Phenothiazinium photosensitisers XI. Improved toluidine blue photoantimicrobials

Article abstract of DOI:10.1016/j.jphotobiol.2016.03.035

The phenothiazinium derivative toluidine blue O (TBO) is widely employed as a photoantimicrobial agent in clinical trialling, particularly in dentistry. However, its activity against a range of pathogenic microbial species is not significantly different t

Full text of DOI:10.1016/j.jphotobiol.2016.03.035

Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 68–71
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Phenothiazinium photosensitisers XI. Improved toluidine
blue photoantimicrobials
⁎
Mark Wainwright , Ciara O'Kane, Sophie Rawthore
School of Pharmacy & Biomolecular Sciences, Liverpool John Moores University, Byrom Street Liverpool L3 3AF, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The phenothiazinium derivative toluidine blue O (TBO) is widely employed as a photoantimicrobial agent in clin-
ical trialling, particularly in dentistry. However, its activity against a range of pathogenic microbial species is not
significantly different to that of the standard photoantimicrobial methylene blue. In the current study, derivatives
of TBO with varying hydrocarbon substitution in chromophore position 2 were synthesised via the established
anilinethiosulphonic route, using the mild oxidant silver(II) carbonate to allow substituent preservation.
The resulting series of analogues demonstrated the expected increases in visible absorption wavelength and lipo-
philicity with increasing hydrocarbon content, as well as decreased aggregation for derivatives with bulkier sub-
stituents, and all produced singlet oxygen on illumination in vitro. Screening against a range of bacterial and
fungal pathogens relevant to infection control showed remarkable increases in activity relative to the parent
compound, particularly against the clinically important Gram-negative bacterium Pseudomonas aeruginosa. In
addition, in order to demonstrate clinical relevance, the photoactivities of the new derivatives against microbial
targets were compared to conventional antibacterial and antifungal drugs, as well as biocides commonly used for
local disinfection. Activity here was also generally greater than that of the conventional agents used for compar-
ison, considerably so relative to the local disinfectant agents.
Received 22 February 2016
Received in revised form 18 March 2016
Accepted 24 March 2016
Available online 8 April 2016
Keywords:
Antibacterial
Antifungal
Disinfectant
Phenothiazinium
Photoantimicrobial
Toluidine blue derivatives
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
established as a planar moiety, this infers that neither the auxochromic
dimethylamino- or amino- groups, nor the methyl at position 2 of the
Toluidine blue
O
(tolonium chloride, TBO, Fig. 1) is
a
ring system cause any deviation from this. Aggregates can be observed
in the visible spectrum of aqueous TBO —– and similarly for other
phenothiazinium cations — as a broad shoulder to lower wavelengths
extending from the λmax [6].
As part of the ongoing programme of photosensitiser discovery, im-
proved derivatives of toluidine blue were sought — particularly for
photoantimicrobial analoguing — by variation of the group at C-2 of
the chromophore. Previous work has demonstrated that the use of in-
creased chromophore methylation leads to greater photoantimicrobial
activity in derivatives of both methylene blue and azure A [11,12].
phenothiazinium derivative related to methylene blue (MB, Fig. 1). It
has been used widely in the biological arena as a stain in the pathology
of both cancer and microbial disease [1,2]. More recently TBO has been
widely examined as a photoantimicrobial agent aimed at oral disinfec-
tion [3,4], and is a lead compound in azine photosensitiser research
and development [5].
Where comparative testing of the photoantimicrobial efficacies of
MB and TBO have been carried out, little overall difference has been
reported [6]. However, as the principal lead compound for
photoantimicrobial research, MB has a significantly fuller pedigree
in terms of the range of bacterial, fungal, viral and protozoal chal-
lenges reported [7]. Logically, both are established to interact with
anionic groups at the external surfaces of the microbial target, e.g.
with lipoteichoic acids [8], and to displace stabilising metal cations
in membrane structures [9].
2. Materials & Methods
2.1. Synthesis
Substituted anilines, p-phenylenediamine derivatives, silver carbon-
ate (5% w/w on celite) and solvents were purchased from Sigma-Aldrich
UK, and used without further purification. Photophysical characterisa-
tion of the products was carried out using a Hewlett Packard 8452A
diode array spectrophotometer. This was also used for the determina-
tion of singlet oxygen yield and of lipophilicity. Accurate molecular ion
masses (m/z) for the derivatives were obtained using a Micromass
LCT TOF mass spectrometer.
The use of TBO in biological staining often utilises the property of
metachromasia. This is exhibited as a change in colour of the stained tis-
sue due to the significant aggregation of the highly planar, substituted
phenothiazinium cation [10]. As the chromophore concerned is
⁎
Corresponding author.
E-mail address: mark_wainwright@hotmail.com (M. Wainwright).
http://dx.doi.org/10.1016/j.jphotobiol.2016.03.035
1011-1344/© 2016 Elsevier B.V. All rights reserved.

