Inhibition of xanthine oxidase by thiosemicarbazones, hydrazones and dithiocarbazates derived from hydroxy-substituted benzaldehydes

Article abstract of DOI:10.1002/cmdc.201100054

Nonpurine xanthine oxidoreductase (XOR) inhibitors represent important alternatives to the purine analogue allopurinol, which is still the most widely used drug in the treatment of conditions associated with elevated uric acid levels in the blood. By cond

Full text of DOI:10.1002/cmdc.201100054

DOI: 10.1002/cmdc.201100054
Inhibition of Xanthine Oxidase by Thiosemicarbazones,
Hydrazones and Dithiocarbazates Derived from Hydroxy-
Substituted Benzaldehydes
Maria Leigh,[a] Daniel J. Raines,[a] Carmen E. Castillo,[a, b] and Anne K. Duhme-Klair*[a]
Nonpurine xanthine oxidoreductase (XOR) inhibitors represent
important alternatives to the purine analogue allopurinol,
which is still the most widely used drug in the treatment of
conditions associated with elevated uric acid levels in the
blood. By condensing mono-, di- and trihydroxybenzaldehydes
with aromatic thiosemicarbazides, aryl hydrazides and dithio-
carbazates, three series of structurally related Schiff bases were
synthesised, characterised and tested for XOR inhibitory activi-
ty. Hydroxy substitution in the para-position of the benzalde-
hyde component was found to confer high inhibitory activities.
Acyl hydrazones were generally less potent than thiocarbonyl-
containing Schiff bases. Within the thiosemicarbazone series,
chloro and cyano substituents in the para-position of the thio-
semicarbazide unit increased activities further, up to potencies
approximately four-times higher than that of the benchmark
allopurinol, as measured under the same assay conditions. In
order to illustrate the potential of the Schiff bases to bind di-
rectly to the molybdenum centre in the active site of the
enzyme, a representative example (H2L) of each inhibitor series
was co-ordinated to a cis-dioxomolybdenum(VI) unit, and the
resulting complexes, [MoO2(L)MeOH], were structurally charac-
terised. Subsequent steady-state kinetic investigations, howev-
er, indicated mixed-type inhibition, similar to that observed for
inhibitors known to bind within the substrate access channel
of the enzyme, remote from the Mo centre. Enzyme co-crystal-
lisation studies are thus required to determine the exact bind-
ing mode. Finally, the coordination of representative inhibitors
to copper(II) gave rise to significantly decreased IC50 values, re-
vealing an additive effect that merits further investigation.
Introduction
Xanthine oxidoreductase (XOR) inhibitors are widely used in
the control of uric acid blood levels.[1] High uric acid concentra-
tions (hyperuricemia) can lead to the formation of monosodi-
um urate crystals in the joints and surrounding tissues, leading
to the inflammation and pain associated with gout. In addition,
monosodium urate may accumulate in the kidneys, resulting in
the development of nephrolithiasis.[2]
hydroxyl radicals or peroxynitrite.[4,5] XO inhibitors may, there-
fore, find additional applications in the treatment of conditions
associated with radical-induced tissue damage and oxidative
stress, including ischemia–reperfusion injury, chronic heart fail-
ure and vascular disease.[2a]
Although discovered more than 40 years ago, the purine an-
alogue allopurinol is still the most commonly prescribed XOR
inhibitor. However, side effects, such as skin rashes, gastroin-
testinal problems and drowsiness, are not uncommon. Severe
hypersensitivity to allopurinol has also been reported, mainly
in patients with renal insufficiency.[2a,6] The implication of toxic
metabolites of purine analogues in adverse effects prompted
the development of alternative structural scaffolds for XOR in-
hibitors.[7] Several of these nonpurine scaffolds have given rise
to derivatives with remarkable in vitro inhibitory activities.[1] Of
these, a number of structurally related drug candidates featur-
ing an aromatic nitrile linked in the meta-position to a five-
membered, unsaturated heterocycle showed hypouricemic ef-
Mammalian XOR exists in two interconvertible forms, xan-
thine dehydrogenase (XDH; EC 1.17.1.4) and xanthine oxidase
(XO; EC 1.17.3.2). Both forms are involved in the catabolism of
dietary and endogenous purines by catalysing the hydroxyl-
ation of hypoxanthine to xanthine and xanthine to uric acid
(Scheme 1).[2b,3] Initially, the XDH form of the enzyme is pro-
[a] M. Leigh, D. J. Raines, Dr. C. E. Castillo, Dr. A. K. Duhme-Klair
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
Scheme 1. Formation of uric acid catalysed by xanthine oxidase.[2a]
duced, which uses NAD+ as a terminal electron acceptor. In tis-
sues, however, XDH can be converted to XO, which uses dioxy-
gen as its final electron acceptor.[2a,4] The latter produces super-
oxide and hydrogen peroxide, which can be converted to
other more damaging reactive oxygen species (ROS), such as
Fax: (+44) 1904 322516
E-mail: anne.duhme-klair@york.ac.uk
[b] Dr. C. E. Castillo
Departamento de Ciencia de los Materiales e Ingenierꢀa Metalfflrgica
Y Quꢀmica Inorgµnica, Facultad de Ciencias, Universidad de Cµdiz
Polꢀgono Rio San Pedro, 11510 Puerto Real, Cµdiz (Spain)
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A.-K. Duhme-Klair et al.
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fects in vivo that merited further progression into preclinical or
even clinical studies.
Extracts derived from plants traditionally used as antigout and
antirheumatic therapeutics have shown that the inhibitory ac-
tivity increases moderately with phenolic content.[15] Reported
work using docking studies has led to the hypothesis that the
phenolic moiety projects into the solvent channel of the
enzyme, potentially stabilising the position of the inhibitor via
hydrogen bonding and electrostatic interactions with key
amino acid residues such as Arg880, Glu1261 and
Thr1010.[13c,16]
Notable compounds within this series of potent nonpurine
XO inhibitors include Y-700 (Pyranostat) synthesised by Ishibu-
chi and co-workers,[8] the particularly potent inhibitor Fyx-
We have recently communicated the first account of promis-
ing XO inhibitory activity exhibited by selected thiosemicarba-
zones derived from 2,4,6-trihydroxybenzaldehyde (Scheme 2;
051,[9] which combines a pyridyl-triazole scaffold that resem-
bles the one initially developed by Baldwin et al.[10] with the
use of a meta-cyanopyridyl fragment similar to the meta-cya-
nophenyl substituent seen in Y-700 and 2-[2-(2-methoxy-
ethoxy)-ethoxy]-5-[5-(2-methyl-pyridin-4-yl)-1H-[1,2,4]triazol-3-
yl]-benzonitrile, a recently reported, related drug candidate
with potent in vivo activity but only moderate pharmacokinet-
ic profile.[11] The first and only nonpurine-type inhibitor to
obtain market approval in the EU and USA in 2008 and 2009,
respectively, is 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-1,3-thia-
zole-5-carboxylic acid (TEI-6720 or febuxostat, brand names:
Adenuric (EU) and Uloric (USA)). Since long-term safety data
are not yet available, and adverse effects, including altered
liver function, have been reported, use of febuxostat in the UK
is recommended in cases where allopurinol is contra-indicated
or poorly tolerated.[12]
Scheme 2. General synthetic approach to Schiff bases using a variety of sub-
stituted benzaldehydes and a selection of NH2-containing compounds. Re-
agents and conditions: a) EtOH, reflux, 4 h, 40–95%.
R1, E=S) and aromatic thiosemicarbazides.[17] In this paper, we
now report further insights into the underlying structure–activ-
ity relationships (SARs) gained by investigating a wider variety
of substituents in the ortho-, meta- and para-positions of both
aromatic components (Rn and R’) of related thiosemicarba-
zones, hydrazones and dithiocarbazates.
In particular, in view of the importance of phenols, as high-
lighted in the literature, the number and position of hydroxy
substituents on the benzaldehyde compounds were investigat-
ed (Scheme 2; R1–5). The synthesis, characterisation and inhibi-
tory activity of a number of such compounds are reported
herein. The SAR data obtained are compared with those re-
ported for other nonpurine XOR inhibitors that contain aro-
matic components with similar substitution patterns, including
febuxostat, Schiff base metal complexes,[18] chalcone deriva-
tives,[19] and naturally occurring XOR inhibitors, such as flavo-
noids.[13c]
As reported in previous studies, the presence of hydroxy
substituents is linked to potent XO inhibition.[13] Naturally oc-
The NH2-containing components chosen for this study con-
tained sulfur and NH groups, as well as hydroxy and cyano aro-
matic substituents. These functionalities are known to contrib-
ute to potent XO inhibition through favourable stabilising hy-
drogen-bond interactions with amino acid residues in the
access channel leading to the active site. Sulfur donors in thio-
semicarbazones and dithiocarbazates were chosen due to the
known thiophilicity of molybdenum and also to allow direct
comparison with oxygen-containing Schiff base counterparts
(hydrazones).[6,9a,20]
curring phenolic compounds, such as luteolin, quercetin and
silibinin, have been shown to inhibit XO effectively, acting as
competitive or mixed-type inhibitors.[13c] Comparison of the
structurally related flavonoids quercetin, luteolin and myricetin,
revealed that the position and the number of hydroxy sub-
stituents greatly influences potency, as the inhibitory activity
decreased in the order of quercetin> luteolin> myricetin.[14]
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Xanthine Oxidase Inhibitors
Results and Discussion
Chemistry
The synthesis of the Schiff bases involved refluxing equimolar
amounts of the appropriate benzaldehyde with the respective
thiosemicarbazide, aryl hydrazide or dithiocarbazate, in etha-
nol. After 4 h, precipitated products were isolated. If no precip-
itate was formed, the products were obtained after partial
evaporation of the solvent or precipitation with a minimum
amount of water. The Schiff bases were washed with ethanol
and dried in vacuo. Evidence of successful condensation lead-
ing to Schiff base formation was obtained by the appearance
1
of the H NMR spectroscopic signature of the new imine bond,
characterised by a singlet resonance of the imine proton ap-
pearing at 8.05–8.65 ppm. The disappearance of the signature
Scheme 3. General synthesis of MoVI Schiff base complexes from [MoO2-
(acac)2]. Reagents and conditions: a) MeOH, reflux, 45–89%.
