Organoruthenium antagonists of human A3 adenosine receptors

Article abstract of DOI:10.1002/chem.201203291

Human A₃ adenosine receptor (hA₃AR) is a membrane-bound G protein-coupled receptor implicated in a number of severe pathological conditions, including cancer, in which it acts as a potential therapeutic target. To derive structure-activity relationships on pyrazolo-triazolo-pyrimidine (PTP)-based A₃AR antagonists, we developed a new class of organometallic inhibitors through replacement of the triazolo moiety with an organoruthenium fragment. The objective was to introduce by design structural diversity into the PTP scaffold in order to tune their binding efficacy toward the target receptor. These novel organoruthenium antagonists displayed good aquatic stability and moderate binding affinity toward the hA₃ receptor in the low micromolar range. The assembly of these complexes through a template-driven approach with selective ligand replacement at the metal center to control their steric and receptor-binding properties is discussed. Scaffold design: A novel class of ruthenium(II)-arene complexes containing chelating N,N-pyrazolo-pyrimidine ligands was rationally developed to be selective antagonists of human A₃ adenosine receptors based on the proven pyrazolo-triazolo-pyrimidine design (see figure). Copyright

Full text of DOI:10.1002/chem.201203291

FULL PAPER
DOI: 10.1002/chem.201203291
Organoruthenium Antagonists of Human A3 Adenosine Receptors
Priyankar Paira,[a] Mun Juinn Chow,[b, c] Gopalakrishnan Venkatesan,[a]
Vamsi Krishna Kosaraju,[b] Siew Lee Cheong,[a] Karl-Norbert Klotz,[d]
Wee Han Ang,*[b] and Giorgia Pastorin*[a, c]
Abstract: Human A3 adenosine recep-
triazolo moiety with an organorutheni-
um fragment. The objective was to in-
troduce by design structural diversity
into the PTP scaffold in order to tune
their binding efficacy toward the target
receptor. These novel organoruthenium
antagonists displayed good aquatic sta-
tor (hA3AR) is a membrane-bound
G protein-coupled receptor implicated
in a number of severe pathological con-
ditions, including cancer, in which it
acts as a potential therapeutic target.
To derive structure–activity relation-
ships on pyrazolo–triazolo–pyrimidine
(PTP)-based A3AR antagonists, we de-
veloped a new class of organometallic
inhibitors through replacement of the
bility and moderate binding affinity
toward the hA3 receptor in the low mi-
cromolar range. The assembly of these
complexes through a template-driven
approach with selective ligand replace-
ment at the metal center to control
their steric and receptor-binding prop-
erties is discussed.
Keywords: adenosine
design nitrogen heterocycles
ruthenium · templated assembly
·
ligand
A
·
·
Introduction
rheumatoid arthritis and asthma.[3] Furthermore, A3AR is
overexpressed in some tumor cell lines, such as astrocytoma,
B16-F10, A378 melanoma, and HL-60 leukemia,[4] as well as
in solid tumors (e.g., a 2.3-fold increase in colon carcino-
mas), and this expression is correlated with disease progres-
sion.[5] Studies have established that this receptor could be a
prospective therapeutic target in cancer therapy. Currently,
the targeting of A3AR in a clinical setting still presents a
major challenge[6] because modulation of this receptor sub-
type may induce both pro- or antiapoptotic effects depend-
ing on the duration and the extent of activation/inactivation
and the type of receptor binding.[1c]
Adenosine is an endogenous purine nucleoside that modu-
lates a wide variety of physiological responses by interacting
with specific adenosine receptors.[1] These receptors are
widely distributed in mammalian tissues and have been clas-
sified into four different G-protein-coupled receptor sub-
types, namely, A1, A2A, A2B, and A3 adenosine receptors.[2]
There is growing evidence that suggests that the A3 adeno-
sine receptor (A3AR) subtype is implicated in pathological
conditions such as cardiac and cerebral ischemia, neurode-
generative diseases, and inflammatory pathologies, including
The possible role of A3AR modulation in the pathogene-
sis of such diseases has led to the advent of A3AR antago-
nists as therapies for various pathophysiological conditions.
In the past few years, there have been concerted efforts to
develop different heterocyclic scaffolds as hA3ARs antago-
nists. These scaffolds include pyridine and dihydropyridine
analogues,[7] flavonoid,[8] isoquinoline,[9] triazoloquina-
[a] Dr. P. Paira, G. Venkatesan, Dr. S.-L. Cheong,
Assoc.Prof. G. Pastorin
Department of Pharmacy
National University of Singapore
3 Science Drive 2, Singapore 117543 (Singapore)
Fax : (+65)6779 1554
E-mail: phapg@nus.edu.sg
ACHTUNGTRENNUNG
[b] M. J. Chow, V. K. Kosaraju, Dr. W. H. Ang
Department of Chemistry
National University of Singapore
3 Science Drive 2, Singapore 117543 (Singapore)
Fax : (+65)6779-1691
AHCTUNGTRENNUNG
Figure 2) centered on the replacement of the conventional
furan ring at the C2 position with a 2-(para-substituted)
phenyl ring and various substituents at the N5 and N8 posi-
tions. These new modifications enhanced the pharmacologi-
cal profile of the antagonists significantly, with hA3 affinity
in the low nanomolar range and improved selectivity against
E-mail: chmawh@nus.edu.sg
[c] M. J. Chow, Assoc.Prof. G. Pastorin
NUS Graduate School for Integrative Sciences and Engineering
Center for Life Sciences (CeLS)
28 Medical Drive, 05-01, Singapore 117456 (Singapore)
[d] K.-N. Klotz
the other adenACTHUNTGRNEUNGosine receptor subtypes. It also provided a
Institut fꢀr Pharmakologie und Toxikologie
Universitꢁt Wꢀrzburg, 97078 Wꢀrzburg (Germany)
convenient replacement of the metabolically unstable furan
ring.[13] Molecular modeling studies indicated stabilizing hy-
drogen-bonding interactions with the asparagine (Asn) 250
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203291.
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fering structural characteristics and binding affinities. This
increasingly prevalent use of ruthenium in the development
of metallocomplexes capable of eliciting biological respons-
es[20] is in part due to recent successes of ruthenium anti-
cancer complexes in preclincal and clinical trials.[21] Notable
examples include the antimetastatic NAMI-A and RuII–
arene–PTA (RAPTA) series of complexes[22] and antitumor-
al KP1019 and {(h6-arene)RuII} complexes with ethylene-
diammine ligACTHNUTRGNEUNG
ands.[23] Ruthenium complexes are not known
to cause systemic toxicities and are well tolerated in vivo,[24]
which is in stark contrast to other heavy metals such as mer-
cury, osmium, and cadmium.
