Antimycobacterial activity of rhodamine 3,4-HPO iron chelators against Mycobacterium avium: Analysis of the contribution of functional groups and of chelator's combination with ethambutol

Article abstract of DOI:10.1039/c5md00456j

Rhodamine-labelled 3-hydroxy-4-pyridinone (3,4-HPO) chelators exhibit antimycobacterial activity, related but not limited to their iron binding capacity. We previously found that bacterial growth inhibition observed for chelators with ethyl substituents on the amino groups of the xanthene ring of rhodamine and a thiourea linkage between rhodamine and the chelating unit (MRH7 and MRB7) was different from that of compounds with methyl substituents and an amide linkage (MRH8 and MRB8). In this work we evaluated the antimycobacterial activity of two new chelators (MRH10 and MRB9) expressly designed to allow: (a) the direct comparison of the influence of the functional groups per se and (b) identification of the finest combination to achieve a higher biological activity. The activity of the chelators was assessed, as previously, by measuring their effect against M. avium. In this study we also report the antimycobacterial effect of MRH7, which proved to be the best performer of all four chelators, in combination with ethambutol, which is one of the antibiotics currently in use to treat mycobacterial infections. The results are indicative that a combination of 3,4-HPO iron chelators with an antibiotic is a promising strategy to fight M. avium infections. The current results are relevant for the choice of the best chelator in our set of compounds and also for the design of novel molecular architectures to target cellular membranes.

Full text of DOI:10.1039/c5md00456j

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DOI: 10.1039/C5MD00456J
Journal Name
ARTICLE
Antimycobacterial activity of rhodamine 3,4-HPO iron chelators
against Mycobacterium avium: analysis of the contribution of
functional groups and of chelator´s combination with ethambutol
pReceived 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Tânia Moniza, Daniel Silvaa, Tânia Silvab, Maria Salomé Gomesc* and Maria Rangeld*
www.rsc.org/
Rhodamine‐labelled 3‐hydroxy‐4‐pyridinone (3,4‐HPO) chelators exhibit antimycobacterial activity, related but not limited
to their iron binding capacity. We previously found that bacterial growth inhibition observed for chelators with ethyl
substituents on the amino groups of the xanthene ring of rhodamine and a thiourea linkage between rhodamine and the
chelating unit (MRH7 and MRB7) was different from that of compounds with methyl substituents and an amide linkage
(MRH8 and MRB8). In this work we evaluated the antimycobacterial activity of two new chelators (MRH10 and MRB9)
expressly designed to allow: (a) the direct comparison of the influence of the functional groups per se and (b)
identification of the finest combination to achieve a higher biological activity. The activity of the chelators was assessed, as
previously, by measuring their effect against M. avium. In this study we also report the antimycobacterial effect of MRH7,
which proved to be the best performer of all four chelators, in combination with ethambutol, which is one of the
antibiotics currently in use to treat mycobacterial infections. The results are indicative that a combination of 3,4‐HPO iron
chelators with an antibiotic is a promising strategy to fight M. avium infections. The current results are relevant for the
choice of the best chelator in our set of compounds and also for the design of novel molecular architectures to target
cellular membranes.
pathogenic microorganisms can be considered as a strategy to
control infections and numerous examples of the use of iron
chelation therapies and the consequently improvement in
Introduction
Bacterial resistance to currently available antibiotics is a
infection susceptibility of several microorganisms have been
reported 3‐12
1, 2
serious health problem
and therefore efforts to develop
.
new molecules that target unconventional mechanisms and
pathways involved in infectious processes are urgently
required. Iron is an essential element for the growth of all
bacteria and also for fungi and protozoa and it has been
demonstrated that limiting the iron available for the growth of
Our group has been particularly interested in mycobacterial
infections, namely those caused by the opportunistic
infectious pathogen Mycobacterium avium that mostly affects
patients with compromised immunity 13. The use of iron
chelators to restrict the iron available for mycobacterial
growth have been reported 14, namely for Mycobacterium
3
tuberculosis 15, 16 and M. avium 17‐20
.
a REQUIMTE‐UCIBIO, Departamento de Química e Bioquímica, Faculdade de
Ciências, Universidade do Porto, 4069‐007 Porto, Portugal
Mycobateria, as many microorganisms, produce various types
of siderophores to acquire iron with different characteristics in
terms of lipophilicity: the lipophilic mycobactins, which remain
cell‐wall‐associated and hydrophilic carboxymycobactins or
b Instituto de Investigação e Inovação em Saúde, IBMC‐Instituto de Biologia
Molecular e Celular, 4150‐180 Porto; CIQ(UP), Departamento de Química e
Bioquímica, Faculdade de Ciências, Universidade do Porto; ICBAS‐ Instituto de
Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal.
c Instituto de Investigação e Inovação em Saúde; IBMC‐Instituto de Biologia
Molecular e Celular, 4150‐180 Porto; ICBAS‐ Instituto de Ciências Biomédicas Abel
Salazar, Universidade do Porto, Portugal.
14,
exochelins which are released to the extracellular medium
21. These siderophore types are hexadentate ligands that
possess similar hydroxamate binding sites and vary only in
lateral chain characteristics, conferring lipophilic character on
mycobactins or hydrophilic on carboxymycobactins 14, 22. The
importance of these molecules for bacterial subsistence in
their host has been demonstrated by reduced growth of
mutant M. tuberculosis strains, in which siderophore
d REQUIMTE‐UCIBIO, Instituto de Ciências Biomédicas de Abel Salazar,
Universidade do Porto, 4050‐313 Porto, Portugal
* Corresponding author
Dr. Maria Rangel
Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto
4050‐313 Porto
Portugal
Tel:00‐351‐220402593
e‐mail address: mrangel@icbas.up.pt; mcrangel@fc.up.pt
production is inhibited 23
.
