Synthesis and properties of benzo[a]phenoxazinium chalcogen analogues as novel broad-spectrum antimicrobial photosensitizers

Article abstract of DOI:10.1021/jm060153i

The goal of this investigation was to develop improved photosensitizers for use as antimicrobial drugs in photodynamic therapy of localized infections. Replacement of the oxygen atom in 5-(ethylamino)-9-diethylaminobenzo[a] phenoxazinium chloride (1) with

Full text of DOI:10.1021/jm060153i

J. Med. Chem. 2006, 49, 5291-5299
5291
Synthesis and Properties of Benzo[a]phenoxazinium Chalcogen Analogues as Novel
Broad-Spectrum Antimicrobial Photosensitizers
,#
James W. Foley,*,† Xiangzhi Song,† Tatiana N. Demidova,‡,§ Fatima Jilal,‡,| and Michael R. Hamblin*,‡,
Rowland Institute at HarVard, HarVard UniVersity, Cambridge, Massachusetts 02142, Wellman Center for Photomedicine, Massachusetts
General Hospital, Boston, Massachusetts 02114, Graduate Program in Cell Molecular and DeVelopmental Biology, Sackler School of Graduate
Biomedical Sciences, Tufts UniVersity School of Medicine, Boston, Massachusetts 02111, Aga Khan Medical School, Karachi, Pakistan,
Department of Dermatology, HarVard Medical School, Boston, Massachusetts 02115, and HarVard-MIT DiVision of Health Sciences and
Technology, 77 Massachusetts AVenue E25-519, Cambridge, Massachusetts 02139.
ReceiVed February 10, 2006
The goal of this investigation was to develop improved photosensitizers for use as antimicrobial drugs in
photodynamic therapy of localized infections. Replacement of the oxygen atom in 5-(ethylamino)-9-
diethylaminobenzo[a]phenoxazinium chloride (1) with sulfur and selenium afforded thiazinium and
selenazinium analogues 2 and 3, respectively. All three dyes are water soluble, lipophilic, and red light
absorbers. The relative photodynamic activities of the chalcogen series were evaluated against a panel of
prototypical pathogenic microorganisms: the Gram-positive Enterococcus faecalis, the Gram-negative
Escherichia coli, and the fungus Candida albicans. Selenium dye 3 was highly effective as a broad-spectrum
antimicrobial photosensitizer with fluences of 4-32 J/cm2 killing 2-5 more logs of all cell types than
sulfur dye 2, which was slightly more effective than oxygen analogue 1. These data, taken with the findings
of uptake and retention studies, suggest that the superior activity of selenium derivative 3 can be attributed
to its much higher triplet quantum yield.
Introduction
evaluation, the PSs used in these studies, hematoporphyrin
derivative (HPD) and Photofrin (a purified fraction of HPD),
were found to suffer from several drawbacks: (a) both have
low coefficients of absorption in the therapeutic window, the
spectral region where light penetrates tissue maximally (600-
900 nm); (b) both consist of a complex mixture of porphyrin
ether and ester monomers and oligomers; and most significantly,
(c) they both cause prolonged cutaneous photosensitization.9
Mechanistically, these drugs cause tumor destruction primarily
by shutting down the microvasculature supporting the tumor
and to a lesser extent by direct photosensitization of the tumor
cells.10
Photodynamic therapy (PDTa) is a medical treatment that uses
the energy of actinic light to selectively inactivate diseased
tissues and infectious pathogens.1 Therapy involves the systemic
or topical administration of a target localizing, nontoxic dye or
photosensitizer (PS) that becomes, or in reaction with molecular
oxygen generates, a cytotoxin when illuminated with light of
the appropriate wavelength.2,3 Because the power and safety of
the method derive from its dual selectivity, that is, inactivation
occurs only where the PS and light are simultaneously present,
effective treatment relies on the availability of PSs having a
high degree of specificity and affinity for selected biological
targets.4 The discovery of PDT dyes having these characteristics
continues to be the focus of considerable interest and research
activity.5
The concept of PDT is not new, having been practiced since
at least the 15th century in the Middle East, where ingested
naturally occurring psoralen PS and sunlight were used to treat
vitiligo.6 Although PDT was rediscovered over 100 years ago
as a means for killing microorganisms, it remained a laboratory
curiosity until the 1960s when the method was shown to be a
safe and efficacious treatment for solid tumors in humans.7,8
However, under the scrutiny of experimental and clinical
The demonstrated potential of PDT as a powerful new
modality for treating localized neoplasia, coupled with the
recognized limitations of HPD and Photofrin, provided strong
incentives for the development of more efficacious second
generation PDT agents. Owing to the successes reported
regarding the use of HPD, it is perhaps not surprising that most
attempts to design these improved drugs have centered on
members of the extended porphyrin family possessing a
tetrapyrrole backbone (i.e., chlorins, purpurins, phthalocyanines,
etc.).5 Less consideration has been paid to an attractive,
contrasting approach that is based on the hypothesis that drugs
derived from structurally dissimilar families with inherently
different physicochemical and pharmacological properties would
exhibit favorable efficacy, toxicity, and mechanistic profiles and,
as a result, prove to be complementary to one another. To this
end, we have investigated the PDT properties of a series of dyes
belonging to the benzophenoxazinium family of cationic chro-
mophores.11,12 We were drawn to this class of dyes because
Lewis et al. had reported that the textile dye Nile Blue A (NBA)
and a select set of derivatives, when administered orally, strongly
stained solid murine tumors while imparting little or no
coloration to the surrounding healthy tissues; significantly, the
dyes appeared to be relatively innocuous, were efficient absorb-
* Corresponding author. Phone: 617-497-4677. Fax: 617-497-4627.
