe
mediated corrosion of structures on land and underwater.4–6 In the
agricultural sector, biofilms are known to decimate crops and
necessitate the use of toxic biocides as control measures.7,8 Medi-
cally, it is estimated that upwards of 80% of infections in the hu-
man body are biofilm mediated.9,10 Given the ubiquity of
biofilms, recently there has been an intense effort to further under-
stand their developmental processes in hopes of alleviating some
of the problems they underpin.
One particular pathogen that has emerged as a threat to the
medical community is the aerobic Gram-negative coccobacilli Aci-
netobacter baumannii. This bacterium, along with Staphylococcus
aureus and Pseudomonas aeruginosa, consistently ranks among
the highest in nosocomial infections.11 Approximately 25% of all
hospital swabs are positive for A. baumannii and recent outbreaks
have occurred in both the US and Europe’s healthcare system.12–15
A. baumannii is known to form robust biofilms that allow the bac-
teria to survive for weeks on inanimate surfaces, making complete
disinfection of hospital and medical instruments difficult.16,17 Mor-
bidity of patients suffering from A. baumannii infections has, in
part, been correlated to the virulence associated with the forma-
tion of biofilms. More specifically, it has recently been disclosed
that morbidity may arise from the virulence associated with outer
membrane protein A (AbOmpA), the bacteria’s most abundant
surface protein, which has been shown to have the ability to induce
apoptosis of epithelial cells through mitochondrial targeting.18 Re-
ports of anti-biotic resistance to carbapenems19 and polymyxins20
are becoming more frequent. It is believed that this is attributed to
changes in porin proteins, development of efflux pumps, and pro-
duction of b-lactamases in the bacteria.19,21 Biofilms easily facili-
tate these adaptations for survival through the selection and
reproduction of bacteria that can withstand harsh environmental
pressures. Clearly, there lies a necessity to develop new therapies
for A. baumannii remediation efforts.
Work in our group has focused on harnessing the innate biolog-
ical activity of sponge-derived marine natural product analogues
whose parent architectures belong to the oroidin class of marine
alkaloids (Fig. 1).22–24 These nitrogen dense molecules contain a
2-aminoimidazole (2-AI) subunit and provide the fundamental
* Corresponding author. Tel.: +1 919 513 2960; fax: +1 919 515 5079.
E-mail address: Christian_Melander@NCSU.edu (C. Melander).
Figure 1. Molecular inspiration for anti-biofilm derivatives.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.089

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J. J. Richards et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4325–4327
basis for our research in hopes of discovering novel small mole-
cules that inhibit and disperse medically relevant bacterial biofilms
across order, class, and phylum.
Recently, we disclosed the synthesis and biological evaluation of
a50-compoundanaloguelibrarybasedontheoroidintemplate.25 An
extensive structure–activity relationship (SAR) study was per-
formed by systematically varying different regions of the template
to tune biological potency within the context of anti-biofilm activity
2-aminoimidazole scaffold.29 While varying the alkylation on the
pyrrole ring was of primary concern, we also sought to delineate
the importance of the pyrrole moiety overall in eliciting a biologi-
cal response. A single atom replacement in the 30-position on the
pyrrole ring was envisioned to afford the imidazole based isostere
6. This molecule could then be directly compared to the activity of
DHS. Access to this derivative was achieved by utilizing identical
chemistry as used in the pyrrole functionalization. All final targets
were then converted into their corresponding hydrochloride salts
before biological testing.
against the c-proteobacterium P. aeruginosa. The most active mem-
ber of the library, dihydrosventrin (DHS), was subsequently assayed
for activity against other proteobacteria. DHS became the first non-
toxic small molecule known to inhibit biofilm formation in a mucoid
variant of P. aeruginosa while also crossing bacterial class to inhibit
and disperse biofilms in the b-proteobacterium Bordetella bronchi-
septica.22 Additionally, DHS was shown to exhibit an IC50 value of
Biofilm inhibition assays were performed with the second gener-
ation oroidin library against A. baumannii utilizing a crystal violet re-
porter assay (Table 1).30 Surprisingly, ethyl derivative 4a was almost
completely inactive, only being able to inhibit A. baumannii biofilm
formation approximately 10% at 500
initially insoluble during the preliminary 500
concentrations(<200 M), solubilitywasnotobservedtohavea lim-
lM. Compounds 4b–4g were
110
lM for inhibition of A. baumannii biofilms.
lM screen. At lower
The SAR studies culminated with several observations about
what features of the molecule were necessary for anti-biofilm
activity. First, the 2-AI motif should remain intact. Second, the opti-
mum chain length between the 2-AI head and pyrrole tail was
three carbons and unsaturation was not necessary for activity.
Third, methylation of the pyrrole ring nitrogen led to increased
activity in conjunction with dibromination of the ring. To this
end, we elected to synthesize an additional library of oroidin deriv-
atives that focused on varying the pendant group located on the
pyrrole nitrogen in hopes of further increasing anti-biofilm activity
in relation to DHS (Fig. 2). It was then decided that A. baumannii
would serve as the target bacterial strain for which the newly gen-
erated compounds would be assayed against for anti-biofilm
activity.
