Synthesis and characterization of ruthenium(II) complexes with the new ligand 2-phenylazopyridine-5-sulfonic acid (Hsazpy): In search for new anticancer agents

Article abstract of DOI:10.1039/b313746e

Isomers of dichlorobis(2-phenylazopyridine)ruthenium(II) [Ru(azpy) ₂Cl₂], especially the so-called α isomer, display remarkably high cytotoxicities against human tumor cell lines. Unfortunately, the parent [Ru(azpy)₂Cl₂] compounds are poorly water-soluble. In this paper the synthesis and characterization of the new water-soluble ligand 2-phenylazopyridine-5-sulfonic acid (Hsazpy) is described. Use of this ligand in reaction with RuCl₃ gave two isomers, which were isolated as α- and β-[NEt₄]₂[Ru(sazpy) ₂Cl₂]. The compounds have been fully characterized by (2D) NMR spectroscopy. The molecular structure of the α isomer of [NEt ₄]₂[Ru(sazpy)₂Cl₂]·2H ₂O has been determined by single-crystal structure analysis. The packing in the crystal structure of α-[NEt₄] ₂[Ru(sazpy)₂Cl₂]·2H₂O shows an interesting hydrogen-bonding pattern in which two water molecules are involved. One water molecule bridges between a Cl ligand and a SO ₃^- group within one ruthenium moiety, the other water molecule forms a bridge between two SO₃^- groups from two different ruthenium centers, resulting in a chain-like structure. Preliminary evaluation of the cytotoxicity by means of the IC₅₀ value in A2780 cell line classifies α-[NEt₄]₂[Ru(sazpy) ₂Cl₂] as non-toxic, but this does not rule out other anticancer activities.

Full text of DOI:10.1039/b313746e

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P A P E R
Synthesis and characterization of ruthenium(II) complexes with
the new ligand 2-phenylazopyridine-5-sulfonic acid (Hsazpy):
in search for new anticancer agents
a
Anna C. G. Hotze,y Huub Kooijman,b Anthony L. Spek,b Jaap G. Haasnoota and Jan Reedijk*a
a
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300
RA, Leiden, The Netherlands. E-mail: reedijk@chem.leidenuniv.nl; Fax: +31-715274671
Bijvoet Center for Biomolecular Research, Crystal and Structural Chemistry,
Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
b
Received (in Montpellier, France) 29th October 2003, Accepted 10th December 2003
First published as an Advance Article on the web 18th March 2004
Isomers of dichlorobis(2-phenylazopyridine)ruthenium(II) [Ru(azpy)2Cl2], especially the so-called a isomer,
display remarkably high cytotoxicities against human tumor cell lines. Unfortunately, the parent [Ru(azpy)2Cl2]
compounds are poorly water-soluble. In this paper the synthesis and characterization of the new water-soluble
ligand 2-phenylazopyridine-5-sulfonic acid (Hsazpy) is described. Use of this ligand in reaction with RuCl3
gave two isomers, which were isolated as a- and b-[NEt4]2[Ru(sazpy)2Cl2]. The compounds have been
fully characterized by (2D) NMR spectroscopy. The molecular structure of the a isomer of
[NEt4]2[Ru(sazpy)2Cl2]ꢀ2H2O has been determined by single-crystal structure analysis. The packing in the
crystal structure of a-[NEt4]2[Ru(sazpy)2Cl2]ꢀ2H2O shows an interesting hydrogen-bonding patꢁtern in which
two water molecules are involved. One water molecule bridges between a Cl liganꢁd and a SO3 group within
one ruthenium moiety, the other water molecule forms a bridge between two SO3 groups from two different
ruthenium centers, resulting in a chain-like structure. Preliminary evaluation of the cytotoxicity by means of
the IC50 value in A2780 cell line classifies a-[NEt4]2[Ru(sazpy)2Cl2] as non-toxic, but this does not rule
out other anticancer activities.