E-mail: foley@rowland.harvard.edu (J.W.F.). Phone: 617-716-6182.
Fax: 617-726-8566. E-mail: hamblin@helix.mgh.harvard.edu (M.R.H.).
† Rowland Institute at Harvard, Harvard University.
‡ Massachusetts General Hospital.
§ Tufts University School of Medicine.
| Aga Khan Medical School.
Department of Dermatology, Harvard Medical School.
# Harvard-MIT Division of Health Sciences and Technology.
a Abbreviations: EtNBA, 5-(ethylamino)-9-diethylaminobenzo[a]phe-
noxazinium chloride; EtNBS, 5-(ethylamino)-9-diethylaminobenzo[a]phe-
nothiazinium chloride; EtNBSe, 5-(ethylamino)-9-diethylaminobenzo[a]-
phenoselenazinium chloride; HPD, hematoporphyrin derivative; NBA, nile
blue A; PDI, photodynamic inactivation; PDT, photodynamic therapy; PS,
photosensitizer; SDS, sodium dodecyl sulfate.
10.1021/jm060153i CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/29/2006

5292 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Foley et al.
ers of 600-700 nm light, and were rapidly eliminated (24 h
for such indications as periodontitis27 and tooth cavity steriliza-
tion.28 Nevertheless, the use of these dyes suffers constraints
that might impair their activities. First, they must be used in
relatively high concentrations to achieve multiple logs of cell
killing with reasonable light fluences. Second, they are much
more effective when the dye remains present in the incubation
mixture during illumination, compared to that when the microbes
are washed free from unbound dye before light delivery.22 Thus,
further efforts need to be directed to the development of more
tenacious, active cationic antibacterial PDT drugs.
The main purpose of this article is to present the results of a
study designed to evaluate the broad-spectrum antimicrobial
sensitizing efficacies of a series of three chalcogen (O, S, Se)
substituted benzo[a]phenoxazinium analogues against a panel
of prototypical human pathogenic microbes consisting of the
Gram-positive bacterium Enterococcus faecalis, the Gram-
negative bacterium Escherichia coli, and the yeast Candida
albicans. We relate these findings to pertinent physical and
photophysical properties of all three chalcogen dyes. Addition-
ally, because EtNBSe (3) is the first and only benzo[a]-
phenoselenazinium dye that has been described in the literature,
we herein present the synthetic approach used to prepare this
novel PS.
post administration).13,14
Although an investigation of the PDT potential of the Lewis
dyes found them to be inefficient PSs both in vitro and in vivo,
they nonetheless proved to be a valuable starting point for the
rational design of drugs with improved photoactivities. One of
the most effective anticancer PDT agents discovered early in
this program involved the replacement of the O atom of an
ethylated-NBA analogue (1, EtNBA) with a S atom to give the
novel PS 5-(ethylamino)-9-diethylaminobenzo[a]phenothiazi-
nium chloride (2, EtNBS).12 This drug, when activated with 650
nm light, showed good efficacy against several murine sarcomas
(EMT-6, RIF), a human carcinoma (FaDu), and a rat glioma
(9L);15 in the case of one study using the EMT-6 model, 100%
of the animals showed a complete response.16 Mechanistic
studies revealed that EtNBS (2) rapidly accumulated in cancer
cells and, unlike HPD, caused inactivation primarily by directly
killing the tumor cells.16 An evaluation of this PDT drug
culminated in a preclinical study that found EtNBS (2)-PDT to
be highly effective against selected spontaneous tumors in pet
cats and dogs.17 In general, the drug was well tolerated and
caused no residual photosensitization 5 days post-treatment for
both species.
We attributed the higher level of phototoxicity shown by
EtNBS (2) compared to that of its O analogue EtNBA (1) to
the heavy atom effect, wherein the increased spin-orbital
coupling constants associated with atoms having high atomic
numbers increases excited triplet state formation, the species
thought to mediate cytotoxicity.6 Consequently, as an extension
of this strategy, we were motivated to synthesize EtNBSe (3),
a chalogen analogue having a still heavier Se atom in place of
the centrally located S atom of EtNBS (2). In an in vitro study
using the EMT-6 mammary sarcoma cell line, a comparative
phototoxicity evaluation of EtNBSe (3) with EtNBS (2), EtNBA
(1), and Photofrin (all at 0.5 mM extracellular concentration)
found that the light dose required to kill 50% of the cells was
<0.5 J/cm2 for EtNBSe (3) and 2.0 J/cm2 for EtNBS (2),
whereas the dose to achieve the same response for EtNBA (1)
and Photofrin could not be determined because of their low
phototoxicities.18 The relative photoactivities of the three
members of the chalogen series correlated with their abilities
to generate single oxygen and with limited intracellular uptake
for Photofrin. Thus, although never studied as rigorously as
EtNBS (2), the data gathered for EtNBSe (3) suggests that it
has the potential to be a superior PDT drug.