1
of the aldehyde proton in the H NMR spectra of compounds
reported herein provided further evidence of Schiff base for-
mation. The imine carbon resonance appeared at 140.80–
149.40 ppm in the 13C NMR spectra, and a n˜(C=N) band ap-
peared at 1578–1643 cmꢀ1 in the Fourier-transform infrared
(FTIR) spectra of the compounds. All Schiff bases proved solu-
ble in acetone, methanol, dimethyl sulfoxide (DMSO) and N,N-
dimethylformamide (DMF).
tra of the ligands as a consequence of coordination to the
MoVI centre. The presence of the coordinated methanol solvent
molecule was indicated by the appearance of resonances at
3.17 and 4.10 ppm.[21e,22,24]
Electronic absorption spectra were recorded for all com-
plexes and their free ligands in DMSO (Table 1). The absorption
bands at 335–366 nm for H2Ln are attributed to n!p* intrali-
Molybdenum(VI) complexation
studies
Table 1. 1H NMR and UV/vis spectroscopic data of selected Schiff base ligands and their MoVI complexes.
Molybdenum
complexes
of
Schiff base ligands with ONO
and ONS donors have been de-
signed and used as models for
studying active sites and reac-
tions of biological importance.[21]
In order to probe the potential
of direct coordination to a MoVI
Compd
H2L1
1H NMR: d [ppm]
UV/vis: lmax [nm], e [molꢀ1 Lcmꢀ1
]
8.36
(CHN)
(OH/NH)
(CHN)
(OH/NH)
(CH3OH)
(CHN)
(OH/NH)
(CHN)
293, 14482
340, 25460
308, 15190
428, 5580
350, 30722
335, 28702
9.82, 9.87, 9.96, 11.66
[MoO2(L1)MeOH]
8.66
9.63, 10.44
3.17, 4.10
8.48
9.99, 11.39, 11.97
8.78
H2L2
292, 11052
326, 23590
304, 14980
421, 7957
centre,
a representative com-
[MoO2(L2)MeOH]
pound from each class of Schiff
bases, derivatives 3b, 3 f and
6a, was reacted with dioxomo-
10.64
3.15, 4.09
8.43
10.53, 11.28
9.01
(OH/NH)
(CH3OH)
(CHN)
(OH/NH)
(CHN)
H2L3
313, 11205
329, 16656
366, 21654
438, 1033
lybdenum(VI)
acetylacetonate
[MoO2(L3)MeOH]
([MoO2(acac)2]; Scheme 3). The
–
(OH/NH)
(CH3OH)
2+
3.17, 4.11
cis-MoO2
moiety of [MoO2-
(acac)2] was chosen to mimic the
oxo and thio groups attached to
the MoVI in the active site of the enzyme.[21b,d,22] All Schiff bases
readily formed mononuclear MoVI complexes, [MoO2(L)MeOH],
with the carbonyl/thiocarbonyl heteroatom, imine nitrogen,
and phenolate oxygen in the ortho-position acting as donor
atoms of the tridentate ligands. The potential of direct coordi-
nation of inhibitors to the MoVI active site of XO can lead to
longer lasting and more potent inhibition, as seen with allo-
purinol and Fyx-051.[9a,23]
gand transitions, while bands ꢁ313 nm arise due to intrali-
gand p!p* transitions.[21d,25] Upon complexation, the n!p*
bands experienced a shift to a shorter wavelength (326–
340 nm), as the two intraligand bands merged. The appear-
ance of a new LMCT band from the ligand O, N or S donors to
the molybdenum centre in [MoO2(Ln)MeOH] at approximately
430 nm, confirmed coordination to the MoVI centre.[24b,26]
Additionally, FTIR spectroscopic data (Table 2) confirmed co-
ordination, with n˜(C=N) shifting to a lower wavenumber as
compared to the free ligand, indicating redistribution of elec-
tron density. A shift was also observed for the phenolic n˜(CꢀO),
indicating participation of the phenolate donor in
The molybdenum complexes were characterised by NMR,
FTIR, UV/vis spectroscopy and X-ray diffraction. Evidence of
complexation was observed through the disappearance of two
singlet resonances assigned to NH and OH in the 1H NMR spec-
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A.-K. Duhme-Klair et al.
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2+
MoO2
bond angles of H2L1
Table 2. IR spectroscopic data of selected Schiff base ligands and their MoVI complexes.[a]
(104.918), H2L2 (105.668) and H2L3
(105.828) and the average Mo=O
Compd
n˜ [cmꢀ1
(C=S/C=O)
]
2+
(OꢀH)
(NꢀH)
(C=N)
(CꢀO)
(Mo=O)asymm
(Mo=O)symm
bond length for the cis-MoO2
H2L1
3450br
3394br
3409br
3500br
3445br
3337br
3159br
3364br
3259m
–
3097m
–
1633sh
1596s
1632s
1605sh
1616sh
1591sh
1210m
–
1661sh
–
1032sh
–
1271m
1225sh
1121sh
1237sh
1326m
1274sh
–
–
of 1.70 ꢁ are consistent with
MoO2(L1)MeOH
H2L2
889sh
–
907sh
–
932sh
–
932sh
–
similar reported cis-dioxomolyb-
denum(VI)
complexes.[21a,c,24,28]
MoO2(L2)MeOH
H2L3
The O(2)ꢀMoꢀO(3) bond angle
MoO2(L3)MeOH
877sh
937sh
of 151.568 for the coordinated
ligand
oxygen
atoms
in
[a] br: broad, sh: sharp, s: strong, m: medium.
[MoO2(L2)MeOH] and the O(1)ꢀ
MoꢀS(1) bond angles of 152.258
and 151.268 in [MoO2(L1)MeOH]
coordination.[22,24a,25] The absence of n˜(OꢀH) and n(NꢀH) in all
the complexes indicates the ligands undergo deprotonation
upon coordination. The existing n˜(OꢀH) and n˜(NꢀH) present in
the complexes are attributed to the coordinated methanol
molecule and the remaining NH group of the thiosemicarba-
zones. The two bands appearing at 877–907 cmꢀ1 and 931–
937 cmꢀ1 are assigned to the asymmetric and symmetric Mo=
O stretching vibrations and confirm the presence of the cis-
and MoO2(L3)MeOH], respectively, are consistent with literature
values.[21c,29] Bond lengths and angles are summarised in
Table 3. These three structurally characterised MoVI complexes
highlight the potential of all three classes of Schiff bases to co-
ordinate directly to a MoVI centre.
˜
Table 3. Selected bond lengths and angles for [MoO2(L2)MeOH] and
[MoO2(L3)MeOH.
MoO2 moiety in the complex, consistent with similar MoVI
2+
complexes reported in the literature.[21d–e,24a,25,27]
[MoO2(L2)MeOH]
[ꢁ]
[MoO2(L3)MeOH]
Diffraction quality red–orange crystals of [MoO2(L2)MeOH]
(Figure 1a) and [MoO2(L3)MeOH] (Figure 1b) were obtained fol-
lowing slow evaporation of methanolic solutions at room tem-
Bond
Bond
[ꢁ]
MoꢀO2
MoꢀO3
MoꢀO4
MoꢀO5
MoꢀO6
MoꢀN1
N1ꢀN2
C7ꢀN1
1.9287(10)
2.0124(10)
2.3601(12)
1.7094(12)
1.7044(10)
2.2301(12)
1.4024(15)
1.2940(18)
Mo-O1
Mo-S1
Mo-O4
Mo-O2
Mo-O3
Mo-N1
N1-N2
C7-N1
1.9239(14)
2.4243(5)
2.3619(15)
1.6929(15)
1.7241(14)
2.2599(16)
1.401(2)
1.292(2)
Angle
(8)
Angle
(8)
O2ꢀMoꢀO3
O6ꢀMoꢀN1
O4ꢀMoꢀO5
O3ꢀMoꢀN1
O2ꢀMoꢀN1
151.56(4)
155.01(5)
172.13(4)
71.77(4)
81.94(4)
O1-Mo-S1
O3-Mo-N1
O2-Mo-O4
S1-Mo-N1
O1-Mo-N1
151.26(5)
159.96(6)
169.26(6)
76.83(4)
82.11(6)
[a] Values in brackets represent the estimated SD.
Biological screening
The XO inhibitory activity of each Schiff base was assessed
spectrophotometrically by monitoring the formation of uric
acid at 295 nm every minute for 10 min at 258C, according to
the method employed by Bergmeyer et al. with minor modifi-
cations.[30] The reaction was initiated by the addition of XO
(2.5 mUmLꢀ1) to a mixture of xanthine (50 mm) and the test
compounds at various concentrations in 50 mm potassium di-
hydrogen orthophosphate buffer (pH 7.5). A negative control
was carried out in the absence of test compounds, while allo-
purinol was used as a positive control and benchmark.
Figure 1. Ortep plots (50% probability ellipsoids) of the molecular structures
of a) [MoO2(L2)MeOH] and b) [MoO2(L3)MeOH]. Hydrogen atoms are omitted
for clarity. Crystallographic data for these structures have been deposited
with the Crystallographic Data Centre (deposition numbers: CCDC 809737,
CCDC 809736).
perature or layering with pentane. The solid-state structures of
the two complexes revealed that the ligands act as meridional-
ly coordinating tridentate ONO and ONS donors. The crystal
structure of the MoVI complex of H2L1 has been reported previ-
ously by the authors (CCDC 786365).[17] All three MoVI centres
display distorted octahedral coordination geometry, consisting
of the phenolate oxygen, imine nitrogen, and thio- or oxo-
group donors from each Schiff base ligand, as well as two cis-
dioxo ligands and a methanol solvent molecule. The cis-
All inhibitors were dissolved in DMSO prior to introduction
to the assay mixture, with the total DMSO concentration limit-
ed to 1% (v/v) in order to minimise the effect on XO activity.
The percent inhibition at each inhibitor concentration was de-
termined as reported in the literature.[31] The inhibitory perfor-
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Xanthine Oxidase Inhibitors
mance of each test compound was evaluated by its IC50 value,
the concentration of inhibitor leading to a 50% decrease in
the enzyme activity. This value was determined from sigmoidal
plots of the inhibition versus log[inhibitor] for each test com-
pound, given in Table 4 for the thiosemicarbazones, Table 5 for
the hydrazones, and Table 6 for the dithiocarbazate Schiff
bases. The positive control, allopurinol, gave an IC50 value of
3ꢂ0 mm (mean ꢂ standard deviation (SD), n=2) under the
assay conditions employed, which is within the values reported
in the literature.[2a]
droxybenzaldehyde moiety, is generally more potent than
series 3, the hydroxy substituent in the para-position is again
highlighted as being vital for potent activity. The important
role that the substituent position has on activity is also con-
firmed on comparison of compounds 1b–4b bearing para-
chloro and 1c–4c with para-cyano substituents on their thio-
semicarbazide moiety, respectively. In general, the presence of
a substituent (b and c) on the thiosemicarbazide ring of each
Schiff base appears to improve the potency compared to their
unsubstituted counterpart (a), for all benzaldehyde moieties
(series 1, 3, 4). Compounds derived from 3-hydroxybenzalde-
hyde (2a–2c), however, display statistically similar inhibitory
activities, irrespective of the thiosemicarbazide used. On com-
parison of the type of aromatic substituent, compounds bear-
ing the cyano substituent appear marginally more potent than
their chloro counterparts (1b<1c, 4b<4c).
