Synthesis of new porphyrin/4-quinolone conjugates and evaluation of their efficiency in the photoinactivation of Staphylococcus aureus

Article abstract of DOI:10.1039/c5ra11070j

Simple methodologies giving access to a new series of pophyrin/4-quinolone conjugates 6 and to the corresponding intra-cyclized derivatives 8 are described. The key steps to obtain 6 involved palladium-catalyzed amination reactions of 6-bromo-4-quinolones containing N-ethyl, N-pentyl and N-ribofuransyl substituents with (2-amino-5,10,15,20-tetraphenylporphyrinato)nickel(ii) followed by demetallation. Compounds 8 were obtained from compounds 4, the nickel(ii) complexes of 6, by an oxidative intracylization approach. The new conjugates were fully characterized and the evaluation of singlet oxygen production showed that these compounds possess good to high capability to generate singlet oxygen. The efficacy of these derivatives to photoinactivate Staphylococcus aureus, a Gram-positive bacteria, was evaluated and the best results were obtained with the N-ethyl derivatives 8a and 6a.

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P. C. Gomes, L. S. M. Forezi, L. Costa, M. C. Souza, F. D. C. S. Boechat, V. F. Ferreira, A. Almeida, M. A. F.
Faustino, G. Neves and J. A. S. Cavaleiro, RSC Adv., 2015, DOI: 10.1039/C5RA11070J.
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Synthesis of new porphyrin/4-quinolone conjugates and evaluation
of their efficiency in the photoinactivation of Staphylococcus aureus
Received 00th January 20xx,
Accepted 00th January 20xx
a,b
a
b
c
Pedro N. Batalha , Ana T. P. C. Gomes , Luana S. M. Forezi , Liliana Costa , Maria Cecília B. V. de
b
b*
b
c
a
Souza , Fernanda C. S. Boechat , Vitor F. Ferreira , Adelaide Almeida , Maria A. F. Faustino ,
Maria G. P. M. S. Neves and José A. S. Cavaleiro
DOI: 10.1039/x0xx00000x
a*
a*
www.rsc.org/advances
Simple methodologies giving access to a new series of pophyrin/4-quinolone conjugates 6 and to the corresponding intra-
cyclized derivatives 8 are described. The key steps to obtain 6 involved palladium-catalyzed amination reactions of 6-
bromo-4-quinolones containing N-ethyl, N-pentyl and N-ribofuransyl substituents with (2-amino-5,10,15,20-
tetraphenylporphyrinato)nickel(II) followed by demetallation. Compounds 8 were obtained from compounds 4, the
nickel(II) complexes of 6, by an oxidative intracylization approach. The new conjugates were fully characterized and the
evaluation of singlet oxygen production showed that these compounds possess good to high capability to generate singlet
oxygen. The efficacy of these derivatives to photoinactivate Staphylococcus aureus, a Gram-positive bacteria, was
evaluated and the best results were obtained with the N-ethyl derivatives 8a and 6a.
1
8
field of research.
Introduction
Staphylococcus aureus is a bacterium frequently implied in
infections, namely superficial infections. For instance, Melo
Tetrapyrrolic macrocycles like porphyrins play an important
1
1
9
and coworkers found that in clinical samples collected from
-3
role in various fields such as catalysis,
4
supramolecular
,5
6-8 patients with diabetic foot infections, S. aureus was the most
common cause of infection and the prevalence and precocity
of multidrug resistant (MDR) isolates, namely methicillin-
resistant (MRSA), were high. From patients undergoing
antibiotic therapy, 93% of the antibiotic regimens were
considered to be inadequate based on the antibiotic
chemistry,
biomimetic models for photosynthesis,
9
,10
electronic materials and medicinal chemistry.
11-13
The medicinal interest on these compounds, namely for
photodynamic therapy (PDT) of cancer cells, is related with
their ability to act as photosensitizers promoting the formation
1
of reactive oxygen species (ROS), such as singlet oxygen ( O
in the presence of light and molecular oxygen. The interaction
of these cytotoxic species with cellular components such as
lipids, proteins and nucleic acids, in the tissue to be treated is
responsible for a cascade of biochemical events that leads to
2
),
susceptibility test results. On the other hand, reliable
2
0
inactivation of S. aureus strains has been attained by PDI and
the photosensitizing activity is independent of the antibiotic-
resistance spectrum of the isolates.
1
1-13
One strategy for obtaining new photosensitizers applicable
to PDI consists on the coupling of porphyrins derivatives with
pharmacologically active molecules, in a way that target
recognition could favour the photosensitizer attachment to
the pathogenic microorganism and potentiating a localized
cellular death.
PDT involving porphyrins as photosensitizers has been
successfully applied to different types of pathologies namely in
the diagnosis of neoplastic diseases, treatment of cancer and
1
3
age related macular degeneration. This approach can be also
used to treat infections. The evaluation of porphyrinic
photosensitizers effectiveness in the photodynamic
inactivation (PDI) of Gram-positive and Gram-negative
2
1-23
photoxidising destructive effect.
Knowing the biological activities associated with 4-
2
4-26
quinolone derivatives, in particular antibacterial ones,
herein we report the synthesis of a series of new porphyrin/4-
quinolone conjugates and of the corresponding intracyclized
derivatives and their efficacy in the photinactivation of S.
aureus, selected as a model of gram-positive bacteria.
14-17
bacteria, bacterial and fungal endospores, viruses
and
6
other antimicrobial resistant microorganisms is an important
8
a
Department of Chemistry and QOPNA, University of Aveiro, 3810-193 Aveiro,
Portugal. E-mail: gneves@ua.pt; jcavaleiro@ua.pt
b
Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Results and discussion
Fluminense, 24010-141 Niterói, Rio de Janeiro, Brazil. E-mail:nandafarma@gmail.com
c
Department of Biology and CESAM, University of Aveiro 3810-193 Aveiro, Portugal
Synthesis of porphyrin/4-quinolone conjugates
The synthetic strategy to obtain the new porphyrin/4-
quinolone conjugates
6
and
8
involved the experimental work
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summarized in schemes 1-4. The key step was based on the chromatography. The use of Xphos (Table 1, entry 1) and DTPB
Buchwald-Hartwig reaction between 2-amino-5,10,15,20- (Table 1, entry 2) provided the desired product in acceptable
tetraphenylporphyrinatonickel(II)
(
1
)
and the 6-bromo-4- yields (54 and 63% respectively), but after 24 h of reaction the
starting porphyrin wasn’t completed consumed. The use of
quinolone substrates (2a-c).
The amino component was prepared through well rac-BINAP (Table 1, entry 3) on the other hand, gave the
established procedures from 5,10,15,20-tetraphenylporphyrin desired conjugate in excellent yield (89%) with complete
(
TPP) through
a
sequence of Cu(II) complexation, consumption of the starting porphyrin, after 6 h. The
is a result of the transfer of a rac-
BINAP phenyl group to the metal ion center, followed by a
regioselective nitration, demetallation, Ni(II) complexation and formation of the byproduct
5
2
7,28
nitro-reduction.
The 6-bromo-4-quinolone substrates (2a-c) (Scheme 1) reductive-elimination step during the catalytic cycle.
were obtained from quinolone ; this intermediate was
prepared from the reaction of para-bromoaniline with diethyl
3
9
3
2
9-
ethoxymethylenemalonate, followed by thermic cyclization.
3
The subsequent reaction of 3 with bromoethane and 1-
2
bromopentane afforded respectively the ethyl 6-bromo-1-
ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (2a) and ethyl
6
-bromo-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxylate
3
3-37
(
2b).
ethyl 1,4-dihydro-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-4-
oxoquinoline-3-carboxylate 2c required the previous
treatment of with the silylating agent N,O-
The synthesis of the protected nucleoside derivative,
(
)
Figure 1. Ligands tested for the palladium catalyzed
amination reaction between 1 and 2a.
3
bis(trimethylsilyl)tri-fluoroacetamide (BSTFA) containing 1% of
trimethylchlorosilane (TMCS), followed by the reaction with 1-
O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence
of trimethylsilyltri-fluoromethanesulfonate (TMSOTf) as
Considering the lower reaction time and the excellent yield
of the desired conjugate 4a we considered the presence of
that secondary compound
5 as a minor drawback and so the
2
9
coupling with the other two bromo-quinolones 2b (Table 1,
entry 4) and 2c (Table 1, entry 5) were performed in the
presence of rac-BINAP. The reactions provided the desired
conjugates 4b and 4c in yields of 67% and 63% respectively,
with the total consumption of the starting porphyrin. In these
cases, unidentified byproducts were also formed and the
longer reaction times required for these two bromo-
quinolones when compared with those from quinolone 2a are
probably related with steric effects caused by the bulky pentyl
and ribofuranosyl groups.
catalyst.