3‐Hydroxy‐4‐pyridinones (3,4‐HPOs) are bidentate oxygen
ligands, which are synthetically versatile allowing the synthesis
See DOI: 10.1039/x0xx00000x
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of chelators of variable denticity. These hard ligands show a units alone do not have an effect 19, 20. For the particular case
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DOI: 10.1039/C5MD00456J
very high capacity to trap Fe(III) providing an O6 coordination of rhodamine labelled chelators it was demonstrated that two
sphere through the binding of the appropriate number of molecular fragments seem to be determinant for a higher
ligands according to its denticity. Chelation of iron by 3,4‐HPO activity: (a) a thiourea linkage and (b) ethyl substituents on the
ligands confers an iron‐binding moiety different from that amino groups of the xanthene ring. Moreover, it was
provided by natural siderophores (catechol or hydroxamate confirmed that the rhodamine fluorophores per se are not
moiety). Various therapeutic applications have been described active in inhibiting M.avium intramacrophagic growth 20
.
for 3,4‐HPO ligands 24 and there are also several reports on the In order to substantiate the previous results and to identify the
effect of these ligands on the inhibition of Gram‐positive and finest combination to achieve a higher biological activity we
7, 11, 12
Gram‐negative bacteria growth
and M. avium infection designed the chelators MRB9 and MRH10 (Figure 1) that
18‐20
.
contain these two functional groups in different positions of
In our previous studies regarding the antimycobacterial activity the molecular framework (in different colour in Figure 1) and
of fluorescent 3,4‐HPO chelators we found that the presence measured their antimycobacterial activity in comparison with
of the fluorophores is determinant for the inhibition of that of chelator MRH7 previously studied.
M.avium intramacrophagic growth since the 3,4‐HPO chelating
Figure 1. Formulae of rhodamine derived 3,4‐HPO iron(III) chelators.
Combination of new molecules with classic antibiotics is an inhibiting mycobacterial arabinofuranosyl transferases which
emerging and promising strategy to overcome bacterial are responsible for glycosylation steps in the biosynthesis of
resistance mechanisms and to restore antibiotic effectiveness lipoarabinomannan (LAM) and arabinogalactan (AG), both
35,
25. The clinical potential of the combination of iron chelators constituents of the mycobacterial cell wall
36. Though
with other antibiotics has been demonstrated to fight bacterial ethambutol is active against M. avium pathogens high doses or
14,
31
32
26‐30, fungal
and protozoal
infections, although, no combinations therapies are required and resistance
reports were found of the use of combinations therapies phenomena have been described 37‐41. Moreover, there is
involving iron chelators and classic anti‐mycobacterial some evidence that combined therapies using ethambutol
antibiotics used in clinics.
have advantages due to the ability of this drug to increase cell
In the present work we report studies in which MRH7 was wall permeability in M. avium and consequently favour the
tested in combination with ethambutol, a drug, [(S,S)‐2,20‐ influx of other drugs tested in combination 42, 43. In addition,
(ethylenediimino)‐di‐1‐butanol], that was first reported in the there are reports of the ability of ethambutol to chelate trace
33
early 1960s and is one of the few well‐known and reliable metals, namely iron and copper, therefore contributing to a
32,
antimycobacterial used in clinics
34. Ethambutol acts by
2 | J. Name., 2012, 00, 1‐3
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better control of the infection additionally to their usual isomer), 8.19 (d, J 8.1 Hz, 1H, H5′ 4′‐isomer), 8.27 (d, J 8.0 Hz,
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described mechanism of action 44‐46
.
1H, H3′ 5′‐isomer), 8.80 (s, 1H, H3′ 4′‐isomer).
DOI: 10.1039/C5MD00456J
In this study we report the in vitro inhibitory effect of the new
chelators and the in vitro inhibitory effect of MRH7, which Synthesis of 3‐hydroxy‐4‐pyridinone ligands. The fluorescent
proved to be the best performer of all four chelators studied bidentate and hexadentate ligands were prepared by the
by our group, when used in combination with ethambutol coupling of fluorophore (rhodamine derivatives) to a protected
against M. avium. Taken together, our results suggest that the bidentate or hexadentate 3‐hydroxy‐4‐pyridinone (3,4‐HPO)
conjugation of hexadentate HPO iron chelators to a second unit 20. The bidentate or hexadentate unit were synthesized in
bioactive molecule is a promising strategy against M. avium our laboratory following the procedures described in the
18,
microorganism and this work is the first report of the used of literature
49. A diagram of the 3,4‐HPO units and
iron chelators in combination with other antibiotics for the fluorophores used to obtain the final fluorescent ligands
treatment of M. avium infection
synthesized in this work and the synthetic procedures are
outlined in Figure 2.
Experimental
Materials and Methods
3,4‐HPO
hexadentate
unit
was
coupled
to
tetramethylrhodamine isothiocyanate (F10) in the presence of
anhydrous N,N‐dimethylformamide (DMF) and triethylamine
to produce
MRH10
Compound
1
, followed by deprotection with BCl3 to yield the
Chemistry
.
General information. Chemicals were obtained from Sigma–
Aldrich (grade puriss, p.a.) or Fluka (p.a.) and were used as
received unless otherwise specified.
2
was obtained by reaction of 3,4‐HPO bidentate
unit with 5(6)‐carboxytetraethylrhodamine
(F9) in the
presence of anhydrous DMF, dicyclohexylcarbodiimide (DCC)
and N‐hydroxysuccinimide (NHS) in order to generate the
activated ester form of the rhodamine derivate and it was
subsequently deprotected with BCl3 to yield the rhodamine
NMR spectra were recorded on a Bruker Avance III 400,
operating at 400.15 MHz for 1H and 100.62 MHz for 13C atoms,
equipped with pulse gradient units, capable of producing
magnetic field pulsed gradients in the z‐direction of 50.0 G/cm
or on a Bruker Avance III Two‐dimensional H/1H correlation
spectra (COSY), gradient selected H/13C heteronuclear single
MRB9
.
1
1
Compound 1. To
a
solution of tetramethylrhodamine
isothiocyanate (F10) (0.0319 g, 7.19x10‐5 mol) in anhydrous
DMF (0.5 mL) and triethylamine (0.01 mL) was added the 3,4‐
HPO hexadentate unit (0.0622g, 5.99x10‐5 mol) and the
mixture was stirred at room temperature in the dark and
under argon atmosphere, for 48 hours. The product was
purified by gradient flash column chromatography, eluting
with chloroform/methanol (7:3) and increasing polarity until
chloroform/methanol (4:6) and ammonia. The sample was
purified by TLC, eluting with acetonitrile/water/ammonia
quantum coherence (HSQC) and 1H/13C heteronuclear multiple
bond coherence (HMBC) spectra were acquired using the
standard Bruker software. NMR and Mass Spectrometry
analyses were performed at Laboratory of Structural Analysis,
Centro de Materiais da Universidade do Porto (CEMUP)
(Portugal). Elemental analyses were performed at the
analytical services of University of Santiago (Spain).
