Biological studies of organotin(IV) complexes with 2-mercaptopyrimidine

Article abstract of DOI:10.1007/s11172-007-0115-z

The known organotin(IV) complexes with 2-mercaptopyrimidine (L) [Me ₂SnL₂] (1), [Buⁿ ₂SnL₂] (2), [Ph₂SnL₂] (3), and [Ph₃SnL] (4) were synthesized using a new approach. The effect of the synthesized compounds on peroxidation of fatty acids (oleic and linoleic) was studied. Complexes 1-4 promote the peroxidation of oleic acid. Their effect on the enzymatic peroxidation of linoleic acid with lipoxygenase was compared with that of cisplatin and in vitro cytoxicity against sarcoma cancer cells was determined. The antiproliferative effect of complexes 2-4 was demonstrated.

Full text of DOI:10.1007/s11172-007-0115-z

Russian Chemical Bulletin, International Edition, Vol. 56, No. 4, pp. 767—773, April, 2007
767
Biological studies of organotin(IV) complexes
with 2ꢀmercaptopyrimidine
a
a
b

b
M. N. Xanthopoulou,a S. K. Hadjikakou,a N. Hadjiliadis, N. Kourkoumelis, E. R. Milaeva, Yu. A. Gracheva,


V. Yu. Tyurin,b I. Verginadis,c S. Karkabounas,c M. Baril,d and I. S. Butlerd
аInorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina,
45110 Ioannina, Greece.
Fax: +30 26510 44831. Eꢀmail: shadjika@uoi.gr, nhadjis@uoi.gr
bDepartment of Chemistry, M. V. Lomonosov Moscow State University,
1 Leninskie Gory, 119992 Moscow, Russian Federation.
Fax: +7 (495) 939 5546. Eꢀmail: milaeva@org.chem.msu.ru
cDepartment of Experimental Physiology, Medical School, University of Ioannina,
45110 Ioannina, Greece
dDepartment of Chemistry, McGill University,
801 Sherbrooke, Montreal Quebec, Canada H2A 2K6.
Fax: +1 (514) 398 6935. Eꢀmail: Ian.Butler@McGill.CA
The known organotin(IV) complexes with 2ꢀmercaptopyrimidine (L) [Me2SnL2] (1),
[Bun2SnL2] (2), [Ph2SnL2] (3), and [Ph3SnL] (4) were synthesized using a new approach. The
effect of the synthesized compounds on peroxidation of fatty acids (oleic and linoleic) was
studied. Complexes 1—4 promote the peroxidation of oleic acid. Their effect on the enzymatic
peroxidation of linoleic acid with lipoxygenase was compared with that of cisplatin and in vitro
cytoxicity against sarcoma cancer cells was determined. The antiproliferative effect of comꢀ
plexes 2—4 was demonstrated.
Key words: bioinorganic chemistry, complexes, organotin(IV) complexes, 2ꢀmercaptoꢀ
pyrimidine, lipoxygenases, antitumor activity.
Biological activity of organotin(IV) compounds has
been verified many years ago.1—3 It has been reported that
a significant role in its manifestation belongs to the bindꢀ
ing of the organotin compounds by thiol groups in bioꢀ
logical systems.4 Although many organotin derivatives are
known to have an efficient antitumor activity,5 its precise
mechanism is still a matter of investigation.1,2,6
It is known that many drugs that inhibit the growth of
tumor cells interact with bases and/or nucleotides of the
double helix of DNA or with metalloenzymes that are
necessary for the rapid growth of malignant cells.7,8 Multiꢀ
nuclear 1D and 2D NMR spectroscopic studies of the
interaction of the organotin(IV) compounds containing
the Et2Sn moiety with 5´ꢀCMP, 5´ꢀdCMP, 5´ꢀUMP,
5´ꢀIMP, and 5ꢀGMP in aqueous solutions showed that at
low pH values (<4) the tin atoms bind to the phosphate
groups of the nucleotides.9 At pH > 9.5 the Et2SnIV groups
react with the O(2´) and O(3´) atoms of the carbohydrate
unit of the nucleotide, while no evidence for the interacꢀ
tion of this fragment with nucleotides was found9 at the
intermediate pH values (4.0—9.5).