Table 4. Inhibitory activity results for thiosemicarbazones.
To investigate whether a meta-cyano substituent would lead
to increased potency, as seen in Y-700, febuxostat and Fyx-051,
3-cyanophenyl thiosemicarbazide was synthesised and con-
densed with 2,4,6-trihydroxybenzaldehyde (series 1) and 4-hy-
droxybenzaldehyde (series 4) to form Schiff bases 1d and 4d,
respectively. On comparison of the inhibitory activity of these
compounds with 1c and 4c bearing the cyano substituent in
the para-position, it is evident that the para substituent is also
important on the NH2-bearing moiety of the Schiff base sys-
tems investigated. Additionally, in order to investigate whether
the hydroxy substituent in compound 4a acts as a hydrogen-
bond donor, 5a was synthesised, with a methoxy substituent
in place of the hydroxy substituent. Analysis of the inhibitory
activity of 5a indicated a poor performance compared with
4a, emphasising the need for hydrogen-bond donors. The hy-
droxy substituent in the para-position may be involved in sta-
bilising the inhibitor in the enzyme active site through the for-
mation of hydrogen bonds with neighbouring amino acid resi-
dues. Under the assay conditions employed, thiosemicarba-
zones 1b, 1c, 3a, 3b, 3c, 4a, 4b and 4c gave IC50 values
lower than the benchmark allopurinol, with 1c, 3b, 3c, 4b
and 4c displaying approximately four-times increased potency.
The common substituents in these promising Schiff bases are
a hydroxy substituent in the para-position of the benzaldehyde
moiety and a para substituent, either chloro or cyano, on the
thiosemicarbazide aromatic ring.
Compd
Rn
R’
Inhibition at 50 mm[a] [%]
IC50 [mm][a]
1a
1b
1c
1d
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
5a
2,4,6-OH
2,4,6-OH
2,4,6-OH
2,4,6-OH
3-OH
3-OH
3-OH
2,4-OH
2,4-OH
2,4-OH
4-OH
H
88ꢂ2
95ꢂ1
89ꢂ1
80ꢂ0
92ꢂ0
94ꢂ1
100ꢂ0
93ꢂ3
95ꢂ4
96ꢂ1
95ꢂ1
98ꢂ0
93ꢂ1
93ꢂ0
7ꢂ1
6ꢂ2
4’-Cl
4’-CN
3’-CN
H
4’-Cl
4’-CN
H
4’-Cl
4’-CN
H
4’-Cl
4’-CN
3’-CN
H
1.8ꢂ0.1
0.6ꢂ0.2
6ꢂ0.2
4.7ꢂ0.2
3.2ꢂ1.0
6.4ꢂ1.5
1.6ꢂ0.7
0.5ꢂ0
0.6ꢂ0.1
1.3ꢂ0.1
0.8ꢂ0.1
0.6ꢂ0
4-OH
4-OH
4-OH
4-OCH3
2.5ꢂ0.3
–
[a] Values represent the mean ꢂSD of duplicate experiments.
The inhibitory activity of all compounds was initially tested
at 50 mm concentrations. Compounds giving rise to a minimum
of 50% inhibition were tested further at a range of concentra-
tions in order to determine the IC50 value. Comparison of the
inhibitory activity of the compounds in each class of Schiff
bases, thiosemicarbazones, hydrazones and dithiocarbazates,
allows SARs to be determined.
Upon inhibitory activity testing of hydrazones 1e–4e, they
were found to be less potent than the thiosemicarbazones.
This can be attributed to the presence of an additional NH
group in the thiosemicarbazones, which can lead to improved
stabilisation through formation of hydrogen bonds with key
amino acids residues. In addition, the presence of a sulfur in
place of the more electronegative oxygen seen in the hydra-
zones, may play a role in the improved potency of the thiose-
micarbazones, owing to the thiophilic nature of the Mo centre
in the active site. Hydrazones of series 1, 2, 3 and 4 were syn-
thesised (Table 5). Initial activity testing at 50 mm gave rise to
ꢄ50% inhibition for series 1 hydrazones, but revealed poor in-
hibitory performance (ꢃ15% inhibition) for hydrazones of
benzaldehyde moieties with fewer hydroxy substituents. The
presence of multiple hydroxy substituents can lead to a great-
SAR analysis
In order to investigate the effect of the number and position
of hydroxy substituents on inhibitory activity, thiosemicarba-
zones 1a–4a, derived from different benzaldehydes
(Scheme 2) but with the same unsubstituted thiosemicarba-
zide, were compared. The inhibitory performance appeared to
increase in the order of 1a <2a <3a ꢃ4a. As the least
potent compound bears three hydroxy substituents (1a) and
the most potent bears one (4a), it can be concluded that the
position is more significant than the number of hydroxy sub-
stituents for the inhibitory potencies of these Schiff base inhib-
itors. Compound series 3, bearing a 2,4-dihydroxybenzalde-
hyde moiety, is more potent than compound series 1, derived
from 2,4,6-trihydroxybenzaldehyde, and series 4, bearing 4-hy-
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1111

A.-K. Duhme-Klair et al.
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Table 5. Inhibitory activity results for hydrazones.
Table 6. Inhibitory activity results for dithiocarbazates.
Compd
Rn
Inhibition at 50 mm[a] [%]
IC50 [mm][a]
Compd
Rn
R’
Inhibition at 50 mm[a] [%]
IC50 [mm][a]
1l
2,4,6-OH
2,4-OH
4-OH
74ꢂ5
87ꢂ3
95ꢂ4
40ꢂ2
12ꢂ3
5ꢂ1
0.7ꢂ0.1
–
1e
1 f
1g
1h
1i
2,4,6-OH
2,4,6-OH
2,4,6-OH
2,4,6-OH
2,4,6-OH
2,4,6-OH
2,4,6-OH
3-OH
3-OH
3-OH
2,4-OH
2,4-OH
2,4-OH
4-OH
4’-OH
4’-Br
4’-Cl
3’-OH
3’-Br
3’-Cl
2’-OH
4’-OH
4’-Br
4’-Cl
4’-OH
4’-Br
96ꢂ4
71ꢂ4
59ꢂ2
60ꢂ2
64ꢂ3
61ꢂ1
41ꢂ9
17ꢂ1
16ꢂ0
14ꢂ0
26ꢂ1
12ꢂ4
8ꢂ3
19ꢂ0
3g
4 f
6a
32ꢂ1
63ꢂ1
2-OH, 5-Br
74ꢂ3
55ꢂ5
[a] Values represent the mean ꢂSD of duplicate experiments.
1j
48ꢂ6
1k
2d
2e
2 f
3d
3e
3 f
4e
85ꢂ12
–
–
–
–
–
–
–
the para-position of the benzaldehyde moiety led to the most
potent compound of the dithiocarbazate class, with 4 f being
approximately four-times more potent than allopurinol under
the assay conditions employed. On comparison of the dithio-
carbazates 1l and 3g, and the unsubstituted thiosemicarba-
zone counterparts 1a and 3a, the presence of a second sulfur
atom does not appear to lead to improved potency. In con-
trast, for 4-hydroxybenzaldehyde Schiff bases, the dithiocarba-
zate 4 f gave a lower IC50 value than its thiosemicarbazone
counterpart 4a. Co-crystallisation studies are, therefore, re-
quired to explain this deviation from the trend, hypothesised
to be attributed to the location and number of polar interac-
tions between inhibitors and the amino acids in the narrow
channel of the active site.
4’-Cl
4’-OH
13ꢂ0
[a] Values represent the mean ꢂSD of duplicate experiments.
er number of stabilising polar interactions as well as improved
solubility. As a consequence of the low inhibition, IC50 values
were not determined for hydrazones 2d–4e. Analysis of the
IC50 values for the 2,4,6-trihydroxybenzaldehyde hydrazones re-
vealed interesting trends in the substitution pattern of the hy-
drazide moiety. The activity of compounds differing only in the
position of the hydroxy substituent on the hydrazide moiety
decreases in the order of 1e (p-OH) >1h (m-OH) >1k (o-OH).
The trend is also repeated for bromo -substituted compounds
as 1 f (p-Br) >1i (m-Br). This again emphasises the para-posi-
tion of aromatic substituents on the hydrazide ring as being
vital for potent inhibition. In terms of the type of the substitu-
ent, as the inhibitory activity increased in the order of 1g (p-
Cl) <1 f (p-Br) <1e (p-OH), the hydroxy substituent was again
highlighted as an important substituent for potent inhibition,
in line with previous studies on flavonoids.[13,14] The trend was,
however, reversed when the meta-substituted hydrazones
were considered, with the inhibitory activity increasing in the
order of 1h (m-OH) <1i (m-Br) <1j (m-Cl). This indicates that
both the position and the type of substituent greatly influences
inhibitory activity and reinforces the aforementioned findings
for the thiosemicarbazones.
Steady-state kinetics
The mode of inhibition of potent Schiff bases was investigated
using Lineweaver–Burk plots. In these steady-state kinetic stud-
ies, the rate of the reaction was monitored as the concentra-
tion of the inhibitor and the substrate, xanthine, were varied.
As reported previously, the in vitro investigation of the mode
of inhibition for 1c, revealed a mixed-type inhibitory behav-
iour, with a Ki value of 0.7ꢂ0.2 mm and K’i value of 1.4ꢂ
0.1 mm.[17] Compounds 4c and 4 f displayed the same mode of
inhibition (Table 7). From this observation, it can be deduced
that both the thiosemicarbazones 1c and 4c and the dithio-
carbazate 4 f inhibit the enzyme by blocking the narrow chan-
nel leading to the molybdopterin active site. The mixed-type
inhibition is in accordance with the majority of nonpurine in-
hibitors reported in the literature, such as Y-700 and febuxo-
From the improved performance of the thiosemicarbazone-
over the hydrazone-type Schiff bases, it is evident that the
presence of a sulfur atom in place of an oxygen atom plays a
significant role in inhibition. It was, therefore, hypothesised
that the presence of two sulfur atoms may lead to improved
potency. S-Benzyldithiocarbazate was, therefore, condensed
with a range of hydroxy-substituted benzaldehydes (Table 6) in
order to investigate effects on inhibition. Determination of the
inhibitory activities of compounds 1l, 3g, 4 f, 6a was under-
taken. As the order of potency increased on going from 1l <
3g <4 f, it appeared that the activity increased as the number
of hydroxy substituents decreased, as seen previously with the
thiosemicarbazones. The presence of a hydroxy substituent in
Table 7. Mode of inhibition (MoI) data.
