Synthesis, X-ray structure, in vitro HIV and kinesin Eg5 inhibition activities of new arene ruthenium complexes of pyrimidine analogs

Abstract

ABSTRACT
Three new ruthenium(II)-arene complexes of the general formula [{(η6-p-cymene)Ru(L)}2](Cl)2), where L are monastrol (L1), ethyl 4-(3-hydroxyphenyl)-6-methyl-2-thioxo-pyrimidine-5-carboxylate (L2) or its 4-bromophenyl analog (L3), have been synthesized and characterized by elemental analysis, 1H, 13C, and 2-D NMR Spectroscopy.

The X-ray diffraction study of complex 1 showed the presence of a
dicationic diruthenium complex where two thioxopyrimidines act as
tridentate μ,κN:κ2S ligand, bridging two Ru ions through the pyrimidine
nitrogen and sulfur atoms. All new complexes were evaluated in vitro
for their antiviral activity against the replication of HIV-1 and HIV-
2 in MT-4 cells using MTT assay. Additionally, complexes 1–3 were
screened for their inhibitory activity against the ATPase enzyme
and the motor-protein Kinesin Eg5. Complex 1 was found to inhibit microtubule-stimulated ATPase activity of kinesin of IC50 = 30 μM(monastrol, IC50 = 10 μM).

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Synthesis, X-ray structure, in vitro HIV and kinesin
Eg5 inhibition activities of new arene ruthenium
complexes of pyrimidine analogs
Wasfi A. Al-Masoudi, Najim A. Al-Masoudi, Bernhard Weibert & Rainer
Winter
To cite this article: Wasfi A. Al-Masoudi, Najim A. Al-Masoudi, Bernhard Weibert & Rainer Winter
(2017) Synthesis, X-ray structure, in vitro HIV and kinesin Eg5 inhibition activities of new arene
ruthenium complexes of pyrimidine analogs, Journal of Coordination Chemistry, 70:12, 2061-2073,
DOI: 10.1080/00958972.2017.1334259
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JOURNAL OF COORDINATION CHEMISTRY, 2017
VOL. 70, NO. 12, 20612073
https://doi.org/10.1080/00958972.2017.1334259
Synthesis, X-ray structure, in vitro HIV and kinesin Eg5
inhibition activities of new arene ruthenium complexes of
pyrimidine analogs
Was A. Al-Masoudia, Najim A. Al-Masoudib, Bernhard Weibertc and Rainer Winterc
aDepartment of Physiology, Pharmacology and Chemistry, College of Veterinary, University of Basrah, Basrah,
Iraq; bDepartment of Chemistry, College of Science, University of Basrah, Basrah, Iraq; cDepartment of
Chemistry, University of Konstanz, Konstanz, Germany
ABSTRACT
Three new ruthenium(II)-arene complexes of the general formula
[{(η6-p-cymene)Ru(L)}2](Cl)2), where L are monastrol (L1), ethyl
4-(3-hydroxyphenyl)-6-methyl-2-thioxo-pyrimidine-5-carboxylate
(L2) or its 4-bromophenyl analog (L3), have been synthesized and
characterized by elemental analysis, 1H, 13C, and 2-D NMR spectroscopy.
The X-ray diraction study of complex 1 showed the presence of a
dicationic diruthenium complex where two thioxopyrimidines act as
tridentate μ,κN:κ2S ligand, bridging two Ru ions through the pyrimidine
nitrogen and sulfur atoms. All new complexes were evaluated in vitro
for their antiviral activity against the replication of HIV-1 and HIV-
2 in MT-4 cells using MTT assay. Additionally, complexes 1–3 were
screened for their inhibitory activity against the ATPase enzyme
and the motor-protein Kinesin Eg5. Complex 1 was found to inhibit
microtubule-stimulated ATPase activity of kinesin of IC50 = 30M
(monastrol, IC50=10M).
1. Introduction
In recent years, ruthenium-based complexes have emerged as promising antitumor and
antimetastatic agents with potential uses in platinum-resistant tumors [1, 2]. Clarke et al.
reported [Ru(NH3)5(purine)]3+ complexes capable of inhibiting DNA and protein synthesis
© 2017 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Anti-HIV activity;
pyrimidines; kinesin Eg5
inhibitors; arene ruthenium
complexes
ARTICLE HISTORY
Received 13 February 2017
Accepted10 May 2017
CONTACT Wasfi A. Al-Masoudi almasoudi59@yahoo.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/00958972.2017.1334259.

