Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides as potential antifungal agents

Article abstract of DOI:10.1016/j.tetlet.2016.07.065

A set of four new benzo[a]phenoxazinium chlorides possessing ethyl, propyl, decyl and tetradecyl groups at the 9-amino function of the heterocycle along with a propyl group at the 5-amino position was efficiently synthesized. These compounds displayed fluorescence with maximum emission wavelengths of 673 and 685?nm, in anhydrous ethanol and water. All the benzo[a]phenoxazines were evaluated against the yeast Saccharomyces cerevisiae in a broth microdilution assay. It was found that their antifungal activity depended on the variation in the lengths of the aliphatic chains. The highest MIC activity of 1.56?μM was obtained for compound 7 comprising a di-alkylated propyl substituent at 9-amino position and a propyl chain at the 5-amino position of the heterocycle core.

Full text of DOI:10.1016/j.tetlet.2016.07.065

Accepted Manuscript
Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides
as antifungal agents
M.Inês P.S. Leitão, B. Rama Raju, Sarala Naik, Paulo J.G. Coutinho, Maria
João Sousa, M.Sameiro T. Gonçalves
PII:
S0040-4039(16)30911-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.07.065
TETL 47927
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2016
11 July 2016
19 July 2016
Please cite this article as: Leitão, M.P.S., Rama Raju, B., Naik, S., Coutinho, P.J.G., Sousa, M.J., Gonçalves, M.T.,
Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides as antifungal agents, Tetrahedron
Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.07.065
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GRAPHICAL ABSTRACT
Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides as
potential antifungal agents
M. Inês P. S. Leitão a,b, B. Rama Raju a,c, Sarala Naik a,c, Paulo J. G. Coutinho c, Maria João Sousab
and M. Sameiro T. Gonçalvesa*
c
aCentre of Chemistry, bCentre of Molecular and Environmental Biology/Department of Biology, and Centre
of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
R
H
n
1
1
2
MIC (µM)
12.5
CH2CH3
(CH2)2CH3
6.25
1.56
*Corresponding author. Fax: +351-253604382; e-mail: msameiro@quimica.uminho.pt
1

Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides as
antifungal agents
M. Inês P. S. Leitão a,b, B. Rama Raju a,c, Sarala Naik a,c, Paulo J. G. Coutinhoc, Maria João Sousab
and M. Sameiro T. Gonçalvesa*
University of Minho
c
aCentre of Chemistry, bCentre of Molecular and Environmental Biology/Department of Biology, and Centre
of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
Abstract: A set of four new benzo[a]phenoxazinium chlorides possessing ethyl, propyl, decyl
and tetradecyl groups at the 9-amino function of the heterocycle along with a propyl group at
the 5-amino position was efficiently synthesised. These compounds displayed fluorescence with
maximum emission wavelengths of 673 and 685 nm, in anhydrous ethanol and water. All the
benzo[a]phenoxazines were evaluated against the yeast Saccharomyces cerevisiae in a broth
microdilution assay. It was found that their antifungal activity depended on the variation in the
lengths of the aliphatic chains. The highest MIC activity of 1.56 µM was obtained for
compound 7 comprising a di-alkylated propyl substituent at 9-amino position and a propyl
chain at the 5-amino position of the heterocycle core.
Keywords: benzo[a]phenoxazinium chloride; antimicrobial drugs; Saccharomyces cerevisiae; Nile
Blue derivatives; NIR probes.
Introduction
Small organic molecules function as fluorescent probes for monitoring and quantification in
chemical and bioanalytical sciences,1,2 being chromophores with emission in red or NIR possessing
good photochemical stability and water solubility of special interest for in vivo and in vitro
studies.3,4 Benzo[a]phenoxazine dyes display good photostability, high molar absorption, strong
fluorescence in the NIR region and modest Stokes shifts (20-60 nm) indicating the potential choice
of these dyes as fluorophores for biological applications.5 Furthermore, non-covalent bonding is a
common feature of benzo[a]phenoxazinium dyes that enables their use for staining purposes in gel
electrophoresis and study of their interactions (intercalative and/or groove binding) with DNA.6
2