Experimental Section
Chemical Synthesis. All solvents were reagent grade and
used as received. 5-Ethylamino-9-diethylaminobenzo[a]phe-
noxazinium (1, EtNBA) and 5-(ethylamino)-9-diethylaminoben-
zo[a]phenothiazinium (2, EtNBS) were available from earlier
studies and had been prepared using previously described
procedures.29-31 All intermediates and dyes were purified using
medium pressure (100 psi) chromatography using Woelm 32-
63 silica gel. Thin-layer chromatography was performed using
commercially prepared silica gel covered glass plates (Whatman
K5F 150A). UV-visible absorption spectra were recorded using
an HP 8453 spectrophotometer. Corrected fluorescence spectra
were recorded using a SPEX Fluorolog2 (solvent measurements)
or a FluoroMax3 (microorganism measurements), SPEX In-
dustries, Edison, NJ; fluorescence quantum yields were mea-
sured relative to the Cresyl Violet standard at 25 °C.32 Quantum
yields for 1O2 formation were determined by measuring the 1O2-
mediated bleaching of 1,3-diphenylisobenzofuran12 relative to
Methylene Blue (0.50)33 at 25 °C. 1H NMR spectra were
obtained using a Varian 400 MHz spectrometer with TMS as
the internal standard; chemical shifts are reported in δ, coupling
constants as J (cps) using standard peak splitting terminology.
HRMS (electrospray ionization (ESI)) were obtained from the
Mass Spectroscopy Facility, Chemical and Chemical Biology
Department, Harvard University.
Bis-(3-N,N-diethylaminophenyl) Diselenide (5). To mag-
nesium turnings (1.94 g, 80 mmol) stirred in fresh, dry ethyl
ether (50 mL) under an argon atmosphere was added dropwise
over a 3 h period an ethyl ether (50 mL) solution of 3-iodo-
N,N-diethylaniline34 (4) (10.0 g, 36.4 mmol) and 1,2-dibromo-
ethane (6.84 g, 36.4 mmol). After an additional hour of stirring,
most of the magnesium had reacted. To the resulting solution,
Grignard reagent was slowly added, via a flexible solids addi-
tion tube, selenium powder (3.35 g, 42.4 mmol, 325 mesh);
the addition rate (15 min) was modulated so as to keep a
controlled, gentle solvent reflux, which results from the exo-
therm of the reaction. The resulting mixture was stirred
overnight at room temperature. The reaction was carefully
quenched with the dropwise addition of 30 mL of water (initial
vigorous reaction) to give a dark orange ether layer and a brown
Because of the well-known increase in multi-antibiotic
resistance among pathogenic microbes of all classes, PDT has
attracted growing attention as a possible treatment for localized
infections.19 Preclinical studies have found that many commonly
used porphyrin PSs are effective at inactivating Gram-positive20
bacteria but are considerably less efficacious against Gram-
negative organisms;21 fungal cells such as Candida are generally
midway in susceptibility between both types of bacteria.22
Cationic PSs, however, have been found to be effective against
both Gram-positive and Gram-negative bacteria,23 which is
consistent with the observation that most dyes used in pathology
to identify bacteria and bacterial spores bear a delocalized
cationic charge as exemplified by the Gram stain/counter-stain
pair, Gentian Violet and Fuchsin,24 and the Wirtz-Conklin pair,
Malachite Green and Safranine-O.25 For example, Methylene
Blue, Toluidine Blue and Dimethylmethylene Blue, phenothi-
azinium dyes closely related to EtNBS (2), are safe and effective
antimicrobial PSs26 that are currently the only PDT agents being
used for clinical antimicrobial applications in the oral cavity

Antimicrobial PhotoinactiVation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5293
1
water layer. Air was bubbled through this stirred mixture
overnight; the exhaust, which contained selenium vapor (TOXIC),
was trapped by bubbling through a solution of aqueous sodium
hypochlorite (bleach). The residue was taken up in ethyl ether
(250 mL) and washed with water and brine and dried over
sodium sulfate. Removal of the solvent in vacuo afforded 7.35
g of a yellow oil that was purified using silica gel chromatog-
raphy using 1% ethyl ether in hexane as eluent to afford 4.61
g (56%) of diselenide 5 as a light yellow oil. TLC analysis
indicated that the product contained a trace of a single impurity
that was below the detection limits of NMR spectroscopy; 5
was used without further purification. 1H NMR (δ/ppm,
CDCl3): 1.10 (t, J ) 7.2, 12H), 3.28 (q, J ) 7.2, 8H), 6.51
(dd, J ) 7.6, 2.0, 2H), 6.90 (m, 4H), 7.06 (dd, J ) 8.4, 7.6,
2H). HRMS (ESI) m/z C20H28N2Se2 (MH+) calcd, 457.0661;
found, 457.0655.
Anal. (C22H24N3SeCl-0.5 H2O) C, H, N. H NMR (δ/ppm,
MeOH-d4): 1.32 (t, J ) 6.8, 6H, N(CH2CH3)2), 1.44 (t, J )
7.2, 3H, NH(CH2CH3)), 3.65 (two overlapping q, J ) 7.2 and
6.8, 6H, both types of N-CH2), 7.13 (dd, J ) 9.6, 2.4, 1H),
7.40 (d, J ) 2.8, 1H), 7.55 (s, 1H), 7.74 (m, 1H), 7.82 (m, 1H),
7.96 (d, J ) 9.6,1H), 8.21 (d, J ) 8.4 1H), 8.99 (d, J ) 8.4,
1H). HRMS (ESI) m/z C22H24N3Se (M+) calcd, 410.1135;
found, 410.1138.
Photostability of Photosensitizers. To ensure that the PSs
of the present study, especially selenium derivative 3, are
resistant to photodegradation by singlet oxygen, a water-cooled
1 cm2 square cuvette containing 3 mL of a methanol/acetic acid;
(100:1, v/v) solution of each dye (OD ) 1.0) was placed in a
cell holder. One face of the cuvette was subjected to the
unfiltered beam emanating from a slide projector (Polaroid 610,
fluence 72 Jcm-2 at an irradiance of 20 mWcm-2 for the 600-
700 nm band). The optical density and the wavelength of
maximum absorption of each solution were compared before
and after illumination; in all cases, the OD of the solutions
decreased less than 4%, whereas the wavelengths of maximum
absorption, a sensitive probe for detecting changes at the 7
chalcogen-substituted ring position,35 remained unchanged,
indicating that at least in the relatively benign environment
afforded by ethanol all of the dyes are relatively stable.
