Synthesis of cephalosporin-3′-diazeniumdiolates: Biofilm dispersing NO-donor prodrugs activated by β-lactamase

Article abstract of DOI:10.1039/c3cc40869h

Use of biofilm dispersing NO-donor compounds in combination with antibiotics has emerged as a promising new strategy for treating drug-resistant bacterial biofilm infections. This paper details the synthesis and preliminary evaluation of six cephalosporin

Full text of DOI:10.1039/c3cc40869h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Synthesis of cephalosporin-30-diazeniumdiolates:
biofilm dispersing NO-donor prodrugs activated
by b-lactamase†
Cite this: Chem. Commun., 2013,
49, 4791
Received 1st February 2013,
Accepted 15th April 2013
Nageshwar Rao Yepuri,a Nicolas Barraud,b Nasim Shah Mohammadi,b
Bharat G. Kardak,a Staffan Kjelleberg,bc Scott A. Ricebc and Michael J. Kelso*a
DOI: 10.1039/c3cc40869h
www.rsc.org/chemcomm
Use of biofilm dispersing NO-donor compounds in combination
with antibiotics has emerged as a promising new strategy for
treating drug-resistant bacterial biofilm infections. This paper details
the synthesis and preliminary evaluation of six cephalosporin-30-
diazeniumdiolates as biofilm-targeted NO-donor prodrugs. Each
of the compounds is shown to selectively release NO following
reaction with the bacteria-specific enzyme b-lactamase and to
trigger dispersion of Pseudomonas aeruginosa biofilms in vitro.
Scheme 1 Proposed mechanism of b-lactamase-triggered NO release and
biofilm dispersion by cephalosporin-30-diazeniumdiolates (e.g., 1).
The discovery of clinically useful therapeutics and novel treatment spontaneous NO donors) since they should localise NO to
strategies against bacterial biofilm infections is one of the greatest biofilm infection sites and minimise exposure of host tissues
challenges in modern infectious disease control.1 In 2006 we to NO (and associated side-effects). We recently described a
reported that low concentrations of the biologically ubiquitous rationally designed cephalosporin-30-diazeniumdiolate NO-donor
gas nitric oxide (NO) trigger biofilm dispersion in the important prodrug 1 which selectively releases NO upon contact with the
human pathogen Pseudomonas aeruginosa and demonstrated that bacterial enzyme b-lactamase (proposed mechanism shown in
the spontaneous NO-donor sodium nitroprusside (SNP) greatly Scheme 1).5 Compound 1 was shown to effect NO-dependent
enhances the efficacy of antibacterial compounds (e.g. tobramycin) P. aeruginosa biofilm dispersal and to act synergistically with
in the removal of P. aeruginosa biofilms.2 Follow-up studies front-line antibiotics (tobramycin and ciprofloxacin) in clearing
showed that NO levels in the pM and low nM range mediate bacterial biofilms. This paper details synthetic efforts towards 1
dispersal in several other single- and multi-species bacterial and and demonstrates the versatility of the chemistry in producing
yeast biofilms3 and that the effects correlate with increases in five analogues carrying variations in the cephalosporin 7-acyl-
bacterial phosphodiesterase activity and associated decreases in amido side chain (R1) and 30-diazeniumdiolate NO-donor portions
cyclic-di-GMP levels.4 These combined results have unveiled a (R2). Each of the analogues is shown to release NO upon reaction
putative new anti-biofilm strategy which uses low concentrations with b-lactamase and to disperse P. aeruginosa biofilms in vitro.
of NO-donor compounds in combination with antibiotics to clear
bacterial biofilm infections.
Alkylation of sodium diazeniumdiolate salts with ester protected
30-chloro-cephalosporins appeared as a viable route towards
Biofilm-targeted prodrugs capable of releasing NO only cephalosporin-30-diazeniumdiolates since terminal oxygen
after reaction with a bacteria-specific enzyme represent ideal alkylation of sodium diazeniumdiolates with alkyl halides was
NO-donors for clinical anti-biofilm applications (as opposed to well established.6 Initial investigations with diphenylmethyl
(DPM)- or p-methoxybenzyl (PMB)-protected 30-chloro-cephalosporin
esters 2 showed that alkylation reactions with sodium diazenium-
diolate 3 did not proceed. Finkelstein conversion of 2 to the
a School of Chemistry, University of Wollongong, NSW 2522, Australia.