Introduction
Since the discovery of the antitumor activity of cisplatin (cis-
diaminedichloroplatinum(II), cis-[PtCl2(NH3)2], many other
metal complexes have been investigated for their possible
application as antitumor drugs.1–3 At present several ruthe-
nium complexes are known for their cytotoxic or antitumor
activity.1 For example, complexes such as trans-(H2ind)-
III
[Ru Cl4(Hind)2] 00 (Hind ¼ indazole),
mer-[Ru(terpy)Cl3]
0
0
IV
(terpy ¼ 2,2 :6 ,6 -terpyridine), and [Ru (H2chd)Cl2] (chd ¼
1,2-cyclohexanediamine tetraacetate) have been reported to
be highly antitumor-active.1 Recently, some Ru(II) arene com-
plexes have also been reported, which show in vitro inhibition
of cancer cell growth and in vivo antitumor activity.4,5
Fig. 1 Schematic representation of the three most common isomers
of the [Ru(azpy)2Cl2] complexes: the a isomer with the coordinating
pairs Npy trans, Nazo cis and Cl cis (left), the b isomer with the coordi-
nating pairs Npy cis, Nazo cis and Cl cis (middle) and the g isomer with
the coordination pairs Npy cis, Nazo cis and Cl trans (right).
The isomeric dichlorobis(2-phenylazopyridine)ruthenium(II)
compounds are under renewed investigation due to their
shown cytotoxicity for several human tumor cell lines.6 As
the ligand 2-phenylazopyridine is an asymmetric ligand, the
moiety [Ru(azpy)2Cl2] can in theory exist in 5 different iso-
meric forms of which three isomers are the most common ones
(see Fig. 1).7,8 The so-called a-isomer (a indicating the coordi-
nating chlorides, the pyridine nitrogens and azo nitrogens in
mutual cis, trans and cis orientations) of the dichlorobis(2-phe-
nylazopyridine)ruthenium(II) complexes, a-[Ru(azpy)2Cl2], has
shown a remarkable high in vitro cytotoxicity.6 Unfortunately,
this compound is not water-soluble (one of the requirements
for further biological testing). Recently, the water-soluble
compound a-[Ru(azpy)2(NO3)2] has been developed, but its
cytotoxicity compared to the dichloro analog was decreased
by approximately a factor of 10.9 In addition, replacing the
Cl ligands of a-[Ru(azpy)2Cl2] by carboxylate ligands resulted
in water-soluble compounds, but these complexes are also less
cytotoxic than the parent dichloro complex.10 For this reason
it was attempted to increase the water solubility of a-[Ru(az-
py)2Cl2] by replacing the azpy ligand by a water-soluble deri-
vative. Water-soluble azpy ligands (Fig. 2) containing, for
example NH2 , NO2 , OH or COOH substituents, are known in
the literature, but only derivatives of bis(2-phenylazopyridine)-
ruthenium(II) compounds with NH2 and NO2 substituents
on the azpy ligand have been reported.11,12 As it is known that
sulfonic acid groups attached to polypyridyl ligands increase
the water solubility, the new ligand 2-phenylazopyridine-5-
sulfonic acid (Hsazpy, shown in Fig. 2) has been synthesized
and used for the synthesis of the corresponding dichlorobis-
(Hsazpy)ruthenium(II) complexes. The only previous example
y Current address: Bijvoet Center for Biomolecular Research, Crystal
and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH,
Utrecht, The Netherlands.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 5 6 5 – 5 6 9
565

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Fig. 2 Schematic representation of the 2-phenylazopyridine (azpy)
and 2-phenylazopyridine-5-sulfonic acid (Hsazpy) ligands, with num-
bering used for the NMR assignments.
of a molecular structure for a sulfonic acid group attached to a
polypyridyl ligand containing the azo functionality has been
reported in the case of a nickel compound.13
Two isomers, a- and b-[NEt4]2[Ru(sazpy)2Cl2] (sazpy is the
anionic form of the 2-phenylazopyridine-5-sulfonic acid
ligand), have been obtained pure and have been characterized
by NMR spectroscopy. The molecular structure of a-
[NEt4]2[Ru(sazpy)2Cl2] has been determined by single-crystal
X-ray diffraction analysis.