2062 W. A. ALMASOUDI ET AL.
in human nasopharyngeal carcinoma cells in vitro [3] and subsequently initiated interest in
ruthenium complexes as potential anticancer pharmaceuticals [4]. Mestroni et al. [5] devel-
oped six-coordinate Ru(II) complexes with dimethylsulfoxide and chloride ligands with anti-
cancer activity. In 1992, Tocher et al. [6] observed the cytotoxicity and anticancer activity of
[(η6-C6H6)RuCl2(metronidazole)]. Following this initial study, Dyson and Sadler [7, 8] have
focused on the antitumor and antimetastatic activity of arene ruthenium complexes such
as (η6-p-MeC6H4Pri)Ru(P-pta)](Cl2) (pta = 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1] decane),
termed RAPTA-C (Figure 1(A)) [9], and [(η6-C6H5Ph)Ru(κ2 N,N-en)Cl]+ PF6− (en = 1,2-ethylen-
ediamine) (Figure 1(B)) and related analogs [10]. Meanwhile, several laboratories have
reported the synthesis and anticancer activities of various kinds of organoruthenium com-
plexes. This eld has been reviewed competently by Süss-Fink [11, 12]. Currently, two ruthe-
nium compounds, NAMI-A [13, 14] (Figure 1(C)) and KP1019 [9, 15, 16] (Figure 1(D)), which
were developed by Sava’s and Keppler’s work have displayed favorable solubilities and anti-
cancer and antitumor activities in clinical trials [17]. Although RAPTA-C exhibits only a low
activity in vitro, it is very active in vivo, where it inhibits lung metastases in CBA mice. Like
NAMI-A, RAPTA-C is also an antimetastatic agent [7] and remains the best anticancer orga-
nometallic compound of this series. Recently, Meggers et al. [18] developed ruthenium anti-
cancer drugs designed to act as an analog of staurosporine, an eective organic drug protein
kinase inhibitor rather than cancer chelation therapy. Even more recently, Li and coworkers
have reported that some dinuclear half-sandwich ruthenium complexes with thiosemicar-
bazone ligands possess higher cytotoxicities against CNE-2, KB, and SGC-7901 cell lines than
cisplatin and oxaliplatin [19].
New mitotic targets such as kinesins have also attracted great interest worldwide as
potential candidates for a new generation of antiproliferative drugs [20, 21]. Most prominent
of these is the kinesin spindle protein (KSP), also known as Eg5, essential for the formation
and separation of bipolar spindles during human cell division [22]. The pyrimidine derivative
monastrol has been reported to be a specic KSP inhibitor under in vivo conditions that
inhibits spindle bipolarity and impairs centrosome separation [23].
Given the antitumor and antimetastatic properties of several arene half-sandwich ruthe-
nium complexes and the bioactivity of monastrol, we hoped that combining these two
ingredients would create complexes with favorable potency for biomedical applications. In
this work, we describe the preparation, characterization, and in vitro anti-HIV and kinesin
Eg5 inhibitory activity of ruthenium(II) complexes of some pyrimidines related to
monastrol.
Figure 1.Some promising anticancer (organo)ruthenium complexes.

JOURNAL OF COORDINATION CHEMISTRY 2063
2. Experimental
2.1. General procedures and physical measurements
Melting points are uncorrected and were measured on a Büchi melting point apparatus
B-545 (Büchi Labortechnik AG, Switzerland). Microanalytical data were obtained using a
Vario Elemental Analyzer (Shimadzu, Japan). NMR spectra were recorded on a Bruker Avance
III DRX 400 or a Bruker Avance DRX 600 spectrometer, with TMS as internal standard and on
the δ scale in ppm. Signal assignments for protons were performed by selective proton
decoupling or by COSY spectra. Heteronuclear assignments were veried by HSQC and
HMBC experiments. TLC plates 60 F254 were purchased from Merck. The chromatograms
were visualized under UV 254–366 nm and iodine (solvent: hexane-ethyl acetate 3:2). [(η6-
p-cymene)RuCl2]2 was prepared according to published procedure [24].
2.2. Synthesis of the ligands: general procedure for preparation of ethyl 4-aryl-6-
methyl-2-thioxo-pyrimidine-5-carboxylate derivatives (L1–L3)
To a stirred mixture of thiourea (1.00 mmol), substituted benzaldehydes E–G (Scheme 1)
(1.00 mmol), ethyl acetoacetate (130 mg, 1.00 mmol), and anhydrous cupric chloride
(10 mol%) were added. The mixture was heated at 80°C for 4 h under stirring. After the
reaction was completed (checked by TLC), a mixture of H2O:EtOH 8:5 (13 mL) was added and
the resulting slurry was stirred at 80°C until total dissolution. After being cooled to room
temperature, the reaction mixture was poured onto crushed ice (30 g) and stirred for
5–10 min. The separated solid was ltered under suction (water aspirator), washed with
ice-cold water (50 mL), and then recrystallized from hot ethanol to aord the pure
product.
2.3. Ethyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-pyrimidine-5-carboxylate
(monastrol) L1))
From 3-hydroxybenzaldehyde (E) (122 mg). Yield: 216 mg (74%); mp 185–186°C (Lit. [23, 25]
184–186°C).
For atom numbering refer to Figure 2.
Scheme 1.Synthesis of ethyl 4-aryl-6-methyl-2-thioxo-pyrimidine-5-carboxylates (L1–L3).