Synthesis of 5(6)‐carboxytetraethylrhodamine (F9). To a
solution of 3‐diethylaminophenol (3.037 g; 1.78×10‐2 mol) in
propionic acid (7 mL), 1,2,4‐benzenetricarboxylic anhydride
(1.755 g; 8.86×10‐3 mol) and p‐toluenesulfonic acid (359 mg;
1.86 ×10‐3 mol) were added and the mixture was heated to
100º C for 6 days. Subsequently the reaction was worked‐up
by adding a mixture of ice/water (300 mL), extracting with
methanol/dichloromethane 85:15 (3×200 mL) and drying the
organic phase with anhydrous sodium sulphate. The product
was purified by gradient flash column chromatography, eluting
with methanol/dichloromethane (3:7) to afford 5(6)‐
carboxytetraethyl‐rhodamine (F9) (168.6 mg; 4%) as a purple
solid with a mixture with a 1:1.1 ratio of 5’‐ 4’‐ isomers (being
the 5’‐isomer the least polar one) as determined through
NMR spectra analysis. The spectral data are accordingly with
(3:2:0.05) to afford
1 (0.0430 g; 48%) as a purple solid. Only 5’‐
isomer was obtained as determined through NMR spectra
analysis. 1H NMR (400.15 MHz, MeOD‐d4, ppm): δ 1.87 (m, 6H,
CCH2CH2); 2.08 (m, 6H, CCH2CH2); 2.33 (s, 9H, 6’’‐CH3); 2.45 (t, J
6,2 Hz, 2H, NCH2CH2CO); 3.19 (s, 12H, CH3‐rhodamine); 3.49 (s,
9H, NCH3); 3.76 (bs, 2H, NCH2CH2CO); 4.45 (s, 6H, CH2NH); 5.07
(s, 6H, CH2C6H5); 6.40 (s, 3H, H‐5’’); 6.77 (bs, 2H, H‐4, H5); 6.89
(d, J 8.9 Hz, 2H, H2, H7); 7.23‐7.44 (m, 15H, ‐ CH2C6H5 and 2H,
H1, H8); 7.71 (bs, 2H, H4’, H6’); 8.07 (d, J 9.5 Hz,1H, H3’). 13C
NMR (100.62 MHz, MeOD‐d4, ppm): δ 20.9 (6’’‐CH3); 30.9
(CCH2CH2); 31.3 (CCH2CH2); 36.3 (CH2NH); 37.4 (NCH3); 40.9
(CH3‐rhodamine); 74.8 (CH2C6H5); 97.2 (C4, C5); 114.7 (C1, C8);
115.0 (C2, C7); 119.4 (C5’’); 124.0 (C4’, 6’); 129.4 – 130.1 (C‐
CH2C6H5 ); 132.5 (C3’); 138.4 (Cq‐C6H5); 143.3 (C2’’); 147.4
(C3’’); 150.9 (C6’’); 158.4 (C4”); 158.8 (C4a, C5a); 160.8 (C1a,
C8a); 175.3 (CONHCH2).
the literature 47, 48
.
1H NMR (400.15 MHz, MeOD‐d4, ppm): δ 1.30 (t, J 7.1 Hz, 24H,
8×CH3‐rhodamine, 4’‐isomer, 5’‐isomer); 3.66 (q, J 7.1 Hz, 16H,
8×CH2‐rhodamine, 4’‐isomer, 5’‐isomer), 6.93 (s, 2H, H4, H5),
6.98‐7.08 (m, 2H, H2, H7), 7.19‐7.25 (m, 5H, H1, H8+ H5′ 4′‐
isomer), 7.87 (s, 1H, H6′ 5′‐isomer), 8.11 (d, J 7.8 Hz, 1H, H4′ 5′‐
MRH10. Compound
1
(0.0400g, 2.67x10‐5 mol) was dissolved
in anhydrous dichloromethane (20 mL), under argon and
cooled to 0ºC. BCl3 (1 mL) was added dropwise and the
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reaction mixture was kept overnight with stirring at room 5’‐isomer); 37.5 (NCH3, 4’‐isomer); 46.7 (CH2CH3‐rhodamine 5’‐
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temperature. Methanol (50 mL) was added and the mixture isomer, 4’‐isomer); 74.7 (CH2C6H5, 5’‐isDoOmI:e1r0);.107349./9C5(MCDH020C465H65J,
was stirred for 1 h. The solid product that formed was 4’‐isomer); 97.1 (C4 + C5, 5’‐isomer, 4’‐isomer); 114.8 (CH);
removed by filtration and the solvent was removed under 114.9 (C2 + C7, 5’‐isomer, 4’‐isomer); 119.2 (C5’’, 5’‐isomer, 4’‐
reduced pressure to afford the crude product. Recrystallization isomer); 129.4 (CH); 129.5 (C6’, 5’‐isomer); 129.6 (C5’, 4’‐
of the crude product from methanol/acetone (1:9) afforded isomer); 129.7 (CH); 129.8 (C4’, 5’‐isomer); 130.0 (CH o‐C6H5);
the hydrochloride salt MRH10 (0,0224g, 61%) as a purple solid 130.2 (C3’, 4´‐isomer); 131.0 (C3’, 5’‐isomer); 132.9 (C1 + C8,
(5’‐isomer). Elemental analysis for (C62H77N11O13S4+.4Cl‐ 5’‐isomer, 4’‐isomer); 133.1 (CH); 133.7 (Cq, 5’‐isomer); 135.6
.11HCl.6H2O): Calculated (found): C 39.88 (39.71); H 5.40 (Cq); 136.4 (Cq); 136.8 (C4’, 4’‐isomer); 138.4 (Cq C6H5, 5’‐
(5.10); N 8.25 (8.21). MS: calculated for C62H74N11O13S+.K+: isomer); 138.5 (Cq C6H5, 4’‐isomer); 142.3 (Cq); 143.1 (C2’’ 5’‐
1251.48
[M+.K+];
found:
matrix‐assisted
laser isomer); 143.3 (C2’’ 4’‐isomer); 145.0 (C5’, 5’‐isomer); 147.6
desorption/ionization time of flight MS: 1251.40 [M+.K+]. 1H (C5’’, 5’‐isomer); 147.7 (C3’’, 4’‐isomer); 151.0 (C2’’, 5’‐
NMR (400.15 MHz, MeOD‐d4, ppm): δ 1.95 (m, 6H, CCH2CH2); isomer); 151.1 (C6’’, 4’‐isomer); 156.9 (C9); 159.3 (C4a, 4’‐
2.22 (m, 6H, CCH2CH2); 2.63 (s, 9H, 6’’‐CH3); 3.02 (m, 2H, isomer); 159.4 (C4a, 5’‐isomer); 162.0 (C1’, 5’‐isomer); 162.2
NCH2CH2CO); 3.31 (s, 12H, CH3‐rhodamine); 3.97 (s, 9H, NCH3); (C1’, 4’‐isomer); 168.6 (CONH, 5’‐isomer); 169.1 (CONH, 4’‐
4.52 m, 2H, NCH2CH2CO); 4.65 (s, 6H, CH2NH); 7.01 (s, 3H, isomer); 172.3 (2’‐CO2H, 5’‐isomer); 172.5 (2’‐ CO2H, 4’‐
H5’’); 6.86‐7.38 (m, 6H, H1 – H8); 7.64‐8.49 (m, 3H, H3’‐ H6’). isomer);175.0 (C4’’, 5’‐isomer); 175.1 (C4’’, 4’‐isomer).