O
O
Br
2
CO Et
Br
2
CO Et
i) for 2a,b
ii) for 2c
N
H
N
O
R
3
2a-c
Bz=
R =
O
OBz
BzO
2a (94 %)
2b (91 %)
2c (78 %)
OBz
In order to test the applicability of the porphyrin/4-
quinolone conjugates as photosensitizers in PDI, it was
necessary to perform the demetallation of derivatives 4a-c
Scheme 1. Synthesis of 6-bromo-4-quinolone substrates 2a-c.
o
Reaction conditions: i. 1) DMF, K CO , rt, 15 min. 2) RBr, 80 C,
2
3
o
(Scheme 3). This procedure was also applied to derivative 5,
2
4 h.; ii. 1) BSTFA/TMCS, CH CN, 70 C, N , 3 h. 2) 1-O-acetyl-
3 2
which has no quinolone nucleus, but was used for further
comparison. With derivative 4c, the deprotection of the β-
ribofuranosyl group was also considered in order to reach a
higher hydrophilic character for biological applications.
2
,3,5-tri-O-benzoyl-β-D-ribofuranose, TMSOTf, rt., 4 h.
In order to select the best ligand, an optimization of the
Buchwald-Hartwig reaction conditions was planned using the
Conjugates 4a,b and derivative
treatment with sulfuric acid in dichloromethane, affording
porphyrins 6a,b and in excellent yields.
5 were demetallated by
β-amino porphyrin
1 and the 6-bromo-4-quinolone 2a. The
ligands selected were XPhos, DTPB and rac-BINAP (Figure 1)
7
and the experiments were performed in the presence of
Due to the ribofuranosyl group sensibility to strong acid
media, a different approach for obtaining unprotected and
demetallated derivative 6c was applied. Therefore, conjugate
4c first underwent deprotection of the nucleoside moiety, by
treatment with potassium carbonate in a mixture of methanol
and ethanol for 48 hours, followed by demetallation with
trifluoroacetic acid (TFA), affording the unprotected nucleoside
Pd(AcO)
2
and sodium t-butoxide (Scheme 2 and Table 1). The
selection of this palladium source and of the base was based
3
on previous works.
8,39
o
All reactions were carried out in dried toluene, at 110 C
and under nitrogen atmosphere, and the reaction progress
was monitored by TLC. The workup involved the washing of
the reaction mixture with distillated water, extraction with
dichloromethane, fractioning the crude residue with silica gel
column and purification with preparative thin layer
6c in 42% yield.
2
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CO
N
2
Et
O
2
`
5
`
Ph
Ni
NH
2
R
Ph HN
8
`
N
N
N
O
7`
Ph HN
Ph
Ph
Br
CO Et
2
N
N
N
Pd(OAc)
KOtBu, toluene,
10 o
2
, ligand
N
+
1
0
5
+
Ph
Ni
N
Ph
1
C
N
N
R
Ni
Ph
Ph
2
a-c
1
Ph
4
a-c
5
Scheme 2. Buchwald-Hartwig reaction between porphyrin 1 and 6-bromo-4-quinolone derivatives 2a-c.
Table 1. Ligand and substituent (R) effects on the time and yield of the Buchwald-Hartwig reaction between 1 and 2a-c.
Entry
R (electrophile)
Ethyl (2a)
Ligand
XPhos
Time (h)
Yield of 4 (%)
Yield of 5 (%)
1 recovered (%)
1
2
3
24
24
6
54
63
89
-
-
7
9
-
Ethyl (2a)
DTPB
Ethyl (2a)
rac-BINAP
8
4
Pentyl (2b)
rac-BINAP
rac-BINAP
24
67
7
2
-
5
72
63
-
(2c)
The possibility of exploring different spectroscopic features
from these conjugates 4a,b via an oxidative intracyclization
reaction was also outlined (Scheme 4). In fact we have shown
that N-arylquinolino[2,3,4-af]porphyrins obtained through the
oxidation of corresponding 2-arylaminoporphyrin show
interesting intense absorption bands in the red region of the
visible spectrum, and this makes them potential candidates in
Ar
Ar
Ph HN
Ph HN
N
N
N
NH
N
N
i
Ph
Ph
Ph
Ni
N
Ph
HN
Ph
Ph
6
a,b and 7
38
4
a,b and 5
various scientific areas such as PDT.
O
O
N
The methodology was tested by using both the nickel(II)
complex 4a (Scheme 4, Route A) and the free-base porphyrin
6a (Scheme 4, Route B). Complex 4a was fully converted into
the corresponding intracyclized derivative after 24 hours in
nitrobenzene under reflux, while it took 48 hours to fully
convert porphyrin 6a into the new conjugate 8a. The higher
reactivity associated with complex 4a is probably due to the
planarity of the porphyrinatonickel(II) system, which could
energetically favor the cyclization process. The intracyclized
intermediate obtained from 4a after nitrobenzene removal,
was demetallated without previous purification, affording
derivative 8a in 60% yield. This procedure was successfully
CO
2
Et
2
CO Et
Ar =
N
7
(94 %)
6
a (85 %)
6b (87 %)
Ph
Ph
O
O
H
N
H
N
CO Et
2
2
CO Et
Ni
N
ii
2 %
N
N
N
4
Ph
OBz
OH
O
Ph
O
4
c
BzO
OBz
6c
HO
OH
Scheme 3. Demetallation/deprotection of derivatives 4a-c and 5.
Reaction conditions: i. 1) CH Cl , H SO , rt, 10 min. 2) K CO (aq)
sat.; ii. 1) K CO , EtOH/MeOH (3:1), rt, 48 h. 2) CHCl , TFA, rt, 1 h.
) K CO sat.
applied to conjugate 4b and derivative
5, affording derivatives
8b and 9 in 56% and 72% yields, respectively. The nucleoside
2
2
2
4
2
3
derivative 4c proved to be rather unstable at the reaction
temperature used and therefore its respective intracyclized
product could not be obtained. When lower temperatures
2
3
3
3
2
3
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1
In the H NMR spectrum of conjugate 4a, for example, it
were applied to the reaction or even when the oxidizing agent
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was added to was possible to identify three AB systems at 8.70 and 8.68
the mixture, the desired product still could not be obtained.
ppm, 8.66 and 8.63 ppm and 8.59 and 8.55 ppm with the same
coupling constant (J = 4.7 Hz) that are related with the
resonances of six β-pyrrolic protons. The three singlets at 8.43,
Route A
8
.35 and 6.48 ppm were assigned to the resonances of H-2’, H-
Ar
Ar
Ar
N
HN
N
3 and NH, respectively. The meso-phenyl protons appeared as
three sets of multiplets at 8.02-7.94 ppm, due to the ortho
protons of 5,10,15-Ph; at 7.90-7.80 ppm, due to the ortho
protons of 20-Ph and at 7.74-7.60 ppm, due to the meta and
para protons of 5,10,15,20-Ph. The protons of quinolone core
were unequivocally assigned according with the signal
multiplicity and the correlations observed in the COSY
spectrum. The resonance of quinolone H-5’ appeared as a
doublet at 7.74 ppm (J = 2.6 Hz) as confirmed by the
correlation with the double doublet at 7.55 ppm (J = 9.0 and
ii
i
N
N
N
Ph
Ni
Ph
Ni
Ph
4
a,b and 5
8a,b and 9
O
N
O
N
Ar =
2
CO Et
2
CO Et
9
(72 %)
2
.6 Hz), assigned to the resonance of H-7’. The correlation
observed between the resonance of H-7’ and the doublet at
.32 ppm (J = 9.0 Hz) allowed the assignment of this signal to
H-8’ proton.
A careful analysis of the HMBC spectrum of 4a allowed the
8
b (56 %)
8
a (60 %)
7
Route B
CO
N
Et
2
CO Et
2
O
O
N
unequivocal assignment of the carbonyl carbon resonances,
being the signals at 173.9 and 166.3 ppm assigned to the C-4’
and to the carbon from the ester carbonyl group, respectively.
HN
iii
2%
N
1
4
Considering the H NMR of conjugate 4b, the main difference
6
a
8a
N
N
when compared with 4a spectrum is due to the resonance of
the extra aliphatic protons due to the pentyl group attached to
N-1 (see Supporting Information).
Ph
Ph
In the case of the porphyrin/ribonucleoside conjugate 4c
+
the expected m/z value at 1345.36388 ([M+H] ) observed in
Scheme 2. Oxidative intracyclization reaction from derivatives
a,b, 5 and 6a. Reaction conditions: i. PhNO , reflux, 24 h.; ii. 1)
CH Cl , H SO , rt, 10 min. 2) K CO sat.; iii. PhNO , reflux, 48 h.
4
2
+
the HRMS-ESI spectrum confirmed its molecular formulae.