These results may indicate that the anticancer activity
of the organotin compounds is possibly related not to the
interaction with DNA at physiological pH values.5b The
present study is devoted to the investigation of the interꢀ
action of organotin(IV) derivatives with the enzyme
lipoxygenase.10 This enzyme catalyzes the oxidation of
arachidonic acid to leukotrienes, which is essential for the
cell life.11 Prostaglandines, the final products of the meꢀ
tabolism of arachidonic acid, play an important role in
the tumorigenesis as angiogenesis factors.12a Linoleic acid
(found in beef and dairy products) was proven to be a
potential mutagen inhibitor.12b
In the present study, for the four known13 organotin(IV)
complexes with 2ꢀmercaptopyrimidine [Me2SnL2] (1),
[Bun SnL2] (2), [Ph2SnL2] (3), and [Ph3SnL] (4), we
2
found a correlation between their activity in lipoxygenase
inhibition and antitumor activity. In vitro cytotoxicity
against sarcoma cancer mesenchymal tissue from
the Wistar rat, whose carcinogenesis was induced by
benzo[a]pyrene,14 was studied for compounds 1—4. The
antitumor activity of the complexes was studied in relaꢀ
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 737—743, April, 2007.
1066ꢀ5285/07/5604ꢀ0767 © 2007 Springer Science+Business Media, Inc.

768
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 4, April, 2007
Xanthopoulou et al.
R´OOH/mmol
1400
tion to the mechanism of this inhibitory activity towards
linoleic acid oxidation to hydroxylinoleic acid by lipoxyꢀ
genase.11b,12a A correlation between the antitumor activꢀ
ity and lipoxygenase inhibition by the studied compounds
was found.
1
2
3
a
1200
1000
800
L1
Results and Discussion
1. General aspects. A new approach was used to synꢀ
thesize organotin(IV) complexes with 2ꢀmercaptopyrimꢀ
idine 1—4 of the general formula RnSnL4–n (cf. Ref. 13).
A solution of RnSnCl4–n in methanol was mixed with an
aqueous solution of 2ꢀmercaptopyrimidine containing an
equimolar amount of potassium hydroxide (Scheme 1).
600
400
200
0
2
4
6
τ•10–3/s
R´OOH/mmol
160
Scheme 1
2
1
140
120
100
80
b
3
L1
60
40
20
0
3
6
9
12
τ•10–3/s
Fig. 1. Kinetic curves of R´OOH accumulation during the oxiꢀ
dation of oleic acid at 65 (a) and 37 °C (b) without additives (L1)
and in the presence of additives (1 mmol L–1): Me2SnL2 (1),
Bun2SnL2 (2), and Ph2SnL2 (3).
R = Me (1), Bun (2), Ph (3)
1
The H, 13C, and 119Sn NMR and FTꢀIR spectra of
these compounds are identical with those reported earꢀ
lier.13 The structures of the complexes R2SnL2 (1—3)13a,c
and Ph3SnL (4) were also confirmed by the Xꢀray diffracꢀ
tion data.
2. Kinetic studies of the peroxidation of oleic acid. The
data on the influence of organotin complexes 1—3 on the
accumulation level of oleic acid hydroperoxides are preꢀ
sented in Fig. 1.