13C NMR (100.62 MHz, MeOD‐d4, ppm): δ 21.3 (2’’‐CH3); 30.6
(CCH2CH2); 31.0 (CCH2CH2); 35.7 (NCH2CH2CO); 36.3 (CH2NH); MRB9. Compound
2
(37.7 mg, 5.50×10‐4 mol) was dissolved in
40.0 (NCH3); 41.1 (CH3‐rhodamine); 44.5 (NCH2CH2CO); 97.5 anhydrous dichloromethane (8 mL), under argon and cooled to
(C4, C5); 114.2 (C5”); 117.9, 123.8, 124.2, 129.2, 132.0 (C1 – 0ºC. BCl3 (0.8 mL) was added dropwise and the reaction
C8); 132.5, 133.5, 134.8 (C3’‐ C6’); 140.8 (C2’’); 144.8 mixture was kept overnight with stirring at room temperature.
(C3’’);150.9 (C4’’); 159.0 (C4a, C5a); 161.2 (C6”); 176.3 Methanol (30 mL) was added and the mixture was stirred for 1
(CH2CONHCH2). UV‐Vis (λmax / nm) 552; ε = 3.2 x 104 mol‐1 dm3 h. The solid product that formed was removed by filtration and
cm‐1; Fluorescence (λmax / nm) 575.
the solvent was removed under reduced pressure to afford the
crude product. Recrystallization of the crude product from
Compound 2. To
a
solution of 5(6)‐carboxytetraethyl‐ methanol/acetone (4:6) afforded the hydrochloride salt MRB9
rhodamine (F9) (168.6 mg, 3.46×10‐4 mol) in anhydrous DMF (37.1 mg, 82%) as a purple solid. Elemental analysis for
(10 mL), 3,4‐HPO bidentate unit (73.3 mg; 2.84×10‐4 mol), DCC (C38H46N4O62+.2Cl‐.CHCl3): Calculated (found): C 55.43 (55.03);
(73.0 mg, 3.50×10‐4 mol) and N‐hydroxysuccinimide (47.7 mg, H 5.61 (5.07); N 6.63 (6.52). MS: calculated for C38H45N4O6 :
40.2×10‐4 mol) were added and the mixture was stirred at 653.33 [M+]; found: ESI ‐ MS: 653.2952 [M+]. H NMR (400.15
+
1
room temperature in the dark and under an argon MHz, MeOD‐d4, ppm): δ 1.31 (t, J 6.7, 24 H, 8× CH2CH3‐
atmosphere, for 2 days. Subsequently, the formed N,N‐ rhodamine 5’‐isomer, 4’‐isomer); 2.63 (s, 3H, 6’’ ‐CH3, 5’‐
dicyclohexylurea (DCU) precipitate was filtered off and the isomer); 2.68 (s, 3H, 6’’ ‐CH3, 4’‐isomer); 3.68 (q, J 6.7, 16H,
solvent removed under reduced pressure. The product was 8×CH2CH3‐rhodamine 5’‐isomer, 4’‐isomer); 4.08 (s, 3H, NCH3,
purified by gradient flash column chromatography, eluting 5’‐isomer); 4.15 (s, 3H, NCH3, 4’‐isomer); 4.97 (s, 2H, CH2NH,
with methanol/chloroform (2:8) to afford
2 (52.5 mg; 25%) as 5’‐isomer); 5.06 (s, 2H, CH2NH, 4’‐isomer); 7.02‐7.11 (m, 9H,
a purple solid. Compound was obtained as a 1.1:1 ratio H5’’, 5’‐isomer, 4’‐isomer + H1, H2, H4, H5, H7, H8); 7.55 (d, J
2
mixture of 4’‐ and 5’‐ isomers as determined through NMR 5.7 Hz, 1H, H6’, 4’‐isomer); 7.88 (br s, 1H, H6’, 5’‐isomer); 8.27
spectra analysis. 1H NMR (400.15 MHz, MeOD‐d4, ppm): δ 1.28 (br s, 1H, H4’, 5’‐isomer); 8.33 (br s, 1H, H5’, 4’‐isomer); 8.41
(t, J 6.9, 24 H, 8× CH2CH3‐rhodamine 5’‐isomer, 4’‐isomer); (br s, 1H, H3’, 5’‐isomer); 8.82 (s, 1H, H3’, 4’‐isomer).13C NMR
2.39 (s, 3H, 6’’‐CH3, 5’‐isomer); 2.45 (s, 3H, 6’’‐CH3, 4’‐isomer); (100.62 MHz, MeOD‐d4, ppm): δ 12.8 (CH2CH3‐rhodamine, 5’‐
3.65 (m, 11H, NCH3, 5’ isomer + 8×CH2CH3‐rhodamine 5’‐ isomer, 4’‐isomer); 21.3 (6’’‐CH3, 5’‐isomer, 4’‐isomer); 37.2
isomer, 4’‐isomer); 3.72 (s, 3H, NCH3, 4’‐isomer); 4.66 (s, 2 H, (CH2NH, 5’‐isomer); 37.3 (CH2NH, 4’‐isomer); 40.0 (NCH3, 5’‐
CH2NH, 5’‐isomer); 4.74 (s, 2H, CH2NH, 4’‐isomer); 5.15 (s, 2H, isomer, 4’‐isomer); 46.9 (CH2CH3‐rhodamine, 5’‐isomer, 4’‐
CH2C6H5, 5’‐isomer); 5.23 (s, 2H, CH2C6H5, 4’‐isomer); 6.45 (s, isomer); 97.3 (C5’’); 114.2 (C4); 114.6 (C5); 114.7 (C2); 115.5
1H, H5’’, 5’‐isomer); 6.51 (s, 1H, H5’’, 4’‐isomer); 6.90 (d, J 2.5 (C7); 130.4 (C4’, 5’‐isomer); 130.6 (C6’, 5’‐isomer); 131.6 (C3’,
Hz, 4H, H4 + H5 4’‐isomer, 5’‐isomer); 6.99 (br s, 4H, H2 + H7, 4’‐isomer); 132.1(C1); 132.2 (C6’, 4’‐isomer); 132.4 (C8); 132.6
5’‐isomer, 4’‐isomer); 7.21‐7.40 (m, 13H, H1 + H8 + m‐, p‐ C6H5 (C5’, 4’‐isomer); 132.9 (C3’, 5’‐isomer); 135.3 (C2’, 5’‐isomer);
5’‐isomer, 4’‐isomer + H6’, 4’‐isomer, + o‐ C6H5, 5’‐isomer); 135.6 (C5’, 5’‐isomer); 136.4 (C2’, 4‐isomer); 138.2 (C1’, 5’‐
7.46‐7.48 (m, 2H, o‐ C6H5, 4’‐isomer); 7.64 (d, J 1.5, 1H, H6’, 5’‐ isomer); 138.6 (C1’, 4’‐isomer); 140.6 (C2’’ 5’‐isomer); 140.8
isomer); 7.99 (dd, J 7.9; 1.8, 1H, H5’, 4’‐isomer); 8.03 (dd, J 8.3; (C2’’, 4’‐isomer); 145.2 (C3’’); 150.7 (C6’’); 157.2 (C3+C6);
1.8, 1H, H4’, 5’‐isomer); 8.10 (br d, J 8.0, 1H, H3’, 5’‐isomer); 159.3 (C9); 159.6 (Cq); 159.7 (Cq); 161.3 (C4a + C5a); 167.3 (2’‐
8.43 (d, J 1.7, 1H, H3’, 4’‐isomer); 13C NMR (100.62 MHz, CO2H); 168.6 (Cq); 168.8 (CONH). UV‐Vis (λmax / nm) 559; ε =
MeOD‐d4, ppm): δ 12.8 (CH2CH3‐rhodamine 5’‐isomer, 4’‐ 5.4 x 104 mol‐1 dm3 cm‐1; Fluorescence (λmax / nm) 583.