These heterocycles has also been reported as lysosome trackers,7 sensors for reversible pH
measurements and NIR live bioimaging purposes.8
In addition, benzo[a]phenoxazine derivatives are important as antifungals,9 antimalarials10
and also function as photosensitizers in photodynamic therapy (PDT) for treatment of Candida
albicans biofilms.11 Compounds possessing amino groups as their ring substituents can also be
functionalized according to various target analytes.12 In our earlier communication, it was reported
that naphtho[2,3-a]phenoxazinium and benzo[a]phenoxazinium chlorides exhibited antifungal
activity that depended on the substituents of the heterocycle.9,13 In continuation of our research
towards fluorescent heterocycles,14-16 the present communication describes the synthesis,
characterization, and preliminary biological studies of a new class of benzo[a]phenoxazinium
chlorides which possess different length of aliphatic alkyl chains.
Photophysical studies were carried out in anhydrous ethanol and aqueous medium. The
antifungal activity of these compounds was determined by using the yeast Saccharomyces
cerevisiae as a model organism. Comparing the MIC values of all the analogues revealed that the
compounds with a di-substitution at 9-amino position and a propyl group at 5-amino position of the
benzo[a]phenoxazines exhibited the best activities.
Results and discussion
The 3-(alkylamino)phenols were obtained by the reaction of 3-aminophenol (S-1) with
appropriate iodo or bromo alkyl halides such as iodoethane, 1-bromopropane, 1-bromododecane
and 1-bromotetradecane in ethanol under reflux conditions (Scheme S1). The reaction afforded two
products, among which the mono N-alklyated product was isolated as the major fraction, namely 3-
(ethylamino)phenol (S-2a), 3-(diethylamino)phenol (S-2b), 3-(propylamino)phenol (S-3a), 3-
(dipropylamino)phenol (S-3b), 3-(decylamino)phenol (S-4a), 3-(didecylamino)phenol (S-4b), 3-
(tetradecylamino)phenol (S-5a) and 3-(ditetradecylamino)phenol (S-5b) in good yields.20
Nitrosophenol precursors 1a-e were obtained by the nitrosation of the required N-alkylated
derivative (S-3a, S-4a, S-5a, S-2b and S-3b, respectively) in an acidic solution of sodium nitrite
under ice cold conditions (Scheme S2).20,21 The other reactant N-propylnaphthalen-1-amine 2 was
prepared by the N-alkylation of naphthalen-1-amine with bromopropane.22 Benzo[a]phenoxazinium
chlorides 3-7 were synthesised by the condensation of N-alkylated nitroso derivatives 1a-e with N-
propylnaphthalen-1-amine 2 in the presence of hydrochloric acid, refluxed in ethanol.23
3

Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 3-7.
After column chromatography purification pure compounds were acquired as blue solids
and characterized by high resolution mass spectrometry, IR and NMR (1H and 13C) spectroscopies.
1H NMR spectra of compounds 3-7 showed triplets (δ 0.87-1.38 ppm) for the terminal methyl
groups of aliphatic chains at 5- and 9-positions. The methylene group adjacent to methyl exhibits
quartet, multiplet or broad singlet (δ 1.75-1.98 ppm) and the methylene group directly attached to
the nitrogen atom shows triplet, multiplet or broad singlet (δ 3.06-3.79 ppm). The aromatic protons
11-H (δ 7.34-7.84 ppm), 4-H (δ 8.31-8.65 ppm) and 1-H (δ 8.79-8.89 ppm) appeared as doublets or
multiplets. 13C NMR spectra of these compounds showed signals of the aliphatic carbons from the
methyl group (δ 11.45-14.08 ppm) and methylene carbons (δ 21.81-54.56 ppm) of the substituents
of 5- and 9-postions. The aromatic signals such as C-11 (δ 131.75-134.10 ppm), C-4 (δ 123.34-
123.86 ppm) and C-1 (δ 125.29-125.59 ppm) were shown in the spectra.
4