M. Wainwright et al. / Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 68–71
69
tetraphenylcyclopentadienone (TPCPD) in dichloromethane. Thus
the decrease in absorption of TPCPD at 500 nm was monitored spectro-
photometrically with time, using methylene blue as a standard
photosensitiser. By assuming that the decrease in absorption of TPCPD
at 500 nm is directly proportional to its reaction with singlet oxygen,
the time for a 50% decrease in absorption caused by each of the toluidine
Fig. 1. Accepted structures for toluidine blue O and methylene blue cations.
blue derivatives under identical conditions (t TBD) thus gives a
½
measure of its photosensitising efficiency. Thus, if the time for the
2.1.1. 2-Amino-5-dimethylaminophenylthiosulphonic Acid
DPIBF absorption to decrease by 50% due to TBO photosensitisation is
N,N-Dimethyl-p-phenylenediamine sulphate (130 mmol) was
added to a mechanically stirred solution of aluminium sulphate
octadecahydrate/water (43.6 g, 65 mmol/100 ml). To this was added a
solution of sodium thiosulfate in water (22.0 g, 139 mmol/80 ml)
followed by zinc chloride in water (8.8 g, 63 mmol/12 ml). The reaction
solution was cooled to 0 °C and aqueous potassium dichromate (5.0 g,
17 mmol in 20 ml water) was added dropwise over a 30 min period.
Following this addition, the mixture was allowed to stir for 2 h. During
the last 30 min the temperature was allowed to rise to 10 °C causing
the formation of a viscous precipitate. This was isolated by filtration
and washed with water followed by acetone. Yield = 15.87 g (49%),
m.p. 190 °C (dec.)
t TBO, relative singlet oxygen yields for the derivatives are given by:
½
t1=2TBO
Relative1O2 yield ¼
t1=2TBD
i.e. the lower the t1/2 value for the derivative, the greater its 1O2 yield.
2.3. Lipophilicity (LogP)
The lipophilicities of the photosensitisers were calculated in terms of
log P, the logarithm of their partition coefficients between phosphate-
buffered saline and 1-octanol. The data were calculated using the stan-
dard spectrophotometric method [14] based on the relationship:
2.1.2. 2-Alkyl-3-amino-7-dimethylaminophenothiazinium Derivatives
2-Amino-5-dimethylaminophenylthiosulphonic acid (4 mmol) and
2-alkylaniline (5 mmol) were refluxed in 120 ml methanol and silver
carbonate on celite (5 g, 50% w/w) was added slowly over 0.5 h. The re-
action mixture was refluxed for a further hour, filtered through a celite
pad and the filtrates evaporated. The residue was extracted with dichlo-
romethane and purified by column chromatography on silica.
ꢀ
ꢁ
8
9
=
A−A1
A1
<
VW
VO
LogP ¼ Log
ꢀ
:
;
where A and A1 are the absorption intensities before and after
partitioning respectively and Vw and Vo are the respective volumes of
the aqueous and 1-octanol phases. Determinations were repeated
three times.
2.1.3. 3-Amino-7-dimethylamino-2-methylphenothiazinium Hydrogen-