Fig. 3 Molecular structure of 2.
molecule. The Ru–Npy and Ru–Nazo distances are comparable
to those in similar structures.9,17 The angle Cl(1)–Ru–Cl(2)
[90.93(9)ꢂ] is also similar to this angle in the related a-[Ru-
(azpy)2Cl2] (89.44ꢂ). The bite angles N(1)–Ru–N(8) and
N(21)–Ru–N(28) of respectively 77.3(3)ꢂ and 77.0(3)ꢂ reveal a
considerable distortion of the octahedron, as was found for the
complexes a-[Ru(azpy)2Cl2] and a-[Ru(azpy)2(NO3)2].9,17
The co-crystallized water molecules contribute to an inter-
esting hydrogen-bonding pattern. One of the water molecules
bridges between two sulfonate groups of two ruthenium cen-
ters, forming an infinite chain-like structure parallel to the
crystallographic b axis; [O(40)–H(40A)ꢀ ꢀ ꢀO(1) (x, 1 þ y, z) with
Results and discussion
Synthetic considerations and general information
Upon reaction of RuCl3 and azpy in methanol the g isomer of
[Ru(azpy)2Cl2] is easily obtained as this isomer precipitates as
the main product (containing also small amounts of the d
isomer).7,8,14,15 The a and b isomers are also formed, but
remain in solution.7,8,14 Synthetic procedures have been
developed to convert the g-[Ru(azpy)2Cl2] isomer into the
corresponding a and b isomers.7,8,14
1
˚
Dꢀ ꢀ ꢀA ¼ 2.841(13) A and O(40)–H(40B)ꢀ ꢀ ꢀO(3) (1 ꢁ x, 2 þ y,
˚
1 ꢁ z) with Dꢀ ꢀ ꢀA ¼ 2.963(13) A]. The other water molecule
forms a bridge between a Clꢁ ligand and the SO3 group
ꢁ
Unfortunately, after reaction of RuCl3 and the ligand
Hsazpy the crude mixture consists of three isomers of [Ru(H-
sazpy)2Cl2] in a ratio of a:b:g ¼ 1:1.2:0.13, which all remain in
solution (the isomers have been identified by NMR, vide infra).
To separate and purify this crude mixture several column
materials and solvents have been tried, but in most of the cases
the compounds remained on top of the column, probably due
to interaction of the compounds with the column material.
Using a basic alumina column and eluting first only with
methanol a green fraction, most likely the g isomer, separated
from the a and b isomers. However, this isomer was not
obtained in quantities sufficient enough to allow its isolation
and characterization. The mixture of a and b isomers could
be obtained from the column by eluting with a tetraethyl-
ammonium chloride solution. Fractional recrystallization
finally allowed separation of the a and b isomers. Sulfonic
acids are easily ionized, which makes it difficult, but not impos-
sible, to observe neutral sulfonic acids rather than the anionic
form.16 The IR spectrum of 2-phenylazopyridine-5-sulfonic
acid shows a signal at 2625 cmꢁ1, assigned to the n(SO3H)
vibration. Important to note is the fact that by using basic alu-
mina, the sulfonic acid group of the ligand is ionized and the
compound was isolated with the counter ions [NEt4]þ, as
shown in the crystal structure (vide infra). The absence of the
O–H vibration in the IR spectrum of a-[NEt4]2[Ru(sazpy)2Cl2]
confirms the ionization of the sulfonic acid group. As antici-
pated the compound a-[NEt4]2[Ru(sazpy)2Cl2] shows a good
water solubility ( > 1 mg mlꢁ1).
