Diverse in vitro and in vivo anti-inflammatory effects of trichlorido-gold(III) complexes with N6-benzyladenine derivatives

Article abstract of DOI:10.1016/j.jinorgbio.2014.02.002

A series of gold(III) complexes involving differently substituted derivatives of a plant hormone N6-benzyladenine (HL1-5) is reported. The complexes have the general formula [Au(HL1-5)Cl₃] a?nH₂O (n = 0 for 1, 3-5; and n = 1 for 2),

Full text of DOI:10.1016/j.jinorgbio.2014.02.002

Diverse in vitro and in vivo anti-inflammatory effects of trichlorido-gold(III)
complexes with N6-benzyladenine derivatives
Radka Krˇikavova´, Jan Hosˇek, Pavel Suchy´ Jr., Ja´n Vancˇo, Zdeneˇk Tra´vn´ıcˇek
PII:
DOI:
Reference:
S0162-0134(14)00041-5
doi: 10.1016/j.jinorgbio.2014.02.002
JIB 9468
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
21 November 2013
23 December 2013
5 February 2014
Please cite this article as: Radka Kˇrikavov´a, Jan Hoˇsek, Pavel Suchy´ Jr., J´an Vanˇco,
Zdenˇek Tr´avn´ıˇcek, Diverse in vitro and in vivo anti-inflammatory effects of trichlorido-
gold(III) complexes with N6-benzyladenine derivatives, Journal of Inorganic Biochemistry
(2014), doi: 10.1016/j.jinorgbio.2014.02.002
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ACCEPTED MANUSCRIPT
Diverse in vitro and in vivo anti-inflammatory effects of trichlorido-
gold(III) complexes with N6-benzyladenine derivatives
Radka Křikavová a, Jan Hošek a, Pavel Suchý, Jr. b, Ján Vančo a, Zdeněk Trávníček a,*
a Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry,
Faculty of Science, Palacký University in Olomouc, 17. listopadu 12, CZ-771 46 Olomouc, Czech
Republic
b Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary
and Pharmaceutical Sciences Brno, Palackého tř. 1/3, CZ-612 42 Brno, Czech Republic
*Corresponding author. Tel.: +420 585 634 352; fax: +420585 634 954; e-mail: zdenek.travnicek@upol.cz (Zdeněk Trávníček).
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Abstract
A series of gold(III) complexes involving differently substituted derivatives of a plant
hormone N6-benzyladenine (HL1-5) is reported. The complexes have the general formula [Au(HL1-
5)Cl3]∙nH2O (n=0 for 1, 3-5; and n=1 for 2), where N6-(2-fluorobenzyl)adenine (HL1), N6-(2-
chlorobenzyl)adenine (HL2), N6-(3-chlorobenzyl)adenine (HL3), N6-(4-chlorobenzyl)adenine (HL4)
and N6-(4-methylbenzyl)adenine (HL5) represent the N9-coordinated ligands. The results of
thorough characterization (elemental and thermal analyses, FT-IR, Raman and NMR spectroscopy,
ESI+ mass spectrometry, conductivity measurements, DFT calculations) showed that the presented
complexes 1-5 involve a central gold(III) atom coordinated in a square-planar geometry by the N9
atom of the purine moiety of HL1-5 and by three chlorido ligands. The complexes (1-5 ) were studied
in vitro for cytotoxicity and anti-inflammatory activity on LPS-activated macrophages (THP-1 cell line),
and in vivo for anti-inflammatory effects (1, 2, 5) using the carrageenan-induced hind paw oedema
model on rats. Surprisingly, the results on the in vitro level revealed that the complexes show
negligible cytotoxicity and anti-inflammatory activity, however, the activity on the in vivo level was
found to be significant, fully comparable with the utilized drug Indomethacin, or even better as
compared to a gold-containing metallodrug Auranofin.
Keywords: Gold(III) complexes; Adenine derivatives; TNF-α; Cytotoxicity; Anti-inflammatory.
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1. Introduction
Medicinal applications of gold-based therapeutic agents have been well established since as early as
2500 BC [1, 2, 3]. Presently, the major clinical use of gold compounds is related to the treatment of a
chronic inflammatory disease rheumatoid arthritis, which applies solely to compounds with gold in
the oxidation state +I, e.g. Auranofin [triethylphosphine-(2,3,4,6-tetra-O-acetyl-β-D-thiopyranosato)-
gold(I)] [4, 5, 6]. The current research interest in the medicinal chemistry of gold compounds is in
particular connected with: a) the above-mentioned clinical success of gold(I) anti-inflammatory
complexes and the efforts to find more efficient agents exhibiting fewer negative side-effects
associated with the treatment (such as metallic taste in the mouth, discolouration of the skin,
dermatitis, nausea, bone marrow damage, etc.); b) the unique chemistry of gold with respect to the
physiological environment – particularly the high affinity for protein thiols and selenols, since many
disease targets, including varied inflammatory illnesses, bone and joint disorders, parasitic diseases
as well as cancer, involve cysteine residues [1]. In relation to this high affinity and distinctive
metabolism of gold drugs, the possible medicinal application of gold compounds could be
significantly widened, as suggested by recent studies and reviews focused on anti-microbial, anti-
malaria, anti-cancer as well as anti-HIV activity [1, 2, 3, 7, 8].
The studies focused on development of potential gold-based drugs are mostly associated with the
two oxidation states of gold, i.e. +I and +III, as they both can exist in physiological conditions. It has
been well established that in the highly reductive environment in vivo, Au(I) species are much more
stable than the Au(III) ones, which are readily reduced to Au(I) or Au(0) by naturally occurring
reductants, such as thiols (cysteine), thioethers (methionine), and/or various proteins [1, 2, 6]. The
fact that gold(III) can be readily reduced, and thus can act as a relatively strong oxidizing agent, has
been connected in recent works with anti-HIV activity of various gold(III) complexes, as in this way
the oxidation of target proteins/enzymes results in their functional inhibition [9]. However, it should
be pointed out that the redox process could in certain conditions be reversible. As recent studies
highlighted, the used Au(I) therapeutics should be in fact considered as pro-drugs. After
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administration, they rapidly enter varied metabolic pathways, one of which involves oxidation of
Au(I) to Au(III) by strong oxidants, such as hypochlorite which is produced in the oxidative
environment in inflamed sites. Moreover, Au(III) has been suggested to dominate the anti-
inflammatory as well as toxic actions of gold species [1,6].