isomer); 20.8 (2’’‐CH3, 5’‐isomer); 20.9 (6’’‐CH3, 4’‐isomer);
37.1 (CH2NH, 5’‐isomer); 37.2 (CH2NH, 4’‐isomer); 37.4 (NCH3,
4 | J. Name., 2012, 00, 1‐3
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ARTICLE
Figure 2. Reaction scheme of the synthetic route for the preparation of the new chelator MRH10 (A) and MRB9 (B)
Electronic spectroscopy: absorption and fluorescence morpholino)propanesulfonic acid (MOPS) buffer solution (pH
measurements. Electronic absorption measurements were 7.4, I=0.1 M NaCl). The percentage of the DMSO stock solution
performed in a Perkin Elmer Lambda 25 spectrophotometer was always less than 1% in the final volume. The results are
equipped with a constant‐temperature cell holder, using the summarized in the experimental section.
conditions T= 25ºC, l=1cm cuvettes and wavelength range 225‐
650 nm.
Biology
Fluorescence measurements were performed in a Varian Cary
Bacteria. M. avium strain 2447, smooth transparent variant
(SmT) was isolated from an AIDS patient and provided by F.
Portaels (Institute of Tropical Medicine, Antwerp, Belgium).
Mycobacteria were grown to mid‐log phase in Middlebrook
7H9 medium (Difco, Sparks, MD) containing 0.04% Tween 80
(Sigma, St. Louis, MO) at 37ºC.
Bacteria were harvested by centrifugation, suspended in a
small volume of saline containing 0.04% of Tween 80 (Sigma)
and briefly sonicated in order to disrupt bacterial clumps. The
suspension was diluted and stored in aliquots at ‐80ºC until
use.
Eclipse spectrofluorometer, equipped with
a constant‐
temperature cell holder, using the conditions T= 25ºC, l=1cm
cuvettes. All spectra were recorded with excitation and
emission slit widths of 5 nm, 650V, with: i)
em from 560 to 700 nm for MRH10; ii) exc=560 nm and em
from 561 to 700 nm for MRB9
exc=553 nm and


.
Stock solutions of the different compounds were obtained by
preparing a concentrated solution of the compound in
dimethylsulfoxide (DMSO). Samples for absorption and
fluorescence measurements were prepared by dilution of a
known volume of the DMSO stock solution in 3‐(N‐
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Bone marrow derived macrophages (BMM). Macrophages
Journal Name
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DOI: 10.1039/C5MD00456J
were derived from the bone marrow of BALB/c mice. Each Statistical analysis. Data obtained in this work were analysed
femur was flushed with 5 mL of Hanks’ balanced salt solution using the software Graph Pad Prism version 6.0. Data are
(HBSS, Life Technologies, Paisley, U.K.). The resulting cell expressed as mean ± SD for the number of experiments
suspension was centrifuged and the cells re‐suspended in indicated in the legends of the figures. Multiple comparisons
DMEM (Life Technologies, Paisley, U.K.) supplemented with 10 were performed using one‐way analysis of variance (ANOVA)
mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 10% followed by Bonferroni multiple comparison posthoc test.
FBS (Fetal Bovine Serum) (Life Technologies), and 10% of L929 Significance was accepted when p value < 0.05 was obtained.
cell conditioned medium (LCCM) as a source of Macrophage
Colony Stimulating Factor (M‐CSF). According to
manufacturer’s information, the iron (Fe(III)) content of DMEM
Results and discussion
Synthesis and characterization of fluorescent 3,4‐HPO
is 0.25 mM. The added FBS contributes an additional 3 mM of
iron. In order to remove fibroblasts, the cells were cultured
overnight, at 37ºC in a 7% CO2 atmosphere, on a cell culture
dish. The non‐adherent cells were collected with warm HBSS
medium, washed and resuspended in DMEM with 10% LCCM
at a cell density of 4x105 cells/ml. One ml of this cell
suspension was distributed per well in 24‐well plates which
were incubated at 37ºC in a 7% CO2 atmosphere. Three days
later, 0.1 mL of LCCM was added. On the 7th day of culture,
the medium was renewed.
chelators
The new fluorescent ligands MRH10 and MRB9 were prepared
using straightforward synthetic protocols 18, 20. For compound
1,
the
commercially
available
fluorophore
tetramethylrhodamine isothiocyanate (F10) was coupled to
the protected 3,4‐HPO hexadentate unit. To obtain compound
2, the fluorophore 5(6)‐carboxytetraethyl‐rhodamine (F9) was
synthetized and coupled to the protected 3,4‐HPO bidentate
unit. The last step is the deprotection reaction for the removal
of the protecting benzyl group with under BCl3, as it is depicted
in Figure 2.