Electronic absorption and emission spectra of 4×10-6 M solutions in acidic anhydrous ethanol
and water were measured for the synthesised benzo[a]phenoxazinium chlorides (Table 1). The
relative fluorescence quantum yields (ΦF) were determined using Oxazine 1 as a standard (ΦF =
0.11 in ethanol)24,25 at 575 nm excitation. In acidified ethanol and water, the absorption maxima
(λabs) for compounds 3-7 lie in the range of 568-645 nm, and molar extinction coefficients (ɛ)
-1
between 24225 and 96350 M cm-1. The emission maxima (λem) was found to be in the range of 633-
685 nm at excitation of 575 nm with moderate to high Stokes shifts (∆λ, 10-65 nm).
Table 1. Photophysical data of compounds 3-7 in anhydrous ethanol and aqueous solution (C = 4×10-6
M) acidified with trifluoroacetic acid (TFA).
Anhydrous ethanol acidified with TFA
Water acidified with TFA
a
a
a
a
∆λa
ΦF
λabs
λem
∆λa
ΦF
εb
εb
Cpd
λabs
λem
623
624
627
637
639
69250
37025
74350
96350
645
647
649
670
673
22
23
22
33
34
0.28
0.53
0.51
0.11
0.28
617
625
568
642
645
35600
31650
24225
36325
40075
652
635
633
681
685
35
10
65
39
40
0.17
0.003
0.007
0.05
3
4
5
6
7
93125
0.04
-1
aUnit: nm; bUnit: M cm-1
It can be seen that compounds 4 and 5 upon elongation of the lateral chain at 9-amino
position almost do not alter the wavelengths of absorption and fluorescence maxima in anhydrous
ethanol and water media. However, comparing the fluorophore 3 with 6, the latter shows greatest
values of λem (670 nm) in anhydrous ethanol and λem (681 nm) in water, which could be mainly due
to the di-alkylation at 9-amino position of the benzo[a]phenoxazinium dye.13,14 The same behaviour
was observed for compound 7. Similarly, compounds 4 and 5 possessing decyl and tetradecyl
groups at 9-amino position showed the highest fluorescence efficiency in ethanolic media, while in
water the compound 3 with an ethyl group at 9-amino position is the one with highest quantum
yield. This was expected to be related to the higher hydrophobicity of compounds 4 and 5.
5

Figure 1. Normalised absorption and emission spectra of compounds 3-7 in anhydrous ethanol.
Figure 1 shows the absorption and emission spectra at 470 nm excitation of the compounds
3-7 in anhydrous ethanol. It can be seen in the absorption spectra (panel A) that
benzo[a]phenoxazinium chlorides exhibited both the acidic (BzH+) and neutral form (Bz) in ethanol
media, corresponding to the second and first bands of the absorption spectrum, as previously
observed with similar type of compounds.16,26,27 With an exception of compound 4, the studied
dyes show a higher fraction of acidic form in ethanolic solution. It has been shown previously that
the fluorescence maximum of neutral form occurs near 600 nm while the acid form occurs in the
range 640-680 nm and the quantum yield of the neutral form is 10-fold lower than the one
corresponding to the acid form.26,27 In dry ethanol medium, at 470 nm excitation, the basic form is
mostly excited with a small fraction of acidic form depending on the 9-amino position.
Nevertheless, the acid form emission has comparable fluorescence intensity due to its higher
quantum yield. The neutral form emission is slightly blue shifted for compounds 4 and 5 appearing
at 580 nm. In addition, another band appears at 540 nm for compounds 4 and 5 as seen in Figure 1
6

(B). This band has been proposed and supported by ab initio calculations as arising from tautomers
with localized positive charge and a slight loss of resonance among the π-electron system.16
Figure 2 shows the absorption spectra of compounds 3-7 under acidic and basic conditions
in anhydrous ethanol. It can be seen that displacement of acid-base equilibrium can be done by the
addition of small amounts of acid (TFA) or base (triethylammonium hydroxide, TEAH) to the
benzo[a]phenoxazine solutions. Upon addition of acid to the ethanolic solution of the fluorophore,
except for compound 4, it was possible to completely shift the equilibrium towards the acidic form
as shown in Figure 2 (A). Similarly, compound 4 shows an additional absorption band around 500
nm indicating a high percentage of tautomerization. The emission spectra at 470 nm excitation
show bands around 645-673 nm that correspond to the acidic form emission and correspond to the
higher quantum yields as observed for similar type of compounds.16,26,27 The peaks around 540 nm
observed for compounds 4 and 5 significantly increased indicating a higher fraction of tautomeric
form, which was obtained upon acidification.
Figure 2. Normalised absorption and emission spectra of compounds 3-7 in anhydrous ethanol
under acidic and basic conditions.
7