Our strategy was to exploit organotransition-metal chem-
istry as a means of directly introducing unique structural el-
ements into the PTP scaffold that are different from classi-
cal organic-based heterocycles. These structural elements
would be organized around a [Ru]–PP scaffold (Figure 2)
Figure 1. Examples of human A3 adenosine receptor (hA3AR) antago-
nists.
residue, p–p stacking interactions between the triazole ring
and phenACHTUNGTRENNUNGylalanine (Phe) 168 residue, and hydrophobic inter-
actions with several other residues of the binding site.
A common feature in the vast majority of reported A3AR
antagonists is their flat multicyclic polyaromatic structure.
While mapping the functional-group dependency on the
PTP scaffold for studies on the structure–activity relation-
ship, we observed that sterically encumbered planar aryl
substituents at the C2 position improved antagonist binding
(see Figure 2 for numbering order). We were therefore in-
terested in exploring the effects of other substitutions, par-
ticularly hydrophobic nonplanar moieties that were structur-
ally distinct from aryl rings, as a means of tuning antago-
nist–receptor interactions at the binding site. We sought to
develop a facile method of introducing such structural ele-
ments into the PTP framework by using templated assembly
of metal–ligand fragments (Figure 1).
The concept of applying organotransition-metal fragments
as structural scaffolds for complex organic frameworks has
been pursued for different purposes.[14] Meggers and co-
workers demonstrated that highly potent active-site inhibi-
tors of kinases can be developed by replacing the glycoside
motif within staurosporine, a known kinase inhibitor, with
different organoruthenium moieties.[15] In this manner, the
ruthenium metal center played a structural role in the or-
ganization of the rigid metal–ligand framework.[16] Metzler-
Nolte and co-workers investigated synthetic analogues of
antibiotic platensimycin by substituting the complex tetracy-
clic cage component with isosteric organometallic frag-
ments.[17] Dyson and co-workers developed a class of highly
potent arene-functionalized metalloinhibitor of glutathione-
S-transferase, which covalently binds at the enzyme dimer
interface through their ruthenium (RuII)–arene compo-
nent.[18] We recently reported a class of selective organoru-
thenium inhibitor of protein tyrosine phosphatase 1B, de-
signed to bind at the enzymatic active site while simultane-
ously interacting with the peripheral structural space.[19] The
hexavalent RuII center enabled the rapid assembly of differ-
ent ligand sets, thus giving rise to metalloinhibitors with dif-
Figure 2. Design of A3AR antagonists based on the PTP scaffold. PP=
pyrazolo-[3,4-d]pyrimidine.
using chelating pyrazolo-[3,4-d]pyrimidine (PP) as a ligand.
Therefore, ligands that are conformationally orthogonal to
the [Ru]–PP metallacyle can be systematically introduced,
thus allowing us to probe the receptor binding space within
the immediate substrate-binding site. In this study, we devel-
oped this new class of organoruthenium hA3AR antagonist
and carried out docking and structure–activity relationship
studies to ascertain the role of different structural elements
in the binding mode at the hA3 receptor subtype.
Results and Discussion
Design of organoruthenium PP scaffold: We adopted a tem-
plate-driven approach to assemble the envisaged organoru-
thenium complexes as hA3AR antagonists. Our key consid-
eration was to design the PTP scaffold in a manner such
that it would bind to organoruthenium precursors in good
yield. The dimeric [{(h6-arene)RuCl2}2] complex was utilized
as the precursor because it is stable and easily prepared with
a variety of different arene ligands. We retained the pyra-
zole ring on the PTP scaffold, which is responsible for the
selective binding at the hA3 receptor, but we retrosyntheti-
cally reduced the aryl–triazole ring into an a-hydrazine–
imine bonding arrangement, so that it would readily chelate
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Templated Assembly of Organoruthenium Antagonists of Adenosine Receptors
FULL PAPER
the organoruthenium center. Reaction with [{(h6-arene)-
RuCl2}2] would result in cleavage of the dimer and displace-
major product. 3-Amino-(N1-substituted)-4-pyrazolecarbo-
nitriles 2a–c were transformed into their corresponding car-
bamates (3a–c) through a reaction with ethyl chloroformate
in pyridine at ambient temperature. A catalytic amount of
DMAP was used as a cocatalyst to favor this acylation reac-
tion. Carbamates 3 were subsequently treated with hydra-
zine hydrate in EtOH at 808C for 6 h to afford the 5-amino-
ACHTUNGTRENNUNG
ment of a Cl ligand, thus inducing a positive charge at the
metal center. This outcome would be accompanied by
change in solvation properties, and we expected the final
compound to precipitate readily from organic solvents upon
formation. We also considered the fact that the arene ligand
would be organized orthogonally from the remaining three
coordination sites upon ligand binding and would provide
the intended unique ligand disposition not easily achieved in
a purely organic framework.
4-imino-2-substituted-4,5-dihydro-2H-pyrazoloCAHTUNGTRNE[NUNG 3,4-d]pyrimi-
din-6(7H)-one (4a–c) in moderate yield. The structures of
AHCTUNGTRENNUNG
1
these compounds were readily confirmed by H NMR and
ESI-MS. A similar reaction protocol was adopted for the
synthesis of substituted hydrazines. N-Carbamate 3a was
treated with phenylhydrazine and benzoic hydrazide in 2-
methoxyethanol at 1208C under reflux for 8 h to give
4-imino-2-methyl-5-(phenylamino)-4,5-dihydro-2H-pyrazolo-
Synthesis and characterization: On the basis of these consid-
erations, we designed a set of PTP mimetics as ligands,
which would retain good interaction at the binding site of
the receptor through the PP motif (as confirmed by the
docking studies reported in the Supporting Information),
while allowing facile coordination to the RuII through the a-
hydrazine–imine chelate. Our strategy of preparing ligands
4a–f is summarized in Scheme 1.
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
benzamide (4e) in good yields (Scheme 1). Ligands 4d and
1
4e were fully characterized by H NMR and ESI-MS. Inter-
mediate 2a was also treated directly with triethyl orthofor-
mate under reflux to give the imidate 3 f, which was further
cyclized with hydrazine hydrate in EtOH at ambient tem-
perature (Scheme 1).[25]
To study the RuII–ligand interaction and its binding affini-
ties on human A1, A2A, and A3 receptors, a new class of
ACTHNUTRGNEUNG
ruthenium complex [(h6-arene)RuCl(k2-PP)]·Cl (5–7) was
developed. The synthesis of the bis-amino complex contain-
ing a bidentate PP ligand [(h6-cymene)RuCl(k2-4a)]·Cl (5a)
AHCTUNGTRENNUNG
was accomplished in one step starting from the RuII complex
[{(h6-cymene)RuCl2}2]. The precursor was treated with slight
excess of ligand 4a in MeOH at ambient temperature for
2 h to yield yellow microcrystalline 5a in high yield
(Scheme 2). The structure of this complex was fully charac-
1
1
terized by H NMR and ESI-MS. In general, the H NMR
resonances for the arene protons of the RuII–arene com-
plexes were shifted downfield relative to the corresponding
starting RuII dimers. There were also characteristic changes
in the splitting pattern of the protons in the arene ligand.