The farꢀIR spectra of complexes 1—4 contain vibraꢀ
tional bands at 380—395 cm–1 assigned to the ν(Sn—S)
stretching vibrations (see Refs 13, 15, and 16), while bands
at 251—249 and 205—192 cm–1 are assigned to ν(Sn—N)
vibrations. The vibrations of the metal—ligand bond in
the Raman spectra are interpreted as follows: the bands at
212—230 cm–1 are due to the ν(Sn—N) stretching vibraꢀ
tions, and the bands at 393— 398 cm–1 are attributed to
the ν(Sn—S) vibrations. The cisꢀN2ꢀ and cisꢀS2ꢀcoordiꢀ
nation of the Sn4+ ion in complexes 1, 3, and 4 (see
Refs 13a,c) is supported by the activity of the Sn—S and
Sn—N bonds in both Raman and IR spectra. Thus, comꢀ
plex 2 has analogous cisꢀN2ꢀ and cisꢀS2ꢀgeometry.
The effect of compounds 1—3 is manifested as an
increase in the content of hydroperoxide R´OOH in the
initial period (see Fig. 1, curves 1—3). The data presented
demonstrate the effect of the nature of the organic group
on the activity of the corresponding complexes R2SnL2.
The initial rate constants for R´OOH formation at 65 °C
are 3.7•10–4 s–1 and 7.1•10–4, 6.4•10–4, and 5.9•10–4 s–1
for oleic acid without additives and with addition of comꢀ
plexes 1, 2, and 3, respectively. Analogous values at 37 °C
are lower because of a decrease in the rate of peroxidation
of the substrate, being 1.9•10–4, 2.1•10–4, 2.0•10–4, and
1.6•10–4 s–1 for oleic acid without additives and with
addition of complexes 1, 2, and 3, respectively.

Organotin(IV) 2ꢀmercaptopyrimidine complexes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 4, April, 2007
769
R´OOH/mmol
R´OOH (%)
a
143.7
1600
4
a
140
1400
134.8
129.4
3
1200
130
L1
1000
120
800
600
400
200
108.0
110
100.0
100
90
0
2
4
6
8
τ•10–3/s
Control Me2SnL2 Bun2SnL2 Ph2SnL2 Ph3SnL
R´OOH (%)
R´OOH/mmol
4
162.5
158.8
b
160
140
120
100
80
160
140
120
100
3
b
137.3
L1
108.7
100.0
60
40
Control Me2SnL2 Bun2SnL2 Ph2SnL2 Ph3SnL
20
Fig. 3. Relative content of R´OOH in oleic acid (% relative to
standard) in the presence of 1 М Me2SnL2, Bun2SnL2, Ph2SnL2,
and Ph3SnL at 65 (a) and 37 °C (b) after storage for 2.5 (a)
and 5 h (b).
0
5
10
15
τ•10–3/s
Fig. 2. Kinetic curves of R´OOH accumulation during the oxiꢀ
dation of oleic acid at 65 (a) and 37 °C (b) without additives (L1)
and in the presence of additives (1 mmol L–1): Ph2SnL2 (3) and
Ph3SnL2 (4).
The formation of the radicals R• and/or
•
[R´OORnSnL4–n
]
can be a reason for the initiation
The number of organic substituents R in compounds
RnSnL4–n also affects the effectiveness of the complexes
as promoters of oxidation, which was observed in the case
of the phenylꢀcontaining derivatives (Fig. 2). Complex 4
exhibits a higher activity at 65 °C (k = 6.9•10–4 s–1) in the
initiation of peroxidation compared to its diphenyl anaꢀ
log. However, the difference in the k values for a similar
pair of the complexes at 37 °C is lower, being 1.6•10–4
and 1.7•10–4 s–1 for Ph2SnL2 and Ph3SnL, respectively.
The comparative data for the total content of R´OOH
in the oxidation of oleic acid in the presence of RnSnL4–n
are presented in Fig. 3. A considerable increase in the
R´OOH content is observed as compared to autooxidation
of oleic acid.