Compd
MoI
Vmax [AUminꢀ1
]
Km [mm]
Ki [mm][a] K’i [mm][a]
0.7ꢂ0.2 1.4ꢂ0.1
1c
Mixed-type Decreases
(0.023–0.013)
Mixed-type Decreases
(0.032–0.021)
Mixed-type Decreases
(0.032–0.014)
Increases
(8.93–12.16)
Increases
(12.76–19.32)
Increases
(18.83–27.90)
4c
4 f
1.1ꢂ0.9 2.4ꢂ1.6
0.4ꢂ0.0 0.8ꢂ0.2
[a] Values represent the mean ꢂSD of duplicate experiments.
1112
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ChemMedChem 2011, 6, 1107 – 1118

Xanthine Oxidase Inhibitors
stat. Co-crystallisation studies of these inhibitors with XO re-
ported in the literature revealed that they are held in place by
a number of polar and nonpolar interactions with neighbour-
ing amino acid residues.[6,9a,20]
vealed that the oxidative half reaction, catalysed by XO, is af-
fected during CuII inhibition, indicating interruption of the
process occurring at the FAD cofactor, where molecular
oxygen is used as the final electron acceptor. Inhibition may,
therefore, occur as a result of slower reoxidation of MoIV to its
active MoVI form. A more recent study by Sau et al. reports evi-
dence for the direct coordination of the CuII to XO via sulfur or
nitrogen ligands.[34b,35] These ligands can arise from amino
acids, such as cysteine, surrounding the FAD. An alternative hy-
pothesis involves the formation of deleterious hydroxy radicals
produced in the Haber–Weiss reaction, catalysed by CuII.[36] Al-
though the exact mechanism of inhibition is unknown, is it evi-
dent that there exists an additive effect in having a Schiff base
ligand and a CuII present, leading to greater inhibitory activity.
Copper(II) complexation studies
The use of transition metal complexes with similar ONS and
ONO donor Schiff base ligands as novel XO inhibitors has been
reported in the literature by Zhu et al.[18a–c] In their studies, CuII
complexes displayed XO inhibitory activities comparable to al-
lopurinol. Further research into the use of transition metals as
inhibitors of the enzyme revealed that copper salts can inde-
pendently inhibit XO.[32] In order to investigate the potential of
increased potency on complexation to copper, thiosemicarba-
zone 3b and hydrazone 1i were used in preparation of the re-
spective copper(II) complexes (Scheme 4).
Conclusions
The synthesis, characterisation, and XO inhibitory activity of
Schiff bases derived from a series of benzaldehydes and three
classes of NH2-containing aromatic compounds was undertak-
en. In particular, the number and position of hydroxy substitu-
ents on the benzaldehyde moiety was investigated. Schiff base
ligands from each class— thiosemicarbazone, hydrazone and
dithiocarbazate—readily formed MoVI complexes upon reaction
with [MoO2(acac)2]. This highlights the potential of direct at-
tachment or close association with the molybdo(VI)-pterin in
the enzyme active site. On testing, thiosemicarbazones 1c, 3b,
3c, 4b and 4c and dithiocarbazate 4 f displayed significantly
increased potency over the benchmark allopurinol under the
employed assay conditions. The SAR study performed on thio-
semicarbazones, hydrazones and dithiocarbazates revealed
that the position rather than the number of hydroxy substitu-
ents is important for potent XO inhibition.
Scheme 4. Synthesis of CuII complexes of thiosemicarbazone 3b and hydra-
zone 1i ligands. Reagent and conditions: a) copper–ligand ratio 1:1, metha-
nol, reflux, 53–80%.
Evidence of complexation was obtained by UV/vis spectros-
copy, IR spectroscopy and elemental analysis. From the charac-
terisation data obtained, it can be concluded that the ligands
behave as tridentate ONO/ONS donors, with chelating pheno-
late oxygen, imine nitrogen, and thio or oxo donors, in accord-
ance to similar reported complexes.[33] The presence of water
was determined by elemental analysis. The complexes were in-
soluble in methanol, ethanol and acetone, but readily soluble
in DMSO. On analysis of the inhibitory activities of the com-
plexes, the IC50 values (Table 8) were approximately three-times
lower than their corresponding free ligands. The improved po-
tency of the copper(II) complexes mirrors the findings reported
by Zhu et al.[18a–b]
A hydroxy substituent in the para-position on the benzalde-
hyde unit gave rise to the most potent Schiff bases. In addi-
tion, the presence of chloro and cyano substituents in the
para-position of the aromatic ring of the thiosemicarbazide
unit led to increased potency over the unsubstituted counter-
parts. Having a substituent in the para-position of aryl hydra-
zides was also significant for potent XO inhibition in hydra-
zones, with the hydroxy substituent giving rise to improved in-
hibition. Substitution of the hydroxy with a methoxy substitu-
ent led to a dramatic loss of activity, indicating the need for
hydrogen-bond donor substituents. Groups such as OH, NH
and CN are postulated to contribute to stabilising polar inter-
actions between the inhibitors and amino acid residues in the
access channel of the enzyme. Oxygen-containing hydrazones
were less potent than sulfur-containing Schiff bases, highlight-
ing the importance of sulfur for potent XO inhibitory activity.
In addition, steady-state kinetic studies revealed mixed-type in-
hibition for both thiosemicarbazone and dithiocarbazate Schiff
bases. The definitive mode of inhibition and the number and
type of stabilising interactions holding these Schiff base inhibi-
tors in place will be determined from co-crystallisation studies
with XO. The initial testing of CuII complexes versus their free
ligands revealed a threefold improvement in inhibition, which
will be investigated further.
Although the role of copper in XO inhibition is still under in-
vestigation, several potential modes of action have been pro-
posed. Steady-state kinetic studies have revealed the noncom-
petitive nature of inhibition of CuII salts, ruling out direct coor-
dination to the MoVI active site.[34] A study by Mondal et al. re-
Table 8. XO inhibitory activity results for Schiff bases 1i and 3b and their
CuII complexes.[a]
IC50 [mm]
3b
1i
Free ligand
Copper complex
0.5ꢂ0
0.2ꢂ0
55ꢂ5
16ꢂ3
[a] Values represent the mean ꢂSD of duplicate experiments.
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A.-K. Duhme-Klair et al.
MED
2H, J=7.0 Hz), 8.65 (s, 1H, CHN), 10.05 (s, 1H, OH), 10.50 ppm (s,
2H, OH/NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=39.2 (CH2), 95.8,
100.1, 128.2, 129.4, 130.2, 137.7, 146.7 (CHN), 161.2 (4Car), 163.5
(4Car), 195.0 ppm (C=S); FTIR (KBr): n˜max =3392 (OꢀH, br), 3200 (Nꢀ
H, br), 3099 (CꢀHar, w), 2969 (CꢀHalkyl, w), 1634 (C=N, sh), 1583 (C=
C, sh), 1293 (CꢀO, sh, s), 1029 cmꢀ1 (C=S, sh); UV/vis (DMSO): lmax
(e)=295 (8203), 368 (27496), 384 nm (23772); HRMS (ESI): m/z [M+
H]+ calcd for C15H15N2O3S2: 335.0513, found: 335.0519 (difference=
0.5 mDa); Anal. calcd for C15H14N2O3S2·1H2O: C 51.13, H 4.58, N
7.96, found: C 50.77, H 4.51, N 7.87.
Experimental Section
Materials and instrumentation: 2,4,6-Trihydroxybenzaldehyde,
2,4-dihydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxy-
benzaldehyde, 4-methoxybenzaldehyde, 5-bromo-2-hydroxybenzal-
dehyde, 4-phenyl-3-thiosemicarbazide, aryl hydrazides and
[D6]DMSO were purchased from Aldrich. Xanthine oxidase and allo-
purinol were purchased from Sigma. Potassium dihydrogen ortho-
phosphate used in the enzyme assays was purchased from Fisher
Chemicals and reagent grade DMSO and was purchased from
Fisher Scientific. 4-(4-Chlorophenyl)-3-thiosemicarbazide was pur-
chased from Alfa Aesar. All reagents were used as supplied. 4-(4-
Cyanophenyl)-3-thiosemicarbazide and 4-(3-cyanophenyl)-3-thiose-
micarbazide were synthesised by refluxing N2H4·H2O (Sigma–Al-
drich) with equimolar amounts of 4-cyanophenyl isothiocyanate
(Alfa Aeasar) and 3-cyanophenyl isothiocyanate (Aldrich) respec-
tively. S-Benzyldithiocarbazate was synthesised as previously re-
ported.[37] 1H and 13C {1H}-decoupled NMR spectra were recorded
on a Jeol EX and ES 400 instrument (1H NMR 400 MHz; 13C NMR
100 MHz). Assignment of resonances was confirmed by HSQC
spectra. Multiplicity abbreviations are reported as follows: s=sin-
glet; bs=broad singlet; d=doublet; t=triplet; dd=double dou-
blet; m=multiplet; q=quartet. Infrared (IR) spectra were recorded
on a Thermo Nicolet Avatar 370 FTIR spectrophotometer in the
region of 4000–400 cmꢀ1: br=broad; sh=sharp; s=strong; m=
medium; w=weak. Melting points were measured on a Stuart Sci-
entific SMP3 apparatus. Elemental analysis of compounds was car-
ried out on an Exeter CE-440 elemental analyser and are within
ꢂ0.4% of the calculated value. Electrospray ionisation mass spec-
trometry (ESI-MS) and high resolution mass spectra (HRMS) were
recorded on a Bruker microTOF electrospray mass spectrometer.
Xanthine oxidase inhibitory activity assays were carried out using a
Jenway 6705 UV/vis spectrophotometer with thermostated cell
holder. Diffraction data was collected on a Bruker Smart Apex dif-
fractometer at 110 K with Mo Ka radiation (l=0.71073 ꢁ).