E-mail: mkelso@uow.edu.au; Fax: +61 2 4221 4287; Tel: +61 2 4221 5085
b School of Biotechnology and Biomolecular Sciences and Centre for Marine
corresponding allylic iodides (NaI–acetone) followed by purification
and reaction with 3 did produce the desired alkylation products 4
under several conditions (ESI,† Scheme S1), however the yields were
consistently low due to double bond migration and formation of
inseparable mixtures containing the undesired D2-isomers 4a as the
major product.7 Addition of 1.0 mol eq. of solid 3 in situ to freshly
prepared allylic iodide–acetone solutions of 2 afforded mixtures
Bio-Innovation, University of New South Wales, 2052, Australia
c Singapore Centre on Environmental Life Sciences Engineering,
Nanyang Technological University, Singapore
† Electronic supplementary information (ESI) available: (a) Experimental proce-
dures, characterisation data and 1H NMR spectra for all compounds. (b) Experi-
mental procedures for amperometric NO release measurements and microtitre
plate biofilm dispersion assays. See DOI: 10.1039/c3cc40869h
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 4791--4793 4791

View Article Online
Communication
ChemComm
PMB-deprotection using neat trifluoroacetic acid–anisole at
0 1C proceeded smoothly for all analogues affording the pure
cephalosporin-30-diazeniumdiolate free carboxylic acids 1, 15–19
in 36–93% yields (Fig. 1). The free acids 1, 15, 17 and 18 could be
converted to their water soluble potassium salts (21, 20, 23 and
24, respectively) by stirring with 1.0 mol eq. aqueous KOH at 0 1C
followed by freeze-drying. Applying the same procedure with
acids 16 and 19 led to decomposition.
Release of NO from cephalosporin-30-diazeniumdiolate free
acids 1, 15–19 in the presence of a commercial b-lactamase
(penicillinase, Sigma) was studied amperometrically at pH 7 in
100 mM Tris buffer (Fig. 2). Compound 18 was found to be
stable over 15 min following addition to buffer, as indicated by
an absence of detectable NO. Compounds 1 and 17 showed
evidence of slight decomposition producing low but detectable
levels of NO, presumably through b-lactam hydrolysis and
expulsion of the NO donor. Compounds 15, 16 and 19 appeared
to be intrinsically less stable generating 2–3 fold higher levels of
NO than 1 or 17 upon addition to buffer.
Treatment with b-lactamase (0.1 U mLꢀ1) triggered immediate
release of NO from each of the acids. Compounds 1 and 18 reached
steady-state NO concentrations of B2 mM within 15 min, while
acids 15, 16, 17 and 19 produced higher NO levels, reaching
steady-state concentrations of B3–4 mM over the same period.
Fig. 1 Synthesis of cephalosporin-30-diazeniumdiolates.
wherein the D3-isomer predominated (i.e., 4 : 4a = 80 : 20 for Adding a further 0.2 U mLꢀ1 of b-lactamase to each of the acids
DPM-protected 2, 90 : 10 for PMB-protected 2). The low solubility of led to additional B2-fold increases in NO and reestablishment
3 in acetone may have reduced solution basicity and minimised of steady-state NO concentrations within 15 min. Formation of
deprotonation at the cephalosporin 2-position. It is well established steady-state NO concentrations and release of NO upon addition
that deprotonation here triggers double bond migration during of a second aliquot of enzyme are consistent with the reaction of
substitution reactions with basic nucleophiles and cephalosporins the b-lactamase with cephalosporin-30-diazeniumdiolates leading
carrying leaving groups at the 30-position.7
to enzyme inhibition. Quenching of the amperometric response
The optimised alkylation conditions were used to prepare upon addition of the free radical scavenger 2-phenyl-4,4,5,5-
two analogues of 4 carrying variations in the 7-acylamido side tetramethylimidazoline-1-oxyl-3-oxide (PTIO) confirmed that
chain (R1: compounds 6, 8, Fig. 1). Compound 4, bearing a the observed signals were due to NO.
thiophenacetyl group at R1 (as found in Cefalotin and other
cephalosporins that have been used clinically), was obtained
pure in 85% isolated yield from 2 following silica gel chromato-
graphy and recrystallisation from MeOH or EtOH. Compound
6, carrying a phenacetyl group at R1 (as found in Cephaloram),
was obtained in 75% yield from 5, while the yield of 8 (R1
=
1-tetrazolylacetyl, as found in Cefazolin and others) was lower
at 14% (from 7) due to appreciable formation of the D2-isomer
8a (42% isolated yield).