2064 W. A. ALMASOUDI ET AL.
2.4. Ethyl-4-(4-hydroxyphenyl)-6-methyl-2-thioxo-pyrimidine-5-carboxylate (L2)
[26]
From 4-hydroxybenzaldehyde (F) (122 mg). Yield: 231 mg (79%); mp 203–205°C. 1H NMR
(400 MHz, DMSO-d6): δ = 10.25 (s, 1H, NH), 9.59 (d, 1H, JH3,H4 = 5.2 Hz, NH), 7.01 (d, 2H,
J = 8.6 Hz, Harom), 6.71 (d, 2H, J = 8.6 Hz, Harom.), 5.06 (d, 1H, JH3,H4 = 5.2 Hz, H-4), 4.00 (q, 2H,
J = 7.1 Hz, CH2CH3), 2.28 (s, 3H, CH3pyrimid), 1.10 (t, 3H, J = 7.1 Hz, CH2CH3). 13C NMR (100 MHz,
DMSO-d6): δ = 14.0 (CH2CH3), 17.1 (C6-CH3), 58.5 (C4pyrimid), 59.5 (CH2CH3), 101.1 (C5pyrimid),
115.1 (C3arom+C5arom), 127.6 (C2arom + C6arom), 134.1 (C1arom), 144.5 (C6pyrimid), 146.9 (C4arom),
165.2 (C=O), 173.7 (C=S). MS (FAB): m/z 292 [M]
+
. Anal. Calcd for C
14
H
16
N
2
O
3
S⋅1/2H
2
O (301.36):
C, 55.79; H, 5.35; N, 9.29. Found: C, 55.71; H, 5.66; N, 9.49.
2.5. Ethyl-4-(4-bromophenyl)-6-methyl-2-thioxo-pyrimidine-5-carboxylate (L3)
From 4-bromobenzaldehyde (G) (184 mg). Yield: 249 mg (70%); mp 187–190°C. 1H NMR
(400 MHz, CDCl3): δ = 10.02, 9.27 (2xs, 2H, NH), 7.56 (d, 2H, J = 8.2 Hz, Harom.), 7.17 (d, 2H,
J = 8.2 Hz, Harom.), 5.37 (d, 1H, JH3,H4 = 5.1 Hz, H-4), 4.10 (q, 2H, J = 7.1 Hz, CH2CH3), 2.36 (s, 3H,
C6pyrimid.-CH3), 1.19 (t, 3H, J = 7.1 Hz, CH2CH3). 13C NMR (100 MHz, CDCl3): δ = 14.1 (CH2CH3),
18.5 (C6pyrimid.-CH3), 55.7 (C4pyrimid.), 60.9 (CH2CH3), 102.7 (C5pyrimid.), 122.5 (C4arom), 128.5
(C2arom + C6arom.), 132.1 (C3arom + C5arom), 141.3 (C1arom.), 142.7 (C6pyrimid.), 165.0 (C=O), 174.7
(C=S). MS (FAB): m/z 354/356 [M]+. Anal. Calcd for C14H15BrN2O2S⋅H2O (373.25): C, 45.04; H,
4.59; N, 7.50. Found: C, 45.52; H, 4.19; N, 7.68.
2.6. General procedure for synthesis of complexes 1–3
To a stirred solution of pyrimidine derivatives (L1–L3) (0.20 mmol) in dry THF (10 mL), solid
[(η6-p-cymene)RuCl2]2 (H) (62 mg, 0.10 mmol) and potassium carbonate (0.20 mmol) were
added. The solution was heated under reux for 6 h under nitrogen. After cooling, the mixture
was evaporated to dryness and the residue was washed with diethylether and dried under
vacuum to give the desired complex.
The atom numbering for the NMR characterization follows that provided in Scheme 2.
Figure 2.JC,H correlations in the HMBC NMR spectrum of ligand L3.