3-[(3-Hydroxyphenyl)methylidene]amino-1-phenylthiourea (2a):
Pale yellow crystalline solid (0.11 g, 0.39 mmol, 77%): Rf =0.70
(MeOH/Et2O 1:10); mp: 202–2038C (lit. 1948C);[38] 1H NMR
(400 MHz, [D6]DMSO): d=6.81 (1H, dd, J=7.8, 1.3 Hz), 7.16–7.35
(6H, m), 7.55 (2H, d, J=7.5 Hz), 8.05 (1H, s, CHN), 9.53 (1H, bs, OH/
NH), 11.74 ppm (1H, bs, OH/NH); 13C{1H} NMR (100 MHz,
[D6]DMSO): d=113.8, 117.3, 118.9, 125.3, 125.8, 128.1, 129.7, 135.3,
139.1, 143.2 (CHN), 157.6 (4Car), 175.9 ppm (C=S); FTIR (KBr): n˜max
=
3394 (OꢀH, br), 3311 (NꢀH, sh, m), 3172 (CꢀHar, w), 1581 (C=N, sh,
m), 1546, 1510 (C=C, sh, s), 1287 (CꢀO, sh, s), 1165 cmꢀ1 (C=S, sh);
HRMS (ESI): m/z [M+H]+ calcd for C14H13N3OS: 272.0852, found:
272.0852 (difference= ꢀ0.3 mDa); Anal. calcd for C14H13N3OS: C
61.97, H 4.83, N 15.49, found: C 61.71, H 4.88, N 15.76.
1-(4-Chlorophenyl)-3-[(3-hydroxyphenyl)methylidene]amino-
thiourea (2b): Beige solid (97 mg, 0.32 mmol, 65%): Rf =0.70
1
(MeOH/Et2O 1:10); mp: 207–2088C; H NMR (400 MHz, [D6]DMSO):
d=6.83 (dd, 1H, J=8.5, 2.0 Hz), 7.22 (t, 1H, J=7.0 Hz), 7.26–7.30
(m, 2H), 7.41 (d, 2H, J=8.5 Hz), 7.61 (d, 2H, J=8.5 Hz), 8.07 (s, 1H,
CHN), 9.57 (bs, 1H, OH/NH), 10.13 (bs, 1H, OH/NH), 11.85 ppm (bs,
1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=113.9, 117.4, 119.0,
127.5, 128.0, 129.3, 129.7, 135.2, 138.1, 143.6 (CHN), 157.6 (4Car),
176.0 ppm (C=S); FTIR (KBr): n˜max =3400 (OꢀH, br), 3281 (NꢀH, br),
1591 (C=N, sh, m), 1550, 1490 (C=C, sh), 1276 (CꢀO, sh, m),
1197 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for
C14H13N3OSCl: 306.0462, found: 306.0457 (difference= 0.9 mDa);
Anal. calcd for C14H12N3OSCl·0.3H2O: C 54.04, H 4.08, N 13.50,
found: C 53.92, H 3.95, N 14.05.
General procedure for Schiff base synthesis: A solution of the ap-
propriate benzaldehyde (1 mmol) in EtOH (10 mL) was treated with
the appropriate thiosemicarbazide, hydrazide or dithiocarbazate
(1 mmol), and the resulting mixture was refluxed for 4 h. The pre-
cipitate that formed after partial evaporation of the solvent or by
addition of a minimum amount of water was isolated, washed with
EtOH and dried. The products obtained were isolated in yields of
40–95%. Characterisation data for compounds 1a–1c, 1e–1k and
[MoO2(L1)MeOH] can be found elsewhere.[17]
1-(4-Cyanophenyl)-3-[(3-hydroxyphenyl)methylidene]amino-
thiourea (2c): Pale yellow solid (33 mg, 0.11 mmol, 62%): Rf =0.52
1
(MeOH/CHCl3 1:8); mp: 197–1988C; H NMR (400 MHz, [D6]DMSO):
d=6.82 (bd, 1H, J=8.5 Hz), 7.23 (t, 1H, J=8.0 Hz), 7.26–7.30 (m,
2H), 7.81 (d, 2H, J=8.8 Hz), 7.94 (bd, 2H, J=8.8 Hz), 8.10 (s, 1H,
CHN), 9.61 (bs, 1H, OH/NH), 10.28 (bs, 1H, OH/NH), 12.02 ppm (bs,
1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=106.8, 113.8, 117.4,
118.9, 119.0, 124.8, 129.6, 132.1, 134.9, 143.4, 144.1 (CHN), 157.5
(4Car), 175.2 ppm (C=S); FTIR (KBr): n˜max =3389 (OꢀH, m), 3296 (Nꢀ
H, sh, m), 2235 (CꢅN, sh), 1606 (C=N, sh), 1583 (C=C, sh), 1276 (Cꢀ
O, sh), 1203 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for
C15H13N4OS: 297.0805, found: 297.0806 (difference= ꢀ0.1 mDa);
Anal. calcd for C15H12N4OS: C 60.79, H 4.08, N 18.91, found: C 60.62,
H 4.08, N 19.25.
1-(3-Cyanophenyl)-3-[(2,4,6-trihydroxyphenyl)methylidene]ami-
no thiourea (1d): Pale orange solid (58 mg, 0.18 mmol, 70%): Rf =
0.43 (MeOH/CHCl3 1:8); mp: 199–2008C (dec); 1H NMR (400 MHz,
[D6]DMSO): d=5.84 (s, 2H), 7.54 (t, 1H, J=8.0 Hz), 7.61 (d, 1H, J=
7.4 Hz), 7.85 (d, 1H, J=7.8 Hz), 8.00 (s, 1H), 8.60 (s, 1H, CHN), 9.91
(s, 1H, OH/NH), 10.02 (s, 1H, OH/NH), 10.12 (s, 1H, OH/NH),
11.74 ppm (s, 1H, OH/NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=
94.7, 99.0, 110.8 (4Car), 118.7, 128.5, 129.5, 130.4, 140.6, 145.2 (CHN),
155.9, 159.4, 161.9 ppm (C=S); FTIR (KBr): n˜max =3335 (OꢀH, sh),
3253 (NꢀH, sh), 2245 (CꢅN, sh), 1643 (C=N, sh, m), 1550 (C=C, sh,
m), 1263 (CꢀO, m), 1050 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+
calcd for C15H13N4O3S: 329.0703, found: 329.0706 (difference=
ꢀ0.3 mDa); Anal. calcd for C15H12N4O3S·1.01H2O: C 51.99, H 4.08, N
16.17, found: C 51.98, H 4.05, N 16.73.
4-Hydroxy-N’-[(3-hydroxyphenyl)methylidene]benzohydrazide
(2d): White crystalline solid (81 mg, 0.32 mmol, 63%): Rf =0.47
(MeOH/Et2O 1:10); mp: >2508C (dec) (lit. 2718C);[39] 1H NMR
(400 MHz, [D6]DMSO): d=6.82 (dd, 1H, J=8.0, 2.0 Hz), 6.84 (d, 2H,
J=8.4 Hz), 7.08 (d, 1H, J=7.6 Hz), 7.19 (bs, 1H), 7.24 (t, 1H, J=
7.8 Hz), 7.80 (d, 2H, J=8.5 Hz), 8.33 (s, 1H, CHN), 9.85 (bs, 1H, OH),
11.59 ppm (bs, 1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=
112.5, 115.1, 117.3, 118.7, 124.2, 129.7, 129.9, 135.8, 147.1 (CHN),
157.7 (4Car), 161.2 (4Car), 162.6 ppm (C=O); FTIR (KBr): n˜max =3455
(OꢀH, br), 3233 (NꢀH, br), 1628 (C=O, sh, m), 1587 (C=N, sh, m),
(2,4,6-Trihydroxybenzaldehyde)dithiocarbazate (1l): Pale orange
solid (0.27 g, 0.81 mmol, 80%): Rf =0.57 (MeOH/CHCl3 1:8); mp:
184–1858C; 1H NMR (400 MHz, [D6]DMSO): d=4.53 (s, 2H, CH2),
5.84 (s, 2H), 7.27 (t, 1H, J=7.4 Hz), 7.33 (t, 2H, J=7.0 Hz), 7.41 (d,
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Xanthine Oxidase Inhibitors
1508 (C=C, sh), 1240 (CꢀO, sh), 1168 cmꢀ1 (CꢀO, sh); HRMS (ESI):
m/z [M+H]+ calcd for C14H13N2O3: 257.0921, found: 257.0921 (dif-
ference= 0.0 mDa); Anal. calcd for C14H12N2O3·1H2O: C 61.31, H
5.14, N 10.21, found: C 61.05, H 5.12, N 10.28.
(MeOH/CHCl3 1:4); mp: >2508C (dec); 1H NMR (400 MHz,
[D6]DMSO): d=6.29 (1H, bd, J=1.8 Hz), 6.31 (1H, bd), 7.80 (2H, d,
J=8.6 Hz), 7.88 (1H, bd, J=8.2 Hz), 7.97 (2H, d, J=8.6 Hz), 8.40
(1H, s, CHN), 9.85 (1H, bs, OH/NH), 9.90 (1H, bs, OH/NH), 10.14
(1H, bs, OH/NH), 11.87 ppm (1H, s, NH); 13C{1H} NMR (100 MHz,
[D6]DMSO): d=102.3, 106.1, 107.7, 111.4, 118.9, 124.4, 128.4, 132.1,
141.6, 143.5 (CHN), 158.3 (4Car), 160.8 (4Car), 174.1 ppm (C=S); FTIR
(KBr): n˜max =3360 (OꢀH, m), 3299 (NꢀH, m), 3164 (CꢀHar, w), 2236
(CꢅN, sh, m), 1633 (C=N, sh), 1550, 1514 (C=C, sh), 1238, 1214 (Cꢀ
O, sh), 1127 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for
C15H13N4O2S:313.0754, found: 313.0750 (difference= 0.4 mDa);
Anal. calcd for C15H12N4O2S: C 57.68, H 3.87, N 17.94, found: C
57.47, H 3.91, N 18.37.