2
2
2
4
2
3
2
1
The H NMR spectrum of this compound presents a more
complex pattern due to the presence of the protected ribose
unit. The multiplets at 8.02-7.95 ppm and at 7.95-7.85 ppm
Structural characterization
The structures of new derivatives 4a-c,
6a-c and 8a,b were assigned to the resonances of the ortho protons of 5,10,15-Ph
1
13
assigned according to their UV-Vis, H and C NMR spectra and 20-Ph, respectively, comprise also the resonances of two
and their molecular formulae were confirmed by HRMS. 2D ortho OBz protons each. The remaining two ortho OBz protons
NMR spectra (COSY, HSQC and HMBC) were also used in order appear as a multiplet at 8.77-8.05 ppm. The most prominent
to unequivocally identify the proton and carbon resonances.
difference observed for the quinolone proton resonances was
The absorption spectra of quinolone–porphyrin type related to H-2’ which appeared as a singlet at lower field than
derivatives 4a-c and 6a-c exhibit intense Soret bands at 411- in conjugates 4a and 4b (8.92 ppm versus 8.43 ppm). The COSY
15 nm and Q-bands at 520-660 nm, being the number of the spectrum analysis was essential for the unequivocal
4
last ones dependent of the absence or presence of the assignments of the ribose moiety protons (Figure 2). The low
nickel(II) ion in the porphyrin core while the derivatives 8a,b field doublet in the aliphatic region at 6.48 ppm (J = 4.6 Hz)
show a visible spectra typical of intracyclized macrocycles (see was attributed to the H-1’’. The correlation between this signal
also SI).
with the triplet at 6.01 ppm (J = 4.6 Hz) allowed its assignment
+
The HRMS-ESI of the porphyrin/quinolone conjugates 4a to H-2’’. The H-3’’ resonance was identified as the triplet at
+
and 4b show protonated molecular ions [M+H] at m/z 5.90 ppm (J = 4.6 Hz) due to the correlations observed with H-
9
29.27363 and 971.32075, respectively, confirming the success 2’’ and with the multiplet at 4.96-4.82 ppm, assigned to the
1
of the Buchwald-Hartwig. The H NMR spectra of these resonances of H-4’’and H-5’’.
conjugates show similar patterns considering the resonances
of the porphyrin and quinolone protons.
4
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function loss, structure disruption and, consequently, cell
1
6,18-20
Ph
Ph
death.
Therefore, the potential photodynamic effect of
1
5
20
N
H
O
N
N
Ni
N
H
N
derivatives 6a-c and 8a,b was first analyzed by evaluating their
5
'
CO2Et
1
ability to generate O
the photodecomposition
diphenylisobenzofuran (DPBF), which reacts with the
produced through a [4+2] cycloaddition mechanism, affording
2
. This was accomplished by monitoring
N
2
'
of the yellowish 1,3-
H
7'
1
0
5
8'
1
O
2
1
''
Ph
Ph
5''
O
H
H
4''
BzO
2''
4
0-46
H
H
3''
the colorless 1,2-dibenzoylbenzene.
DPBF photodecomposition was made by measuring its
absorption decay at 415 nm, on DMF/H O (9:1) solutions
The monitoring of the
4
'
4c
H
H
OBz
H
H
5'
OBz
1
8
O
17
H
CO2Et
''
2
Ph
15
5
''
N
containing the testing conjugates (0.5 µM) and DPBF (50 µM),
while being aerobically irradiated with red light (654 nm ± 20
2
nm) with an irradiance of 9 mW/cm by using a LED array.
H
N
2
N
N
N
7''
H
8
''
H
N
H
1
0
5
Ph
Meso-tetraphenylporphyrin (TPP), a good singlet oxygen
Ph
8a
generator, was used as a positive standard in the same
2
2
conditions. A solution containing DPBF (50 µM), but without
photosensitizer was used to evaluate its photodecomposition
under the irradiation conditions.
Figure 2. Main COSY correlations of compounds 4c and 8a.
1
An important feature of the H NMR spectra of conjugates
Considering that the structural difference between the
synthesized conjugates (6a-c and 8a,b) and the prepared
6a and 6b is the presence of a signal at around -2.60 ppm due
phenyl analogs (
pyridone-3-carboxylate core present in the quinolone scaffold,
derivatives and were also evaluated in order to the further
7 and 9, respectively), relies on the ethyl 4-
to the resonances of the inner NH protons, which confirms the
success of the demetallation step. In the case of
porphyrin/quinolone 6c it is possible to note also the absence
of the signals due to the resonances of the benzoyl groups
confirming the presence of deprotected hydroxyl groups. The
7
9
comparison of their photodynamic profile and to understand
the role of the quinolone portion in the singlet oxygen
generation.
+
HRMS-ESI of the porphyrin/quinolone conjugates 6a-c show
+
the expected [M+H] molecular ions at the m/z values
The results of the photodecomposition of DPBF during the
irradiation period in the presence of each PS are displayed on
Figure 3. The results state that DPBF photodegradation was in
general highly enhanced in the presence of the PS conjugates.
With the exception of nucleoside derivative 6c, it was possible
to observe that all prepared compounds were capable of
generating singlet oxygen. The conjugates 6a,b, and the phenyl
8
73.35446, 915.40094 and 977.36557 respectively.
The molecular formulae of the intracyclized N-(6-
quinolonil)quinolino[2,3,4-af]porphyrins 8a and 8b were also
+
unambiguously confirmed by HRMS-ESI showing the expected
+
[
M+H]
molecular ions at m/z values 871.33852 and
9
13.38539 respectively.
1
Taking into account the H NMR spectrum of derivative 8a
analog
although they are slightly less efficient than TPP. A different
situation can be observed with the cyclic conjugates 8a,b and
that show a singlet oxygen production higher than that
7 display a similar DPBF photodegradation profile
,
the presence of two doublets at 9.69 ppm (J = 4.7 Hz) and 9.66
ppm (J = 8.2 Hz) due to the unshielded protons H-18 and H-5’
respectively is consistent with an intracyclized porphyrinic
9
4
7
3
8
observed for TPP.
core. Correlations observed on the COSY spectra allowed the
identification of the resonance of H-17 as the doublet at 8.83
ppm (J = 4.7 Hz) and of H-4’ as being in the multiplet at 7.85-
7.49 ppm (Figure 2). The electron-withdrawing effect on the
protons H-5’’ and H-7’’, due to the presence of the
heteroaromatic ring attached to the C-6’’, caused their
resonances to appear at a lower field than for derivatives 4a
and 6a, being related to the doublet at 9.12 ppm (J = 2.5 Hz)
and to the double doublet at 8.10 ppm (J = 8.6 and 2.5 Hz),
respectively. The demetallation was also confirmed by the
presence of the singlet at -1.32 ppm, related to the two
internal NH protons. For a more detailed description and NMR
spectra see the Supporting Information.
Figure 3. Time dependent photodecomposition of DPBF (50 µM)
photosensitized by derivatives 6a-c, 7, 8a,b, 9 and TPP in
Singlet oxygen generation studies
The efficiency of a photosensitizer either in PDT or PDI is
DMF/H
0 nm) with an irradiance of 9 mW/cm with and without PS
0.5 µM).
2
O (9:1) upon irradiation with red light LEDs (654 nm ±
strongly related with its ability to generate reactive oxygen
1
species (ROS), namely singlet oxygen ( O
2
2
2
) which can initiate
(
oxidative reactions at cellular components level, causing
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Based on these results it was possible to highlight that
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The studied photosensitizers revealed clear differences
cyclization has
a
positive influence on singlet oxygen amongst them (ANOVA, p < 0.05). The most effective
production of the derivatives probably due to their higher compound was the intracyclized quinolone-conjugate 8a with
3
8,49,50
planarity introduced by the intracyclization process.
In rates of inactivation of more than 3 log soon after 90 min of
addition, it was not found a direct relationship between the irradiation. At the end of the experiment (180 min) the
alkyl groups attached to the N-1 position of the quinolone and inactivation rate was of 5.6 log and after 150 min, of 5.0 log
1
the
2
O production; the low efficiency of nucleoside 6c to (Figure 4). The quinolone-conjugate 6a was also very effective,
produce singlet oxygen is probably related with molecular showing rates of inactivation of 4.3 and 4.9 log, respectively,
aggregation.
after 150 and 180 min. In the presence of 6b the viability of S.
aureus decreased 2.7 log after 150 min of irradiation and 3.4
log after 180 min of irradiation. Compounds 6b and 8b
Microbial photoinactivation studies
The results obtained from the photoinactivation of Gram- produced identical rates of inactivation (ANOVA, p > 0.05)
positive bacterium strain S. aureus (ATCC 6538) using the after 150 min (ca 2.6 log) (Figure 4). The non-quinolone
quinolones conjugates 6a-c
are summarized in Figure 4.