While Au(I) compounds have already been successfully applied in clinic, recently a rising number of
scientific studies has been devoted to Au(III) complexes. The majority of such studies have focused
on the potential cytotoxic activity of gold(III) complexes [7, 8]. The chemical motivation behind this
focus is rather straightforward – Au(III) is isoelectric with Pt(II), and its complexes generally adopt the
square-planar geometry similarly to the widely used platinum-based anticancer drugs cisplatin and
its derivatives. In recent years, the chemists have successfully overcome the low stability of gold(III)
complexes under physiological conditions, connected with their oxidative properties and fast rate of
hydrolysis, by introducing various chelating ligands [1,2]. For example, the compounds [Au(dmdt)X2],
[Au(esdt)X2] [10,11], [AuY2(damp)] [12,13] and [Au(tpp)]Cl [14,15], have shown significant antitumour
activity in vitro as well as in vivo (dmdt = N,N-dimethyldithiocarbamate; esdt =
ethylsarcosinedithiocarbamate; damp
=
2-[(dimethylamino)methyl]phenyl; tpp
=
meso-
tetraphenylporphyrin); X = Cl, Br; Y = Cl, acetate, malonate) [1].
The utilization of N6-benzyladenines as N-donor ligands, which are classified among natural as well
as artificial plant hormones called cytokinins, may be associated with their biological functions. Their
principal functions in the plant life cycle comprise the regulation of cell cycle, differentiation, division
and senescence [16]. Moreover, some derivatives of N6-benzyladenine, such as R-roscovitine, 2-[(R)-
(1-ethyl-2-hydroxyethylamino)]-N6-benzyl-9-isopropyladenine, have attracted further attention for
their ability to influence the human cell cycle, particularly, in the cancer cells, thus exhibiting anti-
tumour activity [17, 18]. In order to improve a biological activity, coordination compounds involving
combinations of a suitable transition metal and N6-benzyladenine derivative are in the focus of
research. The complexes involving such N-donor ligands have been shown to possess significant in
vitro as well as in vivo anti-tumour (with the effective concentrations up to submicromolar level) [19,
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20] and anti-diabetic activity [21]. Moreover, outstanding results concerning in vitro and in vivo anti-
inflammatory activity have been recently reported for gold(I) complexes involving
triphenylphosphine (PPh3) and N6-benzyladenine (HL) derivatives in the coordination sphere, having
the composition [Au(PPh3)(L)]. The described complexes showed similar or better anti-inflammatory
effects as compared with the commercially applied drug Auranofin while being less cytotoxic in vitro
[22].
In relation to the recently reported high anti-inflammatory activity of the Au(I)-PPh3 complexes
involving N6-benzyladenine derivatives, the generally oxidative environment in the inflamed sites
and the suggested role of Au(III) species in the anti-inflammatory action of clinically applied gold-
based drugs outlined above, we decided to prepare and thoroughly characterize the Au(III)
complexes of N6-benzyladenine derivatives and further study their in vitro and in vivo anti-
inflammatory activity on the models of LPS-stimulated THP-1 macrophage-like cell line and
carrageenan-induced hind paw oedema model on rats.
2. Materials and Methods
2.1. Starting materials
Used chemicals and solvents were obtained from commercial sources (Sigma-Aldrich Co.,
Acros Organics Co., Lachema Co. and Fluka Co.) and were used without any further purification. The
organic compounds N6-(2-fluorobenzyl)adenine (HL1), N6-(2-chlorobenzyl)adenine (HL2), N6-(3-
chlorobenzyl)adenine (HL3), N6-(4-chlorobenzyl)adenine (HL4) and N6-(4-methylbenzyl)adenine
(HL5) (see Scheme S1) were synthesized according to the previously published procedure [23]. The
identity and purity of these organic compounds were checked by elemental analysis (C, H, N), FT-IR
and 1H and 13C NMR spectroscopy.
Scheme 1
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2.2. Synthesis of Au(III) complexes
1 mmol of the corresponding N6-benzyladenine derivative (HL1-5) in acetone (70 mL) was
added to a solution of 1 mmol of AuCl3 in acetone (10 mL). The resulting solution was stirred for 4 h
after which it was filtered. The obtained red filtrate was further stirred for about 30 min. Then the
volume of the solution was reduced on the rotary evaporator until a red suspension formed. The red
or dark red products were filtered off, washed with a small amount of acetone and diethyl ether, and
dried at 40 °C under an infrared lamp.
[Au(HL1)Cl3](1): Yield: 70%. Anal. Calc. for C12H10N5FCl3Au: C, 26.4; H, 1.8; N, 12.8; Cl, 19.5. Found: C,
26.0; H, 1.5; N, 12.3; Cl, 19,0%. ΛM (DMF/methanol, S cm2 mol-1): 7.9/5.7. ESI+ MS (methanol, m/z)
244 (calc. 244) HL1+H+, 546 (546) [M+H]+. IR (νNujol/cm–1): 546s, 473m ν(Au–N), 415w, 365vs ν(Au–
Cl), 351w ν(Au–Cl), 347w ν(Au–Cl), 301w, 298w, 261w, 214w. IR (νKBr/cm–1): 3430m ν(N–H), 3203w,
3162w, 3039w ν(C–H)ar, 2936w ν(C–H)al, 1615s ν(C=N)ar, 1591w, 1529w, 1426m ν(C=C)ar, 1365m,
1301w, 1261m ν(C–F)ar, 1227w, 1098w, 982w, 832w, 790w, 653m. Raman (cm-1): 3067m ν(C–H)ar,
2954w ν(C–H)al, 1609m ν(C=N)ar, 1552w, 1415m ν(C=C)ar, 1290m, 1229m ν(C–F)ar, 1180m, 1145w,
1035m, 951w, 775w, 565w, 408w, 318 s ν(Au–Cl), 253m, 168s. 1H NMR (400.00 MHz, DMF-d7, ppm):
 (SiMe4) 8.15 (s, C2H, 1H), 7.96 (s, C8H, 1H), 7.77 (tt, 7.9 Hz, 1.6 Hz, C14H, 1H), 7.30, (qq, 7.5 Hz, 1.6
13
Hz, C15H, 1H), 7.18 (d, 9.2 Hz, C12H, 1H), 7.13 (t, 7.5 Hz, C13H, 1H), 4.98 (bs, C9H, 2H). C NMR
(75.43 MHz, DMF-d7, ppm):  (SiMe4) 160.05 (C11), 155.39 (C6), 151.53 (C2), 148.52 (C8), 145.25 (C4),
133.09, 133.06 (C14), 132.67, 132.47 (C15), 129.21(C10), 124.82, 124.79 (C13), 120.96 (C5), 115.64,
115.43 (C12), 38.15 (C9).
