Synthesis and photodynamic effects of new porphyrin/4-oxoquinoline derivatives in the inactivation of S. aureus

Article abstract of DOI:10.1039/c9pp00102f

New porphyrin/4-oxoquinoline conjugates were synthesized from the Heck coupling reaction of a β-brominated porphyrin with 1-allyl-4-oxoquinoline derivatives, followed by demetallation and deprotection affording the promising photosensitizers 9a-e. Singlet oxygen studies have demonstrated that all the porphyrin/4-oxoquinoline conjugates 9a-e were capable of producing cytotoxic species and found to be excellent photosensitizing agents in the inactivation of S. aureus by the antimicrobial photodynamic therapy (aPDT) protocol.

Full text of DOI:10.1039/c9pp00102f

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Dias , A. T. P. C. Gomes, M. A. Faustino, A. Almeida, A. G. D. Souza , A. R. P. D. Costa, F. D. C. S. Boechat,
M. C. Souza, M. D. G. P. M. S. Neves and J. A. S. Cavaleiro, Photochem. Photobiol. Sci., 2019, DOI:
10.1039/C9PP00102F.
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Number 12
December 2017
Pages 1737-1846
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DOI: 10.1039/C9PP00102F
ARTICLE
Synthesis and photodynamic effect of new porphyrin/4-
oxoquinoline derivatives in the inactivation of S. aureus
Received 00th January 20xx,
Accepted 00th January 20xx
c Fernanda Savacini Sagrillo,a,b Cristina Dias,b Ana T. P. C. Gomes,*c Maria A. F. Faustino,a Adelaide
Almeida,*c Alan Gonçalves de Souza,b Amanda Rodrigues Pinto da Costa,b Fernanda da Costa
Santos Boechat,b Maria Cecília Bastos Vieira de Souza,*b Maria G. P. M. S. Neves*a and José A. S.
Cavaleiro*a
DOI: 10.1039/x0xx00000x
New porphyrin/4-oxoquinoline conjugates were synthesized from the Heck coupling reaction of a β-brominated porphyrin
with 1-allyl-4-oxoquinoline derivatives, followed by demetallation and deprotection affording the promissing
photossensitizers 9a-e. The singlet oxygen studies have demonstrated that all the porphyrin/4-oxoquinoline conjugates
9a-e were capable to produce that cytotoxic species and revealed to be excellent photossensitizer agents in the
inactivation
of
S.
aureus
after
the
antimicrobial
photodynamic
therapy
(aPDT)
protocol.
Gram-positive bacterium is one of the most important human
pathogens both in hospital and community context and has been a
privileged target of therapeutic researches. In fact, numerous
antimicrobial agents, as well as vaccine attempts, were specifically
designed to combat this bacterium.21,22 However, multi resistant
strains, such as methicillin-resistant S. aureus (MRSA) infection has
become a more common cause of healthcare-associated infections
worldwide. MRSA is associated with significant morbidity and
mortality and imposes a huge burden on healthcare resources.22
Introduction
The emergence of resistance by microbial pathogens to
conventional antimicrobial drugs is a serious and growing problem
2
worldwide.1,
Antimicrobial photodynamic therapy (aPDT)
emerged as an effective solution against multidrug resistant strains
and against biofilm formation and destruction.3-5 This therapeutic
approach involves the use of a photosensitizer molecule (PS) that
after being excited by light can react with molecular oxygen
producing cytotoxic reactive oxygen species (ROS) such as singlet
oxygen (1O2) and/or hydroxyl radicals, superoxide and hydrogen
peroxide. These ROS can react with biological molecules (e.g.
nucleic acids, proteins and lipids) causing microbial death.3-6 aPDT
presents several advantages when compared with the use of
traditional antimicrobials. This approach shows to be efficient,
independently of the antimicrobial resistance profile5 and is able to
prevent further development of resistance even after several cycles
of treatment.7-9 In fact, this approach has been efficiently applied to
inactivate several microorganisms, such as Gram-negative10, 11 and
Gram-positive bacteria,12,13 fungi14, 15,16 and viruses.9, 17,18-20
This strain is frequently found in epidemic hospital infections22 and
23
was already found in US patients with HIV infection22,
and in
patients with diabetic foot infections.24 Moreover, in this case,
from patients undergoing antibiotic therapy, 93% of the antibiotic
regimens were inadequate based on the antibiotic susceptibility
test results.24 Fortunately, a literature survey shows that S. aureus
is susceptible to aPDT and the PS efficiency and photosensitizing
activity is independent of the antibiotic resistance spectrum of the
isolates.17, 25
Among the PSs studied in the photoinactivation of microorganisms
mediated by aPDT, porphyrins are, unquestionably, the most
considered class of compounds.17 Nowadays, several research
groups are concerned in the study of new synthetic methodologies
for porphyrin derivatives with appropriate features for their
application in aPDT. One of the strategies used consists in coupling
different compounds with well-established pharmacological
activities.26, 27Under this context, porphyrins can be linked to other
biologically active molecules, aiming to increase their biological
efficacy.28-32
One of the bacteria that deserves special attention due to its
antibiotic resistance development is Staphylococcus aureus.21 This
a. QOPNA & LAQV-REQUIMTE and Chemistry Department, 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 Fluminense, 24010-141 Niterói, Rio de Janeiro, Brazil. E-mail:
mceciliabvs@gmail.com
c. Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal. E-mail:
ana.peixoto@ua.pt; aalmeida@ua.pt
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Having in mind the potentiality already known of porphyrin
derivatives in aPDT and, since 4-oxoquinolines are associated with
34
several biological activities, mainly as antibacterial agents,33, in
this work it is reported the synthesis of new porphyrin/4-
oxoquinoline conjugates through the Heck coupling and their
potential photodynamic effect towards S. aureus. Heck coupling
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Photochemical & Photobiological Sciences
Page 2 of 15
ARTICLE
Journal Name
reaction methodology has been widely used and has already Coupling reactions of porphyrin 2 and 4-oxoquinolines 1a–e.
View Article Online
demonstrated its potential in the functionalization of tetrapyrrolic
DOI: 10.1039/C9PP00102F
macrocycles, generating compounds with interesting properties.35-
38
Study of the reaction conditions according with Table 1
The synthesis of other porphyrin/4-quinolone conjugates was
already reported by our group,28,32 but in those cases the coupling
between the porphyrin macrocycle and the quinolone unit was
made through the 6- and 7-positions of the quinolone nucleus
(Figure 1). Since, N-R1 position of the quinolone moiety is part of
A mixture of porphyrin 239(10 mg, 13.2 mmol), palladium catalyst
(10 mol %), ligand (20 mol %), base (26.4 mmol), 1-allyl-4-
oxoquinoline 1b (7.5 mg, 26.4 mmol) and dried toluene:DMF (1:0.5)
was purged with N2 and stirred at 120 ºC under N2 atmosphere. The
progress of the reaction was monitored by TLC (2-29 h) and it was
stopped when no more evolution was observed (see Table 1). Then,
after reaching room temperature, the mixture was washed with 100
mL of distilled water and extracted with 100 mL of CH2Cl2. The
organic phase was separated and dried with anhydrous sodium
sulphate. The solvent was evaporated, and the crude mixture was
purified by preparative column chromatography using CH2Cl2 firstly
to elute the unreacted porphyrin 2 and then a mixture of CH2Cl2:
MeOH (20:1) to elute the reaction products. The combination of
catalyst/ligand/base that promoted higher yields of porphyrin/4-
oxoquinoline conjugates in the classic conditions was selected to
perform the reaction under microwave (MW) irradiation (see
below).
the enzyme-DNA binding complex and has
a hydrophobic
interaction with the major grove of DNA,34 in this present work we
had prepared novel porphyrin/4-quinolone derivatives linked by the
N-position of the quinolone nucleus (Figure 1) and compared the
aPDT results with the previously reported ones.32
O
CO2
H
Ph
N
H
N
R
N
R
1 = Ethyl; Ribofuranosyl
Tetrahedron 2011,
67, 7336-7342
O
Ph
O
CO2
H
5
8
H
N
4
1
R
R
CO2H
6
N
H
HN
N
This work
R
N
Ph
2
N
7
N
R
RSC Advances 2015,
R
Synthesis of porphyrin/4-oxoquinoline conjugates under MW
irradiation
5, 71228-71239
R
O
N
CO2
H
R
6 or R7 = Cl; Br; H
N
N
H
O
HO2
C
N
N
The porphyrin 2 (10 mg, 13.2 mmol), Pd(OAc)2 (0.3 mg, 10 mol %),
PPh3 (0.7 mg, 20 mol %), KOAc (2.5 mg, 26.4 mmol), 1-allyl-4-
oxoquinoline 1a-e (26.4 mmol) and toluene:DMF (1:0.5, 1.5 mL)
were placed in a MW glass vessel and placed in the microwave
reactor. MW irradiation (250 W) was applied for 15 minutes at 120
ºC. After this period, the reaction mixture was washed with 100 mL
of distilled water and extracted with 100 mL of CH2Cl2. The organic
phase was separated and dried with anhydrous sodium sulphate.