The photoinactivation experiments were performed in inactivation of 6.3 log. However, after 150 min of irradiation
phosphate buffered saline (PBS) using a PS concentration of the inactivation rate was only 3.4 log (Figure 4). The nucleoside
,
8a,b and the phenyl analogs
7
and intracyclized derivative
9 at the end of the experiment was
9
also effective to inactivate the S. aureus with a rate of
-
2
1
0.0 µM under white light with an irradiance of 150 W m . The derivative 6c and compound 7 were the least effective
results obtained in the dark control (bacterial suspension in showing bacterial reduction of ca. 2.1 log after 180 min (Figure
the presence of photosensitizer but protected from light 4).
exposure) and in the light control (bacterial suspension
Having into account that according to the American Society
irradiated with white light in the absence of the of Microbiology, any new approach must achieve a reduction
photosensitizer) indicate that the reduction on bacterial of at least 3 log CFU (killing efficiency of 99.9% or more) to be
concentration was due to the photoinactivation treatment. termed "antimicrobial" or "antibacterial", derivatives 8a
The bacterial concentration in both controls was similar during and even 8b and 6b, can be considered effective compounds
the experiment (ANOVA, p > 0.05). to be used in PDI of Gram-positive bacteria. However, the rate
, 6a, 9,
The concentration of 10.0 µM used in these studies was of inactivation for these five more effective compounds was
based on the results obtained from preliminary assays with significantly different after 90 min of treatment, which is due
1
to the PS nature and O production capability.
conjugate 6a using two different concentrations: 5.0
µ
bacterial inactivation of 2.6 log) and 10.0 M (bacterial
M
2
(
µ
The results of this study show that (1) the insertion of
inactivation of 3.1 log) after 120 min under white light quinolone moiety in the porphyrin core, (2) the alteration of
irradiation (data not shown). Since the results revealed that the planarity of the macrocycle core by intracyclization and (3)
the higher concentration (10.0 µM) was the more effective the type of groups and the chain length in the quinolone
one, all the synthesized compounds were studied at this moiety are key features to be taken into account considering
concentration and the irradiation time was extended to 180 the efficiency of these derivatives in the photoinactivation of S.
min.
aureus.
The high efficiency of the new intracyclized porphyrin/4-
quinolone conjugate 8a when compared with the others
1
tested PS is probably associated with the ability to produce O2
but also with its structural features. Comparing the efficacy of
the intracyclized conjugates 8a (N-ethyl substituted quinolone)
and 8b (N-pentyl substituted quinolone), the high production
1
of O observed for compound 8b is not reflected in a better
2
photoinactivation rate; this suggests that a slight less
hydrophobic character of 8a has also an important role in its
efficiency. In fact the ideal PS must possess hydrophilic and
hydrophobic character, i.e. must be an amphiphilic molecule,
4
8
in order to improve its photodynamic efficiency. The
hydrophobicity is important for the PS to diffuse more easily
across the cell membrane and simultaneously it must have
some hydrophilic character in order to allow its solubilisation
in aqueous medium.
Figure 4. Density variation of S. aureus after 30, 60, 90, 120,
1
50 and 180 min of irradiation, in the presence of 10 µM of
different photosensitizers. Each value represents the mean ±
standard deviation of three independent experiments, with
two replicates each. Error bars represent standard deviations.
6
| J. Name., 2012, 00, 1-3
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a
Table 2. Drug-likeness property/Lipinski’s ‘rule of five’ parameters calculated for compounds 6a-c, 7, 8a,b and 9.
Compound
Molecular Weight
873.03
miLogP
9.88
n-ROTB
n-O/N
n-OH/NH
n-violations
Volume
788.98
839.39
861.93
645.95
778.52
828.93
635.49
TPSA
117.70
117.70
187.62
69.39
6
a
b
10
13
11
6
9
6
3
3
6
3
2
2
2
2
2
4
2
2
2
2
6
915.11
10.09
9.61
6c
977.09
13
5
7
705.87
9.96
8
a
b
871.01
9.77
8
9
110.61
110.61
62.30
8
913.09
10.01
9.82
11
4
9
9
703.85
5
(
a) n-ROTB, number of rotatable bonds; n-O/N, number of hydrogen acceptors; n-OH/NH, number of hydrogen bond donors; TPSA,
topological polar surface area; n-violations, number of violations according to the Lipinski ‘rule of five’.
1
In order to confirm the importance of this feature, the produces a much higher quantity of O
2
than compound 6b,
milogP of the compounds was calculated using Molinspiration the planarity conferred to the macrocycle by the
5
1
WebME Editor 3.81 (Table 2). The compounds presented intracyclization process seems to affect significantly its
values in the range 9.61–10.01 and as expected the N-ethyl interaction with the bacterial cell.
substitution conferred a slight less hydrophobic character than
the N-pentyl substitution.
Experimental
The less hydrophobic character of PS 8a when compared
1
4,15
with 8b can enhance its affinity for bacteria,
and General
1
2
consequently can increase PDI efficiency. The high efficiency
2
13
H and C NMR spectra were recorded on Bruker Avance 300
1
1
13
of the intracyclized derivative
also reflected in the rate of inactivation of S. aureus in the first MHz for H and 125 MHz for C) spectrometers. CDCl
50 min of irradiation. Again, this confirms how important it is CDCl /CD OD were used as solvents and TMS as internal
production and reference and the chemical shifts are expressed in δ (ppm).
2
9 on the production of O is not (300 MHz for H and 75 MHz for C), Bruker Avance 500 (500
1
13
3
or
1
3
3
1
the compromise between an efficient
O
2
1
structural features that confer an amphiphilic character to the Unequivocal H assignments were made using 2D COSY, while
1
13
PS. The results suggest that the O
is adequate to inactivate the bacterium S. aureus.
Compound 6a was the second most efficient to inactivate were optimized for 7 Hz). HRMS were recorded on a VG
2
production capability of 8a
C assignments were made on the basis of 2D HSQC and
HMBC experiments (the delay for long-range JC/H couplings
3
S. aureus. This porphyrin/4-quinolone conjugate is not AutoSpec M mass spectrometer using CHCl as solvent and 3-
intracyclized and consequently is not so efficient to produce nitrobenzyl alcohol (NBA) as matrix.
1
O as compounds 9, 8a and 8b. However, the presence of the
2
short chain ethyl group attached to the N-1’ position probably Coupling reactions of porphyrin 1 and bomoquinolones 2a-c.
1
compensates this less
responsible for a better performance when compared with the
pentyl substituted derivatives 8b and 6b. The low efficiency of bromoquinolone 2a-c (29.2 µmol), KO Bu (3.5 mg, 31,2 µmol),
compound 6c is certainly related with its poor ability to Pd(AcO) (1 mg, 4.5 µmol), rac-BINAP (2.3 mg, 3.7 µmol) and
O
2
production drawback and is General Procedure.
A
mixture of porphyrin
1
(10.0 mg, 14.6 µmol),
t
2
1
o
produce O
2
since it is the less hydrophobic one (miLogP 9.61). dried toluene (5 mL) was purged with N
under N atmosphere until complete consumption of the
generation capabilities, 6a is a much starting porphyrin (6-48 h, monitored by TLC). After reaching
more efficient PS, having derivative given a poor rate of room temperature, the mixture was washed with distilled
inactivation for S. aureus. This fact confirms again the water and extracted with CH Cl . The combined organic
importance of the quinolone moiety for the PDI efficiency, extracts were evaporated under reduced pressure and the
2
and stirred at 110 C
Although quinolone conjugate 6a and phenyl derivative
7
2
1
presented similar O
2
7
2
2
1
considering that due to the O
microseconds, the PS must be close enough to the bacterial CH
membrane or even inside the cell, to cause efficient cell Both fractions were further purified by preparative TLC. The
2
short lifetime, in the order of crude mixture was fractioned in a silica gel column, using first
2
2
Cl as eluent and then a mixture of CH Cl /MeOH (20:1).