[Au(HL2)Cl3]·H2O (2): Yield: 75%. Anal. Calc. for C12H12N5OCl4Au: C, 24.8; H, 2.1; N, 12.1; Cl, 24.4.
Found: C, 24.7; H, 1.7; N, 11.9; Cl, 24.3%. ΛM (DMF/methanol, S cm2 mol-1): 13.2/8.7. ESI+ MS
(methanol, m/z) 260 (calc. 260) HL2+H+, 564 (564) [Au(HL2)Cl3+H]+. IR (νNujol/cm–1): 591vs, 561m,
463s ν(Au–N), 433m, 364m ν(Au–Cl), 341w ν(Au–Cl), 306w, 275s, 260w. IR (νKBr/cm–1): 3401m ν(N–
H), 3274w, 3210w, 3114w, 3061w ν(C–H)ar, 2923w ν(C–H)al, 2854w, 1625vs ν(C=N)ar, 1574w, 1442w
ν(C=C)ar, 1403m, 1358w, 1262m, 1172m, 1146w, 1038m ν(C–Cl)ar, 964w, 747m, 690w, 611w. Raman
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(cm-1): 3064m ν(C–H)ar, 2918w ν(C–H)al, 1616w, 1594m ν(C=N)ar, 1573w, 1511m, 1406m ν(C=C)ar,
1331w, 1291m, 1157m, 1039m ν(C–Cl)ar, 992w, 804w, 759m, 738m, 690w, 542w, 413m, 346vs ν(Au–
1
Cl), 322vs ν(Au–Cl). H NMR (400.00 MHz, DMF-d7, ppm):  (SiMe4) 8.13 (s, C2H, 1H), 7.93 (s, C8H,
13
1H), 7.77 (d, 8.5 Hz, C12H, C15H, 2H), 7.36 (d, 8.5 Hz, C13H, C14H, 2H), 4.90 (bs, C9H, 2H). C NMR
(75.43 MHz, DMF-d7, ppm):  (SiMe4) 155.38 (C6), 151.51 (C2), 148.44 (C8), 141.10 (C4), 138.10 (C10),
133.11 (C15), 133.07 (C12), 129.86 (C14), 120.92 (C5), 43.86 (C9).
[Au(HL3)Cl3] (3): Yield: 65%. Anal. Calc. for C12H10N5Cl4Au: C, 25.6; H, 1.7; N, 12.4; Cl, 25.2. Found: C,
25.2; H, 1.6; N, 12.7; Cl, 24.8. ΛM (DMF/methanol, S cm2 mol-1): 21.6/10.8. ESI+ MS (methanol, m/z)
260 (calc. 260) HL3+H+, 564 (564) [M+H]+, 586 (586) [M+Na]+. IR (νNujol/cm–1): 596m, 565w, 539w,
448m ν(Au–N), 414w, 368s ν(Au–Cl), 338w ν(Au–Cl), 279w, 245w. IR (νKBr/cm–1): 3384m ν(N–H),
3122w, 3066w ν(C–H)ar, 2927w ν(C–H)al, 2854w, 1619vs ν(C=N)ar, 1563w, 1473m ν(C=C)ar, 1404m,
1339m, 1278w, 1178m, 1144w, 1095w, 1078w ν(C–Cl)ar, 956w, 871w, 778w, 684w. Raman (cm-1):
3065m ν(C–H)ar, 2936w ν(C–H)al, 1618w ν(C=N)ar, 1597m, 1571w, 1518w, 1445m, 1401m ν(C=C)ar,
1334m, 1209w, 1167m, 1143w, 1078w ν(C–Cl)ar, 999m, 929w, 751w, 681w, 548w, 415m, 367s ν(Au–
1
Cl), 339vs (Au–Cl). H NMR (400.00 MHz, DMF-d7, ppm):  (SiMe4) 8.18 (s, C2H, 1H), 8.04 (s, C8H,
1H), 7.50 (t, 1.8 Hz, C11H, 1H), 7.42 (d, 7.6 Hz, C13H, 1H), 7.36 (t, 7.7 Hz, C14H, 1H), 7.29 (dd, 7.8, 1.8,
C15H, 1H), 4.92 (bs, C9H, 2H). 13C NMR (75.43 MHz, DMF-d7, ppm):  (SiMe4) 155.15 (C6), 152.32 (C2),
148.18 (C8), 146.05 (C4), 144.49 (C10), 134.03 (C12), 130.70 (C14), 127.92 (C11), 127.25 (C15),
126.70 (C13), 120.01 (C5), 43.98 (C9).