JOURNAL OF COORDINATION CHEMISTRY 2065
2.7. Synthesis of [{(η6-p-cymene)Ru(L)}2](Cl)2) (1)
From L1 (59 mg). Yield: 161 mg (68%) as brown crystalline solid; mp 206–209°C. 1H NMR
(600 MHz, CDCl3): δ = 9.48 (s, 1H, N3H), 7.32 (m, 4H, Harom.), 6.29 (m, 4H, Harom.), 6.03 (2xd, 8H,
J = 7.2 Hz, CH(cymene)), 4.76 (s, 2H, H6pyrimid.) 2.99 (sept., 2H, J = 7.4 Hz, CH(CH3)2), 2.77 (q,
4H, J = 6.8 Hz, CH2CH3), 2.45 (s, 6H, CH3pyrimid.), 2.11 (s, 6H, CH3(cymene)), 1.76 (t, 6H, CH2CH3,
J = 6.8 Hz), 1.45 (d, 12H, J = 7.4 Hz, CH(CH
3
)
2
).
13
C NMR (150.91 MHz, CDCl
3
): δ = 14.1 (CH
2
CH
3
),
17.1 (CH3pyrimid.), 17.7 (CH3(cymene)), 21.5 (CH(CH3)2), 29.1 (CH(CH3)2), 53.9 (C6pyrimid.), 59.5
(CH2CH3), 84.6, 86.3, 100.0, 100.8 (6C(cymene)), 113.2 (C3arom.), 117.1 (C2arom. + C4arom.), 120.4
(C6arom.), 121.4 (C5pyrimid.), 129.4 (C5arom.), 142.8 (C1arom.), 144.7 (C2pyrimid.), 157.5 (C4pyrimid. +
C3
arom.
), 165.2 (C=O). MS (FAB): m/z 1052 ([M–2Cl]
+
). Anal. Calcd for C
48
H
58
N
4
O
6
Ru
2
S
2
Cl
2
⋅3H
2
O
(1176.14): C, 49.02; H, 5.31; N, 4.75. Found: C, 49.01; H, 5.53; N, 4.96.
2.8. Synthesis of bis[(η 6-p-cymene)Ru(L2)] Cl2 (2)
From L2 (59 mg). Yield: 89.69 mg (76%) as orange microcrystalline solid; mp 198–201°C.
1H NMR (600 MHz, CDCl3): δ = 9.87 (s, 1H, NH), 7.20 (d, 4H, J = 8.0 Hz, H2arom. + H6arom), 6.69
(d, 4H, J = 8.0 Hz, H3
arom.
+ H5
arom.
), 6.01 (d, 4H, J = 6.0 Hz, CH(cymene)), 5.73 (d, 4H, J = 6.0 Hz,
CH(cymene)), 4.94 (d, 1H, H2pyrimid.), 4.00 (q, 2H, J = 7.0 Hz, CH2CH3), 2.82 (sept., 1H, J = 7.3 Hz,
CH(CH3)2), 2.44 (s, 3H, CH3pyrimid.), 2.25 (s, 3H, CH3(cymene)), 1.22 (d, 6H, CH(CH3)2), 1.16 (t, 3H,
J = 7.0 Hz, CH2CH3). 13C NMR (150.91 MHz, CDCl3): δ = 14.0 (CH2CH3), 17.2 (CH3pyrimid.), 17.9
(CH
3
(cymene)), 21.5 (CH(CH
3
)
2
), 29.9 (CH(CH
3
)
2
), 53.7 (C6
pyrimid.
), 59.8 (CH
2
CH
3
), 85.5, 86.4, 100.2,
100.6 (6C(cymene)), 117.1 (C3arom. + C5arom.), 120.5 (C5pyrimid.), 129.7 (C2arom. + C6arom.), 135.6
(C1
arom.
), 144.5 (C2
pyrimid.
), 157.8 (C4
pyrimid.
+ C4
arom.
), 165.2 (C=O). MS (FAB): m/z 1052 ([M–2Cl]
+
).
Anal. Calcd for C48H58N4O6Ru2S2Cl2⋅3H2O (1176.14): C, 49.02; H, 5.31; N, 4.75. Found: C, 48.88;
H, 5.49; N, 4.31.
2.9. Synthesis of bis[(η 6-p-cymene)Ru(L3)] Cl2 (3)
From L3 (71 mg). Yield: 61.10 mg (86%) as brown microcrystalline solid; mp 173–175°C. 1H
NMR (600 MHz, CDCl3): δ = 9.97 (s, 1H, NH), 7.41 (d, 4H, J = 8.0 Hz, H3arom. + H5arom.), 7.12 (d,
4H, J = 8.0 Hz, H2arom. + H6arom.), 5.39 (br s., 2H, H6pyrimid.), 5.31 (d, 4H, J = 5.3 Hz, CH(arene)),
5.20 (d, 4H, J = 5.3 Hz, CH(cymene)), 3.48 (q, 4H, J = 7.0 Hz, CH
2
CH
3
), 2.98 (sept., 2H, J = 6.8 Hz,
Scheme 2.Synthesis of ruthenium complexes of ethyl 4-aryl-6-methyl-2-thioxo-pyrimidine-5-carboxylate
(1–3).