The solvent was evaporated and the crude mixture was purified by
preparative column chromatography under the experimental
conditions already described.
R
R
1 = Ethyl; Pentyl; Ribofuranosyl
Figure 1. General structures of the new porphyrin/4-oxoquinoline
conjugates and others previously studied.
Experimental
1
General H and 13C NMR spectra were recorded on Bruker Avance
1
300 (300 MHz for H and 75 MHz for 13C), Bruker Avance 500 (500
The structure and numbering of each new derivatives is presented
in Schemes 3 and 4.
MHz for H and 125 MHz for 13C) spectrometers. CDCl3 was used as
solvent and tetramethylsilane (TMS) as internal reference. Chemical
1
shifts are expressed in δ (ppm) and the coupling constants (J) are
1
expressed in Hertz. Unequivocal H assignments were made using
2-[ethyl-1-(but-2-en-1-yl)-4-oxo-1,4-dihydroquinoline-3-
2D COSY, while 13C assignments were made based on 2D HSQC and
HMBC experiments. HMRS were acquired with a Micromass Q-Tof 2
(Micromass, Manchester, UK), operating in the positive ion mode,
equipped with a Z-spray source, an electrospray probe and a
syringe pump. Source and desolvation temperatures were 80 °C and
150 °C, respectively. Capillary voltage was 3000 V. The spectra were
acquired at a nominal resolution of 9000 and at cone voltages of 30
V. Nebulisation and collision gases were N2 and Ar, respectively.
Porphyrin solutions in methanol were introduced at a 10 μL.min−1
flow rate. The UV–Vis spectra were recorded on an UV-2501 PC
Shimadzu spectrophotometer using CHCl3 as solvent. Flash
chromatography was carried out using silica gel (230–400 mesh)
and preparative thin-layer chromatography was carried out on 20 ×
20 cm glass plates coated with silica gel (1 mm thick). The reactions
were routinely monitored by thin layer chromatography (TLC) on
silica gel precoated F254 Merck plates.
carboxylate]-5,10,15,20-tetraphenylporphyrinatozinc(II) (5a) Yield:
(67 %). H NMR (500 MHz; CDCl3) δ 8.77 (s, 1H, H-3), 8.71-8.70 (m,
1
3H, β-H), 8.67-8.66 (m, 2H, β-H), 8.55 (d, J = 4.6 Hz, 2H, β-H), 8.48 (s,
1H, H-2’), 8.38 (dd, J = 8.1, 1.2 Hz, 1H, H-5’), 8.06-8.04 (m, 6H, H-o-
Ph-5,10,15), 7.92-7.90 (m, 2H, H-o-Ph-20), 7.62-7.55 (m, 10H, H-
m,p-Ph-5,10,15, H-p-Ph-20), 7.47-7.43 (m, 4H, H-m-Ph-20, H-7’, H-
8’), 7.37-7.34 (m, 1H, H-6’), 6.33-6.24 (m, 2H, H-α, H-β), 4.63 (d, J =
4.7 Hz, 2H, CH2), 4.31 (q, J = 7.1 Hz, 2H, OCH2CH3), 1.31 (t, J = 7.1 Hz,
3H, OCH2CH3). 13C NMR (125 MHz; CDCl3) δ 175.4 (C-4’), 165.7
(CO2Et), 150.5, 150.5, 150.3, 150.3, 150.2, 150.0, 149.3 (C-2’), 147.6,
146.0, 143.6, 143.4, 143.1, 140.5, 139.2, 134.4, 133.9, 132.9, 132.3,
132.3, 131.9, 131.9, 131.8, 131.3, 130.6, 130.6 (C-3), 128.7, 128.5,
127.7, 127.4, 127.2, 127.2, 126.4, 126.3, 125.5, 121.4, 120.9, 120.6,
120.2, 120.1, 116.7, 110.4, 61.0 (OCH2CH3), 56.4 (CH2), 14.2
(OCH2CH3). UV-vis (DMF): λmax (log ɛ) 432 (5.38) 476 (4.15) 516
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Photochemical & Photobiological Sciences
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ARTICLE
(3.63) 564 (4.12) 605 (3.73) nm. HRMS (ESI) m/z calcd for 2-[ethyl-1-(but-2-en-1-yl)-7-bromine-4-oxo-1,4-dihydVroiewquAirnticoleliOnnel-ine
C59H42N5O3Zn [M + H]+ 932.2579, found 932.2592.
3-carboxylate]-5,10,15,20-tetraphenylporphyrinatozinc(II)
(5e)
DOI: 10.1039/C9PP00102F
1
Yield: (24 %). H NMR (300 MHz, CDCl3) δ 8.97 (s, 1H, H-3), 8.93-
8.91 (m, 3H, β-H), 8.89-8.87 (m, 2H, β-H), 8.77 (d, J = 4.7 Hz, 1H, β-
H), 8.44 (s, 1H, H-2’), 8.27 (d, J = 8.6 Hz, 1H, H-5’), 8.21-8.18 (m, 6H,
H-o-Ph-5,10,15), 8.10-8.08 (m, 2H, H-o-Ph-20), 7.77-7.72 (m, 10H,
H-m,p-Ph-5,10,15), 7.68-7.65 (m, 3H, H-m,p-Ph-20, H-8’), 7.53 (dd, J
= 8.6, 1.6 Hz, 1H, H-6’), 6.41-6.38 (m, 2H, H-α,H-β), 4.69 (d, J = 3.9
Hz, 2H, CH2), 4.38 (q, J = 7.1 Hz, 2H, OCH2CH3), 1.40 (t, J = 7.1 Hz, 3H,
OCH2CH3). 13C NMR (75 MHz, CDCl3) δ 174.0 (C-4’), 165.7 (CO2Et),
150.9, 150.9, 150.8, 150.7, 150.6, 150.4, 149.5 (C-2’), 148.0, 146.3,
143.2, 143.0, 142.8, 141.0, 140.2, 134.6, 134.1, 132.6, 132.4, 131.9,
131.2 (C-3), 129.7 (C-5’), 128.7, 128.3, 128.0, 127.8, 127.7, 126.9,
126.8, 126.7, 122.0, 121.7, 121.3, 120.9, 119.3, 111.7, 61.2
(OCH2CH3), 56.4 (CH2), 14.6 (OCH2CH3). UV-vis (DMF): λmax (log ɛ)
433 (5.67) 475 (3.74) 524 (3.58) 574 (4.29) 604 (3.86) nm. HRMS
(ESI) m/z calcd for C59H41BrN5O3Zn [M + H]+ 1010.1684, found
1010.1708.
2-[ethyl-1-(but-2-en-1-yl)-6-chloro-4-oxo-1,4-dihydroquinoline-3-
carboxylate]-5,10,15,20-tetraphenylporphyrinatozinc(II) (5b) Yield:
1
(42 %). H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H, H-3), 8.78-8.75 (m,
3H, β-H), 8.73-8.71 (m, 2H, β-H), 8.61 (d, J = 4.6 Hz, 1H, β-H), 8.52 (s,
1H, H-2’), 8.42 (d, J = 2.6 Hz, 1H, H-5’), 8.13-8.09 (m, 6H, H-o-Ph-
5,10,15), 7.99-7.98 (m, 2H, H-o-Ph-20), 7.70-7.63 (m, 10H, H-m,p-
Ph-5,10,15 e H-p-Ph-20), 7.61 (dd, J = 9.1, 2.6 Hz, 1H, H-7’), 7.54 (t, J
= 7,6 Hz, 2H, H-m-Ph-20), 7.47 (d, J = 9.1 Hz, 1H, H-8’), 6.36-6.34 (m,
2H, H-α, H-β), 4.71 (d, J = 3.8 Hz, 2H, CH2), 4.39 (q, J = 7.1 Hz, 2H,
OCH2CH3), 1.37 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (125 MHz,
CDCl3+MeOD) δ 174.2 (C-4’), 165.6 (CO2Et), 150.74, 150.67, 150.57,
150.4, 150.2, 149.6 (C-2’), 147.8, 146.1, 143.8, 143.5, 143.3, 140.5,
137.8, 134.6, 134.0, 133.2, 132.22 (C-α or C-β), 132.16, 131.98,
131.94, 131.5, 130.8 (C-3), 130.2, 127.9, 127.44, 127.39, 127.1 (C-
5’), 126.8, 126.6 (C-7’), 126.5, 121.3, 121.1 (C-α or C-β) 120.8, 120.4,
120.2, 118.7 (C-8’), 111.1, 61.3 (OCH2CH3), 56.3 (CH2), 14.4
(OCH2CH3). UV-vis (DMF): λmax (log ɛ) 433 (5.56) 476 (4.38) 516
(3.81) 564 (4.25) 604 (3.87) nm. HRMS (ESI) m/z calcd for
C59H41ClN5O3Zn [M + H]+ 966.2189, found 966.2207.