Infection and treatment of BMM. On the tenth day of culture,
the macrophages of each well were incubated with 0.2 mL of
DMEM containing 106 M. avium colony forming units (CFU) for
4 h at 37ºC in a 7% CO2 atmosphere. The cells were washed
with warm HBSS to remove extracellular mycobacteria and
reincubated in DMEM with 10% FBS and 10% LCCM with or
without the addition of the compounds to be tested. To
quantify the number of intracellular mycobacteria at time zero
of infection, macrophages from three wells were immediately
lysed with 0.1% of saponin.
The iron chelator solutions were obtained by preparing
concentrated solutions of the different compounds in DMSO
followed by dilution of a known volume of the DMSO stock
solution in DMEM. The percentage of the DMSO stock solution
was always less than 1% in the final volume.
Chelators were added to the culture medium at the
concentrations indicated in each figure (ranging from 5 to 120
μM). Chelators were tested alone and in combination with
ethambutol (ETH) at 2 μg/ mL (7.2 μM). Ethambutol was also
tested alone at the same concentration used in combination
with chelators. Each concentration was tested in triplicates. All
the results were confirmed in at least three independent
assays. The formulae of the bidentate and hexadentate ligands
used in the present study are shown in Figure 1.
The comparison of the results obtained for the synthesis of
and demonstrates that the reaction
1
of
2
tetramethylrhodamine isothiocyanate with the hexadentate
3,4‐HPO lead us to obtain ligand in a 48% yield and in the
reaction of 5(6)‐carboxytetraethyl‐rhodamine with the
bidentate unit we obtained ligand in 76 % yield. Although
both synthetic procedures are very similar, the yield of the
reactions are different and this fact could be related with the
functional group of the fluorophore that reacts with the amine
group pf the 3,4‐HPO unit. In the deprotection reactions, no
major differences are observed and ligands MRH10 and MRB9
was successfully synthetized and the yield of reactions are
respectively 61 and 96%.
1
2
The structures of the ligands
1, MRH10, 2 and MRB9 in
solution were established by NMR analysis (1H and 13C, 1D and
2D experiments, including COSY, HSQC and HMBC spectra for
unequivocal assignment of the most characteristic proton and
carbon chemical shifts). The assignment of the resonance
signals in 13C NMR spectra of the protected and de‐protected
compounds was achieved by analysis of 1H/13C HSQC and
1H/13C HMBC spectra, which provide one and multiple bond
1H‐13C connectivity.
1
The H NMR spectra of ligand
1 revealed that the resonance
Quantification of M. avium inside BMM. After 5 days of
infection, the growth of M. avium 2447 SmT was evaluated by
counting the colony forming units. Macrophages were lysed
using 0.1% saponin and the resulting bacterial suspension was
diluted in water containing 0.04% Tween 80. The dilutions
were plated in Middlebrook 7H10 agar (Difco) and the number
of colonies was counted after 8 days at 37ºC. The difference, in
terms of log10 CFU/well, between time zero of infection and
the last day in culture (day 5), is abbreviated as ‘‘log10
increase’’.
signals of the methyl protons of the rhodamine residue appear
at 3.19 ppm and those of H1‐H8 protons in the spectral are
between 6.77 and 7.44 ppm. The two multiplets at high field
(1.87‐2.08 ppm) were assigned to ‐C(CH2CH2)3 protons. The
resonance signals of NCH2CH2CO protons appear as triplets or
broad singlets at 2.45 and 3.76 ppm. The protons of the
methyl group of the pyridinone ring appear at 2.33 ppm and
the protons of the methyl linked to the nitrogen of the ring
appear at 3.49 ppm.
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The resonance signals, at 6.40 ppm, were assigned to the The aromatic peaks (6.9‐7.4 ppm) were assigned to the
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aromatic CH protons (H5”) of the pyridinone residue and show protons of di‐substituted aromatic ring oDfOrhI:o1d0.a10m3i9n/Ce5rMeDsi0d0u4e5s6.J
HMBC correlation with a carbon at 20.9 ppm assigned as 6’’‐ The signals related to the benzyl group are the singlet at 5.17
CH3. The set observed in the low field spectral area (7.7‐8.1 ppm (5’‐isomer) and at 5.23 ppm (4’isomer) corresponding to
ppm) and assigned to the protons of di‐substituted aromatic the methylene protons of benzyl group while the aromatic
ring of rhodamine residues. Due to the characteristic chemical protons appear as two sets of multiplets comprised between
shift of carbon C3’, the reaction was isomeric selective and 7.21‐7.40 ppm and 7.46‐7.48 ppm. The carbons associated to
only isomer 2’/5’ was obtained.
the methylene of benzyl group are found at 74.7 ppm for the
The signals related with the protecting groups are the singlet 5’‐isomer and at 74.9 ppm for the 4’‐isomer.
at 5.07 ppm that corresponds to the protons of the methyl The quaternary carbons in 13C NMR were assigned resorting to
group and the protons of the benzyl ring appear between 7.23‐ HMBC correlation, namely the signals assigned to 2’‐CO2H at
7.44 ppm. The carbon associated to this methyl group is at 172.3 and 172.5 ppm for 5’‐ and 4’‐isomers respectively due
74.8 ppm, the quaternary carbon appear at 138.4 ppm and the to correlation with the respective H3’proton signals 8.10 (5’‐
last 5 carbons of the benzyl ring appear between 129.4 and isomer) and 8.43 (4’‐isomer). The peak at 168.6 ppm was
130.1 ppm. The resonance signal at 175.3 ppm was found to identified as belonging to the carbon of CONH of 5’‐ isomer,
give long range coupling to the resonance signal of ‐C(CH2CH2)3 due to correlation with the corresponding peaks at 4.66 ppm
and ‐ CH2NH protons and was attributed to carbonyl carbons of (CH2NH2) and with 7.64 ppm (H6’) both associated to 5‐isomer;
the three amide groups.