The binding of the organotin moieties to the sulfur
atom of 2ꢀmercaptopyrimidine prevents possible interacꢀ
tion of the tin atoms with the free SH groups of the proꢀ
teins. However, the complexes RnSnL4–n containing the
Sn—C σꢀbond are reactive with respect to peroxyl radiꢀ
cals R´OO• formed from oleic acid.17a
of the radicalꢀchain process of substrate oxidation.17b
The halfꢀlife period of the radicals formed can affect
the degree of promotion,10 i.e., the more stable the
Cꢀcentered radicals formed in an oxidative medium
•
(Me• < MeCH2CH2CH2 < Ph•),17b the more efficient
promoters of oxidation the corresponding complexes.10 It
has previously been shown10 that an analogous effect is obꢀ
served in the case of organotin complexes with 5ꢀchloroꢀ
2ꢀmercaptobenzothiazole where the Ph2Sn derivatives
were more active than the corresponding Me2Sn comꢀ
plex. However, this effect is not observed for comꢀ
pounds 1—4: complex 1 is more active than compound 3.
These results demonstrate the influence of the halfꢀlife
period of the radicals R• and [R´OORnSnL4–n]• on the
activity of the compounds studied.
3. Study of the peroxidation of linoleic acid with the
enzyme lipoxygenase in the presence of complexes 1—4.
The influence of complexes 1—4 on the oxidation of liꢀ
noleic acid by lipoxygenase was studied in a wide concenꢀ
tration interval. The activity of the enzyme (A, %) in the

770
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 4, April, 2007
Xanthopoulou et al.
Activity (%)
binding site, resulting in a decrease in the rate of the
enzymatic reaction.
100
4. Docking study. In order to investigate the interacꢀ
tion of the complexes with the protein, we performed
computational molecular docking studies. The binding
energy (E) of linoleic acid as substrate S to its binding site
of the enzyme lipoxygenase (E) when complex ES is
formed is –7.89 kcal mol–1 (see Ref. 18a). The correꢀ
sponding binding energies of the complexes as inhibiꢀ
tors (I) in ESI are calculated to be –6.2 (1), –7.9 (3),
and –9.8 kcal mol–1 (4), respectively, whereas the
binding energies of EI are –6.3 (1), –7.8 (3), and
–8.3 kcal mol–1 (4). Thus, the E values for ES, EI, and
ESI suggest that complexes ESI and EI can be formed in
the case of inhibitors 3 and 4, whereas for complex 1 the
energies of the complexes ESI and EI are lower than
those o ES. These results can explain the low inhibitory
activity of complex 1 found.
It is known that the substrateꢀbinding site in comꢀ
plex ES is away from the active site of lipoxygenase
(d(Fe—C) = 19.7 Å, d(Fe—C) = 21.4 Å) and is located in
the region Ala76, Arg533, Asp760, Glu761, Leu249,
Lys110, Met15, Phe108, Ser759, and Val762 (see
Ref. 18a). The docked conformations of inhibitors 1, 3,
and 4 in complexes EI are located away from the active
site of lipoxygenase. The calculated distances between the
tin(IV) atom in the inhibitor and the iron ion in the active
site of lipoxygenase in complexes EI are 18.9 (1), 26.7 (3),
and 20.6 Å (4), respectively. The distances d(Fe—Sn) for
complexes ESI are 30.0 (1), 26.4 (3), and 23.8 Å (4),
respectively. The distance between the tin atom of the
inhibitor and the C(8) atom of linoleic acid are 39.4 (1),
40.3 (3), and 5.2 Å (4), respectively. The CPK presentaꢀ
tion of lipoxygenase is shown in Fig. 5, where the amino
acids of the binding site are represented with space filling
mode, while complexes 1 (Fig. 5, a) and 4 (Fig. 5, b)
docked into the binding site of the enzyme lipoxygenase
in the case of ESI are shown with balls and sticks. It is
shown that complex 1 is docked away from the binding
site of linoleic acid, whereas complex 4 is docked near
to it. The binding site of complex 4 is in the binding
pocket calculated for the inhibitors of lipoxygenase.10
5. Biological tests. Compounds 1—4 were tested for
antitumor activity against sarcoma cells (mesenchymal
tissue) from the Wistar rat induced by benzo[a]pyrene.