3-[(E)-4-(3-oxoprop-1-en-1-yl]-5,10,15,20
tetraphenylporphyrinatozinc(II) (6) 1H NMR (500 MHz, CDCl3) δ
9.11 (s, 1H, H-3), 9.08 (d, J = 8.0 Hz, 1H, CHO), 8.91-8.89 (m, 6H, β-
H), 8.20-8.18 (m, 6H, H-o-Ph-5,10,15), 8.11-8.09 (m, 2H, H-o-Ph-20),
7.83-7.71 (m, 12H, H-m,p-Ph-5,10,15,20), 7.04 (d, J = 15.4 Hz, 1H, H-
α), 6.77 (dd, J = 15.4, 8.0 Hz, 1H, H-β). 13C NMR (125 MHz, CDCl3) δ
194.2 (CHO), 151.2, 151.16, 151.12, 151.1, 150.7, 150.1 (C-β), 149.8
(C-2’), 147.4, 146.4, 142.9, 142.5, 142.4, 138.9, 134.43, 134.41,
134.39, 134.1, 133.9, 132.8, 132.63, 132.61, 132.56, 132.51, 132.3,
132.0, 129.6 (C-α), 129.0, 128.5, 128.4, 128.2, 127.9, 127.6, 127.1,
126.6, 121.9, 121.4, 121.3, 120.8. HRMS (ESI) m/z calcd for
C47H31N4OZn [M + H]+ 731.1789, found 731.1770.
2-[ethyl-1-(but-2-en-1-yl)-7-chloro-4-oxo-1,4-dihydroquinoline-3-
carboxylate]-5,10,15,20-tetraphenylporphyrinatozinc(II) (5c) Yield:
1
(24 %). H NMR (300 MHz, CDCl3) δ 8.95 (s, 1H, H-3), 8.92-8.90 (m,
3H, β-H), 8.89-8.86 (m, 2H, β-H), 8.76 (d, J = 4.7 Hz, 1H, β-H), 8.38 (s,
1H, H-2’), 8.22- 8.16 (m, 6H, H-o-Ph-5,10,15), 8.13-8.08 (m, 3H, H-o-
Ph-20, H-5’), 7.76-7.70 (m, 10H, H-m,p-Ph-5,10,15, H-p-Ph-20), 7.68-
7.61 (m, 3H, H-m-Ph-20, H-8’), 7.49 (dd, J = 8.6, 1.5 Hz, 1H, H6’),
6.35 (s, 2H, H-α, H-β), 4.66 (d, J = 3.1 Hz, 2H, CH2), 4.29 (q, J = 7.1
Hz, 2H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (75
MHz, CDCl3) δ 173.7 (C-4’), 165.4 (CO2Et), 150.9, 150.8, 150.8,
150.7, 150.6, 150.4, 149.4 (C-2’), 147.9, 146.2, 143.3, 143.0, 142.8,
140.8, 139.9, 139.1, 134.6, 134.0, 132.5, 132.4, 131.8, 131.0 (C-3),
129.5, 128.2, 127.7, 127.6, 127.4, 126.8, 126.8, 126.7, 125.7 (C-6’),
121.9, 121.2, 120.8, 120.8, 116.2 (C-8’), 111.5, 61.1 (OCH2CH3), 56.7
(CH2), 14.5 (OCH2CH3). UV-vis (DMF): λmax (log ɛ) 432 (5.45) 476
(4.50) 515 (3.88) 564 (4.18) 604 (3.86) nm. HRMS (ESI) m/z calcd for
C59H41ClN5O3Zn [M + H]+ 966.2189, found 966.2211.
3-[4-(2-(carboxyamino)vinyl]-5,10,15,20-
tetraphenylporphyrinatozinc(II) (7) 1H NMR (500 MHz, CDCl3) δ
8.95 (s, 1H, H-3), 8.93-8.90 (m, 3H, β-H), 8.89-8.87 (m, 2H, β-H),
8.83 (d, J = 4.7 Hz, 1H, β-H), 8.21-8.18 (m, 6H, H-o-Ph-5,10,15), 8.08-
8.05 (m, 2H, H-o-Ph-20), 7.77-7.70 (m, 12H, H-m,p-Ph-5,10,15,20),
6.36-6.33 (m, 2H, H-α, H-β), 4.53 (d, J = 5.0 Hz, 1H, CH2). 13C NMR
(125 MHz, CDCl3) δ 171.1 (NHCO2H), 156.5, 153.4, 150.9, 150.7,
150.5, 150.4, 150.4, 150.3, 148.5, 146.9, 143.1, 143.0, 142.9, 142.7,
137.8, 134.5, 134.1, 132.5, 132.3, 132.2, 132.1, 131.7, 131.2 (C-α or
C-β), 131.0, 130.8, 128.1, 127.6, 127.6, 126.9, 127.7, 124.4 (C-α or
C-β), 121.6, 121.2, 121.1, 120.6, 118.8, 65.7 (CH2). HRMS (ESI) m/z
calcd for C48H33N5O2Zn· M+• 775.1926, found 775.2043.
2-[ethyl-1-(but-2-en-1-yl)-6-bromine-4-oxo-1,4-dihydroquinoline-
3-carboxylate)-5,10,15,20-tetraphenylporphyrinatozinc(II)
(5d)
1
Yield: (46 %). H NMR (300 MHz, CDCl3) δ 8.83 (s, 1H, H-3), 8.80-
8.77 (m, 3H, β-H), 8.75-8.73 (m, 2H, β-H), 8.62 (d, J = 4.7, 1H, β-H),
8.59 (d, J = 2.4 Hz, 1H, H-5’), 8.51 (s, 1H, H-2’), 8.13-8.10 (m, 6H, H-
o-Ph-5,10,15), 8.00-7.98 (m, 2H, H-o-Ph-20), 7.74 (dd, J = 9.0, 2.4
Hz, 1H, H-7’), 7.70-7.62 (m, 10H, H-m,p-Ph-5,10,15, H-p-Ph-20),
7.57-7.52 (m, 2H, H-m-Ph-20), 7.39 (d, J = 9.1 Hz, 1H, H-8’), 6.35 (s,
2H, H-α, H-β), 4.69 (d, J = 2.6 Hz, 2H, CH2), 4.39 (q, J = 7.1 Hz, 2H,
OCH2CH3), 1.38 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (75 MHz,
CDCl3) δ 173.4 (C-4’), 165.8 (CO2Et), 150.8, 150.8, 150.6, 150.5,
150.3, 149.4 (C-2’), 147.9, 146.2, 143.5, 143.2, 143.0, 140.8, 138.2,
135.6, 134.6, 134.1, 132.4, 132.2, 132.2, 132.0, 131.7, 131.0 (C-3),
130.7, 130.6, 128.2, 127.6, 127.6, 126.7, 126.6, 121.7, 121.4, 121.1,
120.6, 119.3, 118.5 (C-8’), 111.6, 61.3 (OCH2CH3), 56.6 (CH2), 14.6
(OCH2CH3). UV-vis (DMF): λmax (log ɛ) 431 (5.53) 475 (3.68) 523
(3.55) 564 (4.22) 604 (3.82) nm. HRMS (ESI) m/z calcd for
C59H41BrN5O3Zn [M + H]+ 1010.1684, found 1010.1697.
Hydrolysis and demetallation of conjugates 5a-e. General
Procedure
Into a flask with reflux condenser were added the conjugates 5a-e
(10 mg), NaOH (160 mg, 4000 mmol), EtOH (4 mL). The reaction was
kept under stirring and reflux for 2 hours. After that time the
mixture was neutralized with 50 mL of dilute citric acid solution and
extracted with 100 mL of CH2Cl2. The organic phase was dried with
anhydrous Na2SO4 and the solvent was evaporated. To the obtained
residue was added CH2Cl2 (4 mL) and TFA (0.5 mL). The mixture was
vigorously stirred at room temperature for 30 minutes, after which
it was neutralized with 100 mL of saturated aqueous solution of
NaHCO3. The organic extracts were dried over anhydrous Na2SO4.
