Cyclometalated ruthenium complexes from naturally occurring quinones: studies on their photophysical features, computational details and trypanocidal activity

Article abstract of DOI:10.1039/c7nj00379j

Phenazinic ligands and Ru(ii)-based complexes were synthesized from natural products lapachol and lawsone and evaluated against T. cruzi, the etiological agent of Chagas disease. These new ruthenium compounds could provide promising trypanocidal drugs. Besides synthesis and trypanocidal activity, this paper reports photophysical features and computational details of the compounds. The fluorescent trypanocidal substances are promising derivatives for further studies aiming to find molecules active against parasites associated with neglected diseases.

Full text of DOI:10.1039/c7nj00379j

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Barreto, C. Demicheli and E. N. da Silva Júnior, New J. Chem., 2017, DOI: 10.1039/C7NJ00379J.
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Cyclometalated ruthenium complexes from naturally occurring
quinones: Studies on their photophysical features, computational
details and trypanocidal activity†
Received 00th January 20xx,
Accepted 00th January 20xx
Clayton M. de Souza,a Rafaela C. Silva,a Philipe O. Fernandes,a José D. de Souza Filho,a Hélio A.
Duarte,a Maria H. Araujo,a Carlos A. de Simone,b Solange L. de Castro,c Rubem F. S. Menna-
Barreto,c Cynthia P. Demichelia and Eufrânio N. da Silva Júnior*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Phenazinic ligands and Ru(II)-based complexes were synthesized from natural products lapachol and lawsone and
evaluated against T. cruzi, the etiological agent of Chagas disease. These new ruthenium compounds could provide
promising trypanocidal drugs. Besides synthesis and trypanocidal activity, this paper reports photophysical features and
computational details of the compounds. The fluorescent trypanocidal substances are promising derivatives for further
studies aiming to find molecules active against parasites associated to neglected diseases.
imidazoles, oxazoles11 and phenazines12 with remarkable
trypanocidal activity were reported.
1. Introduction
The use of metal complexes as chemotherapeutic agents
Chagas disease, caused by the pathogenic protozoa Trypanosoma
cruzi, is primarily transmitted to humans by the faeces of
haemophagic insects, from the family Reduviidae and subfamily
Triatominae.1 In addition to vectorial transmission, blood
transfusion, oral and congenital transmission and, less commonly,
organ transplantation are alternative routes of infection.2 Based on
2010 estimates, approximately 5.7 million people are infected with
T. cruzi worldwide, and up to 70 million are at risk of chronic
infection.3 The treatment of Chagas disease is still the same for five
decades since the introduction of two nitroheterocycles,
benznidazole and nifurtimox.4 Both drugs have shown high
parasitological cure rates during the acute phase, but the
effectiveness declines with the advancement of the infection.
Another limitation is the development of adverse effects, which
leads in several instances to treatment abandonment.5,6
against parasitic diseases is an attractive alternative, since
complexation of transition metals with a proven or potential
biologically active ligand could enhance pharmacological and
chemical properties, such as potency, toxicity, solubility, chemical
stability, and lipophilicity.13 Metal complexes have, in general,
broad structural possibilities, with different modes of coordination
of the organic ligands and different oxidation states for the metal
centre allowing mechanistic and structural possibilities beyond
purely organic frameworks.14a
Ruthenium displays suitable chemical and biological properties
as metal centre such as biologically attainable oxidation states (II,
III), coordination versatility, and octahedral geometry and
appropriate ligand substitution kinetics.14b Rodrigues and
coworkers15 have described nitro-thiosemicarbazones ruthenium(II)
complexes which were 10-fold more active against T. cruzi than
their corresponding ligands. Demoro and collaborators16 reported
the synthesis and evaluation against T. cruzi of ruthenium-based 5-
nitrofuryl-thiosemicarbazones with three potential mechanisms of
action: reactive oxygen species (ROS) generation (5-nitrofuryl),
cruzain inhibition (thiosemicarbazone) and DNA interaction (Ru-
arene). Finally, an interesting example of water-soluble ruthenium
complexes with protozoan activity was described by the Otero
group.17 The complexes displayed high selectivity against T. cruzi,
ability to generate reactive oxygen species besides interaction with
DNA.
