Synthesis of (R)-norbgugaine and its potential as quorum sensing inhibitor against Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmcl.2013.02.051

(R)-Bgugaine is a natural pyrrolidine alkaloid from Arisarum vulgare, which shows antifungal and antibacterial activity. In this Letter, we have accomplished the simple synthesis of norbgugaine (demethylated form of natural bgugaine) employing Wittig olefination and cat. hydrogenation as the key steps and its biological studies are reported for the first time. The synthesized norbgugaine was evaluated for inhibition of quorum sensing mediated virulence factors (motility, biofilm formation, pyocyanin pigmentation, rhamnolipid production and LasA protease) in Pseudomonas aeruginosa wherein swarming motility is reduced by 95%, and biofilm formation by 83%.

Full text of DOI:10.1016/j.bmcl.2013.02.051

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2353–2356
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of (R)-norbgugaine and its potential as quorum sensing
inhibitor against Pseudomonas aeruginosa
a
b
b
a
a
Mahesh S. Majik a, , Deepak Naik , Chinmay Bhat , Santosh Tilve , Supriya Tilvi , Lisette D’Souza
⇑
a Bio-organic Chemistry Laboratory, CSIR – National Institute of Oceanography, Dona-Paula, Goa 403 004, India
b Department of Chemistry, Goa University, Taleigao Plateau, Goa 403 206, India
a r t i c l e i n f o
a b s t r a c t
Article history:
(R)-Bgugaine is a natural pyrrolidine alkaloid from Arisarum vulgare, which shows antifungal and antibac-
terial activity. In this Letter, we have accomplished the simple synthesis of norbgugaine (demethylated
form of natural bgugaine) employing Wittig olefination and cat. hydrogenation as the key steps and its
biological studies are reported for the first time. The synthesized norbgugaine was evaluated for inhibi-
tion of quorum sensing mediated virulence factors (motility, biofilm formation, pyocyanin pigmentation,
rhamnolipid production and LasA protease) in Pseudomonas aeruginosa wherein swarming motility is
reduced by 95%, and biofilm formation by 83%.
Received 4 January 2013
Revised 6 February 2013
Accepted 12 February 2013
Available online 22 February 2013
Keywords:
Norbgugaine
QS inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
Pseudomonas aeruginosa
Wittig reaction
Pyrrolidine alkaloid
Small molecules created by nature possessing impressive bio-
logical activity define a particularly important area of new chemi-
cal space for drug discovery research. Natural products have been
and continue to be a major source of new organic skeleton which
has always inspired and challenged the synthetic chemists.1 In fact,
the structural designs of natural products are highly conserved in
nature. And, it is well known fact that the natural products that
share a common scaffold with different functionalized skeleton
display different bioactivity profiles. Therefore, the scaffolds of nat-
ural products are considered as ‘privileged structures’ for further
modifications.2
2-epi-(+)-pachastrissamine (6) have been isolated and many of
them showed important biological activities.6 As a consequence,
numerous synthetic methods have been developed for the con-
struction of such ring systems.7
Nowadays, the combination of natural products and combinato-
rial chemistry has proven new ground towards development of
new classes of therapeutic agents. The treatment of infectious dis-
eases using new agents has become an important research area
worldwide. Bacteria can grow into surface-associated communities
(biofilm) and this growth cause a significant complexity to the suc-
cessful treatment of infectious diseases.8 In particular, Pseudomo-
nas aeruginosa is the Gram-negative bacterium responsible for
biofilm growth has attracted considerable attentions, as this path-
ogen are able to form biofilms in lungs, kidney, urinary tract, caus-
ing inflammations and septic shock in patients.9 Consequently, the
research for the development of new therapeutic agent and conve-