Bis-(3-N,N-diethylamino-6-nitrosophenyl) Diselenide (6).
To a stirred, cold (5° C) solution of diselenide 5 (2.36 g, 5.2
mmol) in 1 N hydrochloric acid (200 mL) was added an aqueous
solution of sodium nitrite (0.72 g, 11.4 mmol), whereupon the
clear yellow solution rapidly formed an orange precipitate. After
an additional 10 min of stirring, the mixture was extracted twice
with methylene chloride (200 mL). The organic layer was
washed twice with brine and dried over sodium sulfate. The
solvent was removed in vacuo to give 2.22 g of an orange/
brown solid. Crystallization from 2-propanol gave 1.84 g (69%)
of brown crystals. TLC analysis (5% methanol in methylene
chloride) showed that the desired product contained a trace
amount of a blue contaminant that could not be removed by an
additional crystallization. Thus, 6 was used without further
Microbial Strains. The microorganisms studied were Es-
cherichia coli (ATCC 25922), Enterococcus faecalis (ATCC
29212), and Candida albicans (ATCC 18804). Cells were grown
at 37 °C in aerobic conditions in a shaker set at 150 rpm. Brain-
heart infusion broth (Difco, BD Diagnostic Systems, Sparks,
MD) was used for E. coli and E. faecalis, and YM medium
(Difco) was used for C. albicans. Exponential cultures obtained
by reculturing stationary overnight precultures were used for
all experiments. E. coli and E. faecalis were grown in a fresh
medium for approximately 1 h to a density of 108 cells/mL; the
OD values at 650 nm were 0.6. C. albicans was grown for
approximately 4 h to an approximate density of 107 cells/mL
corresponding to an OD of 6 at 650 nm (10 fold dilution
measured). Exact cell numbers were confirmed by counting
colony forming units obtained after serial dilution on square
BHI (or YM for Candida) agar plates.
1
purification. H NMR (δ/ppm, CDCl3): 1.43 (broad m, 12H),
3.70 (broad m, 8H), 7.13 (dd, J ) 9.6, 2.4, 2H), 7.84 (d, J )
2.4 4H), 8.38 (d, J ) 9.6, 2H). HRMS (ESI) m/z C20H26N4O2-
Se2 (MH+) calcd, 515.0464; found, 515.0458.
5-Ethylamino-9-diethylaminobenzo[a]phenoselenazin-
ium Chloride (3, EtNBSe). A stirred solution of bis-nitroso
diselenide (6) (1.70 g, 3.3 mmol) and 1-N-ethylnaphthylamine
(7) (1.49 g, 9.6 mmol) in trifluoroethanol was heated to reflux
temperature. Initially, the reaction solution showed the formation
of an absorption band at 810 nm that was rapidly replaced by
a deep blue band at 660 nm. Reaction was complete in less
than 1 h, whereupon the solvent was removed in vacuo to give
a blue waxy solid. The solid was stirred twice with ethyl ether
to remove excess 7 and dissolved in a mixture of aqueous 1 N
sodium hydroxide and methylene chloride. The color of the
solution turned bright magenta, indicating that deprotonation
of 3 had occurred. The mixture was placed in a separatory
funnel, and the organic layer was washed twice with brine. Dye
3 was regenerated as a deep blue chloride salt by adding 0.5
mL of concentrated hydrochloric acid to the magenta methylene
chloride solution. After removing the solvent and excess
hydrochloric acid in vacuo, the desired product was purified
by column chromatography using a solvent gradient of methanol
(2-7% v/v) in methylene chloride. Fractions that consisted of
a single spot by TLC analysis were combined to give 3 (1.49
g, 55%) as a noncrystalline solid. Because 3 binds tenaciously
to a trace amount of solvent (NMR analysis), which we were
unable to remove using high vacuum and heat, the dye was
twice dissolved in anhydrous ethanol and evaporated to dryness
in order to exchange the residual eluting solvent with this
innocuous alcohol. A sample for combustion analysis was
prepared by adding a small amount of water to a methanol/
methylene chloride (1:15, v/v) solution of the dye and removing
the solvent under vacuum with gentle heating to constant weight.
Photosensitizer Solutions and Light Sources. Stock solu-
tions (2 mM) of the three chalcogen dyes were prepared in water
and stored at 4 °C in the dark for no longer than 1 month before
use. A noncoherent light source with interchangeable fiber
bundles (LumaCare, London, U.K.) was employed. Band-pass
filters (30 nm) allowed total powers of roughly 1 W to be
obtained from 630 ( 15 nm for EtNBA (1), 652 ( 15 nm for
EtNBS (2), and 660 ( 15 nm for EtNBSe (3).
Dye Uptake by Microbial Cells. Suspensions of microor-
ganisms were incubated for 10 min with PSs in the dark at room
temperature. Unbound PSs were washed out by centrifugation
of the mixture of dye and microorganisms for 6 min at 1550g
followed by resuspension of washed pellets in 10 mL of PBS
without Ca2+/Mg2+. Aliquots (200 µL) of these suspensions
were used for photodynamic inactivation (PDI) experiments,
the remaining suspensions were centrifuged again, and the
pellets were dissolved in 3 mL of 10%SDS for at least 24 h.