Upon ligand coordination, the C2v symmetry of the complex
was disrupted. In deuterated dimethyl sulfoxide (DMSO),
5a exhibited four distinct doublets (d=5.45–5.85 ppm) for
cymene CH and two doublets (d=1.14-1.23 ppm) for the
1
isopropyl CH3 group in the H NMR spectra due to the for-
mation of enantiomers. The ESI-MS spectrum of 5a showed
two significant peaks at m/z 451 and 415 for the [M]+ and
[MꢀHCl]+ ions, respectively, as characterized by the unique
Ru isotope pattern. After optimization of the reaction con-
ditions for preparing 5a, the scope of this chemical approach
was expanded to include other arene fragments [{(h6-benze-
ne)RuCl2}2] and [{(h6-triisopropylbenzene)RuCl2}2] and PP
ligands 4a–f to produce a series of Ru complexes 5–7 in
high yields (Schemes 2 and 3). All the structures were char-
Scheme 1. Synthesis of 4-imino-(2-substituted)-2H-pyrazolo
ACHTUNGTREN[NUNG 3,4-d]-pyri-
midine scaffolds (4a–f). DMAP=dimethylaminopyridine. i) 1.5 equiv
A
ACHTUNGTRENNUNG
NH2NH2·H2O, EtOH, reflux 808C, 6 h; ii) 1.5 equiv PhCONHNH2, reflux
808C, 6 h; iii) 1.5 equiv PhNHNH2, reflux 808C, 6 h.
1
acterized by their H NMR and ESI-MS.
For the development of structurally different compounds,
Alkylation of 3-amino-4-pyrazolecarbonitrile 1 with alkyl
or aryl iodide in dry DMF with dry potassium carbonate as
a weak base led to N1-substituted regioisomer 2a–c as a
5a and 5 f were further treated with 1.1 equivalents of 1,3,5-
triaza-7-phosphatricycloACHTUNGTNER[NUNG 3.3.1.1]decane (PTA) in dry MeOH
in an inert atmosphere for 2 h to afford the complexes [(h6-
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W. H. Ang, G. Pastorin et al.
ligand bond lengths in 7 f were
also noticeably lengthened with
respect to 5 f, presumably due
to steric effects that arise from
the bulky triisopropylbenzene
arene ring. All in all, the struc-
tures vindicated our design ap-
proach to preposition the
amido–hydrazide moiety for
RuII chelation.
Aqueous stability: The aqueous
stability of representative orga-
noruthenium inhibitor 5a was
investigated by using UV/Vis
spectroscopy to ensure that our
Scheme 2. Synthesis of [(h6-arene)RuCl
4a)(PTA)]·2Cl (P5a).
(k2-PP)]Cl (5a–d, 6a, 7a, and 7d; PP=4a–e) and [(h6-cymene)Ru(k2-
ACHUTGTNRENNUG CAHTUNGTRENNUGN
ACHTUNGTRENNUNG
organoruthenium
antagonists
would not spontaneously de-
compose or aquate upon disso-
lution in aqueous media, which
would result in multiple species
that could not be identified.
Over a period of 24 h, the UV/
Vis peak profile in water or
100 mm NaCl remained un-
changed with the lmax value,
which remained constant, thus
suggesting that the compound
was stable (see Figure S1 in the
Supporting Information). In the
Scheme 3. Synthesis of [(h6-arene)RuCl(k2-4 f)]Cl (5 f–7 f) and [(h6-cymene)Ru(k2-4 f)
ACHTUNGTRENNNUG ACHTUNRTGENNUNG CAHTNUGTREN(NUGN PTA)]·2Cl (P5 f).
cymene)Ru
(PTA)
(k2-4 f)]·2Cl (P5 f) in good yields (Schemes 2 and 3).
ACHTUNGTRENNUNG(PTA)ACHTUNGTRENNUNG
(k2-4a)]·2Cl (P5a) and [(h6-cymene)Ru-
A
ACHTUNGTRENNUNG
Table 1. Selected X-ray crystallographic data for 5 f and 7 f.[a]
The PTA ligand was introduced to increase the steric en-
cumbrance at the organoruthenium component further. The
structure of these complexes was determined by 1H and
31P{1H} NMR and ESI-MS. The mass spectra of these com-
pounds show a distinct peak at the [M]2+ ion with the usual
Ru isotope pattern. In the 31P{1H} NMR spectra, compounds
P5a and P5 f showed a singlet peak at d=ꢀ34.5 and
ꢀ35.4 ppm, respectively, in comparison with RAPTA-C at
d=ꢀ36.2 ppm.[26]
Complex
5 f·2CH3OH
7 f·H2O
formula
formula weight
T [K]
C18H20Cl2N6O2Ru
534.45
C21H32Cl2N6ORu
556.50
100(2)
223(2)
l [ꢃ]
0.71073
0.71073
crystal size [mm3]
cystal system
space group
a [ꢃ]
0.26ꢄ0.06ꢄ0.06
monoclinic
C2/c
0.22ꢄ0.20ꢄ0.10
monoclinic
P21/n
27.801(4)
9.2910(14)
17.537(3)
94.383(3)
4516.6(12)
8
13.4127(16)
14.2921(17)
14.7053(18)
95.938(3)
2803.8(6)
4
b [ꢃ]
c [ꢃ]
Structural determination of 5 f and 7 f: The structural deter-
mination of 5 f and 7 f in the solid state was carried out by
using X-ray diffraction studies. Single crystals of 5 f and 7 f
were grown by vapor diffusion of diethyl ether into a satu-
rated solution of the compound in MeOH. Key crystallo-
graphic parameters were tabulated in Table 1. The RuII
center assumed a piano-stool configuration supported by the
facially bound arene ring, the bidentate PP moiety, and a Cl
ligand (Figure 3). As far as we know, organoruthenium com-
plexes containing such PTP scaffolds are unprecedented.
b [8]
V [ꢃ3]
Z
1calcd [Mgm3ꢀ
]
1.572
1.318
m [mmꢀ1
V range [8]
]
0.957
0.771
2.31 to 27.50
5174/5/283
0.9448 and 0.7889
R1=0.0623
wR2=0.1146
R1=0.0935
wR2=0.1234
1.049
1.96 to 25.00
4929/15/301
0.9268 and 0.8486
R1=0.0760
wR2=0.1746
R1=0.1032
wR2=0.1878
1.115
data/restraints/parameters
max., min. transmission
final R indices
AHCTUNGTRENN[GUN I>2s(I)]
R indices (all data)
ꢀ
ꢀ
The Ru C and Ru N bond lengths are of typical values and
comparable with the known RuII–arene complexes contain-
ing diamino ligands (Table 2).[23c] The N1-Ru1-N2 bond
angle of 76.89(16)8 is significantly smaller than 908, which
indicated that the five-membered ring is strained. The Ru–
GOF on F2
peak/hole [eꢃꢀ3
]
1.354 and ꢀ1.334
1.093 and ꢀ0.894
2
2
[a] R=Sj jFo jꢀjFc j j/SjFo j, wR2={S[w
A
(Fo )2]}1/2. Good-
ACHTUNGTRENNUNG
2
ness-of-fit (GOF)={S[w
(Fo ꢀFc2)2]/
A
data and p is the number of parameters refined.