Lapachones is part of a class of naphthoquinoidal compounds
with potential trypanocidal activity.7 Lately, we have explored
strategies such as redox centre and C-ring modifications for
preparing new lapachone derivatives with potent trypanocidal
activity.8 Following this approach, we have discovered lapachone-
based triazoles9 and arylamino substituted,10 via C-ring modification
of nor-β-lapachone, that are able to act against T. cruzi. Redox
centre modification was also applied with success by our group and
a.Institute of Exact Sciences, Department of Chemistry, Federal University of Minas
Gerais, Belo Horizonte, 31270-901, MG, Brazil. E-mail: eufranio@ufmg.br; Tel:
+55 31 34095720
In this context, we herein describe for the first time the
synthesis, photophysical, computational features and trypanocidal
activity of cyclometalated ruthenium(II) complexes prepared from
lapachones. Quinoidal-redox centre modification was the strategy
used, allowing the insertion of a metal-redox centre and enabling us
b.Institute of Physics, Department of Physics and Informatics, University of São
Paulo, São Carlos, 13560-160, SP, Brazil
c. Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, 21045-900, RJ, Brazil
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the evaluation of the potential of the new ruthenium-based pattern, two signals at δ 1.81 and 1.67 (for 6) and δ 1.77 and 1.59
compounds against intracellular amastigote forms of T. cruzi.
(for 7) related to diastereotopic hydrogens from methyl groups.
Three signals among δ 4.47 to 3.51 with resonance that integrates
for one proton each one indicates the assignments for C-ring
substituted by iodine and bromine. For compound 10 the main
feature is a singlet at δ 3.38 corresponding to the hydrogens
attached to the C-3 of dihydrofuran ring and the enantiotopic
protons from methyl groups at δ 1.74 (6H). All the expected signals
were observed for compound 13.
2. Results and discussion
Initially, to prepare the phenazines 5-7, 10 and 13 used as ligands
for complexation with ruthenium(II), 1,10-phenanthroline-5,6-
diamine was prepared from 1,10-phenanthroline following
methodology previously reported.18 Lapachol (1) was extracted
from the heartwood of Tabebuia sp. (Tecoma) and purified by a
series of recrystallizations in an appropriate solvent, generally
hexane19 and used to prepare nor-lapachol (8) by Hooker reaction.20
From the natural product 1 and 8, the respective lapachones 2-4
and 9 were prepared via classical reactions: acid cyclization of 1 and
8 with sulphuric acid, β-lapachone (2) and nor-β-lapachone (9) were
synthesized, respectively;21 and the reaction of 1 with iodine and
bromine in dichloromethane afforded 3-iodo-β-lapachone (3) or 3-
bromo-β-lapachone (4), respectively.22 From the quinones 2-4 and
9, the corresponding phenazines 5-7 and 10 were obtained by
simple reaction with 1,10-phenanthroline-5,6-diamine in buffer
solution of AcOH/AcONa (Scheme 1). The naphthoquinoidal
compound (12)23 obtained from C-allyl lawsone (11) was also
considered in this study and it was used in the preparation of
phenazine 13 (Scheme 2). C-allyl-lawsone (11) was prepared from
the natural product lawsone as previously reported.24
The ruthenium(II) complexes 14-18 were prepared by the
reaction
of
the
ligands
5-7,
10
and
13
and
bis(bipyridine)dichlororuthenium(II) in a 1:1 molar ratio in ethylene
glycol reflux afforded the complexes as a crystalline red solids in
high yields (> 90%). To avoid any residue of the starting ligand, all
the complexes were purified in column chromatography, using
alumina as stationary phase, and a mixture of hexane:ethyl acetate
with increasing polarity up to 100% ethyl acetate. Compounds 14-
18 were characterized by 1H NMR. As an example, compound 14
was chosen for X-ray crystallographic analysis. Attempts to
crystallize 14, (BF4)2 as counter ion, were unsuccessful. We decide
for changing the counter ion to PF6 and suitable crystals for X-ray
studies were obtained. Figure 1 shows an Ortep-3 diagram of 14
and, the main crystallographic data are listed in the experimental
section.
Our strategy was based on the modification of dihydropyran
and dihydrofuran rings, starting from accessible semisynthetic
quinones aimed the synthesis of phenazine ligands and their
respective ruthenium(II) complexes with different photophysics and
biological features. This was made since the simple insertion of a
halogen, as for instance, iodine (compounds 3 and 6) or removal a
methyl group (compounds 10 and 13) can modify the trypanocidal
activity as will be discussed in the following topics.