The inhibitory IC50 concentrations found for the tumor
cell after treatment with complexes 1—4 for 24 h are
20—60 (1), 0.65 (2), 1.0—2.0 (3), and 0.1 µmol L–1 (4),
respectively. These results demonstrate the antiproliferaꢀ
tive effect of complexes 2—4 and agree with the data on
the low inhibitory activity of complex 1 and high activity
of complex 4 towards lipoxygenase.
5
6
3
1
2
50
4
0
10
20
30
40
C/µmol L–1
Fig. 4. Activity of the enzyme during the oxidation of liꢀ
noleic acid in the presence of 2ꢀmercaptopyrimidine and comꢀ
plexes 1—4 (1—4), cisplatin (5), and LH (6).
presence of the complexes was calculated using a known
procedure.18a The results characterizing the inhibitory efꢀ
fect of complexes 1—4 at various concentrations are shown
in Fig. 4. The catalytic activity of lipoxygenase is shown
to decrease noticeably at low concentrations of comꢀ
plexes 2—4 (5—75 µmol L–1), whereas no significant deꢀ
crease in activity was observed for cisplatin or the free
ligand 2ꢀmercaptopyrimidine.
The concentrations at which the enzymatic activity is
inhibited by 50% (IC50) for complexes 1—4 are 61.3,
26.2, 20.5, and 16.9 µmol L–1, respectively, indicating a
higher inhibitory activity of complex 4 compared to those
of complexes 1—3. Complex 4 also exhibits a higher inꢀ
hibitory activity than compound 2, whereas complex 1
demonstrates a much lower activity. Thus, the inhibitory
activity of complexes 1—4 depends on both the number
and the nature of substituents bound to the tin(IV) atom.
To determine the inhibition type, we used the steadyꢀ
state kinetic method at various substrate concentrations
(0.01—0.1 mmol L–1) in the absence and in the presꢀ
ence (15 µmol L–1) of the complexes. The experimenꢀ
tal data were processed by the graphical method in
the Lineweaver—Burk coordinates (double reciprocal
method). The Michaelis constant (KM) for the enzyme
was 0.035 mmol L–1 at the Vmax of 27.5 mmol L–1 s–1
(see Ref. 18a), whereas KM was 0.025 and Vmax
=
24.3 mmol L–1 s–1 in the presence of 9 µL of DMSO. For
complexes 1—4, the apparent KM values are 0.054, 0.038,
0.049, and 0.130 mol L–1 with Vmax = 25.7, 19.1, 24.7,
and 22.1 mmol L–1 s–1, respectively. Thus, the comꢀ
pounds studied are characterized by a mixed type of enꢀ
zyme inhibition.18b This agrees with the known18a type of
inhibition for the complexes Ph2SnCl(L) (L is 2ꢀmerꢀ
captonicotinic acid), Ph2SnL2 (L is 2ꢀmercaptobenzoꢀ
thiazole), and Ph3SnL (L is 2ꢀmercaptobenzothiazole,
5ꢀchloroꢀ2ꢀmercaptobenzothiazole, and 2ꢀmercaptoꢀ
benzoxazole).10 According to this mechanism, enꢀ
zyme—inhibitor (EI) and enzyme—substrate—inhibitor
(ESI) complexes are formed.18b This occurs when the
inhibitor binds at the enzyme site away from the substrate
In summary, we studied the interaction of
organotin(IV) complexes with the metalloenzyme necesꢀ
sary for the rapid growth of malignant cells. Organotin(IV)

Organotin(IV) 2ꢀmercaptopyrimidine complexes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 4, April, 2007
771
nonꢀenzymatic peroxidation of oleic acid.10 The observed
regularity is not fulfilled in the case of complexes 1—4; in
this series, compound 1 promotes the nonꢀenzymatic
peroxidation of oleic acid but is a less pronounced inhibiꢀ
tor of lipoxygenase.