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Templated Assembly of Organoruthenium Antagonists of Adenosine Receptors
FULL PAPER
Table 2. Comparison of bond lengths [ꢃ] and angles [8] of 5 f and 7 f.
Complex
5 f
7 f
ꢀ
Ru1 N1
2.052(4)
2.124(4)
2.065(6)
2.129(6)
ꢀ
Ru1 N2
ꢀ
Ru1 Cl1
2.4051(13)
2.169–2.195
76.89(16)
86.30(13)
82.01(12)
2.4142(19)
2.172–2.208
76.7(2)
84.01(18)
80.79(17)
ꢀ
average Ru Carene
N2-Ru1-N1
N2-Ru1-Cl1
N1-Ru1-Cl1
A2A, and A3 adenosine receptors and the corresponding ade-
nylyl cyclase activity in CHO cells that expressed the A2B re-
ceptors, was evaluated through measuring the displacement
of selective radioligands, which were previously bound to
the receptor expressed at the cell surface.[13]
The simple PP scaffold (e.g., 4 f) did not show any affinity
toward any of the adenosine receptors. Moreover, no im-
provement was observed when a carbonyl group was intro-
duced at the C6-position (e.g., 4a; refer to Figure 2 for the
numbering scheme of [Ru]-PP). This was also the case when
groups bigger than a methyl group were introduced as N2
substituents, such as phenylethyl or phenylpropyl groups
(i.e., 4b and 4c, respectively; Figure 2). This result indicated
that, in contrast to the classical PTP scaffold (in which a
clear inverse correlation existed between the molecular
volume of the substituent at N8 and the affinity of the
hA3AR; Figure 2),[12] this position becomes less crucial. As
soon as the free amino group in 4a was substituted with a
benzamide functionality, the affinity at both A2A and A3 ad-
enosine receptors improved to within 14–20 mm (Ki = >100,
14.8, >100, and 19.8 mm for hA1, hA2A, hA2B, and hA3, re-
spectively, for 4e; Figure 2).
Figure 3. Molecular representations of 5 f (top) and 7 f (bottom). All the
atoms are represented by spheres of arbitrary radii.
presence of 0.1 mm glutathione (GSH), to simulate
an endogenous nucleophile, 5a presented a shift in
the lmax value with an isosbestic point at l=284 nm.
These observations suggested that although 5a was
stable under aqueous conditions, it could undergo
ligand-exchange reactions with other nucleophiles.
Table 3. Binding affinity of synthesized compounds at hA1, hA2A, and hA3 adenosine
receptors.[a]
Ki [mm]
[b]
[c]
[d]
[e]
hA1
hA2A
hA2B
hA3
4a
4b
4c
4e
4 f
5a
5b
5c
5d
5 f
6a
6 f
7a
7 f
P5a
P5 f
>100
>100
>100
>100
>100
>100
>100
14.8 (8.88–24.8)
>100
>100
>100
>100
>100
>100
>1
>3
>10
>10
>10
>100
>100
>100
19.8 (15.3–25.6)
>100
3.75 (2.32–6.06)
16.0 (11.5–22.3)
11.4 (7.17–18.2)
>100
17.9 (13.4–24.1)
4.2 (3.04–5.82)
>100
1.9 (1.12–3.23)
2.03 (1.1–3.72)
20.1 (12.6–31.9)
21.5 (15.7–29.4)
The reactivity of monoACTHNUTRGNENGcU atACHTUNGTERNiNUGN onic organoruthenium
complexes were known to be mediated by the aqua-
tion.[27] Replacement of the chloride ligand by bulk
water molecules yielded reactive RuII–aqua com-
plexes that can undergo further ligand-substitution
reactions particularly with the nucleophilic thiol-
containing GSH. Indeed, the {(h6-arene)Ru} moiety
may modulate redox reactions of bound thiol
groups, thus resulting in increased reactivity toward
other nucleophiles.[28] It is, therefore, possible that
such organoruthenium inhibitors can covalently
bind to the target receptors to result in deactivation
of the substrate binding site.
>100
2.79 (1.94–4.01)
18.1 (17.6–18.6)
5.00 (4.81–5.21)
>100
8.86 (7.45–10.5)
2.97 (1.84–4.8)
>100
4.04 (2.33–7.01)
5.01 (4.06–6.17)
>30
>100
51.2 (36.7–71.6)
10.1 (9.2–11.2)
>100
23.5 (16.5–33.6)
>100
>10
>100
>100
7.13 (5.04–10.1)
>1
>10
>100
33.5 (20.9–53.6)
13.8 (12.4–15.3)
>100
>100
>100
[a] Adenylyl cyclase activity of synthesized compounds at the hA2BAR. [b] Displace-
ment of specific [3H]-CCPA binding at human A1 receptors expressed in Chinese ham-
ster ovary (CHO) cells (n=3–6). [c] Displacement of specific [3H]-5’-N-ethylcarboxa-
midoadenosine (NECA) binding at human A2A receptors expressed in CHO cells (n=
3–6). [d] Ki values for inhibition of NECA-stimulated adenylyl cyclase activity in
CHO cells (n=3–6). [e] Displacement of specific [3H]-2-(1-hexynyl)-N6-methyladeno-
Studies on the structure–affinity relationship: The
receptor-binding affinities of the synthesized ligands
4a–f and Ru complexes 5–7 are summarized in
Table 3. The affinity of the antagonists toward their
receptors, expressed in CHO cells for human A1, sine (HEMADO) binding at human A3 receptors expressed in CHO cells (n=3–6).
Chem. Eur. J. 2013, 00, 0 – 0
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W. H. Ang, G. Pastorin et al.
This phenomenon became even more evident when sub-
stitutions with RuII–arene moieties were introduced at the
same position to give rise to RuII complexes [(h6-
reported in Figure S25 of the Supporting Information) into
the receptor binding pocket. Curiously, P5a and P5 f dis-
played a significant increase in selectivity toward hA3AR
(e.g. P5a with Ki = >30, >100, >100, and 20.1 mm for hA1,
hA2A, hA2B, and hA3, respectively, versus 5a with Ki =2.79,
>100, >1.0, and 3.75 mm for hA1, hA2A, hA2B, and hA3, re-
spectively; Figure 2). This suggested that substitutions at the
chloride position could be further explored to increase the
selectivity at the receptor of interest.