Briefly, the Clausen-Kaas variant of the Paal-Knorr pyrrole syn-
thesis was employed to synthesize six sterically and electronically
different pyrroles (Scheme 1).26 Installation of the trichloroacetyl
ester group at the 2-position followed by dibromination of the ring
with excess molecular bromine afforded the requisite pyrrole frag-
ments for construction of the desired library members.27,28 The fi-
nal step employed in the synthesis was amide bond formation
between the functionalized pyrroles and the 4-(3-aminopropyl)-
l
iting effect. These six analogues were subsequently assayed for IC50
values and all were determined to be more potent than the previous
lead compound DHS (Actb IC50 = 110 lM). Of the derivatives with an
unsubstituted phenyl ring (4c, 4e, and 4g), a single methylene spacer
between the pyrrole nitrogen and the phenyl ring (4e) resulted in a
compound approximately 2.5ꢀ more active than no spacer at all
(4c). An additional methylene unit (4g) seemed to elicit only a small
effect by slightly lowering the determined IC50 value. Interestingly,
addition of a methoxy group to the phenyl ring also resulted in a
derivative (4f) that was active. Toleration of the single atom replace-
ment in the pyrrole ring of 6 was not evident as a significant drop in
activity was seen against A. baumannii (ꢁ50% inhibition at 500
in relation to DHS. The most active compound identified from the
inhibition screens was compound 4d (Actb IC50 = 26.8 2.28 M)
lM)
l
which contained an additional bromine atom in the para position
on the phenyl appendage. Growth curve experiments performed in
either the presence or absence of this compound at its determined
IC50 value over a 24-h time period validated that like its predeces-
sors, 4d was not acting as a microbicide and inducing bacterial cell
death before biofilm initiation had begun. Also, analogous to the
observationsmadeinourpreviousstudy,25 modificationofthiscom-
pound through removal of both bromine atoms on the pyrrole moi-
ety while keeping the newly introduced bromine on the phenyl ring
led to a sharp decrease in activity (20% inhibition at 500
shown).
lM, data not
From a medical standpoint, the ability to disperse an existing
biofilm is more important than being able to prevent its formation.
Once a biofilm forms, it is known to be upwards of 1000-times
more resistant to conventional anti-biotics.31 Bacteria within bio-
films also are more resistant to microbicides, antiseptics, and basic
host immune responses. Therefore, compounds that only inhibit
biofilm formation will be less effective in treating a disease caused
by an established biofilm. The most active analogue from the
Figure 2. Template mediated derivatization of the oroidin scaffold.
Table 1
Biological evaluation of second generation oroidin analogues against Acinetobacter
baumannii
b
Compound
A. baumannii (Actb) biofilm
IC50
(lM)
inhibition at 500 l
M (%)a
DHS
4a
4b
4c
4d
4e
4f
>99
10.3 4.61
—
—
—
—
—
—
110
—
40.5 3.80
97.7 10.5
26.8 2.28
35.4 1.69
42.8 4.12
40.7 3.61
—
4g
6
Scheme 1. Synthesis of second generation oroidin library. Reactions and condi-
tions: (a) 2,5-dimethoxytetrahydrofuran, HOAc, reflux; (b) trichloroacetyl chloride,
C2H4Cl2, reflux; (c) Br2, CHCl3, ꢂ10 °C to rt; (d) 4-(3-aminopropyl)-2-aminoimidaz-
ole, Na2CO3, DMAP (cat.), DMF, 50 °C; (e) 2 M HCl in Et2O.
50.7 2.64
a
Assays performed in a minimum of triplicate.
See Supplemental information for dose–response curves.
b

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4327
References and notes
1. Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappinscott, H.
M. Annu. Rev. Microbiol. 1995, 49, 711.
2. Musk, D. J.; Hergenrother, P. J. Curr. Med. Chem. 2006, 13, 2163.
3. O’Toole, G.; Kaplan, H. B.; Kolter, R. Annu. Rev. Microbiol. 2000, 54, 49.
4. Beech, I. B.; Sunner, J. A.; Hiraoka, K. Int. Microbiol. 2005, 8, 157.
5. Flemming, H. C.; Griebe, T.; Schaule, G. Water Sci. Technol. 1996, 34, 517.
6. Lahtinen, T.; Kosonen, M.; Tiirola, M.; Vuento, M.; Oker-Blom, C. J. Ind.
Microbiol. Biotechnol. 2006, 33, 734.
Figure 3. Anti-biofilm activity of analogue 4d.
7. Rodrigues, C.; Takita, M.; Coletta-Filho, H.; Olivato, J.; Caserta, R.; Machado, M.;
de Souza, A. Appl. Microbiol. Biotechnol. 2008, 77, 1145.
8. Rigano, L.; Siciliano, F.; Enrique, R.; Sendin, L.; Filippone, P.; Torres, P.; Questa,
J.; Dow, J.; Castagnaro, A.; Vojnov, A.; Marano, M. Mol. Plant Microbe Interact.