The new phenazinic compounds 5-7, 10 and 13 were
characterized by NMR and data are in accordance with proposed
structures. It was observed in the 1H NMR spectra of phenazine
derivatives the respective signals corresponding to ten hydrogen
atoms related to the aromatic moieties. In the case of compound 5,
a singlet δ 1.52 (6H) was observed corresponding to methylenic
protons and two triplets at δ 3.12 and 2.02 in accordance with the
expected signals from C-ring (the previous quinoidal system) related
to methylene hydrogens. Compounds 6 and 7 showed the same
Scheme 1 Synthesis of phenazinic ligands 5-7 and 10 obtained from lapachol (1). i) To obtain compounds 2 and 9: H2SO4, 0 ºC to rt, 3: I2, Py, CH2Cl2, 0 ºC, 4: Br2, CH2Cl2.
Scheme 2 Synthesis of phenazinic ligand 13 obtained from C-allyl lawsone (11).
2
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Scheme 3 Synthesis of cyclometalated ruthenium(II) complexes 14-18.
We have obtained important information of the complexes by the range of 2.061(5) to 2.076 (5) Å and the N-Ru-N bond angles
X-ray analysis of the compound 14. The compound crystallized with from 79.94(2) to 95.10(2) Å, while the Ru-N bond lengths in the
one acetonitrile solvent molecule in the asymmetric unit. The axial position are 2.045 (5) and 2.064 (5) Å with N-Ru-N bond angle
planarity of the complex is observed, with the exception of the of 175.5(2)°, according to Allen et al.25 The octahedral [PF6]2 anions
dihydropyran ring, which has
a half-chair conformation. The are slightly distorted and the bond lengths P-F are in the range of
puckering parameters calculated for this conformation are: q2 = 1.456(8) to 1.568(5) Å with F-P-F bond angles from 86.7(6) to
0.070(7) Å; q3 = -0.042(6) Å; Q = 0.082(7) Å; θ = 121(4)° and φ = 94.9(7)° (equatorial site) and 1.508(8) to 1.589(5)Å and 177.7(6) to
158(5)°. The Ru ion is bonded for two nitrogen atoms from 179.0(4)° (axial site) respectively, according to Allen et al.25 In the
phenazinic ring ligands, and four nitrogens atoms from two crystal, weak intermolecular C-H…F interactions stabilize the crystal
bipyridine ligand rings, displaying an slightly distorted octahedral packing with interaction distance of 2.505(2) Å and angle of
coordination environment where N3, N4, N6 and N7 occupy the 146.04(5)°. The packing pattern leads to the formation of a three-
equatorial plane, while N5 and N8 are in the axial sites of the dimensional network structure.
octahedron. The Ru-N bond lengths in the equatorial position are in
Figure 1 An ORTEP-3 representation (20% probability displacement ellipsoids) of the asymmetric unit of compound 14.
In order to provide more information on the photophysical and properties of the compounds could be useful for their subcellular
computational features of the synthesized ligands 5-7, 10 and 13 localization and identification of their mechanism of trypanocidal
and Ru(II)-based complexes 14-18, UV/Vis absorption and action (see discussion as following).
fluorescence emission spectra (Figure 2), as well as computational
The absorption and emission spectra were recorded for all the
analysis by DFT (Figure 3), were obtained. The fluorescent compounds aiming solvatochromism studies in different solvents.
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Initially, a dichloromethane solution (5.00 x 106 mol L-1) was as well as strong fluorescent emissions, which were visible to the
prepared and an aliquot of 200 x 10-6 L-1 was moved into a 10.0 mL naked eye, in the green-yellow region (~450-525 nm). The
volumetric flask. After the dichloromethane had completely complexation of the phenazines with ruthenium induces red-shift in
evaporated, the volume of the balloon was completed with one of the fluorescent emissions (bathochromic shift). The complexes have
the solvents used in the studies.
also presented high Stokes shift values and are prospective
Photophysical properties of the ligands and their respective candidates for their practical application in experiments involving
complexes were studied in solvents with different polarities and light technology (Table 1).
protic or aprotic feactures. In general terms, the ligands 5-7, 10 and
13 showed strong absorptions in the ultraviolet-blue (300-475 nm)
Figure 2 UV/Vis absorption and fluorescence emission wavelength (10-6) for phenazines 5-7, 10 and 13 and their respective cyclometalated complex of ruthenium(II) 14-18 in
different solvents.
4
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Table 1. Photophysical data for the studied phenazines and their respective cyclometalated complex of ruthenium(II) in 10-6 solutions.