Figure 3 shows the absorption and emission spectra at 470 nm excitation of the compounds
3-7 in aqueous media. Compound 5 shows an enormous hypsochromic shift of ~100 nm in λabs in
the comparison with other dyes. Comparing with Figure 2, it seems that a long chain in the 9-amino
position promotes the presence of the neutral form in aqueous media. In the emission spectra,
except compound 3, all the other fluorophores exhibited a small bump around 560 nm showing
small amounts of tautomer emission Figure 3(B). When excited at 470 nm, compounds 3, 6 and 7
only show emission on the acid form while compounds 4 and 5 mainly show neutral form emission
red-shifted in comparison to ethanol media (see Figures 1B and 2B).
Figure 3. Normalised absorption and emission spectra of compounds 3-7 in water.
The potential antifungal activity of the synthesised fluorophores 3-7 was investigated using
the yeast Saccharomyces cerevisiae PYCC 4072 as a model organism and a broth microdilution
method for antifungal activity testing.28,29 The minimum inhibitory concentration of growth (MIC)
and log P values, which are theoretically predicted30 are shown in Table 2. Compounds with lower
log P values are hydrophilic, showing higher tendency towards the intracellular environment which
is aqueous in nature. In contrast, these molecules possess lower affinity for the cell membranes. As
8

it can be seen from the data presented (Table 2), there is a general correlation between the
hydrophilicity of the compounds and their antifungal properties. However, this relation is not strict
since compound 6 has the lowest log P value (1.446) and MIC value of 6.25 µM, which is two-fold
higher than that of compound 3 with a log P of 1.888. Compound 5 with the highest log P value and
being highly lipophilic showed virtually no activity.
The antifungal activity of the benzo[a]phenoxazinium dyes was evaluated with a propylamino
group as substituent at 5-position and varying the length and number of alkyl chain at the amine
function of the 9-position. The study showed that the inhibitory properties of the compounds are
dependent on both these changes at the 9-amino position. For instance, compounds 4 and 5, with
one alkyl chain of 10 and 14 carbon atoms, respectively, showed reduced antifungal activities
probably due to their low water solubility. The introduction of a second ethyl group at 9-amino
position in compound 3 resulted in 6 with a better antifungal activity and, moreover, the presence of
N-propyl groups instead of N-ethyl resulted in a 4-times increase of activity, 1.56 µM being the
lowest MIC value observed. The increase in activity with increase of the alkyl chain size of the
substituents at 9-amino position was also observed by replacing a di-methyl by a di-ethyl groups in
benzo[a]phenoxazines with different substituents or even no substituent at 5-amino position.29 In
the present communication we obtained one benzo[a]phenoxazine with even higher activity by
further increasing the length of the 9-amino substituents by one carbon. Hence, the results indicate
there is an optimal medium length for these substituents and that compound 7 possessing a di-
propyl group at 9-amino position and the propylamino at the 5-position of the polycyclic ring is the
best candidate for antifungal activity and for further development of compounds with improved
activity.
9

Table 2. Activity against Saccharomyces cerevisiae PYCC 4072 and log P values of the
benzo[a]phenoxazinium chlorides 3-7.
Compound
MICa,b
log P
R
n
1
H
12.5
25
1.888
6.484
8.408
1.446
2.954
3
4
5
6
7
H
9
H
13
1
>200c
6.25
1.56
CH2CH3
(CH2)2CH3
2
b
aMinimal inhibitory concentration of growth. Experiments were performed in triplicate and and at
c
least two independent experiments were conducted. Insoluble at the highest concentration tested –
400 µM (1% DMSO).
Acknowledgments
Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial
support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and
FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support
to the Research Centres CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)]
and CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)]. A post-doctoral
grant to B. R. Raju (SFRH/BPD/62881/2009) is also acknowledged to FCT, POPH-QREN, FSE.
This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-
0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the
COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI).
References and notes
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10