Overall, this structure–activity relationship study con-
firmed that the RuII–arene moieties increased affinity at
hA3AR relative to the uncoordinated PP ligand. When opti-
mal substituents were introduced into the RuII–arene com-
plexes (see Figure 2; namely 1) a 6-carbonyl unit, 2) a small
alkyl group at N2, and 3) a h6-triisopropylbenzene ligand),
new antagonists with low micromolar affinity at the human
A3 adenosine receptor and a discrete selectivity over the
other adenosine-receptor subtypes would be derived.
arene)RuClACHTUNGTRENNUNG
(k2-PP)]·Cl (5–7). Upon chelation, the Ru center
formed a five-membered metallacycle with the PP ligand,
which spatially mimicked the triazole unit in the PTP scaf-
fold (also confirmed by the docking studies reported in the
Supporting Information). Among the Ru derivatives, those
bearing a carbonyl group to correspond to the C6-position
and a methyl group at the N2-position of the PP ligand
showed the best hA3 affinity values, which were all in the
low micromolar range (e.g., for hA3, Ki =3.75, 4.2, and
1.9 mm for 5a, 6a, and 7a, respectively; Figure 2). The only
exception to this observation was represented by the concur-
rent substitution at the N5-position with an additional aro-
matic ring (for hA3, Ki >100 mm for 5d; Figure 2), which
presumably was responsible for excessive steric hindrance at
the binding site.
The introduction of RuII–arene moieties onto the original
PP scaffold was associated with a more influential role of
the concurrent substituent at the N2-position. Indeed, a sim-
ilar trend for the PTP moiety was observed, because less
sterically encumbered groups (e.g., the methyl group as in
5a, 7a, and 7 f; Figure 2) were more favorable than bulkier
groups (e.g., phenylethyl and phenylpropyl; 5b and 5c, re-
spectively; Figure 2). This suggested that the spatial encum-
brance of the Ru moiety provided a more favorable interac-
tion at the hA3AR relative to the uncoordinated PP ligand.
The substituents on the arene ligand were varied to exam-
ine their influence on the binding affinity at the hA3AR rel-
ative to their parent complexes. Unsubstituted h6-benzene
did not influence the pharmacological profile of the com-
plexes because 6 f remained inactive and 6a, containing a
C6-carbonyl group (Figure 2), showed a moderate affinity.
However, the replacement of the benzene ring with a h6-
cymene or h6-triisopropylbenzene functionality enhanced
the affinity and, in a few cases, also the selectivity toward
hA3AR. For example, RuII complexes containing h6-cymene
exhibited a remarkable increase in the affinity profile, even
in the absence of the C6-carbonyl group (e.g., for 5 f Ki =
8.86, 23.5, and 17.9 mm for hA1, hA2A, and hA3, respectively,
versus 6 f with Ki = >100, >100, >100, and >100 mm for
hA1, hA2A, hA2B, and hA3, respectively; Figure 2). In partic-
ular, the introduction of h6-triisopropylbenzene, provided
that a C6-carbonyl group and a methyl group at N2 were
concurrently present, was responsible for the best affinity at
the hA3AR and a slight increase in selectivity over the other
adenosine receptors (e.g., Ki =1.9, 2.13, 17.6, and 3.8 mm for
hA3, hA1/hA3, hA2A/hA3, and hA2B/hA3, respectively, for 7a;
Figure 2).
Conclusion
We investigated a series of RuII–arene complexes that con-
tain PP chelating ligands to explore the effect of altering the
nature of the bulky groups at the 2-aryltriazole position on
PTP. The binding affinity profiles indicated that sterically
hindered RuII–arene complexes were well-tolerated at the
binding site and exhibited moderate affinity toward the
hA3AR. In particular, [(h6-triisopropylbenzene)RuII] com-
plexes 7a and 7 f exhibited the best profile in terms of both
affinity and selectivity at hA3AR and good aqueous solubili-
ty, overcoming a limitation in the classical tricyclic-PTP scaf-
fold. Our study paves the way for RuII–arene complexes as
promising hA3AR antagonists.
Experimental Section
Materials and methods: Reactions were monitored by TLC analysis on
silica gel (precoated 60 F254 Merck plate). All the organometallic reac-
tions were carried out in a nitrogen atmosphere. Complexes [{(h6-cy-
AHCTUNGTRENNUNG
mene)RuCl2}2], [{(h6-benzene)RuCl2}2], and [{(h6-triisopropylbenzene)R-
uCl2}2] were prepared as described elsewhere.[29] All the other chemicals
were commercial products and were purchased from Sigma-Aldrich.
Column chromatography was performed on silica gel 60 (Merck, 70–230
mesh). IR spectra of the compounds (2 mg, KBr tablets) were recorded
on a Spectrum100 FTIR machine (PerkinElmer). The compounds were
dissolved in HPLC-grade MeOH for the determination of a mass-to-
charge ratio m/z on a LCQ Finnigan MAT electrospray mass spectrome-
ter. H, 13C{1H}, and 31P{1H} NMR spectra were determined in deuterated
1
MeOH (CD3OD) or deuterated dimethyl sulfoxide ([D6]DMSO) on
Bruker DPX Ultrashield NMR (300 or 400 MHz) spectrometers. Chemi-
cal shifts were given in parts per million d downfield relative to the cen-
tral peak of the solvents, and J values (coupling constants) were given in
Hz. The following abbreviations were used: s=singlet, d=doublet, dd=
double doublet, t=triplet, sep=septet, m=multiplet, br=broad.
Lastly, the chloride ligand was replaced with the strong s-
donor PTA species to tune the steric bulk around the RuII
center. Upon substitution, a 1.2–5.6-fold decrease in the
binding affinities were observed. The fact that there was
only a slight decrease (1.2–5.6-fold) in the affinity profile
suggests that the sterically encumbered PTA species would
be accommodated (as observed in the docking studies and
General procedure for the preparation of N-alkylpyrazoles 2: Alkyl
iodide (1.2 mmol) was added dropwise to a suspension of 3-amino-4-pyr-
azolecarbonitriles 1 (100 mg, 0.93 mmol) and anhydrous potassium carbo-
nate (1.5 mmol, 191.8 mg) in DMF (5 mL). The reaction mixture was
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Templated Assembly of Organoruthenium Antagonists of Adenosine Receptors
FULL PAPER
heated to reflux at 1008C for 12 h. After the complete conversion of the
starting material by monitoring with TLC analysis, the reaction mixture
was poured into ice cold water and extracted with ethyl acetate (5ꢄ
25 mL). The organic layers were recombined, dried over Na2SO4, filtered,
and concentrated at reduced pressure to afford the alkylated pyrazoles
2a–c as an inseparable mixture of N1 and N2 isomers. The pure N1
isomer was further purified by fractional crystallization from ethyl ace-
tate/hexane.