nient methods to attenuate bacterial biofilm growth has been
greatly stimulated by medical needs.10 A few natural products have
been witnessed to inhibit bacterial QS or biofilm growth. The hal-
ogenated furanones for example, brominated furanone 7 from the
marine macroalga Delisea pulchra and their derivatives have been
most intensively studied for their QS activities.11 In fact, various
QS inhibiting marine natural products are also exhibiting antifoul-
ing activity. The interference with bacterial quorum sensing (QS)
has been proposed as one potential approach for controlling bio-
fouling.12 The natural N-acyl homoserine lactone (AHL) 8 family
and their derivatives 9–11 can strongly modulate QS in Gram-neg-
ative bacteria (Fig. 2),13 and several of these AHLs also attenuate
The a-substituted amines especially a-substituted pyrrolidines,
piperidines and related ring systems with long chain hydrocarbons
are the common chemical entities that are found abundantly in
nature as well as in many bioactive alkaloids and pharmaceutically
relevant molecules.3 For example, (À)-bgugaine (1) and (À)-irniine
(2) (Fig. 1) are alkaloids isolated from the tubers of Arisarum vulg-
are, a species that is found on the Mediterranean coasts of Morocco
and Spain which show antibacterial activity against Gram-positive
bacteria and antimycotic activity against some Candida and Crypto-
coccus strains.4 (+)-Preussin (3), isolated from the fermentation
broths of Aspergillus ochraceus ATCC 22947 and then from Preussia
sp. possesses a broad spectrum antifungal activity against both fil-
amentous fungi and yeasts.5 Moreover, other long chain containing
compounds such as (+)-deoxyprosopinine (4), (+)-prosopinine (5),
⇑
Corresponding author. Tel.: +91 832 2450458; fax: +91 832 2450607.
E-mail addresses: majikms@gmail.com, mmajik@nio.org (M.S. Majik).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.051

2354
M. S. Majik et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2353–2356
HO
X
H
R
(CH2)11CH3
(R)-Norbgugaine
HO
CH3
N
N
N
H
(CH2)9
H
12
CH3
4
1 (-)-bgugaine,R = -(CH2)12CH3
(+)-deoxyprosopinine, X = H, H
Figure 3. (R)-Norbgugaine as a potential QS inhibitor.
5 (+)-prosopinine,
X = O
2
(-)-irniine, R = -(CH2)8Ph
OH
H
H
H
H2N
OH
a
OH
b
Ph
CHO
COOH
N
N
N
H
C9H19
N
R
14
13
15
Cbz
Cbz
O
CH3
(+)-preussin
6 2-epi-(+)-pachastrissamine, R = -(CH2)12CH3
3
d
c
CH3(CH2)11CH2+PPh3Br-
CH3(CH2)11CH2Br
Figure 1. Selected naturally occurring bioactive compounds with long chain
hydrocarbon.
17
16
(CH2)11CH3
biofilm growth in P. aeruginosa.14 Recently, Blackwell and co-work-
ers observed that the AHL-derived biofilm inhibitors failed to re-
move the preformed biofilms.15 Hence, the applicability of AHLs
as biofilm or QS inhibitor involves few drawbacks interms of their
stability i.e. hydrolytic instability of the lactone group to furnish
inactive hydrolysed AHL.15 However, Horikawa and co-workers
have demonstrated that the presence of the long acyl side chain
H
H
e
(CH2)11CH3
(R)-Norbgugaine
N
N
12
H
18
Cbz
Scheme 1. Reagents and conditions: (a) (i) LiAlH4, THF, reflux, 8 h (88%), (ii) K2CO3,
Benzyl chloroformate, CH3CN, À10–0 °C, 2 h (83%); (b) PCC, CH2Cl2, rt, 5 h (74 %); (c)
PPh3, toluene, reflux, 24 h; (d) n-BuLi, Et2O, N-carbobenzyloxyprolinal 15 (two steps
50%); (e) H2, Pd/C, EtOH, rt, 8 h (80%).
with hydrophobic end is crucial for QS activity of N-acylated-L-
homoserine lactone (AHL).16 Despite the extent of detrimental ef-
fect of biofilms, examples of structural scaffolds that inhibit biofilm
formation are rare in the literature.17 Corresponding to these facts,
the development of alternative hydrolytically stable molecular
scaffolds for QS or biofilm inflection represents an extremely
important goal.
facilitates the deprotection as well as double bond reduction in sin-
gle operation at the final stage of synthesis and thus adding advan-
tage in purification as well as conciseness in approach. Our
synthetic strategy is depicted in Scheme 1.