3. Jose, J.; Loudet, A.; Ueno, Y.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. Org. Biomol. Chem.
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7. Liu, W.; Sun, R.; Ge, J. F.; Xu, Y. J.; Xu, Y.; Lu, J. M.; Itoh, I.; Ihara, M. Anal. Chem. 2013, 85,
7419-7425.
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R.; Battaglia, G.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2013, 135, 14863-14870.
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16, 3274-3282.
10. Mizukawa, Y.; Ge, J-F.; Md, A. B.; Itoh, I.; Scheurer, C.; Wittlin, S.; Brun, R.; Matsuoka,
H.; Ihara M. Bioorg. Med. Chem. 2014, 22, 3749- 3752.
11. Lopes, M.; Alves, C. T.; Raju, B. R.; Gonçalves, M. S. T.; Coutinho, P. J. G.; Henriques, M.;
Belo, I. J. Photochem. Photobiol: B 2014, 141, 93-99.
12. Jose, J.; Loudet, A.; Uneo, Y.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. Org. Biomol.
Chem. 2010, 8, 2052-2059.
13. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Tetrahedron Lett. 2007,
48, 8347-8352.
14. Raju, B. R.; Garcia, A. M. F.; Costa, A. L. S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Dyes
Pigm. 2014, 203-213.
15. Raju, B. R.; Sampaio, D. M. F.; Silva, M. M.; Coutinho, P. J. G.; Gonçalves, M. S. T. Ultrason.
Sonochem. 2014, 360-366.
16. Raju, B. R.; Firmino, D. G.; Costa, A. L. S.; Coutinho, P. J. G.; Gonçalves, M. S. T.
Tetrahedron 2013, 69, 2451-2461.
17. Kiššová, I. B.; Salin, B.; Schaeffer, J.; Bhatia, S.; Manon, S.; Camougrand, N. Autophagy 2007,
3, 329-336.
18. Voeltz, G. K.; Prinz, W. A.; Shibata, Y.; Rist, J. M.; Rapoport, T. A. Cell 2006, 124, 573-586.
19. Huh, W.-K.; Falvo, J. V.; Gerke, L. C.; Carroll, A. S.; Howson, R. W.; Weissman, J. S.; O'Shea,
E. K. Nature 2003, 425, 686-691.
20. Typical procedure for preparation of nitroso precursors 1a-e (described for 1e): To an ice-cold
solution of 3-(dipropylamino)phenol S-3b (0.193 g, 1 mmol) in ethanol (2 mL), 2M hydrochloric
acid (0.4 mL) was added and stirred during 5 min. The solution of sodium nitrite (0.083 g, 1.2
mmol) in water (0.3 mL) was then added dropwise within an interval of 10-15 min. The resulting
11