˚
[O(50)–H(50A)ꢀ ꢀ ꢀO(6) (x ꢁ 1, y, z) with Dꢀ ꢀ ꢀA ¼ 2.867(12) A
and O(50)–H(50B)ꢀ ꢀ ꢀCl(1) (x ꢁ 1, y, z) with Dꢀ ꢀ ꢀA ¼
˚
3.357(10) A] within one ruthenium complex (see Fig. 4).
NMR characterization
The 1H NMR spectrum in acetone-d6 of the crude product
obtained from the reaction mixture directly after the reaction
of RuCl3 and Hsazpy shows three singlets at low field, at
10.17, 10.10 and 9.82 ppm. It is known that the different iso-
mers of [Ru(azpy)2Cl2] show distinct signals in NMR and espe-
cially the H6 signal is used to recognize the particular isomer.15
In general, the H6 resonances of the [Ru(azpy)2Cl2] isomers
appear at lowest field compared to the other pyridine reso-
nances. Moreover, from the synthesis of the [Ru(azpy)2Cl2]
isomers from RuCl3 and azpy it is known that the a, b and g
isomers appear as main isomers and the d isomer only
accounts for 5%. Consequently, the three clearly separated sing-
lets at 10.17, 10.10 and 9.82 ppm, present in the 1H NMR
ꢂ
˚
Table 1 Selected bond distances (A) and angles ( ) of 2
Ru(1)–Cl(1)
Ru(1)–Cl(2)
Ru(1)–N(1)
Ru(1)–N(8)
Ru(1)–N(21)
Ru(1)–N(28)
S(1)–O(1)
2.410(2)
2.393(2)
2.032(7)
2.011(7)
2.057(7)
1.981(7)
1.447(8)
1.430(8)
1.432(8)
1.790(10)
1.433(8)
1.450(10)
1.465(9)
1.287(10)
1.287(12)
Cl(1)–Ru(1)–Cl(2)
Cl(1)–Ru(1)–N(1)
Cl(1)–Ru(1)–N(8)
Cl(1)–Ru(1)–N(21)
Cl(1)–Ru(1)–N(28)
Cl(2)–Ru(1)–N(1)
Cl(2)–Ru(1)–N(8)
Cl(2)–Ru(1)–N(21)
Cl(2)–Ru(1)–N(28)
N(1)–Ru(1)–N(8)
N(1)–Ru(1)–N(21)
N(1)–Ru(1)–N(28)
N(8)–Ru(1)–N(21)
N(8)–Ru(1)–N(28)
N(21)–Ru(1)–N(28)
90.93(9)
89.2(3)
88.0(2)
94.0(3)
171.0(2)
95.3(3)
172.5(2)
84.4(3)
88.3(2)
77.3(3)
176.8(3)
99.8(3)
103.1(3)
94.0(3)
77.0(3)
X-Ray structure determination
S(1)–O(2)
S(1)–O(3)
A projection of the molecular structure of a-[NEt4]2[Ru(saz-
py)2Cl2] (2) is shown in Fig. 3 and selected bond distances
and angles are listed in Table 1. If the coordination pairs Cl,
Npy and Nazo are considered in that order, the configuration
of 2 is cis, trans, cis (a ¼ ctc). The presence of two [NEt4]þ
ions indicates the sazpy ligand to be ionized. The structure
contains further two lattice water molecules per ruthenium
S(1)–C(5)
S(2)–O(4)
S(2)–O(5)
S(2)–O(6)
N(7)–N(8)
N(27)–N(28)
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
566
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 5 6 5 – 5 6 9

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Fig. 4 Hydrogen-bonding pattern in the crystal lattice of a-[NEt4]2[Ru(sazpy)2Cl2]ꢀ2H2O. One of the water molecules bridges two sulfonate
groups of two ruthenium centers, formingꢁan infinite chain-like structure, parallel to the crystallographic b axis. The other water molecule forms
a bridge between a Cl ligand and the SO3 group within one ruthenium complex.
spectrum of the crude reaction mixture [Ru(Hsazpy)2Cl2],
are assumed to correspond to the H6 resonances of the a, b
and g isomers of [Ru(Hsazpy)2Cl2]. The identification of
the three isomers in NMR has been done by NMR spectro-
scopy of several recrystallized fractions containing the three
isomers in different ratios and using the NMR spectra of the
pure a- and b-[NEt4]2[Ru(sazpy)2Cl2] (vide infra). As stated
above, the g isomer could not be isolated and characterized.