Stokes
Stokes
λmax
λmax
Compd
Solvent
λmax
(abs)(nm)
λmax
(abs)(nm)a
(em)(nm)a
Compd
Solvent
shift (nm)
shift (nm)
(em)(nm)a
Ethyl acetate
Acetone
Acetonitrile
DMSO
431
431
430
434
432
464
476
474
486
465
33
45
44
55
33
Ethyl acetate
Acetone
Acetonitrile
DMSO
426
426
425
429
427
453
462
464
475
456
27
36
39
46
29
5
6
THF
THF
Ethyl acetate
Acetone
Acetonitrile
DMSO
425
425
424
428
-
454
461
465
474
-
29
36
41
46
-
Ethyl acetate
Acetone
Acetonitrile
DMSO
447
446
431
447
448
486
496
498
511
486
39
50
67
64
38
7
10
14
16
18
THF
THF
Ethyl acetate
Acetone
Acetonitrile
DMSO
446
445
444
449
447
486
496
497
514
488
40
51
53
65
41
Ethyl acetate
Acetone
Acetonitrile
DMSO
457
459
459
458
461
617
612
605
620
616
160
153
146
162
155
13
15
17
THF
THF
Ethyl acetate
Acetone
Acetonitrile
DMSO
437
453
454
441
454
626
609
608
623
618
189
156
154
182
164
Ethyl acetate
Acetone
Acetonitrile
DMSO
431
453
453
436
435
626
609
608
622
622
195
156
155
186
187
THF
THF
Ethyl acetate
Acetone
Acetonitrile
DMSO
469
468
467
468
471
622
606
609
620
617
153
138
142
152
146
Ethyl acetate
Acetone
Acetonitrile
DMSO
470
468
468
469
471
624
608
608
621
618
154
140
140
152
147
THF
THF
All the metal complexes were completely optimized with no have been calculated through analytical second derivatives. The
symmetry constraint using density functional theory as it is real frequencies assure that a minimal in the potential energy
implemented in the Gaussian09 package.26 The X-ray structure of surface have been found. The highest occupied molecular orbital
the complex 14 was used as starting point for the geometry (HOMO) and the lowest unoccupied molecular orbital (LUMO) was
optimizations. The geometries were fully optimized without estimated from the Kohn-Sham molecular orbital energies (Figure
symmetry constraint using the Berny algorithm. The generalized 3).
gradient approximation for the exchange-correlation (XC) functional
It is worth noting the HOMO orbitals of the ligands 5-7, 10 and
due to Perdew, Burke and Ernzerhof (PBE) was used.27 The Becke´s 13 are centered in the dihydrofuran and dihydropyran rings arising
three-parameter hybrid exchange functional combined with the of the previous lapachone moieties while LUMO orbitals are more
Lee, Yang and Parr (LYP) correlation functional, denoted as distributed in the phenazine rings. For the Ru-based complexes 14-
B3LYP28,29 has also been used for comparison. The LANL2DZ 18 the HOMO orbitals are centered in the metal and coordinated
effective core potential was used for the metal, bromine and iodine ligands and LUMO orbitals are more distributed in the structures.
centers,30-32 while the standard Dunning/Huzinaga valence double This is an important observation corroborating the bathochromic
zeta basis sets33,34 were employed for the other atoms. The shift and higher Stokes shift observed for the cyclometalated
polarizable continuum method (PCM)35 was used to describe the ruthenium complexes.
non-specific interaction of the solvent water. Vibrational analyses
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Figure 3 Molecular orbitals – HOMO and LUMO obtained from B3LYP level for the structures 5-7, 10 and 13-18, considering water as the medium.
Table 2. Activity of compounds on intracellular forms of T. cruzi.a
The phenazines 5-7, 10 and 13 and their respective
cyclometalated ruthenium complexes 14-18 were evaluated against
Compounds
IC50/ 96 h (µM)
11.6 ± 0.9
6.7 ± 1.5
23.0 ± 1.5
the intracellular amastigote forms of T. cruzi (Table 2). The three
novel phenazines 5, 6 (dihydropyran ring) and 10 (dihydrofuran
ring) were active on intracellular forms of the parasite, with EC50/24
h values between 4.9 and 23.0 µM. The phenazines 6 and 10 were
the most active, displaying EC50 values higher than benznidazole
(3.3 µM). The ligands herein, described for the first time with
potential activity against T. cruzi, were useful in the synthesis of
new metal complexes with potential anti-parasitic activity.