2
2
4
8
damage. The similar results obtained in S. aureus inactivation first fraction was purified using a mixture of CH
by 6b and 8b demonstrate that the PS structure planarity also ether (1:1) affording the byproduct . The desired coupling
plays an important role in PDI. Even though compound 8b products (4a-c) were obtained from the second fraction using
2 2
Cl /petroleum
5
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a mixture of CH
2
Cl
Cl
2
/MeOH (20:1). Both products were 2H, J= 4.8 Hz, H-β), 8.66 and 8.59 (AB, 2H, J = 4.9 Hz, 8.36 (s,
/petroleum ether. 1H, H-3), 8.11-8.05 (m, 2H, Ho-OBz), 8.02-7.95 (m, 8H, Ho-Ph-
,10,15 and Ho-OBz), 7.95-7.85 (m, 4H, Ho-OBz and Ho-Ph-20),
crystallized from CH
2
2
5
2
-(3-Ethoxycarbonyl-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl-
7.85-7.78 (m, 1H, Hp-Ph-20), 7.77 (d, 1H, J=2.5 Hz, H-5’), 7.75-
7.61 (m, 11H, Hm,p-Ph-5,10,15 and Hm-Ph-20), 7.60-7.34 (m,
amino)-5,10,15,20-tetraphenylporphyrinatonickel(II) (4a)
1
Yield: (12.1 mg, 89%). H NMR (300 MHz; CDCl
.68 (AB, 2H, J = 4.7 Hz, H-β), 8.66 and 8.63 (AB, 2H, J = 4.7 Hz, 6.41 (s, 1H, N-H), 6.01 (t, 1H, J= 4.6 Hz, H-2’’), 5.90 (t, 1H, J=4.6
H-β), , 8.59 and 8.55 (AB, 2H, J = 4.7 Hz, H-β), 8.43 (s, 1H, H- Hz, H-3’’), 4.96-4.82 (m, 3H, H-4’’ and H-5’’), 4.31-4.06 (m, 2H,
3
) δ 8.70 and 11H, Hm,p-OBz, H-7’ and H-8’), 6.48 (d, 1H, J = 4.6 Hz, H-1’’),
8
1
3
2
’), 8.35 (s, 1H, H-3), 8.02-7.94 (m, 6H, Ho-Ph-5,10,15), 7.90- CO
2
CH
2
CH
3
) and 1.29 (t, 3H, J=7.2 Hz, CO
) δ 173.82 (C-4’), 166.10 (COPh), 165.11
Et), 164.69 (COPh), 145.01 (C-2’), 143.14,
.7 Hz, H-7’), 7.30 (d, 1H, J = 9.2 Hz, H-8’), 6.48 (s, 1H, NH), 142.93, 142.81, 142.47, 142.17, 141.69, 141.34, 140.90,
.43 (q, 2H, J = 7.0 Hz, CO CH CH ), 4.21 (q, 2H, J = 7.1 Hz, 140.69, 140.64, 139.33, 134.10, 133.90, 133.73, 133.68,
CH ), 1.53 (t, 3H, J = 7.1 Hz, NCH CH ) and 1,45 (t, 3H, 133.60, 132.88, 132.31, 132.26, 132.19, 132.00, 131.67,
J=7.1 Hz, CO ) ppm. C NMR (75 MHz; CDCl ) δ 173.9 131.62, 131.00, 130.13, 129.90, 129.83, 129.77, 129.78,
Et), 147.2 (C-2’), 145.2, 143.1, 142.9, 142.8, 129.67, 129.31, 129.01, 128.72, 128.67, 128.63, 128.59,
2 2 3
CH CH ). C NMR
7
.80 (m, 3H, Ho,p-Ph-20), 7.74 (d, 1H, J=2,7 Hz, H-5’), 7.74-7.60 (125 MHz; CDCl
3
(m, 11H, Hm,p-Ph-5,10,15, Hm-Ph-20), 7.48 (dd, 1H, J=9.2 and (COPh), 164.96 (CO
2
2
4
2
2
3
NCH
2
3
2
3
1
3
2
CH
C-4’), 166.3 (CO
2
CH
3
3
(
2
1
1
1
1
1
42.6, 142.1, 141.6, 141.2, 140.9, 140.6, 140.6, 140.2, 139.2, 128.55, 128.39, 128.15, 127.93, 127.79, 127.73, 127.15,
33.6, 133.6, 133.5, 133.1, 132.9, 132.6, 132.2, 132.1, 131.9, 127,06, 126.94, 126.92, 120.31, 120.07, 118.64, 116.49,
31.6, 131.5, 130.9, 130.6, 129.4, 128.6, 127.8, 127.7, 127.7, 116.11, 115.89, 113.56, 113.32 (C-3), 110.79 (C-3’), 90.32 (C-
27.1, 126.9, 120.4, 120.1, 118.6, 116.9, 116.2, 115.8, 113.6, 1’’), 80.83 (C-4’’ or C-5’’), 74.43 (C-2’’), 70.78 (C-3’’), 63.46 (C-
12.3 (C-3), 109.5, 60.9 (CO
CH ), 14.5 (CO CH CH ) ppm. UV-vis (DMF/H
59 6
max (log ε) = 415 (4.94) 537 (4.07) 587 (4.09) nm. HRMS (ESI) (4.14) nm. HRMS (ESI) m/z calcd for C82H N NiO10 [M+H]
2
CH
2
CH
3
), 49.0 (NCH
2
CH
3
), 14.7 4’’ or C-5’’), 60.72 (CO
2 2 3 2 2 3
CH CH ), 14.34 (CO CH CH ) ppm. UV-
(NCH
2
3
2
2
3
2
O (9:1)): vis (DMF/H
2
O (9:1)): λmax (log ε) = 415 (5.21) 538 (4.13) 582
+
λ
+
m/z calcd for
29.27363.
C
58
H
43
N
6
NiO
3
[M+H] 929.27446, found 1345.36407, found 1345.36377.
9
Demetallation of conjugates 4a,b. General Procedure.
2-(3-Ethoxycarbonyl-4-oxo-1-pentyl-1,4-dihydroquinolin-6-yl-
2 2
To a solution of conjugate 4a,b (10.8 µmol) in 6 mL of CH Cl it
amino)-5,10,15,20-tetraphenylporphyrinatonickel(II) (4b)
was added 600 µL of concentrated H
) δ 8.70 and 8.69 vigorously stirred at room temperature for 10 min and then
AB, 2H, J = 4.9 Hz, H-β), , 8.66 and 8.65 (AB, 2H, J = 4.9 Hz, H- neutralized with a saturated aqueous solution of K CO . The
β), , 8.59 and 8.58 (AB, 2H, J = 4.9 Hz, H-β), 8.40 (s, 1H, H-2’), resulting mixture was extracted with CH Cl and the combined
.35 (s, 1H, H-3), 8.04-7.96 (m, 6H, Ho-Ph-5,10,15), 7.93 (d, 2H, extracts were washed with water and dried over anhydrous
J=6.9 Hz, Ho-Ph-20), 7.86 (t, 1H, J=7.5 Hz, Hp-Ph-20), 7.77-7.70 Na SO . The solvent was evaporated under reduced pressure
m, 3H, Hp-Ph-5,10,15), 7.70-7.61 (m, 9H, Hm -Ph-5,10,15,20 and the desired product was obtained pure after purification
and H-5’), 7.55 (dd, 1H, J=9.0 and 2.7 Hz, H-7’), 7.32 (d, 1H, by preparative TLC using a mixture of CH Cl /MeOH (20:1),
J=9.2 Hz, H-8’), 6.49 (s, 1H, NH), 4.43 (q, 2H, J = 7.2 Hz, followed by crystallization with CH Cl /petroleum ether.
CO CH CH ), 4.12 (t, 2H, J=7.0 Hz, H-1’’), 1.95-1.80 (m, 2H, H-
’’), 1.45 (t, 3H, J= 7.2 Hz, CO CH CH ), 1.42-1.32 (m, 4H, H-3’’ 2-(3-Ethoxycarbonyl-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl-
and H-4’’), and 0.93 (t, 3H, J=7.0 Hz, H-5’’) ppm. C NMR (125 amino)-5,10,15,20-tetraphenylporphyrin (6a)
2 4
SO . The mixture was
1
Yield: (9.5 mg, 67%). H NMR (300 MHz; CDCl
3
(
2
3
2
2
8
2
4
(
2
2
2
2
2
2
3
2
2
2
3
1
3
1
MHz; CDCl
3
) δ 173.72 (C-4’), 166.42 (CO
2 3
Et), 147.80 (C-2’), Yield: (8.0 mg, 85%). H NMR (300 MHz; CDCl ) δ 8.84 and 8.79
1
1
1
1
1
1
45.41, 143.16, 142.97, 142.81, 142.71, 142.14, 141.63, (AB, 2H, J = 4.8 Hz, H-β), 8.78 – 8.73 (m, 3H, H-β), 8.61 (d, 1H,
41.27, 141.01, 140.70, 140.65, 140.14, 139.31, 133.68, J=4.8 Hz, H-β), 8.45 (s, 1H, H-2’), 8.36 (s, 1H, H-3), 8.26-8.15
33.59, 133.18, 132.91, 132.86, 132.36, 132.13, 131.97, (m, 8H, Ho-Ph-5,10,15,20), 8.02-7.95 (m, 1H, Hp-Ph-20), 7.90-
31.65, 131.57, 130.92, 130.78, 129.40, 128.65, 127.79, 7.82 (m, 2H, Hm-Ph-20), 7.81 (d, 1H, J = 2.7 Hz, H-5’), 7.79-7.72
27.73, 127.69, 127.13, 126.94, 120.32, 120.12, 118.61, (m, 9H, Hm,p-Ph-5,10,15), 7.68 (dd, 1H, J = 9.2 and 2.7 Hz, H-
16.99, 116.27, 115.85, 113.85, 112.19 (C-3), 109.49, 60.88 7’), 7.39 (d, 1H, J = 9.2 Hz, H-8’), 6.74 (s, 1H, NH), 4.45 (q, 2H, J
(
CO
2
CH
’’), 22.28 (C-3’’ or C-4’’), 14.55 (CO
O (9:1)): λmax (log ε) = 414 (5.12) 534 -2.60 (bs, 2H, internal NH) ppm. C NMR (125 MHz; CDCl
4.05) 588 (4.08) nm. HRMS (ESI) m/z calcd for C61 NiO 173.76 (C-4’), 166.38 (CO Et), 147.32 (C-2’), 142.70, 142.19,
141.97, 140.57, 140.30, 140.25, 134.56, 134.41, 134.25,
34.17, 132.98, 132.74, 132.60, 130.74, 129.61, 128.74,
2
CH
3
), 54.17 (C-1’’), 28.75 (C-2’’ and C-3’’ or C-2’’ and C- =7.2 Hz, CO
2
CH
2
CH
3
), 4.24 (q, 2H, J =7.3 Hz, NCH
2
CH
3
), 1.56 (t,
CH ) and
) δ
4
2
CH CH ), 13.93 (C-5’’) 3H, J=7.3 Hz, NCH
2
3
2
CH
3
), 1,47 (t, 3H, J = 7.2 Hz, CO
2
CH
2
3
13
ppm. UV-vis (DMF/H
2
3
(
[
H
49
N
6
3
2
+
M+H] 971.32141, found 971.32075.