a
Experimental
Materials and instruments. Commercial solvents, 2ꢀmerꢀ
captopyrimidine, and organotin chlorides (Aldrich, Merck) were
used as received. Elemental analysis was carried out on a Carlo
Erba EA MODEL 1108 microanalyzer. IR spectra were recorded
on a Perkin—Elmer Spectrum GX FTꢀIR spectrometer in ranges
of 370—4000 cm–1 (KBr pellets) and 700—30 cm–1 (polyethylene
discs). Micro FTꢀRaman spectra were obtained in the nearꢀIR
laser radiation (Nd3+ : YAG, 1064.1 nm) with a resolution of
2.6 cm–1 on a Bruker IFSꢀ88 FTꢀIR/FRAꢀ105 Raman module
fitted with a Ge proprietary detector and coupled via two
photooptic cables (1.0 m) to a Nikon OptiphotꢀII optical miꢀ
croscope equipped with a Nikon 20X superꢀlongꢀrange objecꢀ
tive. NearꢀIR laser radiation was directed onto the sample
through the objective and collected along the same optical pathꢀ
way in a 180° back scattering mode. Samples were measured as
solid powders dispersed on a glass slide. 1H and 13C NMR specꢀ
tra were recorded on a Bruker AV 250 NMR instrument in
DMSOꢀd6 solutions. Mass spectra (electrospray ionization) of
solutions of the complexes in methanol were recorded on an
Agilent 1100 ESIꢀLCꢀMS spectrometer.
b
Influence of complexes 1—4 on the peroxidation
Fig. 5. Binding sites of inhibitors 1 and 4 in lipoxygenase.
Note. Fig. 5 is available in full color in the onꢀline version of the
journal (http://www.springerlink.com/issn/1573ꢀ9171/current)
and on the webꢀsite of the journal (http://russchembull.ru).
of oleic acid
Oleic acid (Sigma, 99%) was used. The monitoring of the
level of peroxidation of oleic acid as the substrate (R´H) was
performed by the determination of the total content of isomeric
hydroperoxides R´OOH using iodometric titration. Oleic acid
was oxidized using a temperatureꢀcontrolled cell (65 and 37 °C)
with an air flow with a constant rate of 2—4 mL min–1. Before
the addition of complexes, air was passed for 2 h. The concenꢀ
trations of compounds 1—4 were 1 mmol L–1, which is compaꢀ
rable with the initial concentrations of hydroperoxides. The apꢀ
proximation coefficients of the kinetic curves were 0.965—0.984,
and their shape for substrate oxidation in the presence of comꢀ
plexes 1—4 follows the exponential law. It turned out that the
initial rate of hydroperoxide accumulation obeys the pseudoꢀ
firstꢀorder equation.
complexes 1—4 inhibit the peroxidation of linoleic acid
with the enzyme lipoxygenase. The antiproliferative acꢀ
tivity of the complexes studied corresponds to the reguꢀ
larity of inhibition of the lipoxygenase activity. Comꢀ
plex 3 demonstrates the highest activity, and complex 4
(pentacoordinate tin) exhibits the high cytotoxic activity
exceeding that of cisplatin. These results can be comꢀ
pared with the earlier10 obtained data for the complexes
Ph3SnL (L is 2ꢀmercaptobenzothiazole (5), 5ꢀchloroꢀ2ꢀ
mercaptobenzothiazole (6), and 2ꢀmercaptobenzoxꢀ
azole (7)) and R2SnL2 (L is 2ꢀmercaptobenzothiazole,
R = Ph (8), Bun (9), and Me (10)).10 Complex 9 demonꢀ
strates the highest antitumor activity and the highest inꢀ
hibitory activity against lipoxygenase. The results of the
present study confirm the correlation earlier found10 beꢀ
tween the antitumor (antiproliferative) and inhibitory acꢀ
tivities of the organotin complexes with thioamides. The
correlation is independent of the geometry or the number
and type of the aryl and alkyl substituents at the tin(IV) ion
but is determined to a great extent by the ligand nature.