(KBr): n˜ =3699, 3286, 2180, 1692, 1627, 1570, 760 cmꢀ1
; ESI-MS
(MeOH): m/z: 181.0 [M+H]+; HRMS (ESI): m/z calcd for C6H8N6O+
Na+: 203.0657 [M+Na+]; found: 203.0650.
5-Amino-4-imino-2-phenethyl-4,5-dihydro-2H-pyrazoloACTHNUTRGNEU[GN 3,4-d]pyrimidin-
6ACTHNUTRGENUG(N 7H)-one (4b): Product was obtained as a white solid (58.6%, 87.5 mg).
1H NMR (400 MHz, [D6]DMSO): d=3.05 (t, J=7.2 Hz, 2H; CH2), 4.24
(t, J=7.2 Hz, 2H; CH2), 5.33 (br, 2H; NH2), 7.17–7.28 (m, 5H; Ph), 7.93
(s, 1H; pyrazolo-H), 8.25 (s, 1H; CONH), 9.08 ppm (s, 1H; C=NH); IR
(KBr): n˜ =3600, 3350, 2342, 1644, 1620, 1539, 820 cmꢀ1
; ESI-MS
3-Amino-1-methyl-1H-pyrazole-4-carbonitrile (2a): Product was obtained
as a pale yellow solid (67.3%, 76 mg). 1H NMR (300 MHz, [D6]DMSO):
d=3.51 (s, 3H; Me), 6.52 (s, 2H; NH2), 7.49 ppm (s, 1H; pyrazolo-H);
13C{1H} NMR (300 MHz, [D6]DMSO): d=35.6 (Me), 73.1 (C), 116.3 (C),
140.9 (CH), 152.5 ppm (C); ESI-MS (MeOH): m/z: 123.0 [M+H]+.
(MeOH): m/z: 271 [M+H]+; HRMS (ESI): m/z calcd for C13H14N6O+
H+: 271.1307 [M+H+]; found: 271.1302.
5-Amino-4-imino-2-(3-phenylpropyl)-4,5-dihydro-2H-pyrazolo
ACHTUNGTRENUN[NG 3,4-d]py-
A
R
ACHTUNGTRENNUNG
74.6 mg). 1H NMR (400 MHz, [D6]DMSO): d=2.00 (p, J=7.2 Hz, 2H;
CH2), 2.55 (t, J=7.2 Hz, 2H; CH2), 4.02 (t, J=7.2 Hz, 2H; CH2), 5.29 (s,
2H; NH2), 7.16–7.30 (m, 5H; Ph), 7.90 (s, 1H; pyrazolo-H), 8.02 (s, 1H;
CONH), 8.82 ppm (s, 1H; C=NH); IR (KBr): n˜ =3625, 3327, 3249, 2647,
1667, 1630, 1515, 1142, 782 cmꢀ1; ESI-MS (MeOH): m/z: 285 [M+H]+;
HRMS (ESI): m/z calcd for C14H16N6O+H+: 285.1464 [M+H+]; found:
285.1461.
3-Amino-1-phenethyl-1H-pyrazole-4-carbonitrile (2b): Product was ob-
tained as a yellow solid (76.2%, 149 mg). 1H NMR (300 MHz, CD3OD):
d=3.08 (t, J=7.2 Hz, 2H; CH2), 4.15 (t, J=6.9 Hz, 2H; CH2), 7.09–7.29
(m, 5H; Ph), 7.56 ppm (s, 1H; CH); ESI-MS (MeOH): m/z: 213.0 [M+
H]+.
3-Amino-1-(3-phenylpropyl)-1H-pyrazole-4-carbonitrile (2c): Product
was obtained as a brown solid (82.2%, 172 mg). 1H NMR (300 MHz,
CD3OD): d=2.03–2.14 (m, 2H; CH2), 2.56–2.65 (m, 2H; CH2), 3.92–3.97
(m, 2H; CH2), 7.18 (d, J=5.7 Hz, 2H; Ph), 7.28 (t, J=7.2 Hz, 3H; Ph),
7.51 (s, 2H; NH2), 7.84 ppm (s, 1H; CH); ESI-MS (MeOH): m/z: 227.0
[M+H]+.
4-Imino-2-methyl-5-(phenylamino)-4,5-dihydro-2H-pyrazolo-[3,4-d]py-
ACHUTNGRENrNUG imACHTUNGTRENNiNUG ACHTUNRTGENNGdUN in-6ACHTUGNRTEN(NUNG 7H)-one (4d): Phenylhydrazine (0.05 mL, 1.5 equiv) was added
dropwise to ligand 3a (0.25 mmol, 48.5 mg) in dry 2-methoxyethanol
(2 mL) in a two-necked round bottom flask under nitrogen. The reaction
mixture was heated to reflux at 1208C for 8 h. A white solid precipitated
and was filtered. The crude product was washed with acetone and recrys-
tallized from MeOH/hexane as a white solid (52.6%, 33.7 mg). 1H NMR
General procedure for the synthesis of pyrazole-3-yl-carbamate 3: Pyri-
dine (1 mL) was added to 3-amino-(N1-substituted)-4-pyrazolecarboni-
triles 2 (0.491 mmol) and a catalytic amount of DMAP in a two-necked
round-bottom flask at 08C. The solution was stirred in an inert atmos-
phere. Ethyl chloroformate (1.3 equiv) was added dropwise to the reac-
tion mixture, which was further stirred for 10 h. After the complete con-
sumption of the starting material, as monitored by TLC analysis, the re-
action mixture was poured into ice-cold water and extracted with ethyl
acetate (5ꢄ25 mL). The organic layers were recombined, dried over
Na2SO4, filtered, and concentrated at reduced pressure. The residue was
crystallized from ethyl acetate/hexane mixture.
ꢀ
(400 MHz, [D6]DMSO): d=3.61 (s, 3H; N Me), 6.63 (d, J=7.6 Hz, 2H;
CH), 6.85 (t, J=7.2 Hz, 1H; CH), 7.19 (t, J=7.2 Hz, 2H; CH), 7.94 (s,
1H; pyrazole-H), 8.42 (s, 1H; NH), 8.63 (s, 1H; NHCO), 9.05 ppm (s,
1H; C=NH); IR (KBr): n˜ =3511, 3427, 3246, 2984, 1670, 1631, 1602,
1515, 1495, 766 cmꢀ1; ESI-MS (MeOH): m/z: 257.0 [M+H]+; HRMS
(ESI): m/z calcd for C12H12N6O+H+: 257.1151 [M+H+]; found:
257.1153.