2066 W. A. ALMASOUDI ET AL.
CH(CH3)2), 2.35 (s, 6H, CH3pyrimid.), 2.24 (s, 6H, CH3(cymene), 1.31 (d, 12H, J = 6.8 Hz, CH(CH3)2),
1.21 (t, 6H, J = 7.0 Hz, CH2CH3). 13C NMR (150.91 MHz, CDCl3): δ = 14.1 (CH2CH3), 18.2 (CH3pyrimid.),
18.4 (CH
3
(cymene)), 22.6 (CH(CH
3
)
2
), 30.5 (CH(CH
3
)
2
), 55.0 (C6
pyrimid.
), 60.7 (CH
2
CH
3
), 88.7, 89.0,
103.4, 103.5 (6C(cymene)), 122.6 (C5pyrimid.), 125.3 (C4arom.), 128.8, 129.0 (C2arom., C6arom. and
C3arom., C5arom.), 140.3 (C1arom.), 143.3 (C2pyrimid.), 156.5 (C4pyrimid.), 164.8 (C = O). MS (FAB): m/z
1178/1176 ([M–2Cl]+). Anal. Calcd for C48H54Br2N4O4Ru2S2Cl2⋅6H2O (1356.04): C, 42.52; H, 4.91;
N, 4.13. Found: C, 42.43; H, 4.63; N, 4.32.
2.10. Crystallographic structure determination
Complex 1 crystallized as brownish-orange blocks from a CH2Cl2/CH3OH mixture as the
methanol disolvate. X-ray diraction measurements were performed on a STOE IPDS-II dif-
fractometer. The data were processed using the SAINT software [27]. Crystal data, data
collection parameters, and structure renement details are given in Table 1. The structure
was solved by direct methods and rened by full-matrix least-squares techniques. Non-
hydrogen atoms were rened with anisotropic displacement parameters. Hydrogens were
inserted in calculated positions and rened with a riding model. The following computer
programs were used: structure solution, SHELXL [28]; molecular diagrams, ORTEP [29].
2.11. In vitro anti-Kinesin Eg5 assay
The ATPase activity of the Eg5 motor domain was measured using the malachite green assay
as described earlier [30]. The reactions were performed in reaction buer (80 mM Pipes, pH
Table 1.Crystal data and details of data collection for 1.
aR1=∑||Fo| − |Fc||/∑|Fo|.
bwR2 = {∑[w(F2o − F2C]2/∑[w(F2o)2]}½.
cGOF = {∑[w(F2o − F2C]2)2] /(n-p)}½, where n is the number of reflections and p is the total number of parameters refined.
Empirical formula C25H32ClN2O4SRu
Fw 593.10
Wavelength (Å) 0.71073
Crystal system Orthorhombic
Space group Pcan
Unit cell dimensions a = 14.805(3) Å, α = 90°
b = 16.179(2) Å, β = 90°
c = 21.834(3) Å, γ = 90°
V (Å) 5229.7(14) Å3
Z8
λ (Å) 0.71073
Dcalcd 1.507mgm−3
Crystal size 0.3×0.2×0.1mm3
T (K) 100(2)
θ range for data collection (°) 1.865 to 26.117
μ(MoKα) 0.815mm−1
R1a0.0940
wR2b 0.1407
GOFc1.104
ρfin (max/min) 1.144/−0.782 e Å−3
Independent reflections 5160 [R(int) = 0.2634]
Absorption coefficient 0.815mm−1
F(0 0 0) 2440
Index ranges h, k, l- 18/18, ± 19, ± 26
Reflections collected 66,810
Completeness to θ = 25.242° 100.0%

JOURNAL OF COORDINATION CHEMISTRY 2067
6.8; 1 mM EGTA, 1 mM MgCl2, 0.1 mg/mL BSA, 1 mM taxol) supplemented with Eg5 (48 nM)
fusion protein and microtubules (200 nM). Ten minutes after the addition of the compound,
reactions were started by the addition of ATP (50 mM) and incubated at RT for 7 min. The
reactions were stopped by adding perchloric acid (444 mM, Fluka), and the color reaction
was started by adding the developer solution (1 M HCl (Sigma), 33 mM malachite green
(Sigma), 775 mM ammonium molybdatetetrahydrate (Sigma)). After 20 min, the absorbance
at 610 nm was measured using a plate reader (Victor 2, Perkin-Elmer). The IC50 values were
determined in three independent experiments for each compound.
2.12. In vitro anti-HIV assay
Evaluation of the antiviral activity of ligands L1–L3 and compounds H and 1–3 against the
HIV-1 strain (IIIB) and the HIV-2 strain (ROD) in MT-4 cells was performed using an MTT assay
as described previously [31]. In brief, stock solutions (10-times nal concentration) of test
compounds were added in 25-L volumes to two series of triplicate wells to allow simulta-
neous evaluation of their eects on mock and HIV-infected cells at the beginning of each
experiment. Serial ve-fold dilutions of test compounds were made directly in at-bottomed
96-well microtiter trays using a Biomek 3000 robot (Beckman instruments). Untreated control,
HIV- and mock-infected cell samples were included for each sample. HIV-1 (IIIB) [32] or HIV-2
(ROD) [33] stock (50 L) at 100–300 CCID50 (50% cell culture infectious dose) or culture
medium was added to either of the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the eect of test compound on uninfected cells
in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells
[34] were centrifuged for 5 min at 1000 rpm (Minifuge T, rotor 2250; Heraeus, Germany), and
the supernatant was discarded. The MT-4 cells were resuspended at 6 × 10
5
cells per mL, and
volumes of 50 L were transferred to the microtiter tray wells. Five days after infection, the
viability of the mock- and HIV-infected cells was examined spectrophotometrically.
3. Results and discussion
3.1. Synthesis of the ligands
In the past decade, ruthenium arene complexes have received considerable attention as
anticancer agents and because of their favorable properties such as chemical stability and
structural diversity [7–12]. Therefore, our attention was drawn to the synthesis of new ruthe-
nium complexes containing monastrol and closely related conjugated pyrimidine derivatives,
aiming to study their potential application as anti-HIV agents or as inhibitors for kinesin Eg5.
Thus, ligands L1–L3 have been prepared in 70–79% yield, via the Biginelli reaction, i.e. in a
one-pot, three-component cyclocondensation reaction of substituted benzaldehyde E–G,
ethyl acetoacetate and thiourea in the presence of catalytic amounts of CuCl2 (Scheme 1).
The literature reports various Lewis acids as catalyst in optimizing the yield of pyrimidines
[35].
Ligand L
3
was selected for further NMR studies to aid the signal assignment and its HMBC
[36] spectrum revealed a 2JC,H coupling between H4 of the pyrimidine ring at 5.37 ppm and
the aromatic carbon atom 1′ at 141.3 ppm. In addition, 2JC,H coupling between H4 and C5 at
102.7 ppm was observed. Furthermore, a 3JC,H coupling between H4 and the C=S carbon
atom at 174.7 ppm was detected (Figure 2).