[Au(HL4)Cl3] (4): Yield: 75%. Anal. Calc. for C12H10N5Cl4Au: C, 25.6; H, 1.7; N, 12.4; Cl, 25.2. Found: C,
25.2; H, 1.9; N, 11.9; Cl, 25.5%. ΛM (DMF/methanol, S cm2 mol-1): 37.3/15.5. ESI+ MS(methanol, m/z)
260 (calc. 260) HL4+H+, 528 (528) [Au(I)(HL4)Cl+2H2O+H]+, 564 (564) [M+H]+, 586 (586) [M+Na]+. IR
(νNujol/cm–1): 566m, 539m, 521m, 491m ν(Au–N), 407w, 359vs ν(Au–Cl), 329m ν(Au–Cl), 286w, 273m,
251w. IR (νKBr/cm–1): 3425m ν(N–H), 3268w, 3119m ν(C–H)ar, 2925w ν(C–H)al, 2850w, 1629vs ν(C=N)ar,
1546w, 1490m, 1450m ν(C=C)ar, 1406m, 1358w, 1305w, 1269w, 1208w, 1169m, 1139w, 1091m ν(C–
Cl)ar, 1014m, 951w, 801w, 777w, 750w, 637w, 598w, 543w. Raman (cm-1): 3087s ν(C–H)ar, 2942w
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ν(C–H)al, 1618m ν(C=N)ar, 1599m, 1528m, 1428m ν(C=C)ar, 1327w, 1220m, 1227w, 1123w, 1027m
1
ν(C–Cl)ar, 928w, 727w, 634w, 587w, 367m, 336m ν(Au–Cl). H NMR (400.00 MHz, DMF-d7, ppm): 
(SiMe4) 8.15 (s, C2H, 1H), 7.48 (d, 8.6 Hz, C11H, C15H, 2H), 7.39 (s, C8H, 1H), 7.38 (d, 8.6 Hz, C12H,
C14H, 2H), 4.89 (bs, C9H, 2H). 13C NMR (75.43 MHz, DMF-d7, ppm):  (SiMe4) 155.34 (C6), 152.49 (C2),
148.45 (C8), 144.05 (C4), 140.82 (C10), 132.21 (C13), 128.87 (C12,14), 121.06 (C5), 43.83 (C9).
[Au(HL5)Cl3] (5): Yield: 78%. Anal. Calc. for C13H13N5Cl3Au: C, 28.8; H, 2.4; N, 12.9; Cl, 19.6. Found: C,
28.4; H, 2.6; N, 13.2; Cl, 19,9%. ΛM (DMF/methanol, S cm2 mol-1): 18.8/17.1. ESI+ MS (methanol, m/z)
240 (calc. 240) HL5+H+, 542 (542) [M+H]+. IR (νNujol/cm–1): 557s, 532m, 490m ν(Au–N), 400w, 362vs
ν(Au–Cl), 342w ν(Au–Cl), 318w ν(Au–Cl), 287m, 213w. IR (νKBr/cm–1): 3405m ν(N–H), 3210m, 3060m
ν(C–H)ar, 2939m, 2918w ν(C–H)al, 2824m ν(C–H)met, 1617vs ν(C=N)ar, 1514w, 1438m ν(C=C)ar, 1301m,
1256w, 1157w, 1021w, 934w, 809w, 731w, 626m, 590w. Raman (cm-1): 3115w, 3043s ν(C–H)ar,
2992m, 2932w ν(C–H)al, 1615w ν(C=N)ar, 1559w, 1424m ν(C=C)ar, 1373w, 1315vs, 1173w, 999vs,
1
799w, 741m, 582m, 536w, 420w, 337s, 355m ν(Au–Cl). H NMR (400.00 MHz, DMF-d7, ppm): 
(SiMe4) 8.19 (s, C2H, 1H), 7.39 (s, C8H, 1H), 7.34 (d, 8.6 Hz, C11H, C15H, 2H), 7.28 (d, 8.6 Hz, C12H,
13
C14H, 2H), 4.89 (bs, C9H, 2H), 2.30 (s, C13‘H, 3H). C NMR (75.43 MHz, DMF-d7, ppm):  (SiMe4)
155.34 (C6), 152.49 (C2), 148.45 (C8), 144.05 (C4), 140.82 (C10), 133.03 (C11,15), 132.21 (C13),
128.87 (C12,14), 121.06 (C5), 43.83 (C9), 20.21 (C13).
2.3. General methods of characterization
Elemental analyses (C, H, N) were performed on a Flash 2000 CHNO-S Analyser (Thermo
Scientific). The chlorine content was determined by the Schöniger method [24]. Conductivity
measurements of 10-3 M N,N´-dimethylformamide (DMF) and 10-3 M methanol solutions of the
prepared complexes were carried out using a conductometer 340i/SET (WTW) at 25 °C. FT-IR spectra
were recorded on a Nexus 670 FT-IR spectrometer (ThermoNicolet) using KBr pellets (400-4000 cm-1)
and the Nujol technique (150-600 cm-1). Raman spectrometer Nicolet NXR 9650 equipped with the
liquid nitrogen cooled NXE Genie germanium detector (ThermoNicolet) was used to record Raman
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spectra of all the complexes (150-4000 cm-1). The reported FT-IR and Raman signal intensities are
defined as w = weak, m = middle, s = strong and vs = very strong. 1H and 13C NMR spectra of the DMF-
d7 solutions were measured on a Varian 400 MHz NMR spectrometer at 300 K. Tetramethylsilane
1
13
(TMS) was used as an internal reference standard for H and C NMR experiments. Mass spectra of
the methanol solutions of the complexes were obtained by an LCQ Fleet ion trap mass spectrometer
by the positive mode electrospray ionization (ESI+) technique (Thermo Scientific). The theoretic
values were calculated by the QualBrowser software (version 2.0.7, Thermo Fisher Scientific).
Thermogravimetric (TG) and differential thermal analyses (DTA) were performed using a thermal
analyser Exstar TG/DTA 6200 (Seiko Instruments Inc.) in dynamic air conditions (150 mL min-1)
between room temperature (≈25 °C) and 900 °C (gradient 2.5 °C min-1).
2.4. Biological activity testing
2.4.1. Chemicals and biochemicals
The RPMI 1640 medium and penicillin-streptomycin mixture were purchased from Lonza
(Verviers, Belgium). Phosphate-buffered saline (PBS), foetal bovine serum (FBS), phorbol myristate
acetate (PMA), prednisone (98%≤), and Escherichia coli 0111:B4 lipopolysaccharide (LPS) were
purchased from Sigma-Aldrich (Steinheim, Germany). The Cell Proliferation Reagent WST-1 was
obtained from Roche (Mannheim, Germany). An Instant ELISA Kit (eBioscience, Vienna, Austria) was
used to evaluate the production of TNF-α.
2.4.2. Maintenance and preparation of macrophages
For the measurements of biological activity, we used the human monocytic leukemia cell line
THP-1 (ECACC, Salisbury, UK). The cells were cultivated at 37 °C in RPMI 1640 medium supplemented
with 2 mM L-glutamine, 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin in a
humidified atmosphere containing 5% CO2. Stabilized cells (3rd–15th passage) were split into
microtitration plates to get a concentration of 500 000 cells/mL and the differentiation to
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macrophages was induced by a phorbol myristate acetate (PMA) dissolved in dimethyl sulfoxide
(DMSO) at the final concentration of 50 ng/ml and the cells were incubated for 24 h. In comparison
with monocytes, differentiated macrophages tend to adhere to the bottoms of the cultivation plates.