similarly the peak at 169.1 ppm was assigned to carbon of
After the deprotection (MRH10), significant differences in the CONH of 4’‐ isomer because of the observed correlation with
1H and 13C spectra are detected as for example the shift of the the peaks at 4.74 ppm (CH2NH2) and 8.43 ppm (H3’) associated
protons of methyl group linked to the nitrogen of the to 4’‐ isomer. The resonance signals at 175.0 and 175.1 were
pyridinone from 3.49 to 3.97 ppm as also the shift in the assigned to C4’’ of 5’‐ and 4’‐isomers respectively, due to
protons of the methylene group of the linkage (CH2NH) from correlation with respective H5’’ proton signals of 6.45 and 6.51
4.45 to 4.65 ppm. Their respective carbons are also dislocated ppm of both isomers. The carbons at 151.0 and 151.1 ppm s
to low field region, namely NCH3 from 37.4 in the protected were assigned to C6’’ of 5’and 4’‐ ‐isomers respectively, based
ligand and 40.0 in the deprotected form and the shift from on correlations with the respective H3’’, CH2NH and 6’’‐CH3
119.4 to 114.2 ppm for the carbon C5’’.
signals. C3’’ signals were assigned to 147.6 ppm (5’‐isomer)
The carbon at 140.8 show HMBC correlation with H5’’, CH2NH and 147.7 ppm (4’‐isomer) based on correlation with the
and 6’’‐CH3 protons and was attributed to C2’’. The carbon at corresponding H3’and CH2NH protons. 143.1 (5’‐isomer) and
144.8 show HMBC correlation with H5’and CH2NH protons and 143.3 (4’‐isomer) were attributed to C2’’ based on the
was attributed to C3’’. Signals at 150.9 and 161.2 were correlations with corresponding H5’’, NCH3 and CH2NH
attributed as carbons C4’’ and C6’’ due to their HMBC protons.
correlations with H5’’, NCH3 and 6’’CH3 or H5’’, NCH3,
respectively. The resonance signal at 176.3 ppm was found to In the spectra of MRB9 the peaks for the 4’‐ and 5’‐isomers
give long range coupling to the resonance signal of ‐C(CH2CH2)3 present the same chemical shifts for analogous atoms while in
and ‐ CH2NH protons and was attributed to carbonyl carbons of their precursor (compound
2) these appear often fully
the three amide groups.
The 1H NMR spectra of compound
resolved with distinct chemical shifts, being the peaks of 5’‐
indicated that the isomer at higher field than the corresponding ones for 4’‐
2
resonance signals of the ethyl protons of the rhodamine isomer. MRB9 also shows observable increase for chemical
1
residue appear at 1.28 (CH3) and 3.65 (CH2) ppm respectively. shifts of the protons and carbons in the H and 13C spectra
The peaks of H4 and H5 appear as a doublet at 6.90 ppm while respectively, to higher field in comparison to compound 2.
H2 and H7 appear as a broad singlet at 6.99 ppm, being these Among others, it is quite noticeable the change observed for
signals useful of diagnostic of the presence of the rhodamine the methylamino group of the pyridinone (NCH3) from 3.65
moiety. The protons of the methyl group of the 3,4‐HPO group ppm in compound
2 to 4.08 ppm in MRB9 (5’‐isomer) and
appear at 2.39 ppm for the 5’‐isomer and at 2.45 ppm for 4’‐ from 3.72 to 4.15 ppm (4’‐isomer). A similar observation can
isomer. The proportion between the intensities of these was be made for the methylene group of the linkage (CH2NH)
used to determine the ratio of both isomers in the mixture varying from 4.66 in 2 to 4.97 (5’‐isomer), and from 4.74 to
(1:1.1 of the 5’‐ and 4’‐ isomers), being this result coherent 5.06 (4’‐isomer). Their respective carbon signals are also
with the one found for other analogous peaks of the 2 isomers. dislocated to lower field region, namely NCH3 37.4 (5’‐isomer)
The protons of CH2NH attached to the 3,4‐HPO groups appear and 37.5 (4’‐isomer) to 40.0 for both isomers.
at 3.49 ppm.
The observed difference in the chemical shifts of 1H and 13C
The resonance signal at 6.45 ppm was assigned to the nucleus in the spectra of the protected and deprotected
aromatic proton (H5”) of the 5’‐isomer 3,4‐HPO residue and compounds is primarily due to the deprotection of the
shows a HMBC correlation with a carbon at 20.8 ppm of the hydroxyl group and the acidic pH of the deprotection reaction
carbon 6’’‐CH3 of the 5’‐isomer), while the signal at 6.51 was that lead to isolate the final ligands in the enolic form.
attributed to the H5” of the 4’‐isomer correlated to the carbon
6’’‐CH3 of the 4’‐isomer at 20.9 ppm.
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Absorption and fluorescence measurements
avium and the effect is not significantly different between
them.
View Article Online
DOI: 10.1039/C5MD00456J
The absorption electronic spectra of MRH10 and MRB9 show
two sets of bands in different regions of the spectra, which
correspond to π → π* transiꢀons of the π systems of 3,4‐HPO
and the fluorophores that constitute the chelators structure.
The spectral parameters of the new fluorescent chelators are
summarized in experimental section. Transitions in the range
281‐290 nm are associated with ethylene and benzene bonds
of the aromatic ring of the 3,4‐HPO. The other set of
transitions in the range 552 and 559 nm are signed to
tetramethylrhodamine isothiocyanate (F10) or 5(6)‐carboxy‐
tetraethyl‐rhodamine (F9) π systems. The data obtained is in
agreement with the results reported in the literature 18, 20. The
molar absorptivity values obtained are in the magnitude (104),
close to those reported in previous results for similar
The results obtained show that MRH7 seems to be the most
active chelator in the control of the infection, being effective in
most concentration values tested. Chelator MRH10 exhibits a
close performance to that of MRH7 and is significantly better
than that of MRB9.
rhodamine derived ligands 18, 20
.
Evaluation of antimycobacterial activity
Effect of MRH10 and MRB9 chelators on the
intramacrophagic growth of M. avium 2447 SmT in
comparison with MRH7. Intramacrophagic growth of the
bacteria was measured, as CFU (Colony Forming Units), after
five days in culture in the absence or in the presence of the
chelators. The effect of each compound in the
intramacrophagic growth of M. avium was calculated as the
difference in log10 CFU/well between day 0 and day 5. The
antimycobacterial effect of the new chelators MRH10 vs MRB9
was tested and compared with the effect of the MRH7, used
Figure 3. Effect of MRH7, MRH10 and MRB9 on intramacrophagic growth of M.
avium. BMM were infected with M. avium 2447 (SmT). Each chelator was added,
just after infection, at a concentration of 5 (a), 10 (b), 20 (c) and 40 (d) μM for
MRH7 and MRH10 and 15 (a), 30 (b), 60 (c) and 120 (d) μM for MRB9. Infected
BMM were incubated for 5 days to measure the intracellular growth of bacteria.