sulphate (toluidine blue O, 1a)
2.4. Antimicrobial Screening/Comparison
Prepared
as
above
from
2-amino-5-dimethylamino-
phenylthiosulphonic acid and 2-methylaniline. Blue-black powder,
yield = 460 mg, 31%; m/s C15H16N3S requires 270.11, found 270.11;
The photobactericidal efficacies of the derivatives in addition to that
of the known photosensitiser methylene blue were measured against
both Gram positive Staphylococcus aureus (NCTC 6571) and Enterococ-
cus faecalis (NCIMB 13280) and Gram negative Escherichia coli (NCTC
10418), Proteus mirabilis (NCIMB 5887) and a clinical strain of Pseudo-
monas aeruginosa bacteria (courtesy of the Clatterbridge Hospital,
Bebington, UK). Strains were grown in Mueller-Hinton Broth and then
diluted to a concentration of 106 colony-forming units/ml. Aliquots of
the strains were then incubated for 1 h at 37 °C in microtitre trays
with various concentrations of photosensitiser in doubling dilutions
from 100 μM, with zero photosensitiser concentrations in each case
for control purposes. The trays were then either illuminated for 20 mi-
nutes using an array of light-emitting diodes (660 nm) giving a light
dose of 6.2 J cm−2 or alternatively foil-covered to provide dark controls.
From each well showing an inhibition of growth of the micro-organism,
1 μl was sub-cultured on nutrient agar, using the Miles–Misra method,
λmax (MeOH) 629 nm.
2.1.4. 3-Amino-7-(dimethylamino)-2-ethylphenothiazinium Hydrogen-
sulphate (1b)
From 2-amino-5-dimethylaminobenzenethiosulphonic acid and
2-ethylaniline as violet-blue crystals, yield = 273 mg, 18%; m/z,
C
16H18N3S requires 284.40, found 284.42; λmax (MeOH) 634 nm.
2.1.5. 3-Amino-7-(dimethylamino)-2-n-propylphenothiazinium Hydrogen-
sulphate (1c)
From 2-amino-5-dimethylaminobenzenethiosulphonic acid and 2-
n-propylaniline as blue-black powder, yield = 302 mg, 19%; m/z,
C17H20N3S requires 298.43, found 298.40; λmax (MeOH) 636 nm.
2.1.6. 3-Amino-2-tert-butyl-7-(dimethylamino)phenothiazinium Hydrogen-
sulphate (1d)
From 2-amino-5-dimethylaminobenzenethiosulphonic acid and
2-tert-butylaniline as black powder, yield = 487 mg, 30%; m/z,
Table 1
Relevant properties for the toluidine blue derivatives (* relative to toluidine blue;
†Lit. −0.21 [15]).
C
18H22N3S requires 312.45, found 312.41; λmax (MeOH) 637 nm.
2.1.7. 3-Amino-7-(dimethylamino)-2-phenylphenothiazinium Hydrogen-
sulphate (1e)
From 2-amino-5-dimethylaminobenzenethiosulphonic acid and 2-
aminobiphenyl as dark blue powder, yield = 455 mg, 26%; m/z,
R2
λmax
(nm, MeOH)
1O2 yield*
LogP
C20H18N3S requires 332.44, found 332.41; λmax (MeOH) 639 nm.
Me
Et
n-Pr
t-Bu
Ph
629
634
636
637
639
1.00
1.49
1.24
1.79
1.87
−0.20†
−0.46
+0.29
+0.84
+0.63
2.2. Singlet Oxygen Testing
Singlet oxygen production by the photosensitisers was assayed
as in previous work [13], using the decolourisation of 2,3,4,5-