4-Bromo-N’-[(3-hydroxyphenyl)methylidene]benzohydrazide
(2e): White solid (0.14 g, 0.43 mmol, 87%): Rf =0.63 (MeOH/Et2O
1:10); mp: >2508C (dec); 1H NMR (400 MHz, [D6]DMSO): d=6.84
(dd, 1H, J=8.0, 2.0 Hz), 7.10 (d, 1H, J=7.5 Hz), 7.21 (s, 1H), 7.26 (t,
1H, J=7.5 Hz), 7.74 (d, 2H, J=8.5 Hz), 7.86 (d, 2H, J=8.5 Hz), 8.35
(s, 1H, CHN), 9.64 (bs, 1H, OH), 11.86 ppm (bs, 1H, NH); 13C{1H}
NMR (100 MHz, [D6]DMSO): d=112.6, 117.6, 118.9, 125.6, 129.7,
129.9, 131.5, 132.5, 135.5, 148.3 (CHN), 157.7 (4Car), 162.2 ppm (C=
O); FTIR (KBr): n˜max =3487 (OꢀH, br), 3308 (NꢀH, br), 1665 (C=O, s),
1578 (C=N, sh), 1483 (C=C, sh), 1288 cmꢀ1 (CꢀO, s); HRMS (ESI): m/
z [M]+ calcd for C14H11N2O2Br: 319.0077, found: 319.0070 (differ-
ence= 0.7 mDa); Anal. calcd for C14H11N2O2Br·0.49H2O: C 51.27, H
3.68, N 8.54, found: C 51.27, H 3.71, N 8.11.
N’-[(2,4-Dihydroxyphenyl)methylidene]-4-hydroxybenzohydra-
zide (3d): Beige solid (54 mg, 0.20 mmol, 40%): Rf =0.37 (MeOH/
1
Et2O 1:10); mp: >2508C (dec); H NMR (400 MHz, [D6]DMSO): d=
6.30 (s, 1H), 6.34 (d, 1H, J=8.0 Hz), 6.85 (d, 2H, J=8.5 Hz), 7.26 (d,
1H, J=8.0 Hz), 7.79 (d, 2H, J=8.5 Hz), 8.45 (s, 1H, CHN), 10.05 (bs,
2H, OH), 11.59 (bs, 1H, OH/NH), 11.72 ppm (bs, 1H, OH/NH); 13C{1H}
NMR (100 MHz, [D6]DMSO): d=102.7, 107.6, 110.6, 115.1, 123.3,
129.6, 131.4, 148.5 (CHN), 159.5 (4Car), 160.5 (4Car), 160.9 (4Car),
162.2 ppm (C=O); FTIR (KBr): n˜max =3348 (OꢀH, m), 3150 (NꢀH, br),
1650 (C=O, sh), 1633 (C=N, sh), 1603 (C=C, sh), 1259, 1175 cmꢀ1
(CꢀO, sh); HRMS (ESI): m/z [M+H]+ calcd for C14H13N2O4 273.0870,
found: 273.0873 (difference= ꢀ0.4 mDa); Anal. calcd for
C14H12N2O4·0.15CH2CH3OH: C 61.52, H 4.66, N 10.03, found: C 61.52,
H 4.48, N 9.79.
4-Chloro-N’-[(3-hydroxyphenyl)methylidene]benzohydrazide
(2 f): White solid (70 mg, 0.26 mmol, 51%): Rf =0.63 (MeOH/Et2O
1
1:10); mp: 219–2208C; H NMR (400 MHz, [D6]DMSO): d=6.81 (dd,
1H, J=8.0, 1.6 Hz), 7.11 (d, 1H, J=7.6 Hz), 7.21 (s, 1H), 7.26 (t, 1H,
J=7.6 Hz), 7.61 (d, 2H, J=8.4 Hz), 7.94 (d, 2H, J=8.4 Hz), 8.35 (s,
1H, CHN), 9.64 (bs, 1H, OH), 11.90 ppm (bs, 1H, NH); 13C{1H} NMR
(100 MHz, [D6]DMSO): d=112.6, 117.6, 118.9, 128.6, 129.6, 130.0,
132.2, 135.5, 136.6, 148.2 (CHN), 157.7 (4Car), 162.0 ppm (C=O); FTIR
(KBr): n˜max =3406 (OꢀH, br), 3227 (NꢀH, br), 1645 (C=O, sh), 1595
(C=N, sh), 1489 (C=C, sh), 1288 cmꢀ1 (CꢀO, sh); HRMS (ESI): m/z
[M+H]+ calcd for C14H12N2O2Cl: 275.0582, found: 275.0587 (differ-
ence= ꢀ0.5 mDa); Anal. calcd for C14H11N2O2Cl·1.61H2O: C 55.37,
H 4.72, N 9.22, found: C 55.39, H 4.56, N 9.07.
4-Bromo-N’-[(2,4-dihydroxyphenyl)methylidene]benzohydrazide
(3e): Beige crystalline solid (0.12 g, 0.36 mmol, 71%): Rf =0.54
(MeOH/Et2O 1:10); mp: >2508C (dec); 1H NMR (400 MHz,
[D6]DMSO): d=6.32 (d, 1H, J=3.0 Hz), 6.36 (dd, 1H, J=8.5, 3.0 Hz),
7.32 (d, 1H, J=8.5 Hz), 7.75 (d, 2H, J=8.8 Hz), 7.87 (d, 2H, J=
8.8 Hz), 8.48 (s, 1H, CHN), 9.99 (s, 1H, OH/NH), 11.38 (bs, 1H, OH/
NH), 11.97 ppm (bs, 1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO):
d=102.7, 107.8, 110.5, 128.7, 129.5, 131.3, 131.7, 136.7, 149.4
3-[(2,4-Dihydroxyphenyl)methylidene]amino-1-phenylthiourea
(3a): Yellow solid (78 mg, 0.27 mmol, 54%): Rf =0.58 (MeOH/Et2O
1:10); mp: >2008C (dec); 1H NMR (400 MHz, [D6]DMSO): d=6.29
(dd, 1H, J=8.8, 2.4 Hz), 6.32 (d, 1H, J=2.4 Hz), 7.17 (t, 1H, J=
7.5 Hz), 7.34 (t, 2H, J=7.7 Hz), 7.57 (d, 2H, J=7.6 Hz), 7.87 (d, 1H,
J=8.5 Hz), 8.36 (s, 1H, CHN), 9.81 (bs, 1H, OH/NH), 9.85 (s, 1H, OH/
NH), 9.91 (bs, 1H, OH/NH), 11.59 ppm (bs, 1H, NH); 13C{1H} NMR
(100 MHz, [D6]DMSO): d=102.3, 107.8, 111.8, 125.0, 125.5, 128.0,
128.6, 139.3, 140.8 (CHN), 158.3 (4Car), 160.7 (4Car), 174.9 ppm (C=
S); FTIR (KBr): n˜max =3400 (OꢀH, br), 3141 (NꢀH, br), 1610 (C=N, s),
1545 (C=C, m), 1214 (CꢀO, m), 1121 cmꢀ1 (C=S, sh, s); HRMS (ESI):
m/z [M+H]+ calcd for C14H14N3O2S: 288.0801, found: 288.0797 (dif-
ference= 0.4 mDa); Anal. calcd for C14H13N3O2S·0.69H2O: C 56.09,
H 4.84, N 14.02, found: C 56.08, H 4.74, N 14.28.
(CHN), 159.5 (4Car), 160.9 (4Car), 161.5 ppm (C=O); FTIR (KBr): n˜max
=
3432 (OꢀH, br), 3262 (NꢀH, br), 3085 (CꢀHar, w), 1660 (C=O, sh, s),
1632 (C=N, sh, s), 1609 (C=C, sh), 1257 cmꢀ1 (CꢀO, sh); HRMS (ESI):
m/z [M]+ calcd for C14H11N2O3Br: 335.0026, found: 335.0020 (differ-
ence= 0.6 mDa); Anal. calcd for C14H11N2O3Br·0.08H2O: C 49.96, H
3.34, N 8.32, found: C 49.94, H 3.29, N 7.94.
4-Chloro-N’-[(2,4-dihydroxyphenyl)methylidene]benzohydrazide
(3 f): Beige crystalline solid (0.11 g, 0.37 mmol, 74%): Rf =0.52
(MeOH/Et2O 1:10); mp: >2508C (dec); 1H NMR (400 MHz,
[D6]DMSO): d=6.30 (d, 1H, J=2.0 Hz), 6.33 (dd, 1H, J=8.0, 2.0 Hz),
7.28 (d, 1H, J=8.0 Hz), 7.59 (d, 2H, J=8.5 Hz), 7.92 (d, 2H, J=
8.5 Hz), 8.48 (s, 1H, CHN), 9.99 (bs, 1H, OH/NH), 11.39 (bs, 1H, OH/
NH), 11.97 ppm (bs, 1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO):
d=102.5, 107.6, 110.4, 128.5, 129.3, 131.2, 131.6, 136.5, 149.2
1-(4-Chlorophenyl)-3-[(2,4-dihydroxyphenyl)methylidene]amino-
thiourea (3b): Yellow solid (0.11 g, 0.35 mmol, 54%): Rf =0.55
(MeOH/CHCl3 1:8); mp: >2008C (dec); 1H NMR (400 MHz,
[D6]DMSO): d=6.30 (dd, 1H, J=7.0, 2.0 Hz), 6.31 (d, 1H, J=2.0 Hz),
7.38 (d, 2H, J=8.8 Hz), 7.60 (d, 2H, J=8.8 Hz), 7.86 (d, 1H, J=
7.0 Hz), 8.36 (s, 1H, CHN), 9.82 (bs, 1H, OH), 9.87 (bs, 1H, OH), 9.96
(bs, 1H, NH), 11.66 (bs, 1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO):
d=102.3, 107.8, 111.7 (4Car), 127.1, 127.9, 128.3, 128.9, 138.3, 142.2
(CHN), 159.4 (4Car), 160.7 (4Car), 161.3 ppm (C=O); FTIR (KBr): n˜max
=
3409 (OꢀH, br), 3259 (NꢀH, m), 3060 (CꢀHar, w), 1661 (C=O, sh, s),
1632 (C=N, sh, s), 1609 (C=C, sh), 1274 cmꢀ1 (CꢀO, sh); UV/vis
(DMSO): lmax (e)=306 (15140), 340 nm (28608); HRMS (ESI): m/z
[M+H]+ calcd for C14H12N2O3Cl: 291.0531, found: 291.0538 (differ-
ence= ꢀ0.7 mDa); Anal. calcd for C14H11N2O3Cl·0.17CH2CH3OH: C
57.69, H 4.06, N 9.38, found: C 57.69, H 3.80, N 9.10.
(CHN), 158.3 (4Car), 160.8 (4Car), 174.5 ppm (C=S); FTIR (KBr): n˜max
=
3450 (OꢀH, br), 3159 (NꢀH, br), 1633 (C=N, s, sh), 1547 (C=C, s),
1271 (CꢀO, m), 1210 cmꢀ1 (C=S, m); UV/vis (DMSO): lmax (e)=293
(14482), 308 (15190), 350 nm (30722); HRMS (ESI): m/z [M+H]+
calcd for C14H13N3O2SCl: 322.0412, found: 322.0407 (difference=
0.5 mDa); Anal. calcd for C14H12N3O2SCl·0.39H2O: C 51.15, H 3.92, N
12.78, found: C 51.17, H 3.93, N 12.59.