N-(4-Imino-2-methyl-6-oxo-6,7-dihydro-2H-pyrazoloACTHUNRTGNEUNG[3,4-d]-pyrimidin-
Ethyl 4-cyano-1-methyl-1H-pyrazol-3-ylcarbamate (3a): Product was ob-
tained as
[D6]DMSO): d=1.25 (t, J=7.2 Hz, 3H; Me), 3.67 (s, 3H; NMe), 4.18 (q,
5(4H)-yl)benzamide (4e): Ligand 3a (0.21 mmol, 40 mg) was dissolved in
2-methoxyethanol (2 mL) and the solution was taken in a two-necked
round bottom flask under nitrogen atmosphere. Benzhydrazide (32 mg,
1.5 equiv) was added to the reaction mixture, which was heated to reflux
at 1208C for 8 h. A white solid precipitated and was filtered. The crude
product was washed with acetone and recrystallized from MeOH/hexane
as a white solid (56.2%, 32.9 mg). 1H NMR (300 MHz, [D6]DMSO): d=
3.62 (s, 3H; Me), 7.48–7.88 (m, 5H; CH), 7.92 (s, 1H; CH), 8.02 (s, 1H;
NHCO), 8.04 (s, 1H; NHCO), 8.47 ppm (s, 1H; NH); IR (KBr): n˜ =
3627, 3426, 3257, 1739, 1670, 1629, 1542, 788 cmꢀ1; ESI-MS (MeOH): m/
z: 285 [M+H]+; HRMS (ESI): m/z calcd for C13H12N6O2 +H+: 285.1100
[M+H+]; found: 285.1082.
a
yellow solid (87.2%, 83.2 mg). 1H NMR (300 MHz,
ꢀ
J=6.9 Hz, 2H; O CH2), 7.95 (s, 1H; pyrazolo-H), 10.09 ppm (s, 1H;
13
1
ꢀ
CONH); C{ H} NMR (300 MHz, [D6]DMSO): d=15.3 (Me), 37.2 (N
ꢀ
Me), 62.8 (OCH2), 87.4 (C CN), 114.2 (CN), 142.0 (C), 142.1 (CH),
154.0 ppm (CONH); ESI-MS (MeOH): m/z: 195.0 [M+H]+.
Ethyl 4-cyano-1-phenethyl-1H-pyrazol-3-ylcarbamate (3b): Product was
obtained as an orange solid (71.8%, 102 mg). 1H NMR (300 MHz,
CD3OD): d=1.23–1.30 (m, 3H; Me), 3.14 (t, J=7.2 Hz, 2H; CH2), 4.12
ꢀ
(q, J=7.2 Hz, 2H; O CH2), 4.28–4.32 (m, 2H; CH2), 7.18–7.22 (m, 5H;
Ph), 7.87 (s, 1H; pyrazolo-H), 8.55 ppm (s, 1H; CONH); ESI-MS
(MeOH): m/z: 285.0 [M+H]+.
4-Imino-2-methyl-2H-pyrazoloACTHNUGRTENUNG[3,4-d]pyrimidin-5(4H)-amine (4 f): Hydra-
Ethyl 4-cyano-1-(3-phenylpropyl)-1H-pyrazol-3-ylcarbamate (3c): Prod-
uct was obtained as a yellow solid (74.2%, 111 mg). 1H NMR (300 MHz,
CD3OD): d=1.23–1.30 (m, 3H; Me), 2.16 (m, 2H; CH2), 2.56–2.66 (m,
2H; CH2), 4.01–4.03 (t, J=7.5 Hz, 2H; CH2), 4.10–4.29 (m, 2H; OCH2),
7.18–7.28 (m, 5H; Ph), 8.14 (s, 1H; pyrazolo-H), 8.55 ppm (s, 1H;
CONH); ESI-MS (MeOH): m/z: 299.0 [M+H]+.
zine hydrate (0.25 mL, 0.5 mmol) was added to a cold (0–58C) solution
of intermediate 3 f in EtOH (10 mL). The mixture was stirred at room
temperature for 5 h in an inert atmosphere. The resulting precipitate was
filtered and washed with EtOH to provide 4 f as a white solid (82.5%,
76 mg). 1H NMR (300 MHz, [D6]DMSO): d=3.80 (s, 3H; NMe), 5.54 (s,
2H; NH2), 7.65 (s, 1H; C=NH), 7.98 (s, 1H; pyrazole-H), 8.04 (s, 1H;
13
ꢀ
CH=N); C NMR (300 MHz, [D6]DMSO): d=34.7 (N Me), 104.9 (C-
General procedure for the preparation of pyrazolopyrimidine-6-ones
4a–c: N-Carbamates 3 (0.5 mmol) was dissolved in dry EtOH (2 mL) and
the solution was poured in a two-necked round-bottom flask in a nitro-
gen atmosphere. Hydrazine hydrate (1.5 equiv) was added to the reaction
mixture dropwise, which was heated to reflux at 808C for 6 h. A white
precipitate was formed from the reaction mixture and filtered. The crude
solid was washed with acetone and recrystallized from MeOH/hexane.
C=NH), 134.7 (CH-pyrazole), 148.6 (C), 151.4 (CH), 152.6 ppm (C=NH);
IR (KBr): n˜ =3457, 3286, 2938, 2140, 1662, 1609, 1551, 1229, 1157, 855,
767 cmꢀ1; ESI-MS (MeOH): m/z: 165.0 [M+H]+; HRMS (ESI): m/z
calcd for C6H8N6 +H+: 165.0889 [M+H+]; found: 165.0877.
General procedure for the preparation of ruthenium(II)/pyrazolo–pyrimi-
dine complexes 5a–d, 5 f, 6a, 6 f, 7a, 7b, and 7 f: All the compounds are
synthesized by using a similar procedure. Typically, ligand 4a–d or 4 f
(2.1 equiv) was dissolved in dry MeOH (4 mL) and added to [{(h6-cy-
5-Amino-4-imino-2-methyl-4,5-dihydro-2H-pyrazoloACTHNUGRTENUNG[3,4-d]pyrimidin-6-
ACHTUNGTRENNUNG(7H)-one (4a): Product was obtained as a white solid (57.8%, 52 mg).
1H NMR (300 MHz, [D6]DMSO): d=3.59 (s, 3H; NMe), 5.31 (s, 2H;
NH2), 7.85 (s, 1H; pyrazolo-H), 8.03 (s, 1H; CONH), 8.82 ppm (s, 1H;
AHCTUNGTERGUNmNN ene)RuCl2}2] in dry MeOH (3 mL). The solution immediately changed
from red to brown/violet and was stirred at room temperature for 2 h.
After complete conversion of the reagents, monitored by using TLC anal-
ysis, the product was precipitated with excess of diethyl ether. The crude
13
1
ꢀ
C=NH), C{ H} NMR (300 MHz, [D6]DMSO): d=33.7 (N Me), 91.9
ꢀ
(C CN), 134.2 (CH), 153.0 (C), 154.5 (C=NH), 155.1 ppm (CONH); IR
Chem. Eur. J. 2013, 00, 0 – 0
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W. H. Ang, G. Pastorin et al.
product was further washed with diethyl ether followed by acetone to
yield the final product as a yellow–orange powder.