For the next 24 h the cells were incubated with fresh complete RPMI medium, i.e. containing
antibiotics and FBS, without PMA. The medium was then aspirated, and the cells were washed with
PBS and cultivated for next 24 h in a serum-free RPMI 1640 medium. These prepared macrophages
were used for the detection of inflammatory response.
2.4.3. Cytotoxicity testing
THP-1 cells (floating monocytes, 500 000 cells/mL) were seeded into 96-well plates in
triplicate and cultivate in a serum-free RPMI 1640 medium at 37 °C. Measurements were taken 24 h
after the treatment with an increasing concentration of the tested compounds dissolved in DMSO.
Viability was measured by the WST-1 test according to the manufacturer’s manual. The amount of
created formazan (which correlates to the number of metabolically active cells in the culture) was
calculated as a percentage of control cells, which were treated only with DMSO and was set-up as
100%.
2.4.4. Drug treatment and induction of inflammatory response
Differentiated macrophages were pre-treated for 1 h either with a 5 µM solution of the
tested complexes or uncoordinated organic compounds (HL1-5), or 5 µM solution of prednisone, or 5
µM AuCl3 dissolved in DMSO (the final DMSO concentration was 0.1%) or with 0.1% DMSO solution
itself (vehicle); the tested compounds lack a cytotoxic effect at the given concentration (cell viability
>94%). The inflammatory response was triggered by adding 1.0 µg/mL lipopolysaccharide (LPS)
dissolved in water to the pre-treated macrophages, control cells were without the LPS treatment.
Each experiment was repeated three times.
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2.4.5. Evaluation of cytokine secretion by ELISA
Macrophages, which were pre-treated with the tested compounds for 1 h, were incubated
with LPS for next 24 h. After this period, the medium was collected and the concentration of TNF-α
was measured by an Instant ELISA kit according to the manufacture’s manual.
2.4.6. Animals
Wistar - SPF (6-8 weeks male) rats were obtained from AnLab, Ltd. (Prague, Czech Republic).
The animals were kept in plexiglass cages at the constant temperature of 22±1 °C, and relative
humidity of 55±5% for at least 1 week before the experiment. They were given food and water ad
libitum. All experimental procedures were performed according to the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals. In addition, all tests were conducted under
the guidelines of the International Association for the Study of Pain [25]. After a one-week
adaptation period, male Wistar-SPF rats (200-250 g) were randomly assigned to six groups (n = 10) of
animals in the study. The treated groups received the solutions of complexes 1, 2, and 5
(intraperitoneally, i.p.) dissolved in 25% DMSO (v/v in water for injections according to the European
Pharmacopoeia, PhEur, rules). The next three groups included a carrageenan-treated (control group),
Auranofin positive control (Auranofin + carrageenan) and Indomethacin positive control groups
(Indomethacin + carrageenan).
2.4.7. Carrageenan-induced hind paw oedema model
The carrageenan-induced hind paw oedema model was used for the determination of anti-
inflammatory activity [26]. Animals were i.p. pre-treated with complexes 1, 2 and 5 (10 mg/kg),
Auranofin (10 mg/kg), Indomethacin (5 mg/kg) or 25% DMSO (v/v in water for injections PhEur), 30
min prior to the injection of 1% -carrageenan (50 μL) into the plantar side of right hind paws of the
rats. The paw volume was measured immediately after carrageenan injection and during the next 6 h
after the administration of the oedematogenic agent using a plethysmometer (model 7159, Ugo
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Basile, Varese, Italy). The degree of swelling induced was calculated as the percentage of the change
in the right hind paw volume after the carrageenan treatment from the initial right hind paw volume
before the treatment. The anti-inflammatory drugs Auranofin and Indomethacin were used as
positive controls.
3. Results and Discussion
3.1. Syntheses and general characterization of the Au(III) complexes
Red or dark red complexes of the formulas [Au(HL1)Cl3] (1), [Au(HL2)Cl3]·H2O (2), [Au(HL3)Cl3]
(3), [Au(HL4)Cl3] (4) and [Au(HL5)Cl3] (5), where HL1-5 stand for the electroneutral form of the
corresponding
benzyl-substituted
derivatives
of
N6-benzyladenine,
namely
N6-(2-
fluorobenzyl)adenine (HL1), N6-(2-chlorobenzyl)adenine (HL2), N6-(3-chlorobenzyl)adenine (HL3),
N6-(4-chlorobenzyl)adenine (HL4) and N6-(4-methylbenzyl)adenine (HL5) are reported. The
complexes were synthesized by the reactions of AuCl3 and the corresponding organic compound in
the molar ratio of 1:1 in acetone (Scheme 1). The obtained compounds were thoroughly
characterized by elemental analysis, FT-IR, Raman and NMR spectroscopy, ESI+ mass spectrometry,
thermogravimetric (TG) and differential thermal (DTA) analyses. Multiple efforts were performed in
order to prepare single crystals of the complexes 1-5 suitable for X-ray analysis. Apart from
recrystallization of the powder products from DMSO, DMF, methanol or combination of these
solvents and slow evaporation of these solutions as well as of mother liquors after syntheses at room
temperature or 5 °C, diffusion crystallization methods were applied with varied combination of
solvents (diffusion of diethyl ether, ethanol). However, none of these techniques led to the
preparation of single crystals suitable for X-ray analysis.
The complexes 1-5 are well soluble in DMSO, DMF and methanol. The conductivity
measurements were performed for the 10−3 M methanolic as well as DMF solutions of the complexes
1-5, which proved that they behave as nonelectrolytes (ΛM, DMF/methanol: 7.9-37.3/5.7-15.5 S cm2
mol-1).