The 1H NMR spectra of a- and b-[NEt4]2[Ru(sazpy)2Cl2]
are depicted in Fig. 5. The a isomer shows only one set of sig-
nals due to the C2 axis present in the compound. Three signals
are observed belonging to the pyridine H6 (doublet with small
4J coupling), H3 and H4 atoms and three phenyl signals for the
ortho, meta and para resonances. 2D COSY NMR and 2D
NOESY NMR spectroscopy has been used to assign all sig-
nals. In the case of a-[NEt4]2[Ru(sazpy)2Cl2] NOESY NMR
shows a strong NOE cross peak between the H6 and ortho
hydrogen resonances. It has already been mentioned that this
H6-ortho NOE cross peak indicates the a isomer of the
[Ru(azpy)2Cl2] complexes.9,15 Moreover, the a configuration
has been unambiguously proven by the X-ray structure deter-
mination (vide supra). The 1H NMR spectrum of b-
[NEt4]2[Ru(sazpy)2Cl2] shows twice the number of signals, as
this isomer has no C2 axis, which results in two inequivalent
azpy ligands. From all 5 theoretically possible isomers only
the beta isomer has no C2 axis; so already from this 1H
NMR spectrum the configuration has been proven. The
appearance of the NOE cross peak between the two ortho
resonances provides further evidence for the b configuration,
as this NOE interaction is also observed in the case15 of
b-[Ru(azpy)2Cl2]. The NOE cross peak between the H6 and
H60 resonances, which has also been used to indicate the b
isomer, is not observed in the 2D NOESY spectrum of b-
[NEt4]2[Ru(sazpy)2Cl2], probably because these H6 resonances
are somewhat broadened. The origin for this broadening is not
clear as yet.
Cytotoxicity
The cytotoxicity of a-[NEt4]2[Ru(sazpy)2Cl2] has been deter-
mined and compared with the activity of the related and highly
cytotoxic a-[Ru(azpy)2Cl2]. The cytotoxicity has been evalu-
ated by means of IC50 values (the concentration of the drug
required to reduce cellular growth by 50%) in the human ovar-
ium carcinoma cell lines A2780 and A2780CisR (a cisplatin-
resistant cell line). The IC50 values of a-[NEt4]2[Ru(sazpy)2Cl2]
in A2780 and A2780CisR are respectively 112 and 149 mM
which, in comparison to the IC50 value of ca. 0.9 mM in both
cell lines for a-[Ru(azpy)2Cl2], classifies the compound as non-
cytotoxic. Cytotoxicity data are not the only parameter to
identify a potential anticancer agent, as for example the first
ruthenium compound in clinical trials, NAMI-A, is also
devoid of any cytotoxic effect, but appears to be a promising
Fig. 5 1H NMR spectra of the (upper) a and (lower) b isomers of
[NEt4]2[Ru(sazpy)2Cl2] in MeOD.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 5 6 5 – 5 6 9
567

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antimetastatic agent.18 So additional biological experiments
are required for understanding the origin for the activity, or
absence of activity, of bis(2-phenylazopyridine) ruthenium(II)
complexes and its derivatives.
[NEt4][Ru(sazpy)2Cl2]ꢀ2H2O. RuCl3ꢀ3H2O (0.187 g, 0.72
mmol) was dissolved in 21 ml of methanol and N2 gas was
bubbled through for 15 min. The ligand 2-phenylazopyri-
dine-5-sulfonic acid (0.42 g, 1.6 mmol) dissolved in 25 ml of
methanol was added and the mixture was heated under reflux
for 16 h. The solution was concentrated and purified over alu-
mina (basic alumina, diameter 2 cm, length 17 cm). The first
green fraction was separated by eluting with methanol, but
not isolated. A blue fraction remained on top. By eluting with
a tetraethylammonium chloride solution in methanol the blue
fraction was obtained. The solution was concentrated by
rotary evaporation and diethyl ether (1:1) was added slowly
dropwise. After standing for 48 h blue crystals of a-
[NEt4]2[Ru(sazpy)2Cl2] appeared, suitable for X-ray analysis.