5
6
7
10
13
14
15
16
4.9 ± 0.9
69.9 ± 10.4
106.8 ± 10.8
231.6 ± 11.2
41.4 ± 3.7
78.5 ± 11.8
101.4 ± 16.0
3.3 ± 0.3
17
18
We have evaluated the ruthenium complexes 14-18 in the same
experimental conditions. Compounds 16 and 17 were the most
active of the series with EC50/96 h = 41.4 and 78.5 µM, respectively.
Unfortunately, the insertion of ruthenium decreased the
trypanocidal activity in comparison to the original phenazines but
these compounds represent the first complexes designed and
prepared from lapachones with trypanocidal activity. The insertion
of the ruthenium in the active phenazines 5 and 6 was not efficient
to provide more potent compounds. Compound 16, presenting a
good activity profile has a bromine in the dihydropyran ring,
corroborating the association of the insertion of electron-
withdrawing group in the C-ring with different biological features.
Benznidazole
aMean ± SD of at least three independent experiments.
3. Conclusions
In summary, we have synthesized and well characterized five
phenazine ligands and their respective ruthenium(II) complexes
obtained from accessible natural products, lapachol and lawsone.
The photophysical properties for all compounds were studied by
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UV-vis, fluorimetry and DFT calculations evidencing their stability in 4.09 (1H, J = 17.7 and 5.4, dd), 3,91 (1H, J = 17.6 and 9.3 Hz, dd),
the ground and excited states. All the compounds were evaluated 1,81 (3H, s), 1,87 (3H, s). 13C NMR (50 MHz, CDCl3) δ 152.0, 151.6,
against T. cruzi evidencing three phenazines highly active and two 136.6, 132.9, 129.6, 128.1, 125.0, 124.9, 124.1, 123.9, 122.1, 114.4,
cyclometalated complexes with potential activity. This manuscript 109.8, 99.6, 78.8, 58.8, 32.7, 32.4, 31.3, 31.0, 27.8. EI/MS (m/z)
represents the first one to use lapachones as starting material for [M+H]+: 542. Calcd for [C27H20IN4O]+: 542.
preparing luminescent cyclometalated compounds with potential 11-bromo-12,12-dimethyl-11,12-dihydro-10H-benzo[a]pyrano[2,3-
further applications.
c]dipyrido[3,2-h:2',3'-j]phenazine (7). Compound 7 was obtained as
a yellow solid (466 mg, 0.94 mmol, 45% yield); mp 244-245 °C. 1H
NMR (400 MHz, CDCl3) δ 9.20 (2H, J = 15.1 and 8.0 Hz, dd), 9.12 (2H,
J = 10.3 and 3.9 Hz, dd), 9.03 (1H, J = 7.4 Hz, d), 8.18 (1H, J = 7.3 Hz,
d), 7.73 (2H, J = 13.4 and 6.4 Hz, q), 7.61-7.54 (2H, m), 4.48-4.44
(1H, m), 3.73 (1H, J = 17.4 and 5.3 Hz, dd), 3.58-3.51 (1H, m), 1.78
(3H, s), 1.59 (3H, s). 13C NMR (100 MHz, CDCl3) δ 151.8, 151.3,
150.6, 147.4, 141.6, 138.7, 137.8, 132.9, 130.1, 129.4, 128.0, 127.9,
127.3, 127.0, 124.6, 123.6, 123.6, 122.0, 108.9, 78.8, 52.3, 30.0,
27.1, 21.6. EI/MS (m/z) [M+Na]+: 516.9. Calcd for [C27H19BrN4ONa]+:
517.0.
4. Experimental Section
Melting points were obtained on Thomas Hoover and are
uncorrected. Analytical grade solvents were used. Column
chromatography was performed on silica gel (SiliaFlash G60
UltraPure 60-200 µm, 60 Å). 1H and 13C NMR were recorded at
room temperature using a Bruker AVANCE DPX200 and AVANCE
DRX400, in the solvents indicated, with TMS as internal reference.
Chemical shifts (δ) are given in ppm. Electron-impact mass spectra
(70 eV) were obtained using a VG Autospec apparatus (Micromass,
Manchester, UK). Absorption spectra were obtained on a Varian
Cary 100 spectrophotometer at room temperature in the solvents
described above. Fluorescence spectra were obtained on a Varian
Cary Eclipse spectrofluorimeter with a xenon arc lamp as the light
source while using an excitation wavelength (λexc) corresponding
to a higher absorption band. In all experiments, a quartz cuvette
was employed with a 1 cm optical path length.