Complexes 8—10 were found to inhibit the lipoxygenase
activity with the same rate as that of promotion of the
Study of the mechanism of lipoxygenase inhibition
Preparation of solutions. A 0.2 M borate buffer (pH 9) was
used.10,18a A solution of linoleic acid was prepared as follows:
linoleic acid (0.05 mL, Merck) was dissolved in 95% ethanol
(0.05 mL), and water was added to a volume of 50 mL. The
solution prepared (5 mL) was added to the borate buffer (30 mL).
Lipoxygenase from soybean Type IꢀB as a lyophilized powder
(48 000 units mg–1, Aldrich) was used: a solution of 10 000 units
of enzyme for each cm3 of the borate buffer was prepared in an
ice bath.10,18a The enzyme was used in experiment in an amount
of 500 units per 3 mL of the reaction mixture. A lipoxygenase
unit induces an increase in the absorption at 234 nm by
0.001 per 1 min.

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Xanthopoulou et al.
Determination of the lipoxygenase activity. The enzyme activꢀ
stirred for 1 h and the precipitate that formed was filtered off,
washed with cold water (2—3 mL), and dried in vacuo over
silica gel.
Bis(pyrimidineꢀ2ꢀthiolato)dimethyltin(IV) (1). The yield
was 50%; m.p. 257—260 °C. Found (%): C, 32.56; H, 3.86;
N, 15.87; S, 17.12. C10H12N4S2Sn. Calculated (%): C, 32.37;
H, 3.26; N, 15.10; S, 17.28. MS (ESI), m/z: 394
([C10H12N2S2SnNa]+ 394).
Bis(pyrimidineꢀ2ꢀthiolato)diꢀnꢀbutyltin(IV) (2). The yield
was 61%; m.p. 159—163 °C. Found (%): C, 42.31; H, 5.62;
N, 12.55; S, 14.57. C16H24N4S2Sn. Calculated (%): C, 42.2;
H, 5.3; N, 12.3; S, 14.1. MS (ESI), m/z: 479
([C16H24N4S2SnNa]+ 480).
Bis(pyrimidineꢀ2ꢀthiolato)diphenyltin(IV) (3). The yield
was 42%; m.p. 217—222 °C. Found (%): C, 48.36; H, 3.29;
N, 11.31; S, 12.78. C20H16N4S2Sn. Calculated (%): C, 48.50;
H, 3.25; N, 11.31; S, 12.95. MS (ESI), m/z: 519
([C22H18N4S2SnNa]+ 520).
ity was monitored by UV spectroscopy. A solution of the enzyme
(0.05 mL) was added to a cell containing 2 mL of a solution of
linoleic acid and a necessary amount of the buffer. The temꢀ
perature of a solution of the inhibitor was maintained constant
at 25 °C. No preincubation of the enzyme with an inhibitor
solution was carried out. The enzyme activity was determined
from an increase in the intensity of the absorption at 234 nm at
25 °C (ε = 25 000 L mol–1 cm–1 10,18a
)
caused by the oxidation of
linoleic acid. Solutions of compounds 1—4 were prepared in
DMSO, their concentrations being 10–2, 5•10–3, 2.5•10–3, and
10–3 mol L–1. The substrate concentration was maintained conꢀ
stant at a level of 0.3 mmol L–1, and the amounts of solutions
of the buffer and inhibitor were varied according to the necesꢀ
sary final concentration of the inhibitor (5—60 µmol L–1 or
9—18 µL). The total volume of the reaction mixture was 2 mL.