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
The solvent was evaporated under reduced pressure and the (m, 12H, H-m,p-Ph-5,10,15,20), 7.70 (dd, J = 8.6, 1.5VHiezw,A1rtHic,leHO-n6lin’)e,
product was obtained pure after purification by preparative TLC 6.51-6.34 (m, 2H, H-α, H-β), 4.84 (d, J = 5.1 Hz, 2H, CH ), -2.70 (s,
DOI: 10.1039/C9PP00102F
2
using a mixture of CH2Cl2/MeOH (20:1).
2H, NHs). UV-vis (DMF): λmax (log ɛ) 422 (5.39) 518 (4.16) 554 (3.75)
595 (3.65) 651 (3.43) nm. HRMS (ESI) m/z calcd for C57H39BrN5O3 [M
+ H]+ 920.2236, found 920.2242.
2-[1-(but-2-en-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid]-
5,10,15,20-tetraphenylporphyrin (9a) Yield: (53 %). 1H NMR (300
MHz, CDCl3) δ 15.06 (s, 1H, OH), 8.83-8.81 (m, 3H, β-H, H-3), 8.74 (d, Singlet oxygen generation studies
J = 4.8, 2H, β-H), 8.75 (s, 1H, H-2’), 8.67 (d, J = 4.8, 2H, β-H), 8.57
(dd, J = 8.1, 1.4 Hz, 1H, H-5’), 8.21-8.17 (m, 6H, H-o-Ph-5,10,15),
A stock solution of each 4-oxoquinoline porphyrin derivative 9a-e at
0.1 mM in DMF and stock 10 mM solution of 1,3-
8.05-8.02 (m, 2H, H-o-Ph-20), 7.83-7.72 (m, 10H, H-m,p-Ph-5,10,15,
H-p-Ph-20), 7.68-7.58 (m, 4H, H-m-Ph-20, H-7’e H-8’), 7.50-7.46 (m,
1H, H-6’), 6.53-6.34 (m, 2H,H-α, H-β), 4.81 (d, J = 5.3 Hz, 2H, CH2), -
2.72 (s, 2H, NHs). UV-vis (DMF): λmax (log ɛ) 422 (5.50) 518 (4.27)
555 (3.87) 595 (3.76) 651 (3.52) nm. HRMS (ESI) m/z calcd for
C57H40N5O3 [M + H]+ 842.3131, found 842.3124.
a
diphenylisobenzofuran (DPiBF) in DMF were prepared. Aliquots of
2.5 mL of a solution of each porphyrin (0.5 μM) and DPiBF (50 μM)
in DMF were irradiated at an irradiance of 20.0 mW.cm−2, in a glass
cuvette, at room temperature and under gentle magnetic stirring.
As light source it was used an illumination system (LC-122
LumaCare, London) equipped with a halogen/quartz 250 W lamp
coupled to an interchangeable optic fibre probe (400–800 nm) and
2-[1-(but-2-en-1-yl)-7-chloro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid]-5,10,15,20-tetraphenylporphyrin (9b) Yield: (38 using a dichroic cut-off filter (λ > 550 nm) to achieve an emission
%). 1H NMR (300 MHz, CDCl3) δ 14.72 (s, 1H, OH), 8.82-8.73 (m, 7H, radiation from 550 to 800 nm. The absorption decay of DPiBF at 415
β-H, H-3, H-2’), 8.66 (d, J = 4.8, 1H, β-H), 8.54 (d, J = 2.3 Hz, 1H, H- nm was measured at different irradiation times. The production of
5’), 8.20-8.18 (m, 6H, H-o-Ph-5,10,15), 8.06-8.04 (m, 2H, H-o-Ph-20), singlet oxygen was evaluated qualitatively through the DPiBF, a
7.81-7.74 (m, 10H, H-m,p-Ph-5,10,15, H-p-Ph-20), 7.69-7.63 (m, 3H, singlet oxygen (1O2) quencher, since DPiBF decays in a first reaction
H-m-Ph-20, H-8’), 7.59 (dd, J = 9.1, 3.1 Hz, 1H, H-7’), 6.49-6.35 (m, order manner during continuous irradiation in a photosensitised
2H, H-α, H-β), 4.82 (d, J = 4.9 Hz, 2H, CH2), -2.72 (s, 2H, NHs). UV-vis experiment. The irradiation of the PS in the presence of dissolved
(DMF): λmax (log ɛ) 426 (5.39) 519 (4.18) 555 (3.80) 596 (3.69) 652 oxygen will result in the formation of 1O2, which is trapped by DPiBF
(3.46) nm. HRMS (ESI) m/z calcd for C57H39ClN5O3 [M + H]+ resulting in colourless o-dibenzoylbenzene, after the Diels–Alder
876.2741, found 876.2744.
like reaction with 1O2.
2-[1-(but-2-en-1-yl)-7-chloro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid]-5,10,15,20-tetraphenylporphyrin (9c) Yield: (47 %).
1H NMR (300 MHz, CDCl3) δ 14.75 (s, 1H, OH), 8.84-8.81 (m, 4H, β-H,
H-3), 8.79 (d, J = 5.0, 2H, β-H), 8.74 (s, 1H, H-2’), 8.68 (d, J = 5.0, 1H,
β-H), 8.49 (d, J = 8.7 Hz, 1H, H-5’), 8.21-8.17 (m, 6H, H-o-Ph-
5,10,15), 8.09-8.07 (m, 2H, H-o-Ph-20), 7.80-7.74 (m, 10H, H-m,p-
Ph-5,10,15, H-p-Ph20), 7.69-7.55 (m, 2H, H-m-Ph-20), 7.62 (d, J = 1.7
Hz, 1H, H-8’), 7.52 (dd, J = 8.7, 1.7 Hz, 1H, H-6’), 6.49-6.32 (m, 2H, H-
α+H-β), 4.78 (d, J = 5.0 Hz, 2H, CH2), -2.71 (s, 2H, NHs). UV-vis
(DMF): λmax (log ɛ) 427 (5.36) 518 (4.18) 554 (3.78) 595 (3.67) 651
(3.44) nm. HRMS (ESI) m/z calcd for C57H39ClN5O3 [M + H]+
876.2741, found 876.2753.
Photodynamic inactivation studies
Photosensitizers stock solutions
Stock solutions of 500 µM of porphyrin derivatives 9a-e were
prepared in dimethyl sulfoxide (DMSO), protected from light and
were sonicated for 30 min at 25 °C previously to each assay.
Light source
The samples and light control were exposed to white light (400-750
nm) delivered by a LED system (ELMARK – VEGA20, 20W, 1400 lm)
with fluence rate of 25 mW.cm-2.
2-[1-(but-2-en-1-yl)-6-bromine-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid]-5,10,15,20-tetraphenylporphyrin (9d) Yield: (32
%). 1H NMR (300 MHz, CDCl3) δ 14.59 (s, 1H, OH), 8.93-8.72 (m, 7H,
β-H, H-3), 8.62 (d, J = 2.3 Hz, 1H, H-5’), 8.50 (s, 1H, H-2’), 8.22-8.14
(m, 6H, H-o-Ph-5,10,15), 8.06-8.02 (m, 2H, H-o-Ph-20), 7.78-7.59 (m,
13H, H-m,p-Ph-5,10,15,20, H-8’), 7.38 (dd, J = 9.3, 2.2 Hz, 1H, H-7’),
6.44-6.41 (m, 2H, H-α, H-β), 4.85 (d, J = 5.0 Hz, 2H, CH2), -2.72 (s,
2H, NHs). UV-vis (DMF): λmax (log ɛ) 428 (5.37) 520 (4.21) 554 (3.80)
598 (3.69) 653 (3.48) nm. HRMS (ESI) m/z calcd for C57H39BrN5O3 [M
+ H]+ 920.2236, found 920.2249.