2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]dipyrido[3,2-h:2',3'-
j]phenazine (10). Compound 10 was obtained as a yellow solid (286
mg, 0.71 mmol, 34% yield); mp 179-182 °C. 1H NMR (400 MHz,
CDCl3) δ 9.15-9.03 (4H, m), 8.92 (1H, J = 7.8 Hz, d), 7.89 (1H, J = 7.6
Hz, d), 7.71-7.61 (2H, m), 7.59-7.53 (2H, m), 3.38 (2H, s), 1.74 (6H,
s). Several attempts to acquire the 13C NMR spectrum were
performed unsuccessfully due to the low solubility of this
compound. For this reason, DEPT-135 experiment was carried out.
DEPT-135 δ 151.1, 150.4, 132.5, 132.9, 128.8, 127.3, 124.8, 123.2,
123.1, 121.5, 40.7, 28.2. EI/MS (m/z) [M+H]+: 403.1. Calcd for
[C27H20N4OH]+: 403.1.
General procedure to prepare phenazine derivatives 5-7, 10 and
13
2-methyl-1,2-dihydrobenzo[a]furo[2,3-c]dipyrido[3,2-h:2',3'-
j]phenazine (13). Compound 13 was obtained as a yellow solid (610
mg, 1.57 mmol, 75% yield); mp 295- 297 °C. 1H NMR (200 MHz,
CDCl3) δ 9.56-9.42 (1H, m), 9.32-9.23 (3H, m), 8.11-8.06 (1H, m),
7.85-7.69 (4H, m), 7.57-7.51 (1H, m), 4.10 (1H, J = 7.0 Hz, t), 3.84-
3.76 (1H, m), 3.32 (1H, J = 15.5 and 7.4 Hz, dd), 1.44 (3H, J = 6.4 Hz,
d). Several attempts to acquire the 13C NMR spectrum were
performed unsuccessfully due to the low solubility of this
compound. For this reason, DEPT-135 experiment was carried out.
DEPT-135 δ 151.1, 150.4, 132.5, 132.0, 128.8, 127.3, 124.8, 123.2,
123.1, 121.5, 40.9, 28.2. EI/MS (m/z) [M+H]+: 389.1. Calcd for
[C25H17N4O]+: 389.1.
In a round bottom flask of 50 mL was initially added 4,7-
phenanthroline-5,6-diamine (498 mg, 2.4 mmol) and 12 mL of
acetic acid. Thereafter it was added sodium acetate (326 mg, 4.0
mmol) and the respective lapachone (2.1 mmol). The mixture was
stirred for twenty-four hours. After this time, extractions were
performed with ethyl acetate, and then the organic phase
evaporated in a reduced pressure. The red solid obtained was
purified by column chromatography with neutral alumina as
stationary phase and a mixture of hexane and ethyl acetate as
eluent was used. The compounds were obtained as yellow solids.
12,12-dimethyl-11,12-dihydro-10H-benzo[a]pyrano[2,3-
c]dipyrido[3,2-h:2',3'-j]phenazine (5). Compound 5 was obtained as
a yellow solid (341 mg, 0.82 mmol, 39% yield); mp > 330 °C. 1H NMR
(400 MHz, CDCl3) δ 9.19 (2H, J = 8.7 Hz, t), 9.14-9.13 (1H, m), 9.10-
9.08 (1H, m), 9.02 (1H, J = 8.5 Hz, d), 8.21 (1H, J = 7.7 Hz, d), 7.68
(2H, J = 13.7 and 6.8 Hz, dt), 7.56-7.51 (2H, m), 3.13 (2H, J = 6.6 Hz,
t), 2.03 (2H, J = 6.6 Hz, t), 1.53 (6H, s). 13C NMR (100 MHz, CDCl3) δ
151.6, 151.5, 151.0, 147.4, 146.9, 142.8, 138.6, 147.7, 135.4, 132.8,
132.4, 129.9, 129.2, 128.6, 127.5, 127.4, 127.3, 124.6, 123.5, 123.4,
122.0, 110.0, 76.1, 32.3, 26.9, 18.0. EI/MS (m/z) [M+H]+: 417.1.
Calcd for [C27H21N4O]+: 417.1.