mixture was stirred for 3 h and monitored by TLC (dichloromethane/methanol, 9.5:0:5). After
evaporation of the reaction, 2-nitroso-5-(dipropylamino)phenol hydrochloride 1e was obtained as
green solid (0.258 g), and was used in the following step without any purification.
3-(Dipropylamino)phenol S-3b resulted from the reaction of a suspension of 3-aminophenol
(2.0 g, 18.3 mmol) in ethanol (8 mL) and 1-bromopropane (2.50 mL, 27.5 mmol), at reflux
temperature during 4h 30 min, followed by TLC (dichloromethane/methanol 9.5:0.5). After solvent
evaporation and chromatography with dichloromethane and dichloromethane/methanol, mixtures of
increasing polarity, as the eluent, 3-(dipropylamino)phenol S-3b was obtained as a brown solid
(0.715 g, 21%). Mp = 99.7-100.3 oC. TLC (dichloromethane): Rf = 0.22. FTIR (KBr 1%): νmax 3208,
3188, 3053, 2966, 2880, 2711, 2672, 2636, 2508, 1613, 1508, 1488, 1471, 1456, 1431, 1417 cm-1.
1H NMR (CDCl3, 400 MHz): δ 0.84 (6 H, t J = 7.6 Hz, N(CH2CH2CH3)2), 1.59 (4 H, br s,
N(CH2CH2CH3)2), 3.30 (4 H, t J 8.0 Hz, N(CH2CH2CH3)2), 6.87 (br s, 2 H, 4-H, 6-H), 7.05 (1 H, br
s, 2-H), 7.17 (1 H, t J 8.0 Hz, 5-H) ppm. 13C NMR (CDCl3, 100.6 MHz): δ 10.85
(N(CH2CH2CH3)2), 18.86 (N(CH2CH2CH3)2), 58.17 (N(CH2CH2CH3)2), 107.61 (C-2), 113.60 (C-4,
C-6), 130.63 (C-5), 137.52 (C-3), 158.22 (C-1) ppm. HRMS: m/z (TOF EI): calcd for C12H19NO
[M+] 193.1467; found 193.1474.
In the same reaction, 3-(propylamino)phenol S-3a was also isolated as brown liquid (1.410
g, 51%), TLC (dichloromethane): Rf = 0.17. FTIR (KBr 1%): νmax 3250, 2642, 2500, 2415, 1619,
1
1577, 1487, 1426 cm-1. H NMR (CDCl3, 400 MHz): δ 1.05 (3 H, t J 7.2 Hz, NHCH2CH2CH3),
1.75-1.86 (2 H, m, NHCH2CH2CH3), 3.35-3.46 (2 H, m, NHCH2CH2CH3), 6.92-7.01 (3 H, m, 4-H,
6-H, 2-H), 7.35-7.42 (1 H, m, 5-H) ppm. 13C NMR (CDCl3, 100.6 MHz): δ 11.14
(NHCH2CH2CH3), 20.37 (NHCH2CH2CH3), 54.75 (NHCH2CH2CH3), 110.58 (C-2), 114.09 (C-6),
117.63 (C-4), 132.03 (C-5), 137.52 (C-3), 160.19 (C-1). HRMS: m/z (TOF EI): calcd for C9H13NO
[M+] 151.0997; found 151.1003.
21. Crossley, M. L.; Turner, R. J.; Hofmann, C. M.; Dreisbach, P. F.; Parker, R. P. J. Am. Chem.
Soc. 1952, 74, 578-584.
22. Alves, C. M. A.; Naik, S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Tetrahedron Lett. 2009, 50,
4470-4474.
23. General procedure for synthesis of benzo[a]phenoxazines 3-7 (described for 7): To a cold
solution (ice bath) of 2-nitroso-5-(dipropylamino)phenol hydrochloride 1e (0.258 g, 1.0 mmol) in
ethanol (2 mL), N-propylnaphthalen-1-amine 2 (0.185 g, 1.0 mmol) and concentrated hydrochloride
acid (0.027 mL) were added. The reaction mixture was refluxed for 12 h and monitored by TLC
(dichloromethane/methanol 9.5:0.5). After evaporation of the solvent and column chromatography
purification on silica gel with chloroform and chloroform/methanol, mixtures of increasing polarity,
12