Bacterial Strains and Growth Conditions
The genetically transformed bioluminescent Staphylococcus aureus
Xen 31 (S. aureus Xen31)40 was grown on Tryptic Soy Agar (TSA,
Merck) supplemented with 50 mg/mL of ampicillin (Amp) and with
34 mg/mL of chloramphenicol (Cm). Before each assay, one isolated
colony was transferred to 10 mL of Luria Bertani medium
(Liofilchem, Italy) previously supplemented with Amp and Cm and
was grown overnight at 37 ⁰C under stirring (120 rpm). An aliquot
was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under
the same growth conditions till stationary growth phase was
achieved [≈106 relative light unit (RLU). An optical density at 600 nm
(OD600) of 1.6 ± 0.1 corresponded to ≈106 colony forming units
(CFU)/mL. The correlation between CFU/mL and the bioluminescent
2-[1-(but-2-en-1-yl)-7-bromine-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid]-5,10,15,20-tetraphenylporphyrin (9e) Yield: (69
%). 1H NMR (300 MHz, CDCl3) δ 14.75 (s, 1H, OH), 8.84-8.79 (m, 4H,
β-H, H-3), 8.78-8.75 (m, 3H, β-H, H-2’), 8.68 (d, J = 4.9, 1H, β-H),
8.45 (d, J = 8.7 Hz, 1H, H-5’), 8.22-8.17 (m, 6H, H-o-Ph-5,10,15),
8.10-8.07 (m, 2H, H-o-Ph-20), 7.83 (d, J = 1.6 Hz, 1H, H-8’), 7.78-7.74
4 | J. Name., 2012, 00, 1-3
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Photochemical & Photobiological Sciences
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Journal Name
ARTICLE
signal (in RLUs) of bioluminescent S. aureus strain was evaluated. A were collected at time 0 min and after predefined irradiation times
View Article Online
DOI: 10.1039/C9PP00102F
fresh overnight bacterial culture was serially diluted 10-1 to 10-5 in and the bioluminescence was read in the luminometer.
phosphate-buffered saline (PBS.) Non-diluted and diluted aliquots Simultaneously, aliquots of treated and control samples were
were pour plated on TSA medium (0.5 mL) and, simultaneously, collected at time 0 and 90 min, then serially diluted and plated in
were read on a luminometer (0.5 mL) (TD-20/20 Luminometer, duplicate in Tryptic Soy Agar medium. The petri plates were
Turner Designs, Inc., Madison, WI, United States) to determine the incubated for 24 h at 37 ⁰C. Three independent experiments were
bioluminescence signal. The results obtained are presented in done.
Figure 2.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 6. Normal
distributions were checked by the Kolmogorov–Smirnov test and
the homogeneity of variance was verified with the Brown Forsythe
test. For the bioluminescence monitor assay, three independent
experiments with two replicates per assay for each condition were
performed and the differences between the results were analyzed
by two-way analysis of variance (ANOVA) and Dunnet’s multiple
comparison tests. The value of p < 0.05 was considered significant.
For the study of reduction of CFU/mL through the pour plated
methodology, three independent experiments with two replicates
per assay for each condition were performed and the results were
analyzed by two-way analysis of variance (ANOVA) and Bonferroni’s
multiple comparison tests. The value of p < 0.0001 was considered
significant.
7
6
5
4
3
2
3
4
5
6
7
8
9
10
log (CFU. mL-1)
Figure 2. Relationship between the bioluminescence signal and
viable counts of an overnight culture of S. aureus (< 106 CFU. mL)
serially diluted in PBS. All values are the mean of three independent
assays. The error bars represent the standard deviation.
Results and Discussion
Antimicrobial Photodynamic Therapy (aPDT) Procedure
Bioluminescent S. aureus culture was grown overnight and was
tenfold diluted in PBS (pH 7.49) to a final concentration of ≈109
CFU/mL, which corresponds approximately to 106 RLU. The
bacterial suspension was equally distributed in 50 mL sterilized and
acid-washed beakers.
Synthesis of porphyrin/4-oxoquinoline conjugates
The synthetic access to the desired porphyrin-4-oxoquinoline
derivatives 9a-e (Scheme 4) required the preparation of the
precursors 5a-e (Schemes 2 and 3). The key step in the proposed
strategy is the Heck coupling reaction between 1-allyl-4-
oxoquinoline substrates 1a-e and the 2-bromo-5,10,15,20-
Bioluminescence Monitoring
tetraphenylporphyrinatozinc(II)
(2).39
These
oxoquinoline
All the experiments were carried out under white light (400-750
nm) and the S. aureus bioluminescence signal was measured in the
luminometer (TD-20/20 Luminometer, Turner Designs, Inc.,
Madison, WI, United States) at different times of light exposure.
The assays were finished whenever the detection limit of the
luminometer was achieved (ca 2.3 log). Light control (LC) comprised
bacterial suspension exposed to the light and dark control (DC)
comprised bacterial suspension with each PS at the same
concentration (1.0 µM) protected from the light with aluminium
foil, were also evaluated.
derivatives were prepared by the reaction of the adequate anilines
3a-e with diethyl ethoxymethylene malonate (EMME), followed by
thermic cyclization (Scheme 1).41, 42 The subsequent reaction of 4a-e
with allyl chloride afforded the 1-allyl-4-oxo-1,4-dihydroquinoline-3-
carboxylates.43 The porphyrin macrocycle precursor 2 was prepared
according to literature by a controlled bromination of 5,10,15,20-
tetraphenylporphyrin (TPP) with N-bromosuccinimide (NBS),
followed by metalation with Zn(OAc)2, affording 2-bromo-
5,10,15,20-tetraphenylporphyrinatozinc(II) (2) (Scheme 2).39
Photodynamic inactivation assay
Bacterial suspensions were prepared from cultures (≈106 RLU) and
ten-fold diluted in PBS and distributed in sterilized glass beakers.
The appropriate volume of each PS (9a-e) was added to the
suspensions to reach a final concentration of 1.0 µM. Samples and
controls (LC and DC) were incubated under stirring for 15 min and
protect from light. After this period, the samples and LC were
irradiated for 90 min under stirring and the temperature was
controlled at ≈ 37 ⁰C. Aliquots of the treated and control samples
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
n
t
r
e
6
8
rec
ov.
O
O
View Article Online
DOI: 10.1039/C9PP00102F
i
OEt
R
R
y
TBA
B
TBA
B
NH2
N
H
1
2
Pd(OAc)2
Pd(OAc)2
K2CO3
3 h
13
15
19
5
6
5
3a-e
Cs2CO
3
trac
e
29 h
traces
10
32
4a-e
TBA
B
PPh3
TBA
B
3
4
5
Pd(OAc)2
Pd(OAc)2
Pd(PPh3)4
KOAc
KOAc
KOAc
2 h
2 h
3 h
33
42
20
42
1
3
19
9
10
10
12
8
6
5
a
d
b
c
(R = H), (R = 6-Cl), (R = 7-Cl),
ii
e
(R = 6-Br), (R = 7-Br)
18
13
4
14
1
O
O
6
*
15
min
Tra
ces
Pd(OAc)2
KOAc
PPh3
OEt
R
* MW radiation
N
The results obtained show that the yield of the expected
porphyrin/4-oxoquinoline conjugate 5b is strongly dependent of the
reaction conditions and regardless the reaction conditions, its
formation is accompanied by the formation of three unexpected
products: the aldehyde porphyrin derivative 6, the porphyrin
derivative 7 and the fused porphyrin 8. The formation of the by-
products 6 and 7 can be explained by the decomposition of
porphyrin/4-oxoquinoline conjugate 5b and their establishment will
be discussed in the mechanistic consideration part. The formation
of fused porphyrin 8 is due to the thermal cyclization and it was
already reported in the literature.44 From Table 1, it possible to
observe that the combination Pd(OAc)2/Cs2CO3/TBAB (entry 2) is
the one that produced the porphyrin/4-oxoquinoline conjugate 5b
in lower yield (only traces were detected) followed by the
combination of Pd(OAc)2/K2CO3/TBAB (entry 1) that afforded the
desired conjugate in 13% yield. A better performance was observed
when Pd(OAc)2 was used in the presence of KOAc and using TBAB
or PPh3 as ligands (entries 3 and 4). Under these conditions and just
after two hours of reaction, the yield of porphyrin derivative 5b was
dramatically increased to 33% in the presence of TBAB and to 42 %
in the presence of PPh3. No improvement was observed in the
presence of Pd(PPh3)4/KOAc/TBAB (entry 5) where the yield of
conjugate 5b did not go beyond 20 %, even after 3 hours of
reactions.
1a-e
(i) (1) EMME, EtOH, reflux, 2 h. (2) diphenyl ether, 270 ºC;
(ii) (1) DMF, K2CO3, r.t., 15 min. (2) allyl chloride, 80 ºC, 24 h.
Scheme 1. Synthesis of 4-oxoquinoline substrates 1a–e.