It was of particular interest to explore whether diazenium-
diolate salts other than 3 (NO release t1/2 = 2.0 min)8 could
be attached to the cephalosporin scaffold, especially those
with similarly short NO-release half-lives. We predict that
diazeniumdiolates with short half-lives should work best in
clinical anti-biofilm applications since rapid NO release from
the diazeniumdiolate anions following expulsion from the
cephalosporin prodrug would be necessary to limit their diffusion
away from biofilms before releasing NO. Reaction of 30-chloro-
Fig. 2 Amperometric characterisation of NO release from cephalosporin-30-
diazeniumdiolate free acids 1, 15–19 in the presence of b-lactamase. Arrows
indicate addition of the following to reaction vials containing 10 mL Tris buffer
(100 mM) at pH 7.0: (a) 10 mL of 100 mM cephalosporin-30-diazeniumdiolate
free acid, (b) 10 mL 100 U mLꢀ1 b-lactamase, (c) 20 mL 100 U mLꢀ1 b-lactamase,
(d) 80 mL of 10 mM free radical scavenger PTIO.
cephalosporin PMB ester 5 with sodium diazeniumdiolate 9 (t1/2
=
2.8 s)8 under the optimised conditions afforded alkylated product
10 in 66% yield. Reactions of 5 with salts 11 (t1/2 = 1.9 min)9 and
13 (t1/2 = 2.0 min)9 also produced the desired adducts 12 and 14 in
39% and 80% yields, respectively.
c
4792 Chem. Commun., 2013, 49, 4791--4793
This journal is The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
vulnerable planktonic bacteria can be cleared by conventional
antibiotics working in concert with host immune defences.2–4 It
is important to note that NO at the concentrations used are not
toxic to bacteria, but rather induce a genetically programmed
dispersion response. The current work has advanced the strategy
significantly towards clinical utility in describing a novel class of
drug-like biofilm-targeted NO-donor prodrugs (i.e., cephalos-
porin-30-diazeniumdiolates) that make use of the known ten-
dency of cephalosporins to eject leaving groups (in this case
diazeniumdiolate NO donors) from the 30-position via conjugate
elimination12 following b-lactam cleavage. Six examples from
the class were prepared using similar chemistry and each
was shown to release NO upon contact with b-lactamase and
to trigger biofilm dispersion in P. aeruginosa, an important
pathogen responsible for recalcitrant and often fatal broncho-
pulmonary biofilm infections, especially in cystic fibrosis (CF)
patients.11 Studies aimed at identifying optimal cephalosporin-
30-diazeniumdiolate development candidates for these and
other types of biofilm-based chronic infections are on-going
in our laboratories.
Fig. 3 Dispersal of P. aeruginosa biofilms by cephalosporin-30-diazeniumdiolate
free acids 1, 15–19. P. aeruginosa PAO1 biofilms grown in microtiter plates were
pre-treated with imipenem (0.3 mg mLꢀ1) before exposing to various concentra-
tions of compounds (15 min) and quantifying the remaining biofilm mass by
crystal violet staining and measurement of OD550. (n = 2) Control biofilms
treated with imipenem alone produced OD550 readings of B3–3.5.
Dispersion of P. aeruginosa biofilms by cephalosporin-30-
diazeniumdiolate free acids 1, 15–19, along with the D2-isomer
16a, was examined in vitro using microtiter plate biofilm assays
(Fig. 3).5 Briefly, P. aeruginosa PAO1 wild type biofilms were
grown in 24-well plates containing sub-inhibitory imipenem
(0.3 mg mLꢀ1) to induce b-lactamase expression. After 6 h, the
biofilms were treated with test compounds and incubated
for 15 min before washing, staining with crystal violet and
quantifying the remaining biofilms by measuring OD550 of the
homogenized suspensions.