as the eluent, N-propyl-N-(5-(propylamino)-9H-benzo[a]phenoxazin-9-ylidene)propan-1-aminium
chloride 7 was obtained as a blue solid (0.307 g, 73%). Rf = 0.19 (chloroform/methanol, 9.4:0.6).
Mp = 240-242 °C. FTIR (KBr 1%): νmax 3392, 3175, 3051, 2967, 2931, 2872, 1640, 1588, 1547,
1
1495, 1452, 1435 cm-1. H NMR (400 MHz, CD3OD): δ 1.08 (6 H, t J 7.6 Hz, N(CH2CH2CH3)2),
1.14 (3 H, t J 7.2 Hz, NHCH2CH2CH3), 1.75-1.85 (4 H, m, N(CH2CH2CH3)2), 1.86-1.96 (2 H, m,
NHCH2CH2CH3), 3.61 (4 H, t J 8.0 Hz, N(CH2CH2CH3)2), 3.67 (2 H, t J 7.2 Hz, NHCH2CH2CH3),
6.81 (1 H, d J 2.0 Hz, 8-H), 6.88 (1 H, s, 6-H), 7.17-7.27 (1 H, m, 10-H), 7.73-7.83 (2 H, m, 11-H,
3-H), 7.88 (1 H, t J 7.6 Hz, 2-H), 8.31 (1 H, d J 8.0 Hz, 4-H), 8.79 (1 H, d J 8.0 Hz, 1-H) ppm. 13C
NMR (CD3OD, 100.6 MHz): δ 11.45 (N(CH2CH2CH3)2), 11.77 (NHCH2CH2CH3), 21.81
(N(CH2CH2CH3)2), 23.08 (NHCH2CH2CH3), 47.54 (NHCH2CH2CH3), 54.56 (N(CH2CH2CH3)2),
94.51 (C-6), 97.17 (C-8), 116.65 (C-10), 123.86 (C-4), 124.88 (Ar-C), 125.55 (C-1), 130.90 (C-3),
131.41 (Ar-C), 132.53 (Ar-C), 132.91 (C-2), 133.92 (C-11), 135.15 (Ar-C), 149.50 (Ar-C), 153.09
(Ar-C), 155.96 (C-9), 159.36 (C-5) ppm. HRMS: m/z (FAB): calcd. for C25H30N3O [M+]
388.23774; found 388.23834.
24. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24, 709-712.
25. Absorption spectra (200-800 nm) were recorded on a Shimadzu UV-3101PC UV/vis/NIR
spectrophotometer. Fluorescence measurements were performed using a Spex Fluorolog 2
spectrofluorometer, equipped with double monochromators in both excitation and emission. Spectra
were corrected for the instrumental response of the system.
26. Frade, V. H. J.; Gonçalves, M. S. T.; Coutinho, P. J. G.; Moura, J. C. V. P. J. Photochem.
Photobiol: A 2007, 185, 220-230.
27. Alves, C. M. A.; Naik, S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Tetrahedron 2009, 65,
10441-10452.
28. Minimum Inhibitory Concentrations of growth (MIC) were assessed using a broth microdilution
method for antifungal susceptibility testing of yeasts (NCCLS M27-A). The yeast Saccharomyces
cerevisiae PYCC 4072 was used as a model organism. Briefly, cells were cultivated in 96-
microwell plates in RPMI 1640 medium, buffered to pH 7.0 with 0.165
M
morpholenepropanesulfonic acid (MOPS) buffer (Sigma). Initial cell concentration was 0.5×103
cells/mL. Growth was assessed by measuring the absorbance at 640 nm in a microplate photometer
(Molecular Devices SpectraMax Plus) after 48 h of incubation at 30 °C. MIC values were
considered as the lowest concentration of drug that resulted in a inhibition of growth > 80%. Stock
solutions of the compounds were prepared in DMSO and a final dilution was carried out in an
RPMI 1640 medium (Sigma, St. Louis, Mo.). Each drug concentration (from 400 µM to bellow the
13

MIC value, using a two-fold dilution scheme) was tested in triplicate and in at least two
independent experiments.
29. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Bioorg. Med. Chem. 2008,
16, 3274-3282.
30. Calculation of molecular properties and drug-likeness-Molinspiration cheminformatics software
tools (http://www.molinspiration.com).
14

HIGHLIGHTS
Synthesis and photophysical studies of new benzo[a]phenoxazinium chlorides as
potential antifungal agents
M. Inês P. S. Leitãoa,b, B. Rama Rajua,c, Sarala Naika,c, Paulo J. G. Coutinhoc, Maria João Sousab and M.
Sameiro T. Gonçalvesa*
c
aCentre of Chemistry, bCentre of Molecular and Environmental Biology/Department of Biology, and Centre of Physics,
University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
•
A new series of benzo[a]phenoxazinium chlorides, Nile Blue derivatives, was synthesised in
moderate yields.
•
•
•
They exhibit moderate to high fluorescence quantum yields in ethanol and water.
The fluorochromophores displayed interesting antifungal activity.
The best candidate for further development of compounds with improved activity presented a MIC
value of 1.56 µM.
17