2068 W. A. ALMASOUDI ET AL.
3.2. Synthesis of the complexes
Complexes of the general formula [{(η
6
-p-cymene)Ru(L)}
2
](Cl)
2
)·nH
2
O (1–3) (Scheme 2) were
prepared by a typical -chlorido-bridge splitting reaction of [Ru(η6-p-cymene)Cl2]2 (H) with
the corresponding ligands L
1
–L
3
in a 1 : 2 molar ratio in reuxing THF and K
2
CO
3
under nitro-
gen. The complexes precipitated directly from the reaction mixture after concentration of
the solutions under reduced pressure and were isolated in yields of 68 to 86%.
The structures of complexes 1–3 have been identied by their 1H, 13C, mass and 2D NMR
spectra. The
1
H NMR spectra showed the characteristic pattern of the p-cymene moiety with
methyl singlets at 2.08–2.25 ppm, doublets at 1.22–1.43 ppm for the CH(CH3)2 groups and
a septet at 2.82–2.99 ppm for CH(CH3)2 as well as the typical set of two doublets for the
aryl-CH protons in the range of 6.0 to 5.2 ppm. The resonance of proton H6 at the pyrimidine
backbone appeared as a (broad) singlet at 4.77, 4.94 or 5.39 ppm, respectively. The aromatic
protons of the complexes resonated as multiplet or doublets at 6.28–7.41 ppm. The 13C NMR
spectra for complexes 1–3 displayed all expected resonances for the carbon atoms of the
p-cymene ring. The carbon atoms at position 2 of pyrimidine ring (N = C–S) of complexes
1–3 appeared at ca. 144 ppm, and thus at ca. 30 ppm higher-eld compared to the thiocar-
bonyl carbon atom of the corresponding free ligand. This sizable upeld shift is indicative
for complexation of the ligands L
1
–L
3
by the cymene ruthenium half-sandwich fragment via
a thiolate function. In addition, C5 of pyrimidine moiety resonated at ca. 121 ppm, whereas
the carbon resonance of C4 of the same ring appeared at ca. 157 ppm. The expected reso-
nance signals of the appended phenyl ring and the ester function were also observed and
assigned (c.f. Experimental section).
3.3. X-ray crystal structure
The result of the X-ray diraction study of 1 (as methanol disolvate) is shown in Figure 3
with selected bond distances and angles given in Table 2. The C2 symmetric diruthenium
Figure 3.ORTEP view of the molecule of [(η6-p-cymene)Ru(L1)] (1) in the crystal. Displacement ellipsoids
are drawn at 50% probability level.