These suspensions were used for PS uptake measurement. The
fluorescence of dissolved pellets was measured on a spectrof-
luorimeter (FluoroMax3, SPEX Industries, Edison, NJ). For
EtNBA (1), the excitation wavelength was 625 nm, and the
emission spectra of the solution were recorded from 630 to 750
nm. For EtNBS (2), the excitation wavelength was 645 nm,
and the range for emission was 650 to 750 nm. For EtNBSe

5294 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Foley et al.
Scheme 1
Figure 1. Molecular structures of chalcogen-substituted photosensi-
tizers.
Table 1. Physical and Photophysical Properties of Pertinent Dyes
λabs
λfl
c
d
dye
(nm)a
(nm)b
Φfl
1O2
log Pe
1
2
3
632
654
661
660
693
703
0.27
0.21
0.03
0.003
0.03
0.78
2.69
2.76
2.08
(3), the excitation wavelength was 655 nm, and the emission
was recorded in the range from 660 to 750 nm. The fluorescence
was calculated from the height of the peaks recorded. Calibration
curves were made from pure PS dissolved in 10% SDS and
used for determination of sensitizer concentration in the
suspension. Uptake values were obtained by dividing the number
of nmol of sensitizer in the dissolved pellet by the number of
colony forming units obtained by serial dilutions and the number
of PS molecules/cell calculated by using Avogadro’s number.
Photodynamic Inactivation Studies. Illumination was per-
formed either before or after excess dye was washed out.
Aliquots of 200 µL of cell suspension were placed in 96-well
plates and illuminated with appropriate light at room temper-
ature. Fluences ranged from 0 to 80 Jcm-2 at an irradiance of
60-100 mWcm-2. Exact power was measured with a laser
power meter (model FM/GS, Coherent, Santa Clara, CA).
During illumination after defined fluences had been delivered,
aliquots of 20 µL were taken to determine the colony-forming
units. The contents of the wells were mixed before sampling.
a Absorption maximum in ethanol containing 0.1% acetic acid. b Fluo-
rescence maximum in ethanol. c Absolute fluorescence quantum yield.
d Absolute quantum yield for singlet oxygen formation. e Partition coefficient
between 2-octanol and buffered saline at pH 7.4.
N,N-diethylaniline (4) was converted to diselenide 5 by reaction
with selenium powder followed by air oxidation. Treatment of
5 with two equivalents of nitrous acid gave dinitroso diselenide
(6) as an orange solid. Attempts to condense 6 with N-ethyl-
1-naphthylamine (7) in ethanol gave no reaction; increasingly
better yields of EtNBSe (3) were realized in this solvent as
progressively weaker acids, including HCl, acetic acid, and
acetic acid-sodium acetate, were used as the catalyst. This trend
naturally led us to evaluate trifluoroethanol, which is mildly
acidic and nonnucleophilic, as both solvent and benign catalyst,
whereupon the desired dye was formed cleanly and in good
yield. Treatment with an ion exchange procedure ensured that
chloride was the counterion of 3; subsequent column chroma-
tography afforded the chloride salt of EtNBSe (3) as a dark
blue solid (55% yield).
Physical and Photophysical Properties. An inspection of
the molecular structures of the three chalcogen chloride dyes
of the present study, presented in Figure 1, shows that they are
relatively small, planar, and possess a delocalized positive charge
that can be neutralized by the removal of the proton from the
C-5 amino group; the distinguishing feature of each is the nature
of the chalcogen atom residing at the 7-ring position. The generic
molecular structure of the series is closely related to that of
Nile Blue A, a commercial stain, differing by an additional ethyl
moiety on the 5-amino group; we discovered that the inclusion
of this substituent greatly increased the rate of aqueous
dissolubility while concomitantly increasing lipophilicity. We
attribute this seeming paradox to added steric bulk that
accompanies the incorporation of this additional alkyl group,
which decreases the tendency of the dyes to form large, less
water soluble aggregates.
The aliquots were serially diluted 10-fold in PBS without Ca2+
/
Mg2+ to give dilutions of 10-1-10-6 times the original
concentrations and were streaked horizontally on square BHI
agar plates as described by Jett et al.36 The plates were incubated
at 37 °C overnight. Colonies were counted and survival fraction
determined compared to that of the untreated control. Dye in
the absence of light and light alone was also used as controls.
PSs were generally nontoxic for microorganisms in the dark,
and light alone did not cause cell destruction. All experiments
were performed in triplicate.
Statistics. Values are reported as means ( SD. Differences
between means were evaluated by one-way ANOVA in Mi-
crosoft Excel (Microsoft Corp., Redmond, WA). Differences
between the slopes of killing curves were evaluated using the
linear regression analysis function contained in the GraphPad
Prism software (GraphPad Software Inc, San Diego, CA). P
values of less than 0.05 were considered significant.
Chemistry
Physical and photophysical data pertinent to this work is
presented in Table 1. All three dyes are efficient absorbers of
red light with extinction coefficients greater than 50 000 L/mol
cm;18 as expected, the wavelength of maximum absorption shifts
to longer wavelengths as the chalcogen atom is varied from
oxygen to sulfur and selenium.38 Photophysical data are
presented for the dyes dissolved in ethanol rather than water
because in aqueous media, all three chromophores form, in
addition to monomeric species, H-dimers and possibly higher
order aggregates, which we deduced from the appearance of an
additional absorption band in the short red spectral region (data
not shown); in ethanol, the dyes appear to be entirely monomeric
as evidenced by their adherence to Beer’s Law and the absence
of the short red band.39 All three chalcogen chromophores
Benzo[a]phenoxazinium and benzo[a]phenothiazinum dyes
EtNBA (1) and EtNBS (2), available from antecedent studies,
had been synthesized in-house using standard procedures.29-31
Surprisingly, prior to our work, the literature contained no
example of a benzo[a]phenoselenazinum dye and only a single
reference to an unsymmetrical phenoselenazinium dye, which
had been synthesized by Groves et al. via the acid-catalyzed
condensation of a p-nitroso-N,N-dialkylaniline with bis-(3-
aminophenyl) diselenide.37 Although our initial efforts for
preparing EtNBSe (3) using a sequence of reactions identical
to those used in this latter work were unsuccessful, a modifica-
tion of the Groves approach, outlined in Scheme 1, did afford
the PS in high yield. The Grignard reagent derived from 3-iodo-

Antimicrobial PhotoinactiVation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5295
Figure 2. Absorption and fluorescence emission spectra of selenium
analogue 3 in ethanol.