General procedure to prepare ruthenium(II) complex 14-18
In a round bottom flask of 50 mL was added the respective
phenazine (0.19 mmol), bis(bipyridine)dichlororuthenium(II) (91.6
mg, 0.19 mmol) and 20 mL of ethylene glycol. The reaction was
refluxed for twelve hours observing the change of deep red color to
a lighter red. Thereafter it was added 60 mL of water and KPF6 in
excess. After the observation of a precipitation formation, the
product was filtered. The solids obtained was purified by column
chromatography with neutral alumina as stationary phase and a
mixture of ethanol and acetonitrile as eluent was used. The
compounds were obtained as red crystalline solids. Several
unsuccessfully attempts using different deuterated solvents,
including mixtures DMSO-d6:CDCl3, DMSO-d6:MeOD and others
were tested in the attempt of acquisition of 13C NMR spectra. The
11-iodo-12,12-dimethyl-11,12-dihydro-10H-benzo[a]pyrano[2,3-
c]dipyrido[3,2-h:2',3'-j]phenazine (6). Compound 6 was obtained as
a yellow solid (396 mg, 0.73 mmol, 35% yield); mp 319-321 °C. 1H
NMR (200 MHz, CDCl3) δ 9.57 (1H, J = 7.7 Hz, d), 9.50 (1H, J = 7.4 Hz,
d), 9.33-9.31 (1H, m), 9.23-9.20 (2H, m), 8.29-8.27 (1H, m), 7.80-
7.78 (2H, m), 7.76-7.72 (2H, m), 4.65 (1H, J = 8.8 and 5.4 Hz, dd),
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New Journal of Chemistry
identity of the complexes was determined by 1H NMR and Trypanocidal assay
comparison with compound 14 solved by crystallographic analysis.
The trypanocidal activity was evaluated using T. cruzi (Tulahuen
Ruthenium(II) complex (14). Compound 14 was obtained as a strain) expressing the Escherichia coli β-galactosidase gene.42
crystalline red solid (202 mg, 0.18 mmol, 94% yield); mp > 330 °C. Trypomastigote forms were obtained from infected cultures of
1H NMR (400 MHz, CDCl3) δ 9.91 (1H, J = 8.1 Hz, d), 9.81 (1H, J = 8.1 mouse L929 fibroblasts in RPMI-1640 medium, without phenol red
Hz, d), 9.49 (1H, J = 7.8 Hz, d), 8.88-8.83 (4H, m), 8.52 (2H, J = 14.6 (Gibco BRL), containing 10% fetal bovine serum and
2 mM
and 4.9 Hz, dd), 8.28 (3H, m), 8.21-8.14 (4H, m), 8.10-8.06 (4H, m), glutamine (RPMIS). For the trypanocidal assay, L929 cells (4x103
7.90-7.82 (2H, m), 7.66 (2H, J = 6.5 Hz, t), 7.44-7.41 (2H, m), 3.47 cells/well) were plated at 96-well microplates and incubated
(2H, J = 6.4 Hz, t), 2.19 (2H, J = 6.4 Hz, t), 1.56 (6H, s).
overnight at 37 °C with 5% CO2 atmosphere and then infected with
Ruthenium(II) complex (15). Compound 15 was obtained as a trypomastigote forms at 1:10 ratio, as previously described.43
crystalline red solid (192 mg, 0.17 mmol, 92% yield); mp > 330 °C. Briefly, after 2 h of interaction, the cultures were washed to remove
1H NMR (400 MHz, CDCl3) δ 9.87-9.74 (2H, m), 9.45-9.38 (1H, m), non-interiorized parasites and the incubation proceeded for 48
8.93-8.87 (5H, m), 8.57-8.56 (3H, m), 8.32-8.28 (4H, m), 8.24-8.17 h/37 °C to establish infection. Then, the infected cultures were
(6H, m), 8.11-8.06 (4H, m), 7.50-7.48 (1H, m), 7.44-7.41 (1H, m), treated with serial dilutions of the tested compounds in RPMIS for
5.01-4.95 (1H, m), 4.23-4.06 (1H, m), 3.65-3.52 (1H, m), 1.80 (3H, s), 96 h at 37 °C. After this period, 500 μM chlorophenol red glycoside
1.75 (3H, s).
(CPRG) in 0·5% Nonidet P-40 was added to each well; the plate was
Ruthenium(II) complex (16). Compound 16 was obtained as a incubated for 18 h at 37 °C, then the absorbance was measured at
crystalline red solid (184 mg, 0.17 mmol, 92% yield); mp > 330 °C. 570 nm. The measurements were performed at 570 nm at
1H NMR (400 MHz, CDCl3) δ 10.01-9.99 (1H, m), 9.98-9.86 (1H, m), automated microplate reader Spectramax Plus (Molecular Devices).