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Simultaneous TG and DTA analyses provided evidence that the presented complexes are
thermally stable up to 155 °C and are non-solvated apart from complex 2 which is solvated by one
water molecule of crystallization. The weight loss observed on the TG curve (m: calc. 3.0 / found
2.8%) associated with the loss of one crystal water molecule for complex 2 was accompanied by a
small endo-effect at 149 °C on the DTA curve. The dehydration was finished at 155 °C. No stable
intermediates formed during the thermal decay of all the complexes, which was finished by the
formation of elemental gold at 747-908 °C. The differences between the overall weight losses
determined by the thermal analyses and those calculated to Au as the final product of thermal
degradation did not exceed 1.8%. The figures showing the results of TG/DTA analyses of complexes 2
and 5 can be found in Supplementary material (Figure S2).
3.2. Infrared, Raman, NMR and mass spectral characterization
The courses of the infrared and Raman spectral curves of the complexes were qualitatively
similar to those of the corresponding organic compounds HL1-5, which clearly demonstrated the
presence of HL1-5 in the structures of 1-5. The infrared spectra of purine compounds in general are
characteristic by the presence of very intensive bands of the ν(C=N)ar ring vibrations in the region of
1600-1640 cm-1, which were also detected in the spectra of 1-5 at 1615-1629 cm−1 [27]. The multiple
bands observed between 1426 and 1591 cm-1 can be attributed to the ring ν(C=C)ar vibrations
detected in the infrared as well as Raman spectra. Further peaks characteristic of the organic
skeletons were also identified, i.e. the stretching ν(N–H), ν(C–H)ar and ν(C–H)al vibrations at 3384-
3425 cm-1, 3039-3162 cm-1, and 2918-2939 cm-1, respectively. The substitution of hydrogen on the
benzene ring of HL1-5 was confirmed by the presence of the bands assignable to ν(C–F)ar (at 1261 cm-
1), ν(C–Cl)ar (1038-1091 cm-1) and ν(C–H)met (at 2824 cm-1). The spectra measured in the far infrared
region revealed several new peaks as compared to the spectra of the corresponding free organic
molecules HL1-5. The peaks assignable to ν(Au–N) were found at 463-491 cm-1. The previously
published studies report that complexes of the type [Au(L)Cl3] (where L stands for heterocyclic N-
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donor ligands) showed three bands in the far-IR region connected with the vibrational modes of Au–
Cl; the asymmetric ν(Au–Cl) of the chlorine atoms trans to each other (at ca. 350-360 cm-1), the
symmetric ν(Au–Cl) vibration (ca. 340 cm-1) and ν(Au–Cl) of the chlorine trans to the L ligand (310-
320 cm-1). The asymmetric stretching of the Cl–Au–Cl group and ν(Au–Cl) trans to L might also
overlap. In concordance with these findings, strong to medium intensity bands at 359–354 cm-1 and
weak bands at 329-347 cm-1 were detected in the infrared spectra of 1-5 [28,29].
The obtained results of NMR experiments gave evidence of the composition of the gold(III)
_
complexes. The most important coordination shifts (Δδ = δcomplex δligand) are summarized in Table 1.
1
The signals observed in the H NMR spectra for the hydrogen atoms bound to the C2 and C8 atoms
were shifted by 0.04-0.12 ppm upfield, and 0.17-0.81 ppm upfield, respectively. The highest
coordination shifts of carbon atoms were determined for the C4 and C8 atoms, i.e. the closest vicinity
of N9, which were shifted by 4.83-10.27 ppm to lower frequencies, and 8.33-8.86 ppm to higher
frequencies, respectively (see Table 1). Considerable coordination shifts were also detected for the
signals assigned to the C5 atom, which were observed moved by 0.99-1.78 ppm downfield. These
resulting shifts are consistent with the suggested coordination of the organic compounds HL1-5 via
the N9 atom in the complexes 1-5. It should be noted, that another possible coordination site is
represented by N7 as seen in Au(III) complexes involving a purine derivative, whose X-ray structures
have been determined so far, i.e. [AuCl3(1,9-diMeGua)], [AuCl3(O6-Me-9-MeGua)] [30] and
[Au(Hdamp-C1)Cl(Spur)]Cl [31], where 1,9-diMeGua = 1,9-dimethylguanine; O6-Me-9-MeGua = O6,9-
dimethylguanine, HSpur = 6-merkaptopurine and Hdamp-C1 = 2-(dimethylaminomethyl)benzene. The
coordination proceeds via N7, since the N9 site is blocked by methyl substitution in the guanine
derivatives and deprotonated 6-merkaptopurine coordinates in a stable N7,S-chelating mode. On the
other hand, N9-coordination is suggested in Au(III) complex involving adenine, i.e. in [AuCl3(Ade)],
based on results following from a combination of spectral techniques [32]. Accordingly, NMR data
clearly confirm the N9-coordination of the adenine-derived HL1-5 ligands. Additionally, the observed
chemical shifts comply also with the change in the protonation pattern, i.e. a change in the
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tautomeric form of HL1-5. While the most populated tautomer of uncoordinated HL1-5 is
represented by the form with a hydrogen atom bonded to the nitrogen atom N9 (the N9-H
tautomer), the coordination of these molecules to Au(III) leads to a shift from the N9-H tautomer to
the second most favourable N7-H tautomer in 1-5. Such shifts of tautomer equilibria introduced by
metal binding, or in other words, metal-stabilization of less favourable tautomers, have been well
documented for nucleobases [33].
Table 1
ESI+ mass spectrometry proved to be a very useful tool in aiding to elucidate the structural
aspects of the prepared complexes 1-5, as the molecular peaks [M+H]+ were detected for all the
presented complexes thus supporting the suggested compositions. The mentioned [M+H]+ molecular
peaks were found in the 546-564 m/z region for 1-5, while additionally, in the cases of 3 and 4, the
peaks of the [M+Na]+ adducts with the sodium ion were observed at 586 m/z. All the mass spectra of
1–5 contained the fragment corresponding to the relevant N6-benzyladenine derivative, i.e. HL1-
5+H+. In the case of complex 4, also a pseudomolecular ion corresponding to the reduced species of
the composition [Au(I)(HL4)Cl+2H2O+H]+ was identified in the ESI+ mass spectrum. Supplementary
material contains figure Figure S1 displaying the ESI+ mass spectrum of complex 4.
3.3. Quantum chemical calculations
The multiple efforts to prepare single crystals of the presented complexes were unsuccessful.