Figure 3. Photodynamic inactivation of the Gram-positive E. faecalis
(cell density 108/mL). Bacteria were incubated for 10 min with 2 µM
dyes, followed by a wash and illumination with the appropriate
wavelength of red light. The values are means of three independent
experiments, and the bars are SD.
fluoresce, with wavelengths of maximum emission shifted
approximately 40 nm to the red of their respective absorbance
maxima. This relationship is illustrated in Figure 2, which
provides an example of the absorption and emission curves for
selenium dye 3; the other two members of the series have
similarly shaped spectral profiles.
The relative photoactivities of the three PSs studied herein
were gauged by their abilities to generate singlet oxygen (1O2)
when illuminated with a beam of red light with an adjusted
intensity such that each dye absorbed the same number of
photons per unit of time. As shown in Table 1, the quantum
yields of 1O2 formation, measured using the 1,3-diphenylisoben-
zofuran method, rose dramatically as the atomic number of the
chalcogen atom, and hence the spin-orbital coupling constants,
increased from O to Se.
Finally, although previously presented elsewhere,18 because
of the relevance of lipophilicity to the present studies, we list
partition coefficients for the PS between 2-octanol and phosphate-
buffered saline (pH ) 7.4).
Antimicrobial Results
Figure 4. Photodynamic inactivation of Gram-negative E. coli (cell
density 108/mL). Bacteria were incubated for 10 min with 5 µM dyes,
followed by a wash and illumination with red light. The values are
means of three independent experiments, and the bars are SD.
Photodynamic Inactivation of E. faecalis. The Gram-
positive bacterium E. faecalis was killed by all three PSs at a
concentration of 2 µM followed by a wash and application of
red light, but there were large and significant differences
between the degrees of killing observed (Figure 3). Because
preliminary experiments showed that the dyes had very different
activities, we used more light than that used for EtNBSe (3, up
to 32 J/cm2) to kill cells incubated with EtNBS (2, up to 64
J/cm2) and EtNBA (1, up to 80 J/cm2). EtNBSe (3) was by far
the most effective PS, leading to the killing of >99.999% of
cells (5.5 logs, P <0.001) after a delivery of only 8 J/cm2 of
660-nm light; EtNBS (2) was the next most efficient antimi-
crobial PS with a killing of >99.9% (3 logs) after a delivery of
64 J/cm2 of 652-nm light, whereas EtNBA (1) was the least
effective PS, requiring the delivery of 80 J/cm2 of 635-nm light
to kill 95% of the cells.
Photodynamic Inactivation of E. coli. We used a some-
what higher concentration (5 µM) of PS to test the PDT killing
of the Gram-negative E. coli after a wash. This was because
Gram-negative species are generally found to be more resistant
to PDT than Gram-positive species.20 Again EtNBSe (3) was
by far the most effective PS with a delivery of 32 J/cm2 giving
almost total eradication of >99.9999% (>6 logs, P <0.001)
(Figure 4). EtNBS (2) was much less effective, leading to a
killing of slightly more than 99% after 64 J/cm2, whereas EtNBA
(1) was again the least effective, only killing 90% of cells after
80 J/cm2.
Photodynamic Inactivation of C. albicans. Because Candida
cells are 10 times larger than bacterial cells,22 we used an even
higher PS concentration than that used for bacterial cells, that
is, 20 µM with a wash. Under these conditions, EtNBSe (3)
was again the most effective PS by a large margin with only 4
J/cm2 required to kill more than 99% of cells (P <0.001) (Figure
5). EtNBS (2) was less effective, needing a fluence of 64 J/cm2
to give the same degree of cell killing (>98%), whereas EtNBA
(1) was the least effective requiring 80 J/cm2 to kill >97% of
cells.
Effect of Microorganism Wash Pre-PDT. To test whether
the PSs were more or less effective at killing microbial cells
when present in solution during illumination, we compared three
sets of experiments with and without a wash of the unbound
PS from the cell suspension. The killing curves for E. faecalis

5296 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Foley et al.
Figure 5. Photodynamic inactivation of the pathogenic yeast C.
albicans (cell density 108/mL) incubated for 10 min with 20 µM dyes,
followed by a wash and illumination with red light. The values are
means of three independent experiments, and the bars are SD.
incubated with 2 µM EtNBSe (3), E. faecalis incubated with 2
µM EtNBS (2), and E. coli incubated with 5 µM of EtNBSe
(3), all with and without a wash, are presented in the Supporting
Information section. In all three cases, the two killing curves
for wash and no wash are almost identical.