The difference, in terms of log10 CFU/well, between days 0 and 5 was designated
‘‘log10 increase’’. The graphs show the geometric mean ± SD of the log10 increase
per well obtained from three wells for each condition from three of five
independent experiments. Statistical analysis was performed using ANOVA test
to compare the bacterial growth: i) in the absence or presence of each the
herein as a second control, for the same comparable chelators and ii) in the presence MRH7 vs MRH10 vs MRB9, for the same
*
***
concentrations, (a), (b), (c) and (d). Statistical significance: p<0.05 and
concentrations: (a), (b), (c) and (d). Since chelators MRH7 and
MRH10 are hexadentate and MRB9 is bidentate the
concentration of the latter is always three times the
concentration used for the hexadentate ligands. All the
compounds were tested in at least three independent assays
and the results are represented in Figure 3.
#
p<0.001 when compared with control (no chelator); p<0.05 for MRH10 vs
MRB9 or ## p< 0.01 for MRH7 vs MRB9, at concentration (a); && p< 0.01 for MRH7
vs MRB9, at concentration (b); @ p<0.05 for MRH7 vs MRB9, at concentration (c).
Effect of MRH7 in combination with ethambutol (ETH) on the
intramacrophagic growth of M. avium 2447 SmT. The effects
of the chelator MRH7 and of the classic antimycobacterial
antibiotic, ethambutol (ETH) were evaluated per se and in
combination. Different concentrations of ethambutol were
tested in preliminary studies and the chosen value (7.2 μM)
represents the concentration for which the effect of the
antibiotic alone is virtually null. The experiments were
performed using a fixed concentration of the antibiotic
combined with MRH7 in the concentrations of 5, 10 and 20
µM. The antimycobacterial activity of MRH7, ETH and their
combination was compared with the control (no treatment)
and all the conditions were compared between them. The
results obtained are represented in Figure 4.
The results show that all the fluorescent chelators exhibit
antimycobacterial effect and this activity seems to be dose‐
dependent. In comparison with the control, MRH7 has a
significant effect at lower concentrations such as 10 µM while
the new chelator MRH10 only induces a reproducible
inhibitory effect leading to a significant decrease on M. avium
growth at concentrations up to 20 μM. Moreover, MRB9 is
only effective at the highest concentration tested (120 µM).
Comparison of the effect of the three chelators shows that:
(a) at the lowest concentration, no significant differences are
observed for the activity of MHR10 and MRH7 and the effect
of the latter is significantly higher than the obtained for MRB9
.
The results of the combination of MRH7 with ETH reveal that
the effect is significantly different from the control for all the
concentration range, even at the lowest concentration (5 µM)
of MRH7. Also, the effect of the combination of MRH7 with
ETH at 10 and 20 µM is significantly higher than the effect
obtained with ETH per se.
(b and c) at intermediate concentrations MRH7 exhibits the
highest activity and its effect is significantly greater than that
obtained for MRB9. No significant differences on the inhibition
of bacterial growth are apparent between MHR10 and MRB9.
(d) at the highest concentration tested, all ligands induce a
significant decrease on the intramacrophagic growth of M.
8 | J. Name., 2012, 00, 1‐3
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Considering the combination of MRH7 with ETH at the highest chelator used to obtain a significant biologicalVeiefwfeArctitclewOhnliinlee
concentration of chelator (20 µM), the effect is also simultaneously improving the activity ofDEOTHI: .10.1039/C5MD00456J
significantly higher than that obtained for MRH7 per se, at the To the best of our knowledge the present work is the first
same concentration.
report revealing the advantageous combination of iron
These data reinforce our hypothesis of the advantageous use chelators and classic antibiotics in the treatment of
of the combination of iron(III) chelators and the clinical used intracellular infections such as M. avium infections.
antimycobacterial antibiotic, ETH, as a strategy to fight M.
avium infections.
Acknowledgements
This work received financial support from the European Union
(FEDER funds through COMPETE) and National Funds (FCT,
Fundação para a Ciência e Tecnologia) through project Pest‐
C/EQB/LA0006/2013 and also, under the framework of QREN,
through Project NORTE‐07‐0124‐FEDER‐000066. The Bruker
Avance II 400 spectrometer is part of the National NMR
network and was purchased under the framework of the
National Programme for Scientific Re‐equipment, contract
REDE/1517/RMN/2005, with funds from POCI 2010 (FEDER)
and (FCT). To all financing sources the authors are greatly
indebted. To all financing sources, the authors are greatly
indebted. T. Moniz and T. Silva also thank FCT for the Ph.D.
Figure 4. Effect of MRH7, ETH and their combination on intramacrophagic
grants (SFRH/BD/79874/2011 and SFRH/BD/77564/2011,
respectively).
growth of M. avium. BMM were infected with M. avium 2447 (SmT). Each
compound was added, just after infection, at a concentration of 5, 10 and 20 μM
for MRH7 and 7.2 µM for ETH. Infected BMM were incubated for 5 days to
measure the intracellular growth of bacteria. The difference, in terms of log10
CFU/well, between days 0 and 5 was designated ‘‘log10 increase’’. The graphs
show the geometric mean ± SD of the log10 increase per well obtained from
three wells for each condition from four independent experiments. Statistical
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0.001 when compared with ETH 2 µg/ mL; @ p<0.05 when compared with MRH7
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Conclusions
The present results substantiate the premise that the
inhibitory effect of rhodamine labelled 3,4‐HPO chelators is
strongly dependent on the fluorophore and confirm that the
importance of the thiourea linkage, the ethyl substituents on
the amino groups of the xanthene ring and the advantage of
their associated inclusion in the molecular framework.
9. C. Ratledge and L. G. Dover, Annual Review of Microbiology,
2000, 54, 881–941
The relevance of the concomitant presence of the two
functional groups for the chelators interaction with
membranes has been suggested in an NMR and MD simulation
study in which we demonstrate that the presence of N‐ethyl
groups in the xanthene structure and the thiourea link in the
structure of MRB7, as opposed to N‐methyl groups in the
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DOI: 10.1039/C5MD00456J
Chelator MRH7 (thiourea linkage; ethyl substituents) and its co-administration with ethambutol
are the best choices for a higher antimycobacterial effect.