Three series of experiments were carried out to determine
the KM and Vmax values. The inhibitor concentration was conꢀ
stant (15 µmol L–1 in DMSO). The substrate concentrations
(Pyrimidineꢀ2ꢀthiolato)triphenyltin(IV) (4). The yield
was 38%; m.p. 225—226 °C. Found (%): C, 57.43; H, 3.90;
N, 6.20; S, 6.00. C22H18N2SSn. Calculated (%): C, 57.3; H, 3.9;
N, 6.1; S, 6.9. MS (ESI), m/z: 485 ([C21H17N2SSnNa]+ 484).
were 0.01, 0.025, 0.05, 0.075, and 0.1 mmol L–1
.
Computational methods — docking study. The docking proꢀ
gram was ArgusLab.19a This program was also applied for visualꢀ
ization and molecular modeling of the compounds. The threeꢀ
dimensional coordinates of lipoxygenase was obtained through
the Internet at the Research Collaboratory for Structural
Bioinformatics (RCSB) Protein Data Bank.19b The coordinates
of the complexes were obtained by Xꢀray diffraction analysis.13
The ArgusLab program implements an efficient gridꢀbased dockꢀ
ing algorithm, which approximates an exhaustive search within
the free volume of the binding site cavity. The conformational
space was explored by the geometry optimization of the flexible
ligand (rings are treated as rigid) in combination with the increꢀ
mental construction of the ligand torsions. Thus, docking ocꢀ
curs between the flexible ligands and a rigid protein. The ligand
orientation is determined by a shape scoring function based on
the enhanced modification of the XScore(HP) method,19с and
the final positions are ranked by lowest interaction energy valꢀ
ues. Prior to docking, the ground state was optimized using the
structures of the complexes determined by Xꢀray diffraction and
using the PM3 parametrization19d implemented in the ArgusLab
program package to confirm no significant divergence in the
conformations of the complexes due to crystal packing effects.
Biological tests. Determination of in vitro cell toxicity as the
cell proliferation/survival (%) was measured at intervals of 24
and 48 h. Cell lines were maintained at 37 °C in Dulbecco´s
modified Eagle´s medium (DMEM) containing 10% fetal calf
serum (FCS) (incubated at 37 °C, 5% CO2). The initial concenꢀ
trations of the tested compounds were 1 µmol L–1. Exponenꢀ
tially growing cancer cells were seeded in six culture plates at
50 000 mL–1, stored for 24 h, and treated with solutions (100 µL)
of the tested compounds. After 24 and 48 h, the cells were
washed with a buffer solution, detached from the support with
trypsin—EDTA, and counted in a Bauer Slide hemocytometer.10
Synthesis of complexes 1—4. A 1 М solution of KOH (3 mL
(3 mmol) for 1—3 and 1.5 mL (1.5 mmol) for 4) was added to a
suspension of 2ꢀmercaptopyrimidine (0.224 g (2 mmol) to obꢀ
tain 1—3 and 0.112 g (1 mmol) for 4) in water (8 mL) as transꢀ
parent solutions. Then a solution of an organotin compound
(Me2SnCl2, 0.220 g (1 mmol) for 1, Bun2SnCl2, 0.304 g (1 mmol)
for 2, Ph2SnCl2, 0.334 g (1 mmol) for 3, and Ph3SnCl, 0.385 g
(1 mmol) for 4) in methanol (3 mL) was added. The mixture was
This work was financially supported by the Russian
Foundation for Basic Research (Project No. 06ꢀ03ꢀ
32731), the Russian Academy of Sciences (Program
No. 10 "Biomolecular and Medicinal Chemistry"), the
National Foundation of Greece and the European Social
Fund (Educational Program of the Hellenic Ministry of
Education), the program Heraklitos of the operational
program for education and initial vocation training of the
3rd community support framework of the Hellenic Minꢀ
istry of Education funded by 25% from national sources
and 75% from the European Science Foundation (ESF),
the Graduate Program in Bioinorganic Chemistry coorꢀ
dinated by Prof. N. Hadjiliadis, and the NATO Grant for
the exchange of scientists awarded to N. Hadjiliadis and
I. S. Butler.
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Received January 24, 2007;
in revised form April 13, 2007