1
2
-[3-Ethoxycarbonyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranos-1-yl)-
-oxo-1,4-dihydroquinolin-6-yl-amino)-5,10,15,20-
127.75, 127.69, 127.68, 127.47, 127.43, 127.15, 126.89,
126.76, 126.66, 121.48, 120.68 (C-7’), 119.97, 117.92, 116.92,
116.87 (C-8’), 116.60, 114.32 (C-5’), 110.86, 109,82, 60.91
4
tetraphenylporphyrinatonickel(II) (4c)
1
Yield: (12.4 mg, 63%). H NMR (300 MHz; CDCl
3
) δ 8.92 (s, 1H, (CO
2
CH
2
3 2 3
CH ), 48.93 (NCH CH ), 14.75 and 14.56 ppm. UV-vis
H-2’), 8.70 and 8.69 (AB, 2H, J = 4.8 Hz, H-β), 8.67 and 8.66 (AB, (DMF/H
2
O (9:1)): λmax (log ε) = 411 (5.34) 459 (4.65) 524 (4.27)
8
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5
71 (3.98) 600 (3.98) 659 (3.52) nm. HRMS (ESI) m/z calcd for 143.00, 142.89, 142.71, 142.60, 142.26, 141.78, 141.39,
+
[M+H] 873.35477, found 873.35446.
C
58
H
45
N
6
O
3
140.91, 140.70, 140.67, 139.37, 133.71, 133.63, 133.61,
33.18, 132.95, 132.54, 132.41, 132.30, 132.23, 132.05,
1
2
-(3-Ethoxycarbonyl-4-oxo-1-pentyl-1,4-dihydroquinolin-6-yl-
131.74, 131.70, 130.95, 129.63, 129.19, 128.53, 127.88,
127.81, 127.26, 127.11, 127.09, 126.99, 121.31 (C-7’), 120.18,
amino)-5,10,15,20-tetraphenylporphyrin (6b)
1
Yield: (8.6 mg, 87%). H NMR (300 MHz; CDCl
3
) δ 8.84 and 8.80 118.76, 117.15 (C-5’ or C-8’), 116.49, 115.81, 112.90 (C-3),
(AB, 2H, J = 4.8 Hz, H-β), 8.78 – 8.74 (m, 3H, H-β), 8.62 (d, 1H, J 111.91 (C-8’ or C-5’), 108.76, 92.55 (C-1’’), 84.68 (C-2’’), 75.76
=
4.8 Hz, H-β), 8.40 (s, 1H, H-2’), 8.36 (s, 1H, H-3), 8.25-8.16 (m, (C-3’’), 69.00 (C-4’’), 61.44 (CO ), 60.35 (C-5’’) and
2
CH
2
CH
3
8
H, Ho-Ph-5,10,15,20), 8.03-7.95 (m, 1H, Hp-Ph-20), 7.91-7.83 14.22 (CO CH CH ) ppm. UV-vis (DMF/H O (9:1)): λmax (log ε) =
3
2
2
2
(
m, 2H, Hm-Ph-20), 7.82 (d, 1H, J = 2.7 Hz, H-5’), 7.78-7.70 (m, 415 (5.09) 536 (4.02) 583 (4.05) nm. HRMS (ESI) m/z calcd for
+
9
7
7
2
H, Hm,p-Ph-5,10,15), 7.65 (dd, 1H, J = 9.2 and 2.7 Hz, H-7’),
61 49 6 7
C H N O [M+H] 977.36572, found 977.36557.
.35 (d, 1H, J = 9.2 Hz, H-8’), 6.73 (bs, 1H, NH), 4.45 (q, 2H, J =
.2 Hz, CO
2
CH
2
CH
3
), 4.12 (t, 2H, J = 7.0 Hz, H-1’’), 1.95-1.83 (m, Oxidative cyclization and demetallation of porphyrin/4-
CH CH ), 1.43-1.35 (m, 4H, quinolone conjugates 4a,b. General Procedure A.
H, H-2’’), 1.46 (t, 3H, J = 7.2 Hz, CO
2
2
3
H-3’’ and H-4’’), 0.94 (t, 3H, J = 7.0 Hz, H-5’’) and -2.61 (bs, 2H, A solution of conjugate 4a,b (11.5 µmol) in nitrobenzene (1
1
internal NH) ppm. C NMR (125 MHz; CDCl
3
3
) δ 173.82 (C-4’), mL) was refluxed until complete consumption of the starting
1
1
1
1
1
1
66.41 (CO
40.30, 140.25, 138.02, 137.33, 134.56, 134.42, 134.25, temperature, the solution was solubilized in CH
34.17, 132.78, 133.00, 132.60, 130.75, 129.63, 128.75, nitrobenzene removed through elution with CH
2
Et), 147.85 (C-2’), 142.69, 142.19, 141.97, 141.59, material (24 h, monitored by TLC). After reaching room
2
Cl
2
and the
2
over a
2
Cl
27.75, 127.69, 127.66, 127.46, 127.15, 126.89, 126.76, short silica gel column. The crude product was recovered by
26.66, 121.48, 120.68, 119.97, 117.92, 116.99, 116.60, elution with CH Cl /MeOH (20:1) and solubilized with CHCl (6
14.29, 110.76, 109.51, 60.92 (CO CH CH ), 54.18 (C-1’’), 28.74 mL). Concentrated H SO (600 µL) was then added to the
2
2
3
2
2
3
2
4
(
C-2’’ and C-3’’ or C-2’’ and C-4’’), 22.28 (C-3’’ or C-4’’), 14.55 mixture which was vigorously stirred under room temperature
CO CH CH ) and 13.93 (C-5’’) ppm. UV-vis (DMF/H O (9:1)): for 10 min. The mixture was neutralized with a saturated
max (log ε) = 411 (5.30) 523 (4.21) 571 (3.97) 600 (3.95) 656 aqueous solution of K CO followed by extraction with CH Cl
The combined extracts were washed with water and dried
over anhydrous Na SO . The solvent was evaporated under
reduced pressure and the products were purified through
preparative TLC using a mixture of CH Cl /MeOH (20:1),
followed by crystallization with CH Cl /petroleum ether.
(
2
2
3
2
λ
2
3
2
2
.
+
51 6 3
(3.56) nm. HRMS (ESI) m/z calcd for C61H N O [M+H]
9
15.40172, found 915.40094.
2
4
Deprotection and demetallation of conjugate 4c.