In order to select the best reaction conditions for the Heck coupling
reaction, an optimization was planned using the Zn(II) β-
bromoporphyrin 2 and the 1-allyl-4-oxo-1,4-dihydroquinoline-3-
carboxylate 1b (Scheme 2 and Table 1). The catalysts selected for
this study were palladium(II) acetate [Pd(AcO)2] or Pd(PPh3)4 and
the couplings were performed in the presence of the different
bases K2CO3, Cs2CO3 and KOAc using as ligands tetra-n-
butylammonium bromide (TBAB) or triphenylphosphine (PPh3). All
reactions were performed using a solvent system comprised by dry
toluene and DMF (1:0.5) at 120 °C under nitrogen atmosphere and
the progress of the reactions were monitored by TLC (Scheme 2 and
Table 1). After the workup the crude residue was fractionated by
silica gel column and all the fractions were further purified by
preparative thin layer chromatography.
Cl
O
O
N
O
O
N
H
Cl
O
OEt
Ph
Ph
Zn
Ph
X
EtO
In the attempt to minimize the formation of the secondary products
by reducing the reaction time, an extra assay was performed under
MW radiation using the best catalytic combination,
Pd(OAc)2/K2CO3/PPh3. Under these conditions the porphyrin
derivative 5a was also isolated in 42 % but a considerable reduction
on the reaction time from 2h to 15 min was achieved (Table 1, entry
6) So, these conditions were selected to couple the porphyrin
derivative 2 with the remaining 4-oxoquinoline substrates 1a, 1c–e
(Table 2, Scheme 3)
H
N
N
N
N
1b
iii
N
N
N
N
N
Zn
Ph
Ph
M
Ph
Ph
Ph
.
6
5b
Ph
Ph
TPP
X
M
= H; = 2H
,
O
i., ii.
OH
2
,
X
M
= Br; = Zn
NH
Ph
i.
iii
ii.
NBS, CHCl3, ; Zn(OAc)2,CHCl3, ;
. Catalyst, Base, Ligand, Toluene:DMF (1:0.5),
classic conditions
N
N
Zn
Ph
Zn
Ph
7
8
Scheme 2. Heck coupling reaction between β-bromoporphyrin 2
and 1-allyl-4-oxoquinoline derivative 1b
Table 1. Catalyst, base and ligand effects on the time and yield of
the Heck reaction between quinoline derivative 1b and β-bromo
porphyrin 2.
E
Cat.
Base
Lig.
Tim
% 5b
%
% 7
%
% 2
6 | J. Name., 2012, 00, 1-3
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ARTICLE
R
V7ie' w6A' rticle Online
6'
2'
R
6'
R
7'
8'
DOI: 10.1039/C95P' P00102F
7'
8'
5'
5'
8'
O
O
O
O
N
O
N
N
O
O
N
H
H
2'
O
OEt
Ph
Ph
HO
H 
EtO
H 
H
2'
R

Ph
Ph
Br
EtO
H

N
N
N
N
N
NH
N
1) NaOH /EtOH 1M, 1 h, reflux
H
Ph
3
Ph
Zn
Ph
Ph
1a-e
N
N
N
N
N
N
N
(2) CHCl3, TFA, r.t., 15 min.
(3) NaHCO3/H2O sat
HN
Zn
Ph
6
7
8
+
+
+
Zn
Ph
Ph
Ph
a (R= H, 53 %)
b (R = 6-Cl, 38 %)
c (R = 7-Cl, 47 %)
d (R = 6-Br, 32 %)
e (R = 7-Br, 69 %)
N
9
Pd(OAc)2/KOAc/PPh3
Toluene:DMF (1:0.5)
MW, 15 min
5
Ph
Ph
2
5a-e
Ph
Ph
Scheme 4. Deprotection and demetallation of porphyrin/4-
oxoquinoline conjugates 5a-e.
a
b
c
d
e
(R = H), (R = 6-Cl), (R = 7-Cl), (R = 6-Br), (R = 7-Br)
Scheme 3. Heck coupling reaction between β-bromo porphyrin 2
and 1-allyl-4-oxoquinoline derivatives 1a-e.
The structures of new conjugates 5a-e and the by-products 6-8
were confirmed by using 1D (1H and 13C spectra) and 2D [(1H,1H)
COSY, (1H,13C) HSQC and (1H,13C) HMBC] NMR techniques, high-
resolution mass spectrometry (HRMS-ESI) and UV-Vis spectroscopy;
the structures of the free-bases 9a-e were confirmed by 1H NMR
Table 2. Results obtained in the Heck coupling reactions between
quinoline derivative 1a-e and β-bromoporphyrin 2.
1
and confirmed by UV-Vis spectroscopy, H NMR and HRMS-ESI (see
experimental part).
Entry
1-allyl-4-
% 5
% 6
% 7
% 8
% 2 recov.
The HRMS-ESI+ of the porphyrin/4-oxoquinoline conjugates 5a-e
show molecular ions at the m/z values 932.2592 for 5a, 966.2207
for 5b, 966.2211 for 5c, 1010.1697 for 5d and 1010.1708 for 5e
corresponding to their [M+H]+, confirming the success of the Heck
coupling reaction of porphyrin 2 with the 1-allyl-4-oxoquinoline
derivative 1a-e.
oxoquinoline
derivative
1
2
3
4
5
1a
1b
1c
1d
1e
67
42
24
46
24
9
5
4
1
2
3
13
8
5
Traces
1
1
3
8
10
2
22
25
37
8
The 1H NMR spectra of the porphyrin/4-oxoquinoline conjugates
5a-e show a similar profile, being the principal difference the signals
due to the resonances of H-5’, H-6’/H-7’ and H-8’ of the
oxoquinoline moiety.
Also, with these 4-oxoquinoline substrates the formation of the
expected conjugates 5a, 5c-e was accompanied by the formation of
the by-products 6-8 with total yields varying between 7 and 27%.
The yields obtained for the desired porphyrin-quinoline derivatives
5a-e show that the best performance was achieved with the non-
substituted oxoquinoline 1a (67%), followed by the substituted
oxoquinolines 1b and 1d with the halogen atoms (chloro and
bromo) in position 6 that afforded the conjugates 5b and 5d in
moderate yields (42 and 46% of yield, respectively). The presence of
the halogen atom at position 7 is responsible for a decrease in the
efficiency of the coupling since both derivatives 5c and 5e were
isolated in 24% yield. So, these results suggest that the electronic
features of the oxoquinoline moiety are affecting the reactivity of
these quinolones in the Heck coupling.
1
For example, the H NMR spectra of substance 5b (Figure 3) is in
accordance with an asymmetric molecule. The resonances of the
seven β-pyrrolic protons appear as a singlet at 8.79 ppm (H-3), two
multiplets at 8.78-8.75 ppm (3 β-H) and 8.73-8.71 ppm (2 β-H) and a
doublet at 8.61 ppm (J = 4.6 Hz, 1 β-H). The meso-phenyl protons
appeared as four multiplets: at 8.13-8.09 ppm due to the resonance
of the ortho protons at 5, 10 and 15 positions; at 7.99 -7.98 ppm
due to the resonance of the ortho protons at 20 position; at 7.70-
7.63 ppm due to the resonance of the meta-phenyl and para-phenyl
protons at 5, 10 and 15 positions and the resonance of the para-
phenyl protons at 20 position and a triplet at 7.54 ppm ( J = 7.6 Hz,
2H), due to the resonance of the meta-phenyl protons at 20
position.
Finally, the porphyrinic acid derivatives 9a-e were obtained in
moderate yields (32-69%) after submitting the porphyrin/4-
oxoquinoline conjugates 5a-e to basic hydrolysis and demetallation
under acidic conditions (Scheme 4).
In what concerns to the signals related to oxoquinoline ring, the
multiplet at 6.36-6.34 ppm is related to the resonances of H-α and
H-β and the doublet at 4.71 ppm (J = 3.8 Hz) is related to the
resonance of the CH2, which confirms the success of the Heck
reaction. The resonance of H-5’ appeared as a doublet at 8.42 ppm
(J = 2.6 Hz), as confirmed by the correlation with the double doublet
at 7.61 ppm (J = 9.1 and 2.6 Hz), assigned to the resonance of H-7’.