In the view of these observations and our research interest to-
wards the syntheses of small bioactive molecules,18 have encour-
aged us to undertake the synthesis of pyrrolidine derivative 12
and demonstrate its potential as QS inhibitor (Fig. 3). We reasoned
that combining structural attributes of these compounds (Figs. 1
and 2) into simple molecular scaffold could give molecule with
heightened QS properties. Herein, we have described the synthesis
of norbgugaine, the N-demethylated form of natural pyrrolidine
alkaloid and explored its biological properties associated with quo-
rum sensing inhibition in Pseudomonas aeruginosa.
The synthesis commenced from commercially available L-pro-
line 13 which was converted to N-carbobenzyloxy prolinal 15
using the literature procedure.20 Treatment of tridecyl bromide
16 with PPh3 in refluxing toluene for 24 h gave the corresponding
phosphonium salt 17 which was further treated with n-BuLi to
generate in situ the desired phosphorane. Furthermore, the freshly
prepared (S)-N-carbobenzyloxy prolinal was subjected to Wittig
olefination in one-pot without isolating the intermediate phos-
phorane (Scheme 1) to give olefin 18. The double bond geometry
of olefin was assumed to be cis based on 1H NMR values [5.33–
5.36 (m, 2H)]. In the end, the Cbz–deprotection and double bond
reduction was achieved in one-pot by reaction of olefin 18 with
H2, Pd/C in ethanol, which resulted in a facile formation of the title
Previously, we had reported the facile approach to the synthesis
of (R)-bgugaine using Wittig olefination.19 Herein, we have utilized
the analogous approach for the synthesis of norbgugaine 12,
wherein Cbz is a choice of protecting group instead of Boc. This
Br
O
O
O
O
O
Br
C11H23
C9H19
N
H
N
H
O
O
O
O
9
7
8
Br
N-acyl-L-homoserine lactones (AHL)
Brominated furanone
CH3
CH3
CH3
O
O
O
O
OH
11
10
Alkylated butenolides
Figure 2. Structures of some natural furanones, and synthetic analogue in quorum sensing systems. Substructures of interest to present work are shown in bold.

M. S. Majik et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2353–2356
2355
Figure 4. (A) Effect of norbgugaine 12 on growth of P. aeruginosa. (B) Effect of norbgugaine 12 on motilities in P. aeruginosa. (C) Effect of norbgugaine 12 and salicylic acid on
biofilm formation in P. aeruginosa.
compound 12. This completed the first synthesis of norbgugaine in
6 steps using easily available -proline as the starting material.
0 to 0.5 mM.23 The compound 12 showed a minimal effect on the
growth rate of P. aeruginosa as indicated by growth curve experi-
ment (Fig. 4A). Whereas, it was found to exhibit significant effect
on motility, particularly swarming as well as swimming motilities
in P. aeruginosa (Fig. 4B). Former was reduced by 95% (p <0.0005)
and the latter by 33% (p <0.0005). However, no significant reduc-
tion was seen in twitching motility. The compound also showed
significant reduction in rhamnolipid production by 42%, pyocyanin
production by 37% and Las A production by 34% (p <0.0005)
(Table 1).
Furthermore, the effect of norbgugaine 12 on biofilm formation
in P. aeruginosa was assessed via static biofilm inhibition assay. P.