JOURNAL OF COORDINATION CHEMISTRY 2069
complex 1 crystallized in the Pcan space group belonging to the orthorhombic crystal system
with four identical molecules within the unit cell. The arene ruthenium moieties adopt the
archetypical three-legged, piano-stool structure. Bond angles subtended by the ligands
forming the legs and the ruthenium atom are all close to 90° (N1–Ru1–S1 = 87.90(19)°,
C21–Ru1–S1 = 89.8(3)°, S1–Ru1–S1′ = 83.07(7)° and attest to the overall pseudo-octahedral
coordination of the metal atom. The 4-aryl-6-methyl-2-thioxopyrimidine-5-carboxylate
ligand is present in its unideprotonated, monoanionic form and adopts the iminothiolate
tautomeric structure as indicated by the short C(2)–N(1) bond length of 1.284(11) Å and a
C–S single bond length C2–S1 of 1.764(10) Å. The latter value is within the range of a Car–S
single bond (1.773(9) Å), while the C(2)–N(1) bond is appreciably shorter than the delocalized
intracyclic Caryl = N double bond of pyrimidine heterocycles (1.333(13) Å) [37]. The ligand L
acts as a tridentate bridging ligand in the μ,κN:κ2S binding mode, coordinating to one Ru
atom via the pyrimidine N1 nitrogen atom and bridging two ruthenium atoms via the thi-
olate donor S1. The central Ru2S2 ring is distinctly puckered with a dihedral angle of 30.2°
between the Ru
2
S aps. The long Ru⋯Ru distance of 3.522 Å demonstrates that the present
complex belongs to the class of dinuclear ligand-bridged, half-sandwich ruthenium com-
plexes with no direct bonding interactions between the Ru atoms [38]. The central Ru2S2
ring is annealed to two kite-shaped, almost planar four-membered rings comprising atoms
Ru1, S1, C2 and N1, which are almost planar with an angle sum of 351.7° and an interplanar
angle between the S1,Ru1,N1 and the S1,C2,N1 planes of 2.7°. These four-membered rings
are in a cis-arrangement and are symmetry-related by a twofold rotational axis passing
through the midpoint of the Ru2S2 ring. This arrangement forces the two four- and the
annealed six-membered thiooxopyrimidine rings into a cofacial, slightly tilted arrangement
with an interplanar angle of 13.3° and a centroid-to-centroid distance of 3.313 Å. The thioox-
opyrimidine rings are notably planar despite the sp3 hybridization of carbon atom C6. The
overall structural features and arrangement of the core of the three annealed four-mem-
bered rings resembles closely those published very recently for diruthenium thiosemicar-
bazone complexes [19]. This overall similarity also pertains to the Ru–S and the Ru–N bond
lengths of 2.422(3) and 2.423(3) Å or 2.101(7) Å, which all fall in the range of the seven related
diruthenium complexes with thiosemicarbazone ligands (range 2.3999(1) to 2.434(4) Å and
2.0371(1) to 2.1287(1) Å). The Ru–C(cymene) bond lengths vary from 2.174(12) Å to 2.233(9) Å.
In 1, the longest Ru–C bond involves the carbon atom C(17) as it is observed for many
cymene ruthenium complexes. We note here that other related diruthenium thiosemicar-
bazone-bridged complexes usually prefer the N,N binding mode of the ligand with
Table 2.Selected bond lengths [Å] and angles [°] for 1.
Bond lengths Bond angles
Ru1–S1′2.422(3) S1–Ru1–S1′83.07(7)
Ru1–S1 2.423(3) Ru1–S1–Ru1′93.25(7)
Ru1–C2 1.766(9) S1–Ru1–N1 67.08(19)
Ru1–N1 2.102(7) S1′–Ru–N1 87.92(19)
C2–S1 1.766(9) S1–C2–N1 110.1(7)
C2–N1 1.282(10) C2–N1–Ru1 103.8(6)
N3–C4 1.370(12) N1–C2–N3 125.4(8)
C4–C5 1.381(12) C2–N1–C6 121.8(7)
C5–C6 1.514(12) N1–C6–C5 111.4(7)
N1–C6 1.479(10) C4–C5–C6 120.7(8)
C2–N3–C4 119.5(7)