The correlation observed between the resonance of H-7’ and the
doublet at 7.47 ppm (J = 9.1 Hz) allowed the assignment of this
signal to H-8’ proton. Furthermore, the signals related to the ethyl
group of the ester appear as a quartet at 4.39 ppm (J = 7.1 Hz)
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related to OCH2 protons, correlated with a triplet at 1.37 ppm (J = signal at 194.3 ppm due to the carbonyl carbon ofVtiehweAratilcdleeOhnyldinee
DOI: 10.1039/C9PP00102F
group. According to the HSQC spectra, it was also possible to assign
7.1 Hz), related to the CH3 protons.
the signals at 150.1 and 129.6 ppm to the carbons C-β and C-α,
Based on the heteronuclear (1H-13C) HSQC and HMBC spectra it was respectively.
possible to identify the resonances of some carbons. For example,
for compound 5b the signals at 174.2 and 165.6 ppm were related The structure of the by-product 7 was also confirmed by 1H and 13
C
to the carbon of the ketone carbonyl (C-4’) and of the ethyl ester NMR and two bidimensional studies (COSY and HSQC). From the 1H
group (CO2Et), respectively; In the aliphatic region of this spectrum, NMR and COSY studies it was possible to assign in the aromatic
the carbon resonances of the ethyl group were identified at 61.3 region the signals related to the β-pyrrolic protons and the H-o,m,p-
ppm (OCH2CH3) and 14.5 ppm (OCH2CH3). Through these spectra it Ph protons at the meso-positions of the porphyrin macrocycle (see
was also possible to identify the resonances of the carbons C-α and experimental part). The most important feature of these spectra is
C-β at 132.22 and 121.1 ppm, although it was not possible to reach the multiplet at 6.36-6.33 ppm, related to the resonance of H-α and
their specific assignments. The resonances of the methylene carbon H-β) and the doublet at 4.53 ppm (J = 5.0 Hz) related to the
(CH2) appears at 56.3 ppm and the signals at 149.6, 127.1, 126.6 resonance of CH2. 13C, HSBC and HMQC NMR studies allowed the
and 118.7 ppm were related to the hydrogenated carbons of the 4- unequivocally assignment of the signal at 171.1 ppm to the carbonyl
oxoquinoline nucleus (C-2', C-5', C-7 'and C-8', respectively). The carbon of the amide group and the correlation between these
signal at 130.8 ppm was unequivocally assigned to the C-3 carbon of carbonyl group with the signal at 65.7 ppm, that was assigned to
the porphyrin macrocycle.
CH2. However, it was not possible to distinguish one from the other,
carbons C-α and C-β were assigned to signals at 131.2 and 124.4
ppm.
Mechanistic considerations
The mechanism of the formation of the porphyrin/4-oxoquinoline
conjugates 5a-e followed the catalytic cycle described for the Heck
coupling reaction.45, 46 However, in all the reactions the formation
of the unexpected aldehyde 6 and of the allylcarbamic acid 7 was
observed. We believe that the formation of these compounds is
related to the thermal decomposition of the porphyrin/4-
oxoquinolineconjugates 5a-e and followed the mechanism
proposed in Scheme 5. In fact, when porphyrin/4-
oxoquinolineconjugates 5b was submitted to same temperature
conditions used for the Heck coupling reactions, it was observed the
formation of the by-products 6 and 7.
Figure 3. 1H NMR of porphyrin/4-oxoquinoline conjugate 5b.
The formation of derivative 6 can be justified by the oxidative step i
of the porphyrin/4-oxoquinolineconjugates
5
leading to
intermediate A. The hydrolysis of this iminium intermediate can
then afford the aldehyde 6 and the non-substituted oxoquinoline 4
(Scheme 5, via i.).
In what concerns the deprotected and demetallated derivatives 9a-
e, it was possible to confirm the success of both processes by the
appearance of the signal at 14.72 ppm assigned to the proton of the
carboxylic group and the signal at -2.72 ppm due to the inner NH
protons of the porphyrinic macrocycle.
The structures of the by-products were elucidated by adequate
spectroscopic techniques, namely 1H and 13C NMR spectroscopy.
The resonance of the protons was unequivocally assigned according
with the signal multiplicity and the correlations observed in the
COSY spectrum. In the NMR spectrum of the by-product 6, it was
possible to identify a singlet at 9.11 ppm related to the resonance
of H-3. The doublet at 7.04 ppm (J = 15.4 Hz) and a double doublet
at 6.77 ppm (J = 15.4 and 8.0 Hz) are related to H-α and H-β,
respectively. The signal related to the resonance of H-β was also
unequivocally assigned due to its correlations with the doublet at
9.08 ppm (J = 8.0 Hz), related to the proton of the CHO group. In the
13C NMR spectrum of this by-product it was possible to identify a
8 | J. Name., 2012, 00, 1-3
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ARTICLE
O
O
OEt
View Article Online
DOI: 10.1039/C9PP00102F
O
R
1.501
1.001
0.501
0.001
O
O
R
O
R
H
N
H
O
R
O
OEt
N
OEt
+
OEt
Ph
H
N
N
Ph
H
Ph
H
O
Ph
H
DFiBF
9a
N
H
N
N
O
[O]
Zn
Ph
H
H
Zn
N
Ph
Zn
Ph
Zn
Ph
i.
5
- H+
A
9b
6
O
9c
ii.
H
H
R
R
R
OH
OH
O
R
OH
9d
O
O
O
O
OEt
OEt
O
Et
H
H
OEt
9e
N
HN
HN
H
N
Ph
Ph
Ph
O
H
O
O
Ph
O
H
N
N
N
N
prototropism
Ph
Zn
Ph
Zn
Ph
Zn
Zn
Ph
Keto-enolic balance
0
200
400
600
D
C
Irradiation Time (s)
B
B`
H2
O
R
R
O
O
OEt
OEt
OH
O
O
HO
H
H
H
HN
Figure 4. Time-dependent photodecomposition of DPiBF at 50 μM
photosensitized by porphyrin/4-oxoquinolone derivatives 9a-e at
0.5 μM in DMF:H2O (9:1) upon irradiation with white light filtered
through a cut-off filter for wavelengths < 550 nm, with an irradiance
of 20.0 mW.cm−2.
Ph
F
N
Zn
Ph
7
Scheme 5. Proposed mechanism for formation of derivatives 6 and
7.
It is evident from figure
4 that in all cases a significant
In the case of the formation of derivative 7 the Michael type
addition of water to the C-2 carbon of the 4-oxoquinoline nucleus
of conjugate 5 would lead to intermediate B (Scheme 5, via ii.),
which by prototropism affords intermediate B`. This intermediate
after the ring-opening of the hydroxyquinoline ring affords
tautomers C and D. The hydrolysis of these intermediates furnished
the by-product 7 accompanied by the elimination of the ketoester F
(Scheme 5, via ii.).
photodegradation of DPiBF is observed. This indicates that all
porphyrin/4-oxoquinolinederivatives demonstrate a photooxidizing
ability. Despite the slight differences, it is possible to highlight that
1
derivative 9e (R = 7-Br) is the best generator of O2, followed by 9b
(R = 6-Cl) and 9c (R = 7-Cl). Porphyrin/4-oxoquinolones 9d (R = 6-Br)
and 9a without any substituent at 6- and 7- position of the
quinolone nucleus, were the less efficient 1O2 generators. These
results suggest that the positive effect in the generation of 1O2
induced by the presence of the halogen atom, in the case of
bromine is correlated with its position in 4-oxoquinoline nucleus.
Singlet oxygen generation studies
The photodynamic activity of a PS is strongly related to its capability
to produce reactive oxygen species (ROS), namely the O2. The PSs
Photodynamic effect of porphyrin/4-oxoquinoline derivatives 9a-e
in S. aureus
1
that produce ROS induce processes that are responsible for
oxidative reactions at cellular components level, causing function
loss, structure disruption and, consequently, cell death.3-5
The photodynamic efficiency of the porphyrin/4-oxoquinoline
derivatives 9a-e was evaluated in the presence of the recombinant
bioluminescent S. aureus. This Gram-positive bacterium is an
Hence, the potential photodynamic effect of the new porphyrin/4-
oxoquinoline conjugates 9a-e was first analysed by evaluating their
excellent bacterial model to monitor the efficiency of
a
1
ability to generate O2. To monitor the generation of this cytotoxic
photoinactivation process since its light output is a highly sensitive
reporter of its metabolic activity.49 This bioluminescent strain
possesses a stable copy of the Photorhabdus luminescens luxABCDE
operon on the bacterial chromosome.40 In order to evaluate the
effect of each PS on the S. aureus photoinactivation efficiency, the
porphyrin/4-oxoquinolinederivatives 9a-e were tested at the PS
concentration of 1.0 μM. The photodynamic experiments were
carried out under PAR white light (380–700 nm) irradiation at an
irradiance of 250 W.m-2 for 90 min. The results obtained are
summarized in Figure 5.
species was used an indirect method based on the
photodecomposition of 1,3-diphenylisobenzofuran (DPiBF). In this
1
method, DPiBF, a yellow compound, reacts with the O2 produced
through a [4+2] cycloaddition process being converted to 1,2-
dibenzoylbenzene, which is colourless. Since DPiBF absorbs at 415
nm, it is possible to follow the capability of the photosensitizer to
generate 1O2 by measuring the absorption decay at that
wavelength.23,25 Hence, aerated solutions of the porphyrin/4-
oxoquinolineconjugates 9a-e and DPiBF (100-fold molar excess) in
DMF/H2O (9:1) were exposed to filtered white light (cut-off < 550
nm) while monitoring the 415 nm absorption of DPBF.29, 47, 48
The results of the photodecomposition of DFiBF during a period of
irradiation in the presence of each one of the compounds 9a-e are
shown in Figure 4.