Yield 48 mg (8%). From the filtrate a second blue fraction
was isolated, which after slow re-crystallization from metha-
nol–ether resulted in pure b-[NEt4]2[Ru(sazpy)2Cl2].
Concluding remarks
In this work the synthesis and characterization of the new
water-soluble ligand 2-phenylazopyridine-5-sulfonic acid
(Hsazpy) and the corresponding ruthenium(II) compounds,
isolated as [NEt4]2[Ru(sazpy)Cl2] salts, have been described.
The a and b isomers have been obtained pure and have been
characterized by NMR spectroscopy, which showed once more
the usefulness of NMR for the characterization of isomeric
azpy-like complexes. The packing in the crystal structure of
a-[NEt4]2[Ru(sazpy)2Cl2] showed the incorporation of two
water molecules, one forming a bridge between two acceptors
within the Ru moiety, the other linking the Ru moieties into an
infinite chain parallel to the crystallographic b axis. The IC50
values of a-[NEt4]2[Ru(sazpy)2Cl2] against the human ovarium
carcinoma cell lines A2780 and A2780cisR exceed 100 mM,
which identifies the compound as being non-cytotoxic. For a
better understanding of the reason for the absence of cytotoxi-
city for these compounds, more biological experiments are
required.
a-[N(Et)4]2[Ru(sazpy)2Cl2], 2. Anal. calcd. for RuC38H56
N8O6S2Cl2ꢀ2H2O: C, 46.0; H, 6.09; N 11.28, Cl 7.14, S 6.46;
found: C, 46.2; H, 5.92; N 11.05, Cl 7.22, S 6.99. IR (CsI): n
(SO3 asym stretch) 1231, 1219; (SO3 sym stretch) 1040, 1024.
1H NMR (300 MHz, MeOD): d 9.84 (d, H6), 8.56 (d, H3),
8.43 (d, H4), 7.33 (t, p), 7.21 (t, m), 7.00 (d, o).
b-[N(Et)4]2[Ru(sazpy)2Cl2]. This isomer was only obtained
in a sufficient amount to characterize by NMR spectroscopy.
1H NMR (300 MHz, MeOD): d 10.02 (s, H6), 8.74 (d, H3),
8.65 (d, H4), 8.57 (d, H30), 8.39 (d, H40), 7.81 (d, o0), 7.67 (s,
H60), 7.52 (t, p0), 7.42 (m, p), 7.39 (m, m0), 7.24 (t, m), 6.70
(d, o).
Experimental
General
1H NMR spectra were recorded on a Bruker 300 DPX spectro-
meter. Spectra were recorded in MeOD and calibrated on the
residual solvent peak. Elemental analyses (C, H and N) were
carried out on a Perkin Elmer 2400 CHNS analyzer. Mass
spectra were obtained by the chemical services of the Gorlaeus
Laboratories with a Finnigan MAT 900 instrument equipped
with an electrospray ionization (ESI) interface.
Hydrated RuCl3 was used as received from Johnson and
Matthey, Inc. Basic aluminium oxide (alumina Woelm B Super
I) was used. The synthesis of 2-aminopyridine-5-sulfonic acid
was performed as described in the literature.19 The synthesis
of the ligand 2-phenylazopyridine-5-sulfonic acid from nitro-
sobenzene and 2-aminopyridine-5-sulfonic acid was performed
as a modification of the synthesis of 2-phenylazopyridine.8
The synthesis of the crude mixture of isomers of the dichloro-
bis(2-phenylazopyridine-5-sulfonic acid)ruthenium(II) com-
plexes was done analogously to the synthesis of the crude
product of the [Ru(azpy)2Cl2] complexes, as described in the
literature.7,8 The isolation of the pure isomers required more
complicated purification steps than described for the [Ru(az-
py)2Cl2] complexes and will be discussed below.