(2,4-Dihydroxybenzaldehyde)dithiocarbazate (3g): Pale yellow
solid (25 mg, 0.079 mmol, 16%): Rf =0.71 (MeOH/Et2O 1:10); mp:
190–1928C (lit. 175–1778C);[40] 1H NMR (400 MHz, [D6]DMSO): d=
4.43 (s, 2H, CH2), 6.25 (d, 1H, J=1.5 Hz), 6.28 (dd, 1H, J=7.0,
1.5 Hz), 7.22–7.37 (m, 6H), 8.40 (s, 1H, CHN), 10.00 (bs, 1H, OH/NH),
1-(4-Cyanophenyl)-3-[(2,4-dihydroxyphenyl)methylidene]amino-
thiourea (3c): Yellow solid (51 mg, 0.16 mmol, 65%): Rf =0.18
ChemMedChem 2011, 6, 1107 – 1118
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1115

A.-K. Duhme-Klair et al.
MED
10.17 (bs, 1H, OH/NH), 13.21 ppm (bs, 1H, NH); 13C{1H} NMR
(100 MHz, [D6]DMSO): d=37.4 (CH2), 102.5, 108.5, 110.6, 127.3,
128.6, 129.3, 137.0, 145.9 (CHN), 159.2 (4Car), 161.7 (4Car),
193.8 ppm (C=S); FTIR (KBr): n˜max =3346 (OꢀH/NꢀH, br), 3104 (Cꢀ
Har, m), 2972 (CꢀHalkyl, m), 1627 (C=N, sh, s), 1506 (C=C, sh), 1238,
1219 (CꢀO, sh), 1027 cmꢀ1 (C=S, sh, s); HRMS (ESI): m/z [M+H]+
calcd for C15H15N2O2S2: 319.0569, found: 319.0566 (difference=
0.4 mDa); Anal. calcd for C15H14N2O2S2·0.86CH3OH: C 55.06, H 5.08,
N 8.10, found: C 55.06, H 5.04, N 7.65.
1163 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for
C15H13N4OS: 297.0805, found: 297.0804 (difference= 0.0 mDa);
Anal. calcd for C15H12N4OS·0.2H2O: C 60.06, H 4.17, N 18.68, found:
C 60.01, H 4.22, N 18.41.
4-Hydroxy-[(4-hydroxyphenyl)methylidene]benzohydrazide (4e):
Beige solid (0.20 g, 0.76 mmol, 76%): Rf =0.23 (MeOH/CHCl3 1:8);
mp: >2508C (lit. 2738C);[39] 1H NMR (400 MHz, [D6]DMSO): d=6.84
(bt, 4H, J=8.2 Hz), 7.54 (d, 2H, J=8.0 Hz), 7.79 (d, 2H, J=8.8 Hz),
8.32 (s, 1H, CHN), 10.00 (bs, 2H, OH), 11.45 ppm (s, 1H, NH); 13C{1H}
NMR (100 MHz, [D6]DMSO): d=115.7, 115.0, 124.1 (4Car), 125.5
(4Car), 128.8, 129.6, 147.2 (CHN), 159.3 (4Car), 160.6 (4Car),
162.6 ppm (C=O); FTIR (KBr): n˜max =3362 (OꢀH, br, m), 3186 (NꢀH,
br, m), 3038 (CꢀHar, m), 1609 (C=O, s), 1584 (C=N, sh), 1509 (C=C,
sh, s), 1237 cmꢀ1 (CꢀO, m); HRMS (ESI): m/z [M+H]+ calcd for
C14H13N2O3: 257.0921, found: 257.0918 (difference= 0.3 mDa);
Anal. calcd for C14H12N2O3·0.4H2O: C 63.82, H 4.90, N 10.63, found:
C 63.87, H 4.98, N 10.36.
3-[(4-Hydroxyphenyl)-methylidene]amino-1-phenylthiourea (4a):
Pale yellow crystalline solid (0.20 g, 0.74 mmol, 54%): Rf =0.71
(MeOH/CHCl3 1:8); mp: 195–1968C (lit. 188–1908C);[41] 1H NMR
spectroscopic data are in agreement with the reported values;[41]
13C{1H} NMR (100 MHz, [D6]DMSO): d=115.6, 125.0 (4Car), 125.2,
125.7, 128.0, 129.5, 139.2 (4Car), 143.3 (CHN), 159.5 (4Car),
175.4 ppm (C=S); FTIR (KBr): n˜max =3293 (OꢀH, m), 3210 (NꢀH, br),
3168 (CꢀHar, m), 2798 (CꢀHalkyl, w), 1606 (C=N, sh), 1541, 1515 (C=C,
sh, s), 1196 (CꢀO, sh, s), 1063 cmꢀ1 (C=S, sh, w); HRMS (ESI): m/z
[M+H]+ calcd for C14H14N3OS: 272.0852, found: 272.0857 (differ-
ence= ꢀ0.4 mDa); Anal. calcd for C14H13N3OS: C 61.97, H 4.83, N
15.49, found: C 61.56, H 4.85, N 15.77.
(4-Hydroxybenzaldehyde)dithiocarbazate (4 f): Bright yellow solid
(0.13 g, 0.42 mmol, 43%): Rf =0.50 (MeOH/CHCl3 1:8); mp: 176–
1
1778C; H NMR (400 MHz, [D6]DMSO): d=4.47 (s, 2H), 6.82 (d, 2H,
J=8.6 Hz), 7.26 (t, 1H, J=7.2 Hz), 7.33 (t, 2H, J=7.2 Hz), 7.42 (d,
2H, J=7.2 Hz), 7.53 (d, 2H, J=8.6 Hz), 8.14 (s, 1H, CHN), 10.09 (bs,
1H, OH), 13.22 ppm (bs, 1H, NH); 13C{1H} NMR (100 MHz,
[D6]DMSO): d=37.5 (CH2), 115.9, 124.2 (4Car), 127.2, 128.5, 129.3,
1-(4-Chlorophenyl)-3-[(4-hydroxyphenyl)methylidene]amino-
thiourea (4b): Pale yellow solid (0.19 g, 0.60 mmol, 58%): Rf =0.57
(MeOH/CHCl3 1:8); mp: 199–2008C (lit. 165–1668C);[42] 1H NMR
(400 MHz, [D6]DMSO): d=6.81 (d, 2H, J=8.2 Hz), 7.41 (d, 2H, J=
8.4 Hz), 7.63 (d, 2H, J=8.4 Hz), 7.73 (d, 2H, J=8.2 Hz), 8.07 (s, 1H,
CHN), 9.95 (s, 1H, OH/NH), 10.02 (s, 1H, OH/NH), 11.74 ppm (s, 1H,
NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=115.6, 124.9 (4Car),
127.2, 127.9, 129.1 (4Car), 129.5, 138.2 (4Car), 143.7 (CHN), 159.6
(4Car), 175.4 ppm (C=S); FTIR (KBr): n˜max =3301 (OꢀH, br), 3172 (Nꢀ
H, br), 2986 (CꢀHar, w), 1602 (C=N, sh), 1555, 1515, 1490 (C=C, sh,
s), 1269 (CꢀO, sh, m), 1197 (C=S, sh, m), 1093 cmꢀ1 (CꢀCl, sh);
HRMS (ESI): m/z [M+H]+ calcd for C14H13N3OSCl: 306.0462, found:
129.4, 147.1 (CHN), 160.2 (4Car), 195.2 ppm (C=S); FTIR (KBr): n˜max
=
3354 (OꢀH, br), 3127 (NꢀH, br), 3029 (CꢀHar, w), 2964 (CꢀHalkyl, w),
1607 (C=N, sh, s), 1577, 1496 (C=C, sh, s), 1200 (CꢀO, s), 1025 cmꢀ1
(C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for C15H15N2OS2:
303.0620, found: 303.0628 (difference= ꢀ0.8 mDa); Anal. calcd for
C15H14N2OS2: C 59.58, H 4.67, N 9.26, found: C 59.34, H 4.66, N 9.23.
3-[(4-Methoxyphenyl)methylidene]amino-1-phenylthiourea (5a):
White crystalline solid (0.20 g, 0.70 mmol, 85%): Rf =0.48 (MeOH/
CHCl3 1:15); mp: 179–1808C (lit. 1798C);[42] 13C{1H} NMR (100 MHz,
[D6]DMSO): d=55.3 (CH3O), 114.1, 125.2, 125.7, 126.6, 128.0 (4Car),
129.3, 139.1 (4Car), 142.9 (CHN), 160.9 (4Car), 175.6 ppm (4Car);
HRMS (ESI): m/z [M+H]+ calcd for C15H16N3OS: 286.1009, found:
286.1008 (difference= 0.1 mDa); Anal. calcd for C15H15N3OS: C
63.13, H 5.30, N 14.72, found: C 62.99, H 5.29, N 14.58. Spectro-
scopic data are in agreement with reported values.[43]
306.0459
(difference=
0.3 mDa);
Anal.
calcd
for
C14H12N3OSCl·0.45H2O: C 53.57, H 4.14, N 13.38, found: C 53.59, H
4.27, N 13.13.
1-(4-Cyanophenyl)-3-[(4-hydroxyphenyl)methylidene]amino-
thiourea (4c): Bright yellow solid (0.10 g, 0.35 mmol, 72%): Rf =
0.67 (MeOH/CHCl3 1:8); mp: 215–2168C; 1H NMR (400 MHz,
[D6]DMSO): d=6.82 (d, 2H, J=8.8 Hz), 7.73 (d, 2H, J=8.4 Hz), 7.81
(d, 2H, J=8.8 Hz), 7.98 (d, 2H, J=8.8 Hz), 8.10 (s, 1H, CHN), 10.19
(bs, 1H, OH), 11.93 ppm (bs, 1H, NH); 13C{1H} NMR (100 MHz,
[D6]DMSO): d=106.5 (4Car), 115.6, 119.0 (4Car), 124.7 (4Car), 124.7,
129.7, 132.2, 143.6 (4Car), 144.4 (CHN), 159.8 (4Car), 174.7 ppm (C=
S); FTIR (KBr): n˜max =3450 (OꢀH, br), 3294 (NꢀH, br, m), 2231 (CꢅN,
sh), 1608 (C=N, sh), 1581, 1535, 1516 (C=C, sh, s), 1277 (CꢀO, sh),
1187 cmꢀ1 (C=S, sh); HRMS (ESI): m/z [M+H]+ calcd for
C15H13N4OS: 297.0805, found: 297.0800 (difference= 0.5 mDa);
Anal. calcd for C15H12N4OS·1H2O: C 57.31, H 4.49, N 17.82, found: C
57.24, H 4.48, N 18.14.