That is why, in the endeavour to confirm the composition of 1-5 arising from the indirect analytical
methods described above and to predict the molecular structure of 1, the results of spectroscopic
and other analyses were summarized, together with the known structures of similar complexes, and
the built model was optimized at the DFT-level of theory using the hybrid B3LYP functional with the
LACVP+* basis set using a Spartan´10 (version 1.1.0v4) software [34,35,36]. The optimized geometry
of complex 1 is shown in Figure 1. The gold(III) atom is tetra-coordinated by one N9-coordinated N6-
(2-fluorobenzyl)adenine (Au1−N9 = 2.084 Å) and by three chloride ligands (Au1−Cl1 = 2.324 Å,
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Au1−Cl2 = 2.378 Å and Au1−Cl3 = 2.381 Å). Only 18 tetra-coordinated [Au(L)Cl3] complexes, where L
represents an organic ligand coordinated via a heterocyclic nitrogen atom, have been deposited
within the Cambridge Structural Database (version 5.34, update May 2013) [37]. A comparison of
bond distances showed that the calculated Au–N bond length (Au1−N9 = 2.084 Å) is well comparable
with those crystallographically determined [1.980(20)–2.107(10) Å]. On the other hand, the
calculated Au–Cl bond lengths were found to be slightly longer, as they equal 2.324–2.381 Å as
compared to 2.195(11)–2.363(2) Å for the CSD-deposited compounds. This might be connected with
the fact that the calculations of the optimized geometry were performed in vacuum. The geometry
around the gold(III) atom can be described as distorted square-planar; the angles Cl1–Au–N9, and
Cl2–Au–Cl3 which are equal to 179.63°, and 175.65°, respectively.
Figure 1
3.4.
In vitro cytotoxicity
The cytotoxic effects of the tested complexes 1-5 as well as of uncoordinated N6-
benzyladenine derivatives HL1-5 were evaluated on the THP-1 cell line. None of the used compounds
showed a cytotoxic effect up to the concentration of 20 µM. The lowest observed viability (84 % ±
6 %) was found for complex 5 at the concentration of 20 µM. Previously prepared Au(I) complexes
also involving benzyl-substituted derivatives of N6-benzyladenine [22] showed EC50 lower than 2.5
µM. This comparison indicates higher cytotoxic potential of the Au(I) complexes of N6-benzyladenine
derivatives than the Au(III) ones. Similar observation was reported by Sivaram et al. [38] whose work
compared cytotoxicity of Au(I) and Au(III) complexes bearing benzimidazole- and pyrazole-derived
carbenes on the NCI-H1666 non-small cell lung cancer cell line.
Cytotoxicity as well as further anti-inflammatory activity testing was performed in DMSO. In
1
order to confirm the stability of the tested complexes in this solvent, time-dependent H NMR
spectra of representative complex 1 were measured in DMSO. The obtained results clearly showed
that the complex behaves in DMSO solutions quite similarly as in DMF solutions (as described above),
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as the character of the spectra was analogical. Moreover, even after 48 hours no significant shift,
splitting or appearance of new peaks (connected with the coordination of DMSO) were observed.
Based on these results, we can propose that the title complexes are stable in DMSO solutions in the
time span of the biological testing.
3.5.
In vitro anti-inflammatory effect
Gold containing complexes are known for their anti-inflammatory potential. However, these
properties have been primarily studied for compounds with gold in the oxidation state (+I). Yet,
multiple works dealing with the mechanism of action of Au(I) anti-inflammatory drugs suggest that
one possible metabolic pathway of gold(I) species could involve oxidation of Au(I) to Au(III) in the
highly oxidative environment in the inflamed sites. We have recently reported significant anti-
inflammatory potential of Au(I) complexes involving N6-benzyladenine derivatives [22]. In order to
verify if this biological effect could be observed also for the Au(III) complexes involving similar
ligands, we determined the ability of the herein presented complexes 1-5 to attenuate the LPS-
induced TNF-α secretion in macrophage-like cell line THP-1 (Figure 2). The obtained results indicated
a moderate effect of the tested complexes on the TNF-α secretion attenuation, but the effect was
lower than for positive control prednisone. Au(III) complexes showed a highly decreased anti-
inflammatory effect in comparison with the previously published Au(I) complexes bearing similar
ligands [22].
For comparison, we also tested the starting compounds, i.e. the uncoordinated organic
compounds HL1-5 and AuCl3. It was shown that only AuCl3 significantly attenuated production of
TNF-α, while the free HL1-5 ligands exhibited no effect.
Figure 2
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3.6. The effect of Au(III) complexes on carrageenan-induced oedema in vivo
The in vivo tests of anti-inflammatory activity were inspired by the previous very positive
results of anti-inflammatory activity demonstrated by the gold(I) complexes, [Au(L)(PPh3)], involving
triphenylphosphine and similar N-donor ligands [22]. The other reason for realization of the in vivo
experiments was based on the results of in vitro testing, which revealed reasonable in vitro anti-
inflammatory activity of the tested gold(III) complexes. From the group of five gold(III) complexes (1–
5), three were chosen (1, 2, 5) to fulfil the requirements of the principle of three R´s (the principles of
reduction, refinement and replacement on which all animal testing is based) and also to open the
possibility to at least qualitatively evaluate the effect of substitution of the N-donor ligands on the
anti-inflammatory activity in vivo. For these reasons, the complexes 1, 2 and 5 were selected for the
pre-treatment of laboratory animals in the carrageenan-induced hind paw oedema model on rats. In
this experiment, the effects of the tested complexes on one of the symptoms of acute inflammation
(i.e. the formation of oedema) were investigated. Due to the similar chemical composition and
intended purpose, we chose Auranofin as the primary standard of anti-inflammatory activity.
According to the literature, it has been used in biological experiments in diverse dosage regimen. In
our experiments, we used it in the same dosages as the tested compounds; at 10 mg/kg in the form
of the fine suspension in 25% DMSO (v/v in water for injections PhEur) applied intraperitoneally 30
min before the intraplantar injection of -carrageenan solution. As a secondary standard, NSAID
Indomethacin was used in the dose of 5 mg/kg and applied intraperitoneally. The changes in the hind
paw volume were monitored plethysmometrically for 6 h after the carrageenan injection and
calculated as percentual changes of the initial volume for every individual animal. The
comprehensive overview of the antiedematous activity time-dependent profiles of the tested
compounds is summarized in Figure 3.