Microbial Uptake. We asked whether the observed large
differences in effectiveness of antimicrobial PDT of the three
dyes depended on photochemical efficiencies, the degree of cell-
dye binding, or both. We therefore examined the uptake of dye
in terms of molecules per cell by carrying out 10 min incubations
with the same concentration of dyes that was used for photo-
inactivation followed by a wash to remove residual incubating
solution. The resulting bacterial pellets were dissolved in 10%
SDS to give a homogeneous solution and comparison of the
resulting fluorescence intensities with a calibration curve
prepared with known concentrations of dye in the same solvent
yielded the amount of dye in the pellet. Figure 6A shows that
for E. faecalis the cell uptake was in the order EtNBSe (3) >
EtNBS (2) > EtNBA (1). The differences between EtNBA (1)
and both EtNBS (2) and EtNBSe (3) were significant, but the
difference between EtNBS (2) and EtNBSe (3) was not
significant. A similar order of cell uptake (but higher values
due to higher dye concentrations being used) was found for E.
coli, with EtNBSe (3) > EtNBS (2) > EtNBA (1); in this case,
the differences between all three values were significant. For
the fungus C. albicans, we used an even higher concentration
of dye (20 µM) because the eukaryotic cells are 10 times bigger
than the prokaryotic bacterial cells. The uptake of EtNBA (1)
was significantly lower than that found for EtNBSe (3).
By comparing the amount of dye that remained in solution
with the dye that was in the bacterial pellet, it was possible to
calculate the fraction of total dye added that was bound to the
cells as shown in Figure 6B. The binding of EtNBSe (3) to C.
albicans and E. coli and the binding of EtNBS (2) to C. albicans
were essentially quantitative, whereas approximately 75% of
the dye was cell bound in the case of EtNBA (1) to C. albicans,
EtNBS (2) to E. coli, and EtNBSe (3) to E. faecalis. Thus, the
affinity of dyes for cells increased in the order EtNBA (1) <
EtNBS (2) < EtNBSe (3), and the affinity of cells for dyes
increased in the order E. faecalis < E. coli < C. albicans.
Figure 6. (A) Uptake of the three PSs by the three microbial species.
The concentration was 2 µM for E. faecalis, 5 µM for E. coli, and 20
µM for C. albicans. Cells were incubated for 10 min followed by a
wash and dissolving the pellet in 10% SDS and fluorescence quantifica-
tion. The values are means of three independent experiments, and the
bars are SD. *, **, ***, P <0.05, 0.01, 0.001, respectively vs EtNBA
(1); ## P, <0.01 vs 1. (B) Fraction of added PS that is bound to cells.
Conditions as in Figure 6A. Fluorescence was determined in the
dissolved bacterial pellet and in the remaining in supernatant.
powerful antibiotic drugs has provided the impetus for the
discovery of new approaches for controlling pathogens. In this
study, we show that a series of chalcogen-substituted phenox-
azinium derivatives originally developed as anticancer PDT
agents are also effective broad-spectrum antibacterial PSs. The
physical and photophysical attributes that characterize the series
and make these dyes well suited for this purpose are as follows.
(1) They are strong absorbers of red light; (2) when illuminated
with red photons, they become, or in reaction with molecular
oxygen generate, a cytotoxin; (3) they are lipophilic and bear a
delocalized positive charge; (4) they are relatively stable to
photodegradation when illuminated in alcohol at moderate light
fluences; and (5) they emit red or near-infrared fluorescence.
Additionally, they are water soluble, which is beneficial for drug
administration purposes, and two members of the series, 1 and
2, evaluated in prior extensive animal model studies, appear to
have little or no toxicity in the dark.
The ability for an antimicrobial PS to absorb light in the 600-
900 nm region of the spectrum is critically important for in
vivo applications because these are the wavelengths that
penetrate tissues most effectively. All three chalcogen analogues
investigated herein meet this criterion with the oxygen-, sulfur-,
and selenium-substituted PSs having absorption maxima at 634,
653, and 661 nm, respectively.
Discussion
The evolution of disease causing bacteria into phenotypes
that are becoming increasingly resistant to even the most

Antimicrobial PhotoinactiVation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5297
Photosensitizers usually inactivate biological targets via their
excited triplet states primarily because the millisecond lifetimes
of this species are long enough to participate in bimolecular
chemistry, whereas the nanosecond lifetimes of the correspond-
ing excited singlet state generally precludes this behavior. A
triplet PS can inactivate biomolecules in one of two ways: (1)
it can react via an electron transfer reaction directly with the
target or with some other species to produce cytotoxic radicals,
or (2) it can transfer its electronic energy to ground state oxygen
to generate highly cytotoxic singlet oxygen, 1O2, with regenera-
tion of the ground-state dye.40 Regardless of which mechanism
a PDT agent employs, an efficient PS must have a high triplet
quantum yield. Because it is well established that in aerated
alcoholic solvents the triplet states of phenothiazine dyes such
as those studied herein generate singlet oxygen with 100%
efficiencies33 owing to their π-π* character, we chose to
determine the triplet quantum yields of EtNBA (1), EtNBS (2),
and EtNBSe (3) indirectly by measuring their respective singlet
oxygen quantum yields in ethanol. EtNBA has an extremely
low triplet yield (0.003), which is not surprising because this
dye lacks any of the structural features that promote intersystem
crossing. Because it is well-known that the incorporation of a
heavy atom into a molecule with a low intrinsic intersystem
crossing rate constant will increase the probability of such
transitions roughly in proportion to the square of the spin-
orbital coupling constant (ú) of the atom where the transition
occurs,41 ignoring the small contributions from C and N (ú )
13, 76 cm-1, respectively), we found that replacing the central
O atom (ú ) -79.6 cm-1) with a somewhat heavier S atom (ú
) -184 cm-1) resulted in a small but significant, increase in
the triplet yield (0.03) and that as expected the replacement by
urinary tract pathogen, there is increasing concern about its