9.60-9.59 (1H, m), 8.91-8.85 (4H, m), 8.57-8.53 (2H, m), 8.45-8.43 All stock solutions of the compounds were made in dimethyl
(1H, m), 8.29 (2H, J = 8.0 Hz, t), 8.21-8.11 (7H, m), 8.11 (1H, s), 7.99- sulfoxide, never exceeding 1% of the solvent. Benznidazole was
7.98 (2H, m), 7.66 (2H, J = 6.5 Hz, t), 7.45-7.39 (2H, m), 4.98-4.95 used as
a positive control. The half maximal inhibitory
(1H, m), 4.24-4.16 (1H, m), 4.06-3.99 (1H, m), 1.77 (6H, s). concentration (EC50) was calculated based on the percentage of T.
Ruthenium(II) complex (17). Compound 17 was obtained as a cruzi growth inhibition by comparing the optical density (O.D.) data
crystalline red solid (168 mg, 0.17 mmol, 90% yield); mp > 330 °C. with those obtained from those infected cell cultures exposed only
1H NMR (400 MHz, CDCl3) δ 10.02-9.88 (2H, m), 9.71-9.57 (2H, m), to the solvent.
8.91-8.86 (7H, m), 8.56-8.51 (3H, m), 8.30-7.99 (23H, m), 7.68-7.65
(4H, m), 7.42 (3H, s), 3.71 (2H, s), 1.77 (6H, s).
Data for compound 14
Ruthenium(II) complex (18). Compound 18 was obtained as a
crystalline red solid (166 mg, 0.17 mmol, 91% yield); mp > 330 °C. C49H39F12N9OP2Ru; M = 1160.9; monoclinic, space group P21/c; a =
1H NMR (400 MHz, CDCl3) δ 9.57-9.35 (2H, m), 9.21 (1H, J = 16.1 Hz, 12.9980(3) Å, b = 17.7660(4) Å, c = 24.1930(5) Å; α = γ = 90º, β =
d), 8.65-8.54 (4H, m), 8.20-7.71 (14H, m), 7.42-7.24 (2H, m), 3.74- 120.52°; V = 4812.6(2) Å3; Z = 4; Dc = 1.602 g.cm-1; F(000) = 2344; T
3.67 (1H, m), 3.42 (2H, J = 14.0 Hz, q), 1.58 (3H, s).
= 293(2) K; orange block, size 0.25 x 0.20 x 0.18 mm; 11002
independent measured reflections, refinement based on F2 to give
R1 [F2> 4σ(F2)] = 0.054; w2 = 0.088 for 6595 observed reflections,
and 668 parameters.
X-Ray analysis of compound 14
The structure of the compound 14 has been determined from X-ray
diffraction on an Enraf-Nonius Kappa-CCD diffractometer (95 mm Acknowledgements
CCD camera on κ-goniostat) using graphite monochromated MoKα
This research was funded by grants from the Conselho Nacional de
radiation (0.71073 Å), at room temperature. Data collections were
carried out using the COLLECT software36 up to 50° in 2θ. Final unit
cell parameters were based on 25687 reflections. Integration and
scaling of the reflections, correction for Lorentz and polarization
effects were performed with the HKL DENZO-SCALEPACK system of
programs.37 The structure of 14 was solved by direct methods with
SHELXS-97.38 The models were refined by full-matrix least squares
on F2 using SHELXL-97.39 The program ORTEP-340 was used for
graphic representation and the program WINGX41 to prepare
materials for publication. All H atoms were located by geometric
considerations placed (C–H = 0.93-0.97 Å) and refined as riding with
Uiso(H) = 1.5Ueq (C-methyl) or 1.2Ueq (other). The reference number
is CCDC 1477601. Copies of the available material can be obtained,
free of charge on application to the Director, CCDC, 12 Union Road,
Cambridge CH21EZ, UK (fax: +44-1223-336-033 or e-mail:
deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
Desenvolvimento Científico e Tecnológico (CNPq 480719/2012-8,
474797/2013-9, PVE 401193/2014-4, PQ-SR CNPq 302613/2013-7),
FAPEMIG (Edital 01/2014) and Programa Pesquisador Mineiro,
CAPES, FAPERJ, CAPES/PROCAD and PAPES/Fiocruz. Prof. CAS would
like to thank the Institute of Physics of University of São Paulo (São
Carlos), for kindly allowing the use of the KappaCCD diffractometer.
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TABLE OF CONTENTS GRAPHIC
Ligands and Ru(II)-based complexes were synthesized from natural products and
evaluated against T. cruzi. These compounds could provide promising trypanocidal
drugs.