2007, 20, 1222.
inhibition assays, 4d (Fig. 3), was therefore assayed for the ability
to disperse existing A. baumannii biofilms. Gratifyingly, 4d dis-
played significant dispersion activity with an EC50 value of
41.3 4.50 lM, making it one of the most potent known com-
9. Blackwell, H. E. Chem. Biol. 2005, 12, 721.
10. Davies, D. Nat. Rev. Drug Discov. 2003, 2, 114.
pound to date that disperses existing A. baumannii biofilms.22,32,33
In conclusion, a second generation oroidin library was synthe-
sized and assayed for anti-biofilm activity against the emerging
pathogen A. baumannii. Numerous derivatives of the library
showed greater potency than the previous first generation lead
compound DHS. Specifically, these derivatives are highlighted by
incorporation of an alkyl-based phenyl chain off the pyrrole ring
nitrogen or an N-arylated dibromopyrrole derivative. The most ac-
tive analogue, 4d, was also shown to have exceptional activity in
dispersing preformed A. baumannii biofilms, making it a notewor-
thy addition to the limited number of compounds which share this
ability.22,32,33 Future work in this area includes mechanistic studies
on how these compounds elicit their anti-biofilm effects and the
identification and development of other unique biologically active
scaffolds that incorporate a 2-aminoimidazole motif.
11. Falagas, M. E.; Karveli, E. A. Clin. Microbiol. Infect. 2007, 13, 117.
12. Shelburne, S.; Singh, K.; White, C.; Byrne, L.; Carmer, A.; Austin, C.; Graviss, E.;
Stager, C.; Murray, B.; Atmar, R. J. Clin. Microbiol. 2008, 46, 198.
13. Moro, M.; Nizzero, P.; Biancardi, A.; Baldan, R.; Scarpellini, P.; Curti, C.; Cichero,
P.; Ossi, C.; Mancini, N.; Colombo, S.; Marazzi, M.; Mazzuconi, R.; Cirillo, D. J.
Hosp. Infect. 2008, 68, 97.
14. Hawley, J. S.; Murray, C. K.; Griffith, M. E.; McElmeel, M. L.; Fulcher, L. C.;
Hospenthal, D. R.; Jorgensen, J. H. Antimicrob. Agents Chemother. 2007, 51, 376.
15. Forster, D.; Daschner, F. Eur. J. Clin. Microbiol. 1998, 17, 73.
16. Tomaras, A. P.; Dorsey, C. W.; Edelmann, R. E.; Actis, L. A. Microbiology 2003,
149, 3473.
17. Kawamura-Sato, K.; Wachino, J.; Kondo, T.; Ito, H.; Arakawa, Y. J. Antimicrob.
Chemother. 2008, 61, 568.
18. Choi, C.; Hyun, S.; Lee, J.; Lee, J.; Lee, Y.; Kim, S.; Chae, J.; Yoo, S.; Lee, J. Cell.
Microbiol. 2008, 10, 309.
19. Qi, C.; Malczynski, M.; Parker, M.; Scheetz, M. J. Clin. Microbiol. 2008, 46, 1106.
20. Reis, A.; Luz, D.; Tognim, M.; Sader, H.; Gales, A. Emerg. Infect. Dis. 2003, 9, 1025.
21. Mah, T. F. C.; O’Toole, G. A. Trends Microbiol. 2001, 9, 34.
22. Richards, J.; Huigens, R.; Ballard, T.; Basso, A.; Cavanagh, J.; Melander, C. Chem.
Commun. 2008, 14, 1698.
23. Richards, J.; Ballard, T.; Melander, C. Org. Biomol. Chem. 2008, 6, 1356.
24. Huigens, R. W.; Richards, J. J.; Parise, G.; Ballard, T. E.; Zeng, W.; Deora, R.;
Melander, C. J. Am. Chem. Soc. 2007, 129, 6966.
25. Richards, J.; Ballard, T.; Huigens, R.; Melander, C. ChemBioChem 2008.
doi:10.1002/cbic.200700774.
Acknowledgments
Financial support for this work is gratefully acknowledged from
Burroughs-Wellcome (predoctoral fellowship to J.J.R.), NCSU, Agile
Sciences, and the University of North Carolina Competitiveness Re-
search Fund.
26. Clausen-Kaas, N. Acta Chem. Scand. Ser. B 1952, 667.
27. Jaramillo, D.; Liu, Q.; Aldrich-Wright, J.; Tor, Y. J. Org. Chem. 2004, 69, 8151.
28. Bailey, D. M.; Johnson, R. E. J. Med. Chem. 1973, 16, 1300.
29. Olofson, A.; Yakushijin, K.; Horne, D. A. J. Org. Chem. 1998, 63, 1248.
30. O’Toole, G.; Kolter, R. Mol. Microbiol. 1998, 28, 449.
31. Rasmussen, T. B.; Givskov, M. Int. J. Med. Microbiol. 2006, 296, 149.
32. Rogers, S.; Melander, C. Angew. Chem. Int. Ed. 2008, 47, 5229.
33. Huigens, R.; Ma, L.; Gambino, C.; Moeller, P. D. R.; Basso, A.; Cavanagh, J.;
Wozniak, D. J.; Melander, C. Mol. Biosyst. 2008. doi:10.1039/b719989a.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.06.089.