Figure 3
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The highest activity was found for complex 1, whose time-dependent profile of anti-oedematous
activity copied almost perfectly the profile of Indomethacin and proved to be significantly higher
than Auranofin (P<0.05). As it turned out, however, the biggest drawback of complex 1 was its
toxicity in the applied dose, leading to the sudden death of all animals 5 h after the injection of
carrageenan. Complex 5 was less effective and less toxic than complex 1, its activity profile was
however shifted by about 5% upwards, especially in the middle stages of the experiment. It can be
stated that, in general, its activity was comparable to Auranofin. Complex 2 showed only weak
activity in this experiment.
Although it is complicated to objectively transform the results of this type of pharmacological
experiment to numeric output useful for QSAR, we decided to describe the structure-activity
relationship at least in a qualitative manner. It can be observed that there is an evident link between
the hydrophobicity of the N-donor ligands (and probably also the complexes) and the results of anti-
inflammatory activities determined both in vitro and in vivo. With the increasing lipophilicity, the
biological effect decreases in the following order N6-(2-fluorobenzyl)adenine > N6-(4-
methylbenzyl)adenine > N6-(2-chlorobenzyl)adenine. The increase in lipophilicity of complexes can
also have various side-effects on different physical properties (such as lowering of solubility) which
can interfere with the bioavailability of complexes from biologically applicable vehicles.
4. Conclusions
A series of new gold(III) complexes (1-5) with benzyl-substituted derivatives of N6-
benzyladenine (HL1-HL5) of the general formula [Au(HL1-5)Cl3]∙nH2O (n=0 for 1, 3-5; and n=1 for 2)
was prepared and characterized. The complexes were tested in vitro for their cytotoxic and anti-
inflammatory activity on LPS-activated macrophages derived from the THP-1 cell line. The complexes
were shown to be non-cytotoxic up to the concentration of 20 µM. The results of the in vitro testing
evaluating the ability of the complexes to influence the LPS-induced TNF-α secretion in macrophage-
like THP-1 cell line showed that the compounds only moderately attenuated the secretion.
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Representative complexes 1, 2 and 5 were tested for their anti-inflammatory activity in vivo on the
carrageenan-induced hind paw oedema model on rats. The anti-oedematous activity was the most
pronounced for complex 1 involving N6-(2-fluorobenzyl)adenine, as its activity was comparable with
Indomethacin and significantly better than Auranofin. The positive in vivo anti-inflammatory action of
complex 1 was negatively affected by a finding that the complex showed considerable toxicity, which
decreases the possibility of the utilization of this complex as a gold-containing metallodrug. On the
contrary, the results confirmed that the remaining complexes deserve deeper attention and study to
understanding mechanisms of action and relationships between the structure and anti-inflammatory
activity.
Acknowledgment
We acknowledge the financial support from the Operational Program Research and
Development for Innovations - European Regional Development Fund (CZ.1.05/2.1.00/03.0058), the
Operational Program Education for Competitiveness European Social Fund (CZ.1.07/2.3.00/20.0017)
of the Ministry of Education, Youth and Sports of the Czech Republic and Palacký University
(PrF_2013_015) for financial support. The authors thank Dr. Roman Buchtík for the mass spectra
measurements, Dr. Pavel Štarha for TG/DTA experiments, and Mr. Tomáš Šilha for performing
synthetic optimizations.
Appendix A. Supplementary material
The supplementary material file associated with this article contains the structural formulas of the
N6-benzyladenine derivatives HL1−5 (Scheme S1), figures depicting the ESI+ mass spectrum of
complex 4 (Figure S1) and TG/DTA curves for the complexes 2 and 5 (Figure S2). Supplementary data
can be found, in the online version of this article, at doi:
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Table 1. Selected coordination shifts (Δδ = δcomplex δligand, ppm) calculated from 1H and 13C NMR
spectra for the gold(III) complexes 1–5.
1H NMR
C8H
13C NMR
C5
Complex
C2H
C2
C6
C4
C8
1
2
3
4
5
-0.12 -0.28
-0.12 -0.32
-0.07 -0.17
-0.09 -0.81
-0.04 -0.79
-1.64 0.30 1.78
-1.69 0.22 1.35
-0.90 -0.20 1.01
-0.72 -0.22 0.99
-0.77 -0.32 1.39
-6.44
8.42
-10.27 8.33
-4.83
-6.98
-6.59
8.41
8.75
8.86
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Figure/Scheme Legends
Scheme 1. Schematic representation of the synthesis of gold(III) complexes (1–5) and the N6-
benzyladenine (HL1-5) derivatives given with the atom numbering scheme of the organic
compounds.
Fig. 1 Geometry of [Au(HL1)Cl3] (1) optimized at the B3LYP/LACVP+* level of theory.
Fig. 2. Effects of the tested complexes (1–5), N6-benzyladenine derivatives used as ligands (HL1–
HL5), AuCl3, and the reference drug prednisone (Pred.) on the LPS-induced TNF-α secretion in
macrophages derived from the THP-1 cell line. Cells were pre-treated with the compounds (5 μM), or
the vehicle (Veh.; DMSO) only. After 1 h of incubation, the inflammatory response was induced by
LPS [except for the control cells (CTRL)]. The results are expressed as means ± S.E. for three
independent experiments. * significant difference in comparison to vehicle-treated cells (P<0.05), **
significant difference in comparison to vehicle-treated cells (P<0.01).
Fig. 3 A time-dependent profile of anti-oedematous activity of the tested compounds. A single
asterisk besides the symbol for complex 1 at 300 min means that the applied dose of the complex led
to toxic death of all laboratory animals behind this time-point.
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Graphical abstract
Synopsis:
Au(III) complexes of the type [Au(HL1-5)Cl3]∙nH2O involving N6-benzyladenine derivatives were
prepared and fully characterized. They showed negligible in vitro cytotoxicity, moderate in vitro anti-
inflammatory activity on LPS-activated THP-1 macrophages, and significant in vivo anti-oedematous
activity, even exceeding the activity of gold-containing metallodrug Auranofin.
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