(5-Bromo-2-hydroxybenzaldehyde)dithiocarbazate (6a): Pale
yellow solid (0.34 g, 0.89 mmol, 90%): Rf =0.52 (MeOH/CHCl3 1:15);
mp: 201–2028C (lit. 1958C);[44] UV/vis (DMSO): lmax (e)=313
(11205), 329 (11776), 366 (21654), 381 nm (20076); Anal. calcd for
C15H13N2OS2Br: C 47.25, H 3.44, N 7.35, found: C 47.19, H 3.43, N
7.24. Spectroscopic data are in agreement with reported values.[45]
General procedure for [MoO2(L)MeOH] synthesis (L1 =3b, L2 =
3 f, L3 =6a): [MoO2(acac)2] was synthesised according to a litera-
ture procedure.[46] Equimolar amounts of the ligand and [MoO2-
(acac)2] were refluxed for 2 h in MeOH. Following partial evapora-
tion of the solvent, the MoVI complex was isolated, washed with
MeOH and dried in vacuo. Diffraction quality crystals were grown
in MeOH/pentane or obtained after slow evaporation of a solution
of the complex in MeOH.
1-(3-Cyanophenyl)-3-[(4-hydroxyphenyl)methylidene]amino-
thiourea (4d): Yellow solid (58 mg, 0.19 mmol, 77%): Rf =0.60
1
(MeOH/CHCl3 1:8); mp: 193–1948C; H NMR (400 MHz, [D6]DMSO):
d=6.82 (d, 2H, J=8.4 Hz), 7.56 (t, 1H, J=8.0 Hz), 7.64 (d, 1H, J=
8.0 Hz), 7.74 (d, 2H, J=8.6 Hz), 8.00 (d, 1H, J=8.2 Hz), 8.09 (s, 1H),
8.11 (s, 1H, CHN), 9.97 (s, 1H, OH/NH), 10.15 (s, 1H, OH/NH),
11.87 ppm (s, 1H, NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=110.7
(4Car), 115.6, 118.6 (CꢅN), 124.8 (4Car), 128.5, 129.3, 129.6, 130.2,
140.1 (4Car), 144.1 (CHN), 159.7 (4Car), 175.3 ppm (C=S); FTIR (KBr):
n˜max =3450 (OꢀH, br), 3273 (NꢀH, m), 3100 (NꢀH, br), 2231 (CꢅN,
sh, m), 1607 (C=N, sh, m), 1543, 1515 (C=C, sh, s), 1276 (CꢀO, sh),
MoO2(L1)MeOH (L1 =3b): Dark red microcrystalline solid (33 mg,
0.070 mmol, 45%): Rf =0.50 (MeOH/CHCl3 1:8); mp: >2508C (dec);
1H NMR (400 MHz, [D6]DMSO): d=3.17 (d, 3H, J=5.3 Hz, CH3OH),
4.10 (q, 1H, J=5.1 Hz, CH3OH), 6.26 (d, 1H, J=2.2 Hz), 6.48 (dd,
1H, J=8.7, 2.2 Hz), 7.32 (d, 2H, J=8.0 Hz), 7.50 (d, 1H, J=8.7 Hz),
7.76 (d, 2H, J=8.0 Hz), 8.66 (s, 1H, CHN), 9.63 (bs, 1H, OH/NH),
10.44 ppm (bs, 1H, OH/NH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=
1116
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1107 – 1118

Xanthine Oxidase Inhibitors
48.6 (CH3OH), 104.1, 109.8, 112.9 (4Car), 120.7, 125.5 (4Car), 128.4,
135.7, 139.8 (4Car), 155.2 (CHN), 159.1 (4Car), 160.7 (4Car), 163.7 ppm
(CꢀS); FTIR (KBr): n˜max =3394 (MeOꢀH, br), 3364 (NꢀH, br), 1596 (C=
N, s), 1516 (C=C, s), 1225 (CꢀO, sh), 932 (O=Mo=Osymm, sh),
889 cmꢀ1 (O=Mo=Oasymm, sh); UV/vis (DMSO): lmax (e)=266 (28546),
340 (25460), 428 nm (5580); Anal. calcd for C15H14N3O5SClMo: C
37.55, H 2.94, N 8.76, found: C 37.47, H 2.28, N 9.17. Diffraction
quality dark red needles were obtained from MeOH/pentane.
Xanthine Oxidase activity assay:[30] The assay solution contained
xanthine (1 mL of 0.15 mm in H2O) and test compounds at various
concentrations made up to 3 mL with 50 mm potassium phosphate
monobasic buffer (pH 7.5). The reaction was initiated by addition
of the appropriate amount of xanthine oxidase (XO) to give rise to
a reaction rate for the control reaction of 0.03–0.04 AUminꢀ1. The
absorbance was monitored every minute for 10 min on a Jenway
6705 UV/Vis spectrophotometer with a thermostated cuvette
holder set at 258C. The inhibitors and positive control (allopurinol)
were dissolved in DMSO, with the final DMSO concentration main-
tained at 1% (v/v) of the total assay mixture. At this level, the
DMSO had no appreciable effect on XO activity. The IC50 value was
determined from analysis of dose–inhibition curves of inhibition
(%) versus log[inhibitor] using Origin 6.1 software, with each assay
performed in duplicate and the IC50 value quoted as the mean ꢂ
standard deviation (SD).
MoO2(L2)MeOH (L2 =3 f): Dark red crystalline solid (91 mg,
0.20 mmol, 62%): Rf =0.54 (MeOH/CHCl3 1:8); mp: >2508C (dec);
1H NMR (400 MHz, [D6]DMSO): d=3.15 (s, 3H, CH3OH), 4.09 (bs, 1H,
CH3OH), 6.30 (d, 1H, J=2.0 Hz), 6.52 (dd, 1H, J=8.6, 2.0 Hz), 7.56
(dd, 3H, J=8.6, 2.0 Hz), 7.95 (d, 2H, J=8.6 Hz), 8.78 (s, 1H, CHN),
10.64 ppm (s, 1H, OH); 13C{1H} NMR (100 MHz, [D6]DMSO): d=48.6
(CH3OH), 104.6, 110.4, 112.5 (4Car), 129.0, 129.1 (4Car), 129.5, 136.0,
136.4, 156.0 (CHN), 161.4 (4Car), 164.5 (4Car), 166.2 ppm (CꢀO); FTIR
(KBr): n˜max =3500 (OꢀH, br), 3199 (OꢀH, br), 1605 (C=N, sh, s), 1550
(C=C, sh, s), 1237 (CꢀO, sh), 932 (O=Mo=Osymm), 907 cmꢀ1 (O=Mo=
Oasymm); UV/vis (DMSO): lmax (e)=326 (23590), 421 nm (7957); HRMS
(ESI): m/z [MꢀMeOH+H]+ calcd for C14H10N2O5ClMo: 418.9323,
found: 418.9311 (difference= 1.2 mDa); Anal. calcd for
C15H13N2O6ClMo·0.25H2O: C 39.76, H 3.00, N 6.18, found: C 39.75, H
3.51, N 5.77. Diffraction quality dark red needles were obtained
from MeOH/pentane.
Acknowledgements
We thank the UK Biotechnology and Biological Sciences Research
Council (BBSRC) and Sareum Ltd (Cambridge, UK) for financial
support, Dr. Adrian Whitwood, Ms. Abeda Jamadar, Ms. Emma
Dux and Mr. Robert Thatcher for crystal structure determination,
and Dr. Nicola J. Smith for S-benzyldithiocarbazate synthesis.
[MoO2(L3)MeOH] (L3 =6a): Bright orange solid (0.15 g, 0.29 mmol,
89%): Rf =0.48 (MeOH/CHCl3 1:15); mp: 218–2198C; 1H NMR
(400 MHz, [D6]DMSO): d=3.17 (d, 3H, J=5.1 Hz, CH3OH), 4.11 (q,
1H, J=5.1 Hz, CH3OH), 4.44 (s, 2H), 6.93 (d, 1H, J=8.8 Hz), 7.27 (t,
1H, J=7.3 Hz), 7.34 (t, 2H, J=7.3 Hz), 7.47 (d, 2H, J=7.3 Hz), 7.70
(dd, 1H, J=8.8, 2.5 Hz), 8.06 (d, 1H, J=2.5 Hz), 9.01 ppm (s, 1H,
CHN); 13C{1H} NMR (100 MHz, [D6]DMSO): d=37.3 (CH2), 48.6
(CH3OH), 112.0 (4Car), 120.8, 121.7 (4Car), 127.4, 137.0, 137.9, 136.7,
137.0, 137.9 (4Car), 158.7 (4Car), 158.8 (CHN), 171.0 ppm (C=S); FTIR
(KBr): n˜max =3337 (OꢀH, br), 1591 (C=N, sh, s), 1541 (C=N, sh, s),
1484, 1465 (C=C, sh, s), 1274 (CꢀO, sh, s), 937 (O=Mo=Osymm),
887 cmꢀ1 (O=Mo=Oasymm); Anal. calcd for C16H15N2O4S2BrMo: C
35.64, H 2.80, N 5.19, found: C 35.86, H 2.80, N 5.00; UV/vis
(DMSO): lmax (e)=329 (16656), 362 (13484), 380 (10051), 438 nm
(1033). Diffraction quality orange needles were obtained from slow
evaporation of a solution of [MoO2(L3)MeOH] in MeOH.
Keywords: copper complexation · inhibitors · molybdenum
complexation · schiff bases · xanthine oxidase
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Synthesis of [Cu(L5)H2O]: A solution of 3b (49 mg, 0.15 mmol) in
MeOH (5 mL) was treated dropwise with a solution of Cu(OAc)2
(28 mg, 0.15 mmol) in MeOH (2 mL). The mixture was refluxed for
4 h. [Cu(L5)H2O] precipitated out as
a brown solid (49 mg,
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NꢀH, br), 1599 (C=N, sh, s), 1522 (C=C, sh, s), 1221 cmꢀ1 (CꢀO, sh);
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ChemMedChem 2011, 6, 1107 – 1118
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ˇ
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Published online on April 8, 2011
1118
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1107 – 1118