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irradiation (light control) or by any of the tested compounds (dark
View Article Online
7.1
6.1
5.1
4.1
3.1
2.1
1.1
DOI: 10.1039/C9PP00102F
control).
LC - Bacteria
9a
9
DC - 9a
9b
9c
9d
9e
DC - 9b
DC - 9c
DC - 9d
DC - 9e
7
0
90 min
****
5
3
1
0.1
0
****
10 20 30 40 50 60 70 80 90
Irradiation Time (min)
****
****
****
Figure 5. Bioluminescence monitoring of S. aureus treated with
porphyrin/4-oxoquinolinederivatives 9a-e at 1.0 µM after 10, 30, 60
and 90 min of irradiation with white light at an irradiance of 250
W.m-2. The values are expressed as the means of three independent
experiments; error bars indicate the standard deviation; DC dark
control; LC light control.
9a
9c
9b
9d
9e
eria
LC-Bact
Figure 6. Photodynamic inactivation of S. aureus treated with
porphyrin/4-oxoquinoline derivatives 9a-e at 1.0 µM after 90 min of
irradiation with white light at an irradiance of 250 W.m-2. The
values are expressed as the means of three independent
experiments; error bars indicate the standard deviation; DC dark
control; LC light control. ****(p < 0.0001) significantly different
from time 0.
The results confirmed that the porphyrin/4-oxoquinoline
derivatives 9a-e are able to cause a significant decrease in the
bioluminescent signal emitted by S. aureus (p < 0.05). In fact,
porphyrin derivatives 9a, 9b and 9d caused a reduction of the
bioluminescence signal of 4.1 log (p < 0.05) after 90 min of
irradiation, reaching the detection limit of the method. Under the
same light conditions, porphyrin derivatives 9c and 9e showed to be
slightly less effective, inducing reductions of 3.2 and 2.7 log (p <
0.05) on bacterial bioluminescence, respectively. The control
samples (light and dark controls) did not affect the bioluminescence
signal, showing that the viability of S. aureus is not affected by
irradiation or by the presence of any porphyrin/4-
oxoquinolinederivatives 9a-e tested in the dark.
The results also shown that the halogen atom localization at the 4-
oxoquinoline nucleus in derivatives 9b-9e seems to be an important
feature for their photodynamic efficacy: the ones with the halogen
atom at the 6- position in quinolone nucleus (9b and 9d) were the
most efficient in the photoinactivation of S. aureus. On the
contrary, porphyrin/4-oxoquinolinederivatives 9c and 9e, with the
halogen atom at the 7- position in quinolone nucleus, were the less
efficient ones. It is important to highlight the result obtained for
derivative 9a, since this compound is also one of the best PSs in the
inactivation of S. aureus. Since this efficacy is not directly related
with the ability of these compounds to generate oxygen singlet and
also considering their amphiphilicity (miliLoP: 9a: 9.75; 9b and 9c:
9.86; 9d and 9e: 9.88)50 we believe that compounds 9a, 9b and 9d
are the ones with the better adequate structural features to
generate the toxic singlet oxygen in close proximity to the target
cells.
The results obtained showed that, as previously mentioned,
porphyrin/4-oxoquinoline derivatives 9a, 9b and 9d were the most
efficient PSs, causing a viability bacterial decrease of 5.4, 6.3 and
6.4 log (p<0.0001) of S. aureus, respectively, after 90 min of
irradiation (135 J.cm-2). Also, in this case, conjugates 9c and 9e
promoted the lowest decrease in photoinactivation of S. aureus,
3.2 and 4.7 log (p < 0.0001), respectively. These results also
confirm that the irradiation (light control) or any of the tested
compounds (dark control) does not affect the viability of the
microorganisms. These results prompt us to consider porphyrin/4-
oxoquinolinederivatives 9a-e at 1.0 μM to be effective PS, since,
according to the American Society for Microbiology, a compound
can be designated antimicrobial if achieve a reduction of at least 3
log10 CFU.51
As already mentioned, our research group recently reported the
synthesis and the photodynamic effect of porphyrin/4-quinolone
conjugates with N-ethyl, N-pentyl and N-ribofuransyl substituents.32
These derivatives were tested also against S. aureus bacteria and it
was found that using a 10 µM concentration of each PS and an
irradiance of 15.0 mW cm-2 (135-162 J.cm-2), the viability of the
bacteria was reduced about 4.9, 3.4 and 2.1 log CFU, for N-ethyl, N-
pentyl and N-ribofuransyl-substituted derivatives, respectively.32
The results now achieved with these new porphyrin/4-oxoquinoline
derivatives 9a-e are very encouraging, since with a 10-fold lower
concentration it was achieved better inactivation rates against that
Gram-positive bacterium.
In order to confirm the bioluminescence reduction of S. aureus with
the reduction of CFU/mL, the previous study was accomplished with
the pour plated methodology. Thus, aliquots of the samples and
controls were collected at times 0 and 90 min of the aPDT protocol,
serially diluted in PBS and pour plated in duplicate in TSA. The
results obtained are presented in Figure 6. LC and DC were also
evaluated and no significant variation in the S. aureus viability was
observed, indicating that S. aureus was not affected either by
Conclusions
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In this work new porphyrin/4-oxoquinoline conjugates 5a-e were
obtained by Heck reaction between β-bromoporphyrin 2 and 1-
allyl-4-oxoquinolines 1a-e. The best conditions for these reactions
were established using Pd(OAc)2 as catalyst, PPh3 as ligand and
KOAc as base. The use of MW radiation allowed the reduction of
the reaction time to 15 minutes without changes in yields. In these
reactions the aldehyde porphyrin derivative 6, porphyrin
allylcarbamic acid 7 and the fused porphyrin 8 were also obtained
and their mechanisms of formation were assigned. The ester group
of the 4-oxoquinolines conjugates 5a-e were deprotected trough
alkaline hydrolysis and demetallated affording porphyrin/4-
oxoquinoline acids 9a-e in good yields. The new conjugates were
fully characterized, and the evaluation of singlet oxygen production
showed that these compounds are capable to generate that
cytotoxic species. The photodynamic effect of these derivatives in
the photoinactivation of bioluminescent S. aureus, was evaluated
and the results demonstrate that all compounds are capable to
inactivate S. aureus, in particular, porphyrin/4-oxoquinoline
derivatives 9a, 9b and 9d promoted the higher photoinactivation
rates (up to 5 log).
Conflicts of interest
There are no conflicts to declare.
View Article Online
DOI: 10.1039/C9PP00102F
Acknowledgements
Thanks are due to the University of Aveiro, to FCT/MEC for the
financial support to QOPNA Research Unit (FCT
UID/QUI/00062/2019) and Centre for Environmental and Marine
Studies (CESAM) Unit (UID/AMB/50017/2019) and the co-funding
by the FEDER, within the PT2020 Partnership Agreement and
Compete 2020, and also to the Portuguese NMR Network. Thanks
are also due to Universidade Federal Fluminence, FAPERJ, CNPQ,
CAPES (finance Code 001), and UFF for financial support and
fellowships. Fernanda Savacini Sagrillo thanks CAPES for granting
the scholarship for a sandwich doctorate (PDSE) at the University of
Aveiro. C. J. Dias also thanks FCT for her research fellow
(BI/UI51/7965/2017).
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Contents List
New porphyrin/4-oxoquinoline conjugates were synthesized and revealed to be excellent
photosensitizer agents in the inactivation of S. aureus after the antimicrobial photodynamic
therapy protocol.

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DOI: 10.1039/C9PP00102F
Graphical Abstract
118x91mm (150 x 150 DPI)