A solution containing conjugate 4c (17 mg, 12,6 µmol), K
3.5 mg, 25.3 µmol), EtOH (1 mL) and MeOH (2 mL) was stirred
2
2
2
CO
3
2
2
(
in the dark under room temperature for 48 h. Then, the N-(3-Ethoxycarbonyl-1-ethyl-4-oxo-1,4-dihydroquinolin-6-
solvent was evaporated under reduced pressure and to the yl)quinolino[2,3,4-af]-5,10,15-triphenylporphyrin (8a)
1
(2 mL) and TFA (300 µL). Yield: (6.0 mg, 60%). H NMR (300 MHz; CDCl
obtained residue was added CHCl
3
3
) δ 9.69 (d, 1H, J
The mixture was vigorously stirred under room temperature = 4.7 Hz, H-18), 9.66 (d, 1H, J = 8.2 Hz, H-5’), 9.12 (d, 1H, J = 2.5
for 1 h, after which it was neutralized with a saturated Hz, H-5’’), 8.83 (d, 1H, J = 4.7 Hz, H-17), 8.75 (d, 1H, J = 5.0 Hz,
aqueous solution of K
combined organic extracts were washed with water and dried 1H, J = 5.0 Hz, H-β), 8.61 (d, 1H, J = 4.5 Hz, H-β), 8.32-8.22,
over anhydrous Na SO . The solvent was evaporated under 8.20-8.14 and 8.14-8.04 (3bs, 6H, Ho-Ph-5,10,15), 8.10 (dd, 1H,
reduced pressure and the product was obtained pure after J = 8.6 and 2.5 Hz, H-7’’), 7.85-7.75, 7.75-7.69, 7.69-7.59 and
purification by preparative TLC using mixture of 7.54-7.49 (4m, 14H, H-3, H-2’, H-3’, H-4’, H-8’’, Hm,p-Ph-
/MeOH (20:1), followed by crystallization with 5,10,15), 4.46 (q, 2H, J = 7.1 Hz, CO CH CH ), 4.42-4.34 (bs, 2H,
/petroleum ether. NCH CH ), 1.69 (t, 3H, J = 7.2 Hz, NCH CH ), 1,44 (t, 3H, J = 7.1
Hz, CO CH CH ) and -1.32 (bs, 2H, internal NH) ppm. C NMR
(75 MHz; CDCl ) δ 173.6 (C-4’’), 165.8 (CO Et), 149.1 (C-2’’),
dihydroquinolin-6-yl-amino)-5,10,15,20-tetraphenylporphyrin (6c) 145.8, 142.4, 142.4, 142.2, 139.0 (C-8a’’), 138.2, 138.1, 136.2,
2 3 2 2
CO and extracted with CH Cl . The H-β), 8.69 (d, 1H, J = 4.4 Hz, H-β), 8.67 (s, 1H, H-2’’), 8.65 (d,
2
4
a
CH
CH
2
Cl
Cl
2
2
2
3
2
2
2
3
2
3
1
3
2
2
3
2-[3-Ethoxycarbonyl-1-(β-D-ribofuranos-1-yl)-4-oxo-1,4-
3
2
1
Yield: (5.2 mg, 42%). H NMR (300 MHz; CDCl
3
/CD
3
OD) δ 9.13 135.3 (C-5’), 134.7, 134.6, 134.5, 134.0, 133.7 (C-7’’), 133.6,
(s, 1H, H-2’), 8.72-8.65 (m, 3H), 8.63 (d, 1H, J = 4.9 Hz, H-β), 132.5, 131.5, 131.4, 129.4 (C-5’’), 128.6, 128.54, 127.8, 127.4,
8.59 (d, 1H, J = 4.9 Hz, H-β), 8.50 (d, 1H, J = 4.9 Hz, H-β), 8.36 127.2, 126.8, 126.8, 126.7, 126.6, 124.2 (C-18), 123.3, 122.2,
(
s, 1H, H-3), 7.98 (d, 6H, J = 6.7 Hz, Ho-Ph-5,10,15), 7.93-7.86 122.1, 122.1, 118.7, 117.3, 116.8, 116.7, 115.2, 111.9, 110.3,
m, 1H, Hp-Ph-20), 7.82-7.52 (m, 15H, Ho,m-Ph-20, Hm,p-Ph- 101.3, 61.20 (CO CH CH ), 49.2 (NCH CH ), 14.7 (CO CH CH
,10,15, H-5’ and H-8’), 7.40-7.35 (m, 1H, H-7’), 6.49 (s, 1H, N- and 14.4 (NCH CH ) ppm. UV-vis (DMF/H O (9:1)): λmax (log ε) =
H), 6.06 (d, 1H, J=2.0 Hz, H-1’’), 4.39 (q, 2H, J = 6.9 Hz, 412 (5.04) 451 (4.74) 593 (4.15) 610 (4.15) 663 (4.13) nm.
(
2
2
3
2
3
2
2
3
)
5
2
3
2
+
CO
2
CH
2
CH
3
), 4.33-4.19 (m, 3H, H-2’’, H-3’’ and H-4’’), 4.10 (dd, HRMS (ESI) m/z calcd for C58
H
43
N
6
O
3
[M+H] 871.33912, found
1
H, J = 12.8 and 2.0 Hz, H-5’’), 3.94-3.84 (m, 1H, H-5’’) and 1.43 871.33852.
1
3
(t, 3H, J = 6.9 Hz, CO
2
CH
2
CH
3
). C NMR (125 MHz;
Et), 144.71, 143.20,
CDCl /CD OD) δ 174.81 (C-4’), 167.44 (CO
3
3
2
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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DOI: 10.1039/C5RA11070J
ARTICLE
RSC Advances
o
N-(3-Ethoxycarbonyl-4-oxo-1-pentyl-1,4-dihydroquinolin-6-
inoculated into 100 mL of BHI and was grown for 18 h at 37 C,
at 170 rpm stirring. After that, an aliquot of this culture was
yl)quinolino[2,3,4-af]-5,10,15-triphenylporphyrin (8b)
1
Yield: (5.9 mg, 56%). H NMR (500 MHz; CDCl
3
) δ 9.70 (d, 1H, J subcultured in 100 mL of fresh BHI and was grown for 18 h
o
4.8 Hz, H-18), 9.67 (d, 1H, J = 9.8 Hz, H-5’), 9.12 (d, 1H, J = 2.4 under stirring (170 rpm) at 37 C to achieve the stationary
=
8
Hz, H-5’’), 8.84 (d, 1H, J = 4.8 Hz, H-17), 8.76 (d, 1H, J = 4.8 Hz, phase, corresponding to a concentration of approximately 10 -
9
H-β), 8.70 (d, 1H, J = 4.8 Hz, H-β), 8.66 (d, 1H, J = 4.8 Hz, H-β), 10 colony forming units per mL (CFU mL ).
-1
-1
8.63-8.60 (ms, 2H, H-β and H-2’’), 8.32-8.21, 8.21-8.15 and
8.14-8.06 (2bs and 1m, 7H, Ho-Ph-5,10,15 and H-7’’), 7.85-7.52 Photosensitizer stock solutions
(m, 14H, H-3, H-2’, H-3’, H-4’, H-8’’, Hm,p-Ph-5,10,15), 4.47 (q, For biological assays, 500 μM stock solutions of
2
2
1
H, J = 7.0 Hz, CO
H, H-2’’), 1.51-1.43 (m, 7H, H-3’’ and H-4’’ and CO
.01 (t, 3H, J = 6.7 Hz, H-5’’) and -1.31 (bs, 2H, internal NH) saline (PBS, 4 g of NaCl, 0.1 g of KCl, 0.72 g of Na
2
CH
2
CH
3
), 4.26 (bs, 2H, H-1’’), 2.07-2.00 (m, photosensitizers were prepared in dimethylsulfoxide (DMSO),
CH CH and diluted to the final concentrations in phosphate buffered
HPO , 0.12 g
to a final volume of 500 mL and pH 7.4 ± 0.2),
49.6 (C-2’’), 145.8, 145.4, 142.4, 142.2, 139.1, 138.0, 136.2, depending on the experiment.
35.2 (C-5’), 134.7, 134.6, 134.5, 134.1, 133.6, 132.5, 131.4,
2
2
3
)
2
4
1
3
3 2 2 4
ppm. C NMR (75 MHz; CDCl ) δ 173.6 (C-4’’), 165.8 (CO Et), of KH PO
1
1
1
1
1
1
29.3, 128.6, 128.5, 127.8, 127.4, 127.2, 126.8 (C-17), 126.8, Photoinactivation assay
26.7, 126.6, 124.2 (C-18), 123.3, 122.2, 122.1, 118.8, 117.3, Staphylococcus aureus (ATCC 6538) photoinactivation by the
16.8, 115.2, 111.6, 110.3, 101.4, 61.2 (CO
’’), 28.8 (C-3’’ or C-4’’), 28.7 (C-2’’), 22.3 (C-3’’ or C-4’’), 14.5 was achieved by exposing the S. aureus bacterium in
CH CH ) and 14.0 (C-5’’) ppm. UV-vis (DMF/H O (9:1)): laboratory conditions, during defined time intervals, under
max (log ε) = 412 (5.21) 452 (4.90) 593 (4.31) 610 (4.31) 663 stirring (100 rpm), to white light at an irradiance of 150 W m ,
2 2 3
CH CH ), 54.5 (C- seven porphyrin derivatives, at the concentration of 10.0 µM,
(CO
2
2
3
2
-2
λ
+
(4.30) nm. HRMS (ESI) m/z calcd for C61
H
49
N
6
O
3
[M+H]
provided by an illumination system (LC-122 LumaCare,
London) equipped with a halogen 250 W quartz-type lamp and
coupled to an interchangeable fiber optic probe (400-800 nm),
measured with a Coherent FieldMaxIITop energy meter
913.38607, found 913.38539.