We thank the Australian National Health and Medical
Research Council (NHMRC, Project Grant 568841), the University
of Wollongong and the University of New South Wales for funding
this work.
Notes and references
1 G. Ramage, S. Culshaw, B. Jones and C. Williams, Curr. Opin. Infect.
Dis., 2010, 23, 560.
2 N. Barraud, D. J. Hassett, S.-H. Hwang, S. A. Rice, S. Kjelleberg and
J. S. Webb, J. Bacteriol., 2006, 188, 7344.
Compounds 1 and 15–19 all showed dose-dependent biofilm
dispersion responses in the range 5–100 mM. Compounds 1 and
17 appeared as the most potent members of the series reducing
biofilm mass at 5 mM by 31% and 28%, respectively. The other
compounds showed no significant effect at this concentration.
At 10 mM, compound 1 reduced biofilm mass by 78%, with only
slight further reductions being observed at higher concentrations
(87% at 50 mM, 91% at 100 mM). A similar dose dependency was
observed with 17. While less potent, the other four analogues 15,
16, 18 and 19 all reduced biofilms by more than 70% at 100 mM.
As expected, the D2-isomer of 16 (i.e., 16a) showed no dispersion
effect at any concentration, consistent with its inability to undergo
the conjugate elimination reaction to expel the diazeniumdiolate
anion following b-lactam cleavage.
Bacteria encased in biofilms are known to exhibit upwards
of 10–1000-fold higher resistance to biocides and traditional
antibiotics and to be less susceptible to host immune defences
than their free-swimming planktonic counterparts.10 As a
result, chronic bacterial infections tend to be biofilm-based.
In our anti-biofilm strategy, low concentrations of NO-donors
are used to first trigger biofilm dispersion such that the more
3 N. Barraud, M. V. Storey, Z. P. Moore, J. S. Webb, S. A. Rice and
S. Kjelleberg, Microb. Biotechnol., 2009, 2, 370.
4 N. Barraud, D. Schleheck, J. Klebensberger, J. S. Webb, D. J. Hassett,
S. A. Rice and S. Kjelleberg, J. Bacteriol., 2009, 191, 7333.
5 N. Barraud, B. G. Kardak, N. R. Yepuri, R. P. Howlin, J. S. Webb,
S. N. Faust, S. Kjelleberg, S. A. Rice and M. J. Kelso, Angew. Chem.,
Int. Ed., 2012, 51, 9057.
6 T. B. Cai, D. Lu, M. Landerholm and P. G. Wang, Org. Lett., 2004,
6, 4203; X. Wu, X. Tang, M. Xian and P. G. Wang, Tetrahedron Lett.,
2001, 42, 3779; X. Tang, M. Xian, M. Trikha, K. V. Honn and
P. G. Wang, Tetrahedron Lett., 2001, 42, 2625; J. E. Saavedra,
T. M. Dunams, J. L. Flippen-Anderson and L. K. Keefer, J. Org.
Chem., 1992, 57, 6134.
7 W. F. Richter, Y. H. Chong and V. J. Stella, J. Pharm. Sci., 1990,
79, 185; C. F. Murphy and R. E. Koehler, J. Org. Chem., 1970,
35, 2429; S. Mobashery and M. Johnson, J. Org. Chem., 1986,
51, 4723.
8 http://star.ncifcrf.gov/nitricoxide/default.aspx?url=searchNO.
aspx&st=3.
9 J. E. Saavedra, M. N. Booth, J. A. Hrabie, K. M. Davies and
L. K. Keefer, J. Org. Chem., 1999, 64, 5124.
10 D. Davies, Nat. Rev. Drug Discovery, 2003, 2, 114; D. J. Musk and
P. J. Hergenrother, Curr. Med. Chem., 2006, 13, 2163.
11 N. Høiby, T. Bjarnsholt, M. Givskov, S. Molin and O. Ciofu, Int. J.
Antimicrob. Agents, 2010, 35, 322.
12 T. P. Smyth, M. E. O’Donnell, M. J. O’Connor and J. O. St Ledger,
Tetrahedron, 2000, 56, 5699.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 4791--4793 4793