presence in wound infections, and its ability to develop extended
spectrum beta-lactamase resistance is causing increased inter-
national concern.46,47 E. coli is generally considered to be a good
model Gram-negative organism to test PDT because it is
midway in susceptibility between the easy to kill Hemophilus
and the difficult to kill Pseudomonas. C. albicans is increasingly
found in nocosomial infections in burns48 and to a lesser extent
in surgical wounds.49
When evaluated against these prototypical cell types, EtNBSe
(3) was found to be a highly effective, broad-spectrum
antimicrobial PDT drug. It was active against Gram-positive
and Gram-negative bacterial and fungal cells and was equally
effective whether or not the cells were washed of free dye. A
possible reason for this latter observation is the almost quantita-
tive binding of the dye to the cells after a 10 min incubation
(75-98%). Cell-dye binding was also found to be stronger
for EtNBS (2) than for EtNBA (1). As note above, phenothi-
azinium dyes such as Toluidine Blue O and Methylene Blue
have previously been used as antimicrobial PSs. However, much
higher concentrations of these dyes must be used to achieve
the degree of microorganism inactivation found using selenium
analogue 3. The reason this higher concentration of dye is
needed for phenothiazinium salts is probably the fact that the
binding between the dye and the cell is weaker and that the
cell uptake is correspondingly lower. In addition, it has been
found that PDT using Methylene Blue and Toluidine Blue to
kill Gram-positive and Gram-negative bacteria and fungi is much
more effective when the free dye of the incubating solution is
left in the cell suspension than when it is washed out. This
observation may be explained by the fact that the dye that
remains in solution can generate reactive oxygen species in the
vicinity of the microbial cells that can damage the outer
structures of the cell, directly causing cell death, or by allowing
more dye to bind or penetrate the organism, thus accelerating
the PDT effect.
a much heavier Se atom (ú ) -825 cm-1 42
resulted in a
)
dramatic improvement in the triplet yield (0.78); these values
are similar to results previously obtained in methanol.18
On the basis of an extensive study of Gram-positive and
Gram-negative antibacterial agents, Hansch et. al. concluded
that the most important factor in determining activity was
lipophilic character as expressed by their oil-water partition
coefficients.43 With this in mind, in Table 1, we list partition
coefficient data showing that all three chalcogen dyes are
lipophilic, with values greater than log 2. In comparison, under
identical conditions, the PDT antibacterial drug Methylene Blue
has a log P ) 0;44 the difference in lipophilicity between the
dyes of this study and Methylene Blue might explain the
superior microbial affinities of the former materials as reflected
in chromophore washing studies (vide infra).
The high efficiency of EtNBSe (3) compared to that found
for EtNBS (2) and EtNBA (1) is presumably due to the much-
increased yields of an excited triplet state species, which can
be cytotoxic in its own right or can react with ubiquitous
molecular oxygen generating singlet oxygen or superoxide,
depending on whether the reaction occurs via energy or electron
transfer, respectively. This conclusion is supported by the
observation that although uptake by the cells was slightly higher
for EtNBSe (3) compared to that for EtNBS (2), there is a very
large difference in killing efficacy between the two (2-5 logs
depending on cell type). Significantly, EtNBSe (3) killed 5 logs
more of the Gram-negative E. coli than EtNBS (32 J/cm2), and
it is well-known that Gram-negative bacteria are, in general,
most resistant to antimicrobial PDT.19-21
Because most PDT agents emit a fluorescent signal upon
excitation with visible light, they offer the valuable benefit of
doubling as real-time in situ luminescent reporters of the location
and concentration of the drug. Such is the case with the PSs of
the present investigation; 1 and 2 are strong emitters that can
easily be detected with a fluorescence microscope, whereas 3,
because of its relatively low fluorescence quantum yield, is more
challenging to detect visually. Nevertheless, even selenium
analogue 3 emits with an intensity that can easily be detected
by modern electronic imaging instrumentation of the type used
in our quantitative studies.
Conclusion
A benzo[a]phenoselenazinium dye (3) has been found to be
an effective broad-spectrum antimicrobial PDT agent in vitro.
The dye has many of the characteristics that constitute the
hallmarks of an effective antibacterial PS: it absorbs red light
efficiently, it binds strongly to both Gram-positive and Gram-
negative bacteria as well as Candida fungi, it is highly phototoxic
to these pathogens, and retains this level of activity even under
washing conditions. The high photodynamic activity and the
broad-spectrum of targets inactivated by EtNBSe (3) supports
further testing of this novel antimicrobial PS in animal models
of localized infections.
The microorganism species chosen to evaluate the photoac-
tivities of the dyes in the present study are important pathogens
in the type of infections, such as wounds and burns, likely to
be amenable to PDT. E. faecalis is typical of Enterococci that
frequently develop vancomycin resistance and are important
nocosomial pathogens in postsurgical wounds and burns.45
Although E. coli is generally thought of as an intestinal or

5298 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Foley et al.
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Meso-substituted cationic porphyrins as efficient photosensitizers of
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Acknowledgment. J.W.F. and X.S. were supported by the
Rowland Institute at Harvard University. F.J. and M.R.H. were
funded by the National Institute of Allergy and Infectious
Disease (grant number R01AI050875 to M.R.H.). T.N.D. was
supported by a Wellman Center of Photomedicine graduate
student fellowship.
Supporting Information Available: Elemental analysis of key
compound 3 and Figures showing photodynamic inactivation of
E. faecalis using photosensitizers 2, 3, and E. coli using PS 3 with
and without a wash. This material is available free of charge via
the Internet at http://pubs.acs.org.
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