Oxidative cyclization of conjugate 6a. Procedure B.
A solution of conjugate 6a (10 mg, 11.5 µmol) in nitrobenzene combined with a Coherent PowerSens PS19Q energy sensor
1 mL) was refluxed until complete consumption of the starting before each photodynamic inactivation assay. Dark and light
material (48 h, monitored by TLC). After reaching room controls were also included in the experiment. In the light
temperature, it was added CH Cl to the reaction mixture and control (LC), the bacterial suspension without photosensitizer
the nitrobenzene was removed through elution with CH Cl was exposed to the same irradiation protocol. In the dark
over a short silica gel column. The crude product was control (DC), the beaker containing the bacterial suspension
recovered by elution with CH Cl /MeOH (20:1) and purified and the photosensitizer at 10.0 µM was covered with
through preparative TLC using a mixture of CH Cl /MeOH aluminum foil to protect it from any light exposure. Both test
20:1), followed by crystallization with CH Cl /petroleum and light and dark control beakers were submitted to a pre-
(
2
2
2
2
2
2
2
2
(
2
2
ether. Derivative 8a was isolated in 30% (3.0 mg) yield through irradiation period in the dark during 15 min, at room
this procedure and several unidentified minority byproducts temperature, to promote the porphyrin/quinolone binding to
were observed on the TLC analysis.
S. aureus cells. Sub-samples of 1.0 mL of test and LC and DC
samples were aseptically taken at times 0, 30, 60, 90, 120, 150
and 180 min. The Petri plates were kept in the dark
Singlet oxygen generation
A solution of each PS at a concentration of 0.5 μM in DMF/H
2
O
immediately after spreading and also during the incubation.
o
9:1) and 50.0 μM of the singlet oxygen quencher 1,3- After 48 h of incubation at 37 C the number of colonies was
(
diphenylisobenzofuran (DPBF) was irradiated in a glass counted and the rate of bacterial inactivation was evaluated
5
4
cuvette, under magnetic stirring at an irradiance of 9.0 mW through the quantification of the number of bacteria. Three
−2
cm with a LED array. The LED array is composed of a matrix independent experiments were carried out with two replicates
of 5 x 5 LED making a total of 25 light sources with an emission each.
peak centred at 654 nm and a bandwidth at half maximum of ±
20 nm. The production of singlet oxygen was evaluated Statistical analysis
qualitatively through the absorption decay of DPBF at 415 nm Statistical analyses were performed by using SPSS (SPSS 15.0
5
for 10 min at defined intervals. The irradiation of the PS in for Windows, SPSS Inc., USA). Normal distributions were
2
the presence of dissolved oxygen will result in the formation of assessed by the Kolmogorov-Smirnov test. The significance of
1
2
O , which is trapped by DPBF resulting in colorless o- all porphyrins and irradiation time on bacterial inactivation
1
Alder like reaction with O
dibenzoylbenzene, after the Diels
–
2
.
was assessed by two-way univariate analysis of variance
ANOVA) model with the Bonferroni post-hoc test. A value of p
(
Bacterial growth conditions
< 0.05 was considered as significant.
Staphylococcus aureus ATCC 6538 was grown on Brain-Heart
Infusion (BHI, Liofilchem, Italy). Before each photodynamic
inactivation assay one colony of S. aureus was aseptically
Conclusions
1
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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The new pophyrin/4-quinolone conjugates
PleaseRd So Cn oA t da vd aj un s ct ems argins
View Article Online
DOI: 10.1039/C5RA11070J
ARTICLE
6
were obtained 17. L. Costa, J. P. C. Tomé, M. G. P. M. S. Neves, A. C. Tomé, J. A. S.
Cavaleiro, M. A. F. Faustino, Â. Cunha, N. C. M. Gomes and A.
efficiently via a palladium-catalyzed amination approach that
Almeida, Antivir. Res., 2011, 91, 278.
afforded the intracylized conjugates
8 under oxidative
1
8. J. Almeida, J. P. C. Tomé, M. G. P. M. S. Neves, A. C. Tomé, J. A.
S. Cavaleiro, Â. Cunha, L. Costa, M. A. F. Faustino and A.
Almeida, Photoc. Photobio. Sci., 2014, 13, 626.
conditions. The structures of all conjugates were confirmed by
adequate spectroscopic techniques and the ability to generate
singlet oxygen was accessed by an indirect method. In general 19. J. J. Mendes, A. Marques-Costa, C. Vilela, J. Neves, N. Candeias,
P. Cavaco-Silva and J. Melo-Cristino, Diabetes Res. Clin. Pr.,
they show good to high ability to generate singlet oxygen, one
2
012, 95, 153.
of the most important requirements for a photosensitizer. The
efficacy of these derivatives to photoinactivate the Gram-
positive bacterium S. aureus was also evaluated and the best
bacterial inactivation rates were obtained with the N-ethyl
2
0. A. Almeida, Â. Cunha, M. A. F. Faustino, A. C. Tomé and M. G. P.
M. S. Neves, In Photodynamic Inactivation of Microbial
Pathogens: Medical and Environmental Applications., ed.
Hamblin MR, Jori G, Royal Society of Chemistry, Cambrige,
2
011, pp. 83-160.
derivatives 8a and 6a. These compounds showed an important
1
relationship between O
21. F. C. Santos, A. C. Cunha, M. C. B. V. de Souza, A. C. Tomé, M. G.
P. M. S. Neves, V. F. Ferreira and J. A. S. Cavaleiro, Tetrahedron
Lett., 2008, 49, 7268.
22. A. T. P. C. Gomes, A. C. Cunha, M. R. M. Domingues, M. G. P. M.
S. Neves, A. C. Tomé, A. M. S. Silva, F. C. Santos, M. C. B. V. de
Souza, V. F. Ferreira and J. A. S. Cavaleiro, Tetrahedron, 2011,
2
production and their PDI efficiency.
Synthesis of similar compounds and the biological assessment
considering other types of bacterial pathogens are underway.
6
7, 7336.
2
2
3. M. M. Bastos, A. T. P. C. Gomes, M. G. P. M. S. Neves, A. M. S.
Silva, O. A. Santos-Filho, N. Boechat and J. A. S. Cavaleiro, Eur. J.
Org. Chem., 2013, 1485.
Acknowledgements
Thanks are due to the University of Aveiro (Aveiro, Portugal), and
Universidade Federal Fluminense (Niterói, RJ, Brazil). We also thank
FCT/MEC for the financial support to the QOPNA research Unit (FCT
UID/QUI/00062/2013), through national funds and where applicable to
4. A. Ahmed and M. Daneshtalab, J. Pharm. Pharm. Sci., 2012, 15,
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6. C. Mugnaini, S. Pasquini and F. Corelli, Curr. Med. Chem., 2009,
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those co-financed by
the
FEDER, within the PT2020 Partnership,
to
2
7. H. K. Hombrecher, V. M. Gherdan, S. Ohm; J. A. S. Cavaleiro, M.
G. P. M. S. Neves and M. F. Condesso, Tetrahedron, 1993, 49,
the Portuguese NMR Network and to the FCT-CAPES collaborative
programme. Pedro Netto Batalha thanks CAPES for his research grant and
for funding his research stay in Aveiro, Portugal (BEX-7131/13-5).
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1
1
1
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DOI: 10.1039/C5RA11070J
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2 | J. Name., 2012, 00, 1-3
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Synthesis of new porphyrin/4-quinolone conjugates and evaluation of
their efficiency in the photoinactivation of Staphylococcus aureus
a,b
a
b
c
Pedro N. Batalha , Ana T. P. C. Gomes , Luana S. M. Forezi , Liliana Costa , Maria
b
b*
b
Cecília B. V. de Souza , Fernanda C. S. Boechat , Vitor F. Ferreira , Adelaide
c
a
a*
a*
Almeida , Maria A. F. Faustino , Maria G. P. M. S. Neves , José A. S. Cavaleiro
a
Department of Chemistry and QOPNA, University of Aveiro, 3810-193 Aveiro,
Portugal
b
Programa de Pós-Graduação em Química, Instituto de Química, Universidade
Federal Fluminense, 24010-141 Niterói, Rio de Janeiro, Brazil.
c
Department of Biology and CESAM, University of Aveiro 3810-193 Aveiro, Portugal
Table of Contents
O
CO
N
2
Et
O
Br
2
CO Et
R
N
N
R
R
Ph HN
2
N
2
CO Et
N
O
Ph
NH
2
M
Ph
N
4
6
, M = Ni
, M = 2H
Ph
8
N
N
N
N
Ph
Ni
Ph
Ph
1
The synthesis of new porphyrin/4-quinolone conjugates and their evaluation as
potential photosensitizers in the photoinactivation of Staphylococcus aureus is
described.
1