2070 W. A. ALMASOUDI ET AL.
concomitant formation of ve-membered chelate rings and signicantly shorter Ru–N bonds
of 2.077(5) to 2.093(5) Å [38, 39].
3.4. In vitro anti-HIV activity
Compounds L1–L3, H, and 1–3 were evaluated for their in vitro anti-HIV-1 (strain IIIB) and
anti-HIV-2 (strain ROD) activity and monitored by the inhibition of the virus-induced cyto-
pathic eect in the MT-4 cells, based on an MTT assay [31]. The results are summarized in
Table 3, in which the data for lamuvidine [40] and nevirapine (BOE/BIRG587) [41] are included
for comparison. None of the tested compounds were active against inhibition of HIV-1 and
HIV-2. However, the antimitotic agent (monastrol, L1) and the 3-bromophenyl analog L3
exhibited EC50 values of 11.24 and 11.68 g/mL, respectively, with no selectivity observed
(SI < 1). It is noteworthy to mention that the ligands L1 and L3 showed much more HIV
inhibitory activity than their ruthenium-cymene complexes 1 and 3 (EC50 > 75.45
and > 43.55 g/mL), respectively, which means that ruthenium coordination has a detrimen-
tal eect on their potency to HIV inhibition.
3.5. In vitro anti-kinesin Eg5 activity
Kinesin-5 motor proteins act to separate the spindle poles during formation of the bipolar
mitotic spindle [42]. Monastrol (MA) has been reported as one of these antimitotic agents
that is capable of arresting cells in mitosis by specically inhibiting Eg5 [25, 43]. Like many
enzyme inhibitors, monastrol might be substrate competitive, inhibiting the ATP hydrolysis
cycle of Eg5 by directly competing with ATP [42, 44], or by microtubule binding. Alternatively,
Table 3.In vitro anti-HIV-1a and HIV-2b activity of some pyrimidines and their ruthenium(II)-cymene
complexes.
aAnti-HIV-1 activity measured on strain IIIB.
bAnti-HIV-2 activity measured using strain ROD.
cCompound concentration required to achieve 50% protection of MT-4 cells from HIV-1 to HIV-2 induced cytopathogenic
effects.
dCompound concentration that reduces the viability of mock-infected MT-4 cells by 50%.
eSelectivity index (CC50/EC50).
Compd. Virus strain EC50 (g/mL)cCC50 (g/mL)dSIe
L1IIIB> 11.24 > 11.24 < 1
ROD > 11.24 > 11.24 < 1
L2IIIB> 66.50 > 66.50 < 1
ROD > 66.50 > 66.50 < 1
L3IIIB> 11.68 11.68 < 1
ROD > 11.68 11.68 < 1
HIIIB> 125 > 125.00 X1
ROD > 125 > 125.00 X1
1IIIB> 75.45 75.45 < 1
ROD > 75.45 75.45 < 1
2IIIB> 85.35 85.35 < 1
ROD > 85.35 85.35 < 1
3IIIB> 43.55 > 43.55 < 1
ROD > 43.55 > 43.55 < 1
Lamuvidine IIIB0.58 0.13 > 34
ROD 2.27 0.13 > 9
Nevirapine IIIB0.075 > 4 > 80
ROD > 4 > 4 X1

JOURNAL OF COORDINATION CHEMISTRY 2071
monastrol might inhibit the motor domain allosterically, either by inhibiting ATP hydrolysis
or by uncoupling partner head interactions to inhibit motor, but not ATPase activity [45].
Organometallic ruthenium-arene compounds bearing a maltolate ligand have been
shown to be nearly inactive in cytotoxicity assays. However, dinuclear ruthenium-arene
derivatives were found to show cytotoxic activity against cancer cells, which increases with
the spacer length between the metal centers [46]. For this reason, complexes 1–3 have been
screened for their inhibition of Eg5 activity using an in vitro malachite green ATPase assay
(enzyme-coupled assay) [47]. Complex 1 exhibited IC50 value of 30 M and turned out to
show the best performance of all complexes of this series. None of the present compounds
matches, however, the criteria of a selective inhibitor of Eg5 in this assay in comparison to
monastrol (IC50 = 10 M, Figure 4).
4. Conclusion
A series of η6-p-cymene ruthenium complexes with 4-aryl-2-thioxopyrimidine ligands were
synthesized and characterized. Spectroscopic data agree with binding of the ligands as
unideprotonated N–S chelates. The molecular structure of complex 1 showed a ligand-
bridged diruthenium structure with a central Ru2S2 core and two cofacially arranged
four-membered Ru,S,C,N rings, which are constructed from two (p-cymene)Ru fragments
and two bridging tridentate 2-thiooxpyrimidine ligands in a μ,κN:κ2S binding mode. The
in vitro anti-HIV-1 and HIV-2 activities of the ligands and the complexes were investigated,
and the compounds were found to be inactive. Additionally, the complexes have been
screened for their inhibitory activity against protein kinesin Eg5. None of these compounds
matched the criteria of a selective inhibitor of Eg5 in this assay in comparison to monastrol
(IC50 = 10 M).
Supplementary material
Crystallographic data have been deposited at the Cambridge Crystallographic Data Center as supple-
mentary publication No. CCDC-1521005. Copies of the data can be obtained free of charge from www.
ccdc.cam.ac.uk/data_request/cif.
Figure 4.Inhibition concentrations (IC50) in M of monastrol and the new ruthenium complexes 1–3 for
Eg5-driven microtubule motility.

2072 W. A. ALMASOUDI ET AL.
Acknowledgement
We thank Prof. C. Pannecouque of Rega Institute for Medical Research, Katholieke Universiteit, Leuven,
Belgium, for the anti-HIV screening. W. Al-Masoudi would like to thank Basrah University for the sabbat-
ical leave. Miss A. Friemel and Mr U. Haunz, Chemistry Department, Konstanz University, Germany are
acknowledged for the NMR experiments. The authors also thank Prof. Dr Thomas Mayer and Dr Martin
Möckel from the Department of Biology at the University of Konstanz for anti-Kinesin Eg5 screening.
Disclosure statement
No potential conict of interest was reported by the authors.
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