70
M. Wainwright et al. / Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 68–71
Table 2
Photoantimicrobial activity for TBO analogues (MBC — minimum bactericidal concentra-
tion; MB — methylene blue; Levo — levofloxacin, standard antibacterial; Fluc — flucona-
zole, standard antifungal; BPO — benzoyl peroxide, standard topical acne treatment).
R2
MBC (μM)
S. aureus
E. coli Enterococcus Proteus
Prop. Ps.
Candida
(Gram+) (−)
faecalis
mirabilis acnes aeruginosa albicans
(+)
(−)
(+)
(−)
Me
Et
n-Pr 3.13
t-Bu 3.13
Ph
MB
Levo 6.26
Fluc
BPO
7.36
3.13
7.36
1.47
1.32
3.13
3.13
25
3.13
-
-
7.36
1.35
0.33
0.33
-
100
100
-
14.71
5.50
5.70
3.13
-
0.20
3.13
-
0.78
-
0.39
0.78
-
50
-
-
7.36
3.13
0.78
0.39
-
25
25
-
12.5
3.13
0.78
3.13
0.78
3.13
-
3.13
25
Fig. 2. Aqueous visible absorption by TBO (black), t-BuTB (grey dashes) and PhTB
(mid-grey), showing variation in the aggregational, lower wavelength, shoulder
(photoantimicrobial concentration approx. 15 μM).
-
-
25
-
-
-
56
-
and incubated for 18 h at 37 °C. The minimum bactericidal concentra-
tions were then determined as the lowest concentration for each
photosensitiser giving no bacterial growth.
Similarly, the LogP values of the derivatives increased, as expected,
with hydrocarbon content (Table 1), with most resulting compounds
being in the range normally considered to be amphiphilic, and all
retaining water solubility.
All derivatives were shown to produce singlet oxygen in reasonable
yields in the in vitro test (Table 1), although this may not reflect activity
against target cells, since this is dependent on the eventual cellular en-
vironment/biomolecular binding of the photosensitiser.
Inspection of the visible spectra of the alkyl toluidine blue deriva-
tives revealed a lessening of the lower-wavelength shoulder to the
main peak absorption in the red region with increasing alkyl group
size at C-2 (TBO and the t-butyl derivative shown in Fig. 2). As noted,
the λmax was shifted with increasing hydrocarbon group size, via
hyperconjugation, as expected. The decreased shoulder with increased
alkyl size indicates a drop in aggregational behaviour, logically due to
the difficulty in packing caused by the extra asymmetrical volume in-
curred. This volume change is demonstrated in the energy-minimised,
space-filling models of TBO and its 2-tert-butyl derivative in Fig. 3. How-
ever, the 2-phenyl derivative demonstrated intermediate aggregation,
with the shoulder more emphasised (Fig. 2). While the phenyl moiety
is relatively large, even compared to tert-butyl, it is assumed that it
adopts a coplanar orientation with the phenothiazinium chromophore
at higher concentrations, allowing aggregation to occur.
The standard antibacterial agent levofloxacin and several disinfec-
tants (chlorhexidine digluconate, cetyltrimethylammonium bromide,
benzalkonium chloride and Triclosan, purchased from Sigma-Aldrich,
Gillingham UK) were screened for activity in the same way as above.
Antifungal screening was carried out similarly against the yeast Can-
dida albicans (NCPF 8179), using Sabouraud broth and agar. The stan-
dard antifungal agent fluconazole (Sigma-Aldrich, Gillingham, UK)
was included in this test for comparison.
3. Results & Discussion
3.1. Photosensitisers
Toluidine blue and its analogues were synthesised in straightfor-
ward manner, in low-moderate yields (around 30%), and normally re-
quiring purification by column chromatography.
Given the importance of photosensitiser action wavelength in
avoiding endogenous light absorption – typically by blood – the deriva-
tives exhibited the expected increases in λmax with electron release of
the groups added at C-2 of the phenothiazinium chromophore (Table 1).
Fig. 3. Increased asymmetric molecular volume due to 2-tert-butyl group substitution (b) compared to TBO (a) (energy minimised structures using ChemDraw 3D software).

M. Wainwright et al. / Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 68–71
71
4. Conclusions
The established phenothiazinium dyes, having suitable light absorp-
tion and photosensitising profiles, have been investigated throughout
much of the renaissance of research into the photodynamic therapy of
cancer and photoantimicrobial action [20]. Lead compounds such as to-
luidine blue are structurally simple with relatively straightforward syn-
theses, allowing facile analogue production.
MB has been reported to produce more singlet oxygen than TBO in
various formulations in vitro [21], thus the improved performance of
the new derivatives in the current work is encouraging in this respect,
and is, indeed, reflected in the increased photoantimicrobial efficacies
reported here.
Fig. 4. Photobactericidal efficacies vs. standard bacteria of a range of photosensitisers
measured against standard disinfectants (abbreviations as above, except: CX
chlorhexidine digluconate; CTAB cetyltrimethylammonium bromide; BAC
—
—
—
benzalkonium chloride; Tric — triclosan).
The toxicology of new drug candidates is, of course, paramount in es-
tablishing a route forward towards clinical trialling. One of the better
candidates from the current work is currently beginning initial small
scale testing in this respect and this work will be reported at a later date.
3.2. Antimicrobial Efficacy
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