aeruginosa strain demonstrated decreasing biofilm density with
increasing concentration of norbgugaine 12 from 0.0 to 4.0 mM
(Fig. 4C). A leveling off in decreasing biofilm densities with an aver-
L
Quorum sensing (QS) is cell to cell signaling mechanism that
enables bacteria to collectively control gene expression and regu-
late diverse physiological processes.21 QS is involved in expression
of virulence genes in various bacteria, indicating the possible role
of quorum sensing as a drug target. Molecules that target quorum
sensing act by disarming pathogens by attenuating virulence fac-
tors rather than killing them.22 This feature could greatly reduce
the emergence of multi-antibiotic resistant bacteria often associ-
ated with traditional antimicrobial chemotherapy. In the present
study, the synthesized norbgugaine 12 was evaluated for its poten-
tial inhibition of QS mediated virulence factors (motility, pyocya-
nin pigmentation, rhamnolipid production and LasA protease) in
P. aeruginosa (ATCC 27853) at a variable concentration range from
Table 1
Effect of norbgugaine 12 on QS mediated virulence factors (swarming, pyocyanin pigmentation, biofilm formation, rhamnolipid production and LasA) in P. aeruginosa in
comparison with salicylic acid24
Virulence parameters (% inhibition)
Compound
Swarming motility
Swimming motility
Twitching motility
Pyocyanin pigmentation
Biofilm formation
Rhamnolipid production
LasA
Nor-bgugaine
Salicylic acid
95
60
33
40
15
35
37
83
48
42
34
NA⁄
NA⁄
NA⁄
NA⁄—not applicable

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M. S. Majik et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2353–2356
Science of Synthesis and Its Impact on Society; Wiley: New York, 2008; (c)
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age percentage of 83% was observed at a concentration of 1.8 mM
and above. In comparison with salicylic acid,24 a well known bio-
film inhibitor, our synthesized compound 12 showed two times
better biofilm inhibition i.e. norbgugaine showed decrease in bio-
film formation by 83% while salicylic acid reduced by 48% at
4 mM. Additionally, as P. aeruginosa growth was not inhibited by
the pyrrolidine derivative 12 (Fig. 4A), it is possible to conclude
that the inhibition on virulence factor production is caused by an
anti-QS mode of action.
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In summary, a concise and simple synthesis of (R)-norbgugaine
has been achieved for the first time using L-proline through Wittig
reaction as a crucial step. Furthermore, purification by column
chromatography was required in only one step, which makes an
efficient route to this alkaloid. The majority of infectious diseases
are associated with the formation of biofilms. In this study, we
found that norbgugaine, inhibits various motilities, pyocyanin pig-
mentation, LasA protease rhamnolipid production, and biofilm for-
mation in Pseudomonas aeruginosa. Swimming repression suggests
that norbguganine affects the flagella related functions, which
might lead to swarming inhibition. Rhamnolipids are known to
modulate swarming motility in P. aeruginosa25 and repression of
rhamnolipid formation affects colony wetness which may be the
reason for almost the complete inhibition of swarming motility ob-
served in the present case. Biofilm formation and swarming motil-
ity have been shown to be closely connected and regulated by a
large set of overlapping genes. Moreover, swarming motility has
been shown to be important for the early stages of biofilm forma-
tion. Therefore, it can be expected that the biofilm repression in the
presence of norbgugaine may be mediated by a mechanism similar
to that for swarming inhibition. Efforts are on in our laboratory to
identify the exact mode of action of norbgugaine in P. aeruginosa
and give the role QS plays in P. aeruginosa virulence. Thus, norbg-
ugaine has immense potential to act as a next generation therapeu-
tic agent.
15. (a) Frei, R.; Breitbach, A. S.; Blackwell, H. E. Angew. Chem., Int. Ed. 2012, 51,
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Acknowledgments
16. Horikawa, M.; Tateda, K.; Tuzuki, E.; Ishii, Y.; Ueda, C.; Takabatake, T.; Miyairi,
S.; Yamaguchi, K.; Ishiguro, M. Bioorg. Med. Chem. Lett. 2006, 16, 2130.
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18. (a) Majik, M. S.; Tilve, S. G. Tetrahedron Lett. 2010, 51, 2900; (b) Majik, M. S.;
Parameswaran, P. S.; Tilve, S. G. J. Org. Chem. 2009, 74, 6378; (c) Majik, M. S.;
Parameswaran, P. S.; Tilve, S. G. J. Org. Chem. 2009, 74, 3591; (d) Majik, M. S.;
Parameswaran, P. S.; Tilve, S. G. Helv. Chim. Acta 2008, 91, 1500.
19. Majik, M. S.; Parameswaran, P. S.; Tilve, S. G. J. Chem. Res. 2008, 121.
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23. See Supplementary data for detail procedure.
The authors thank the Director, CSIR-National Institute of
Oceanography for constant encouragement. Financial assistance
provided by the Ocean Finder Grant No. OLP 1201 is highly
acknowledged. Author (M.S.M.) is grateful to CSIR–NIO for the
award of Scientist Fellow-QHS.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
051.
24. Chow, S.; Gu, K.; Jiang, L.; Nassour, A. J. Exp. Microbiol. Immunol. 2011, 15, 22.
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