Structure determination
Crystal data for 2. [C22H16Cl2N6O6RuS2][C8H20N]ꢀ2H2O,
Mr ¼ 993.05, black plate-shaped crystal (0.05 ꢃ 0.20 ꢃ 0.25
mm3), monoclinic, space group P21 (no. 4) with a ¼
ꢂ
˚
9.3614(12), b ¼ 9.8737(12), c ¼ 24.449(4) A, b ¼ 90.81(3) ,
3
U ¼ 2259.6(5) A , Z ¼ 2, Dc ¼ 1.4596(3) g cmꢁ3, F(000) ¼
˚
1036, m(Mo Ka) ¼ 0.615 mmꢁ1
, 20733 reflections mea-
sured, 8023 independent, Rint ¼ 0.0582, 1.00ꢂ < y < 25.26ꢂ,
T ¼ 150 K, Mo Ka radiation, graphite monochromator,
˚
l ¼ 0.71073 A, Nonius Kappa CCD diffractometer on a rotat-
ing anode, no absorption correction. The structure was solved
by automated direct methods.20 The measured crystal turned
out to be a pseudo-merohedral twin. The twin operation is a
2-fold rotation axis parallel to the a axis. The crystal was also
an inversion twin, leading to a total of 4 components, related
¯
by symmetry operations 1, 2, m and 1 with respect to the a
axis. Since b is close to 90ꢂ a refinement model21 of complete
overlap of the twin components led to satisfactory results.
¯
The ratio in which the four components 1, 2, m and 1 were
present refined to 0.28(4):0.15(3):0.42(5):0.15(4). The water
hydrogen atoms were placed at calculated positions corre-
sponding to ideal H-bond geometry. Refinement of 535 para-
meters converged at a final wR2 value of 0.1348, R1 ¼ 0.0577
[for 7131 reflections with Fo > 4s(Fo)], S ¼ 1.057, ꢁ1.08 <
Syntheses
2-Phenylazopyridine-5-sulfonic acid (Hsazpy), 1. 2-Amino-
pyridine-5-sulfonic acid (5.0 g, 0.029 mol) was added to a solu-
tion of 13.5 g NaOH in 13.5 ml of water containing 1.8 ml of
benzene. The solution was heated up to 100 ꢂC and nitrosoben-
zene (3.2 g, 0.030 mol) was added over a 20 min period. The
mixture was stirred and heated under reflux for 30 min. The
mixture was cooled in ice and the precipitate was isolated by
filtration. The compound was dissolved in diluted hydrochloric
acid (1:1), a black residue was filtrated and benzene was added
to the filtrate in order to dissolve unreacted nitrosobenzene. A
precipitate was formed in the water layer, which was collected
by filtration. Yield 1.05 g (14%). ESI-MS: m/z 264 [M þ H]þ.
IR (CsI): n(SO2–OH), 2625, n(SO3 asym stretch) 1216, n(SO3
sym stretch) 1042. 1H NMR (300 MHz, MeOD): d 9.10 (d,
H6), 8.88 (d, H4), 8.35 (d, H3), 8.14 (d, o), 7.21 (m, m þ p).
Dr < 1.36 e Aꢁ3.z
˚
Acknowledgements
This work was supported in part by the Council for Chemical
Sciences of the Netherlands Organization for Scientific
Research (CW-NWO). We thank Johnson Matthey Chemicals
(Reading, UK) for a generous loan of RuCl3ꢀ3H2O. Addi-
tional support from COST Action D20 allowing regular
z CCDC reference numbers 228185 . See http://www.rsc.org/
suppdata/nj/b3/b313746e/ for crystallographic data in .cif or other
electronic format.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
568
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 5 6 5 – 5 6 9

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