[(h6-cymene)RuCl(k2-4a)]·Cl (5a): [{(h6-cymene)RuCl2}2] (10 mg,
ACHTUNGTRENNUNG
6.0 Hz, 1H; CH-cymene), 5.79 (d, J=6.0 Hz, 1H; CH-cymene), 5.96 (d,
J=6.0 Hz, 1H; CH-cymene), 7.97 (s, 1H; CH), 8.55 (s, 1H; CH), 8.72 (d,
J=8.0 Hz, 1H; NH), 10.05 (s, 1H; NH), 11.12 ppm (d, J=8.0 Hz, 1H;
NH); IR (KBr): n˜ =3465, 3249, 3118, 2966, 1662, 1608, 1542, 1425, 1230,
1174, 821 cmꢀ1; ESI-MS (MeOH): m/z: 435.0 [M]+, 399.0 [MꢀHCl]+;
HRMS (ESI): m/z calcd for [C16H22N6RuClꢀHCl]+: 399.0871 [MꢀHCl]+;
found: 399.0870; single crystals suitable for X-ray diffraction studies were
obtained by vapor diffusion of diethyl ether into a saturated solution of
the compound in MeOH.
16 mmol) and 4a (2.1 equiv, 34 mmol, 6.1 mg) were used. The solution
turned from red to yellow–brown after stirring for 2 h. The product was
obtained as a pale-yellow solid (88.5%, 14.0 mg) with HPLC purity
(96.4%, eluent: 80% ACN/H2O, Rt =1.74 min). 1H NMR (400 MHz,
[D6]DMSO): d=1.14(d, J=7.2 Hz, 3H; Me2CH), 1.23 (d, J=6.8 Hz, 3H;
Me2CH), 2.15 (s, 3H; Me), 2.84 (sep, J=6.8 Hz, 1H; CH), 3.75 (s, 3H;
NMe), 5.45 (d, J=6.0 Hz, 1H; CH-cymene), 5.67 (br, 2H; CH-cymene),
5.85 (d, J=6.0 Hz, 1H; CH-cymene), 7.80 (d, J=9.6 Hz, 1H; NH), 8.07
(s, 1H; CH), 9.79 (d, J=10.4 Hz, 1H; NH), 10.08 (s, 1H; NHCO),
12.77 ppm (br, 1H; NH); IR (KBr): n˜ =3733, 3647, 3029, 1732, 1667,
1614, 1355, 1221, 1177, 821 cmꢀ1; ESI-MS (MeOH): m/z: 451.0 [M]+, 415
[MꢀHCl]+; HRMS (ESI): m/z calcd for [C16H22N6ORuClꢀHCl]+:
415.0820 [MꢀHCl]+; found: 415.0779.
ACTHNUTRGNEUNG
[(h6-benzene)RuCl(k2-4a)]·Cl (6a): Complex [{(h6-benzene)RuCl2}2]
(10 mg, 20 mmol) and 4a (2.1 equiv, 42 mmol, 7.0 mg) were used. The solu-
tion turned from red to deep brown after stirring for 2 h. The product
was obtained as a pale-yellow solid (80.4%, 13.8 mg) with HPLC purity
(99.3%, eluent: 60% ACN/H2O, Rt: 1.36 min). 1H NMR (400 MHz,
[D6]DMSO): d=3.74 (s, 3H; NMe), 5.81 (s, 6H; CH-benzene), 7.86 (d,
J=8.4 Hz, 1H; NH), 8.02 (s, 1H; CH), 10.00 (d, J=8.8 Hz, 1H; NH),
10.20 (s, 1H; NHCO), 12.71 ppm (br, 1H; NH); IR (KBr): n˜ =3497,
3385, 3178, 2760, 1642, 1597, 1557, 1400, 1239, 844, 758 cmꢀ1; ESI-MS
(MeOH): m/z: 394.9 [M]+, 358.9 [MꢀHCl]+; HRMS (ESI): m/z calcd for
[C12H14N6ORuClꢀHCl]+: 359.0194 [MꢀHCl]+; found: 359.0192.
ACHTUNGTRENNUNG
[(h6-cymene)RuCl(k2-4b)]·Cl (5b): [{(h6-cymene)RuCl2}2] (10 mg,
16 mmol) and 4b (2.1 equiv, 34 mmol, 9.2 mg) were used. The solution
turned from red to yellow–brown after stirring for 2 h. The product was
obtained as a brown solid (82.4%, 15.5 mg) with HPLC purity (99.8%,
eluent: 80% ACN/H2O, Rt: 2.04 min). 1H NMR (400 MHz, [D6]DMSO):
d=1.12 (d, J=6.8 Hz, 3H; Me2CH), 1.21 (d, J=6.8 Hz, 3H; Me2CH),
2.12 (s, 3H; Me), 2.80 (sep, J=6.8 Hz, 1H; CH), 3.00 (t, J=7.2 Hz, 2H;
CH2), 4.14 (t, J=8.0 Hz, 2H; CH2), 5.35 (d, J=5.6 Hz, 1H; CH-cymene),
5.60 (br, 1H; CH-cymene), 5.79 (d, J=6.0 Hz, 1H; CH-cymene), 5.83 (d,
J=5.2 Hz, 1H; CH-cymene), 7.17–7.25 (m, 5H; Ph), 7.27 (2 brs, 2H;
NH), 7.79 (s, 1H; CH), 8.55 (s, 1H; NHCO), 9.51 ppm (d, J=10.2 Hz,
1H; NH); IR (KBr): n˜ =3640, 3349, 3058, 2930, 1726, 1664, 1442, 1213,
ACTHNUTRGNEUNG
[(h6-benzene)RuCl(k2-4 f)]·Cl (6 f): Complex [{(h6-benzene)RuCl2}2]
(10 mg, 20 mmol) and 4 f (2.1 equiv, 42 mmol, 6.4 mg) were used. The solu-
tion turned from red to deep brown after stirring for 2 h. The product
was obtained as a brown solid (82.5%, 13.6 mg) with HPLC purity
(98.3%, eluent: 60% ACN/H2O, Rt: 2.14 min). 1H NMR (400 MHz,
[D6]DMSO): d=3.96 (s, 3H; NMe), 6.02 (s, 6H; CH-benzene), 8.04 (d,
J=8.4 Hz, 1H; NH), 8.18 (s, 1H; CH), 9.04 (s, 1H; CH), 10.23 (d, J=
8.4 Hz, 1H; NH), 11.8 ppm (s, 1H; NH); IR (KBr): n˜ =3659, 3303, 2987,
1705, 1617, 1555, 1473, 1194, 807, 759 cmꢀ1; ESI-MS (MeOH): m/z: 379.0
1127, 1052, 781 cmꢀ1
;
ESI-MS (MeOH): m/z: 540.9 [M]+, 504.8
[MꢀHCl]+; HRMS (ESI): m/z calcd for [C23H28N6ORuClꢀHCl]+:
[M]+,
342.8
[MꢀHCl]+;
HRMS
(ESI):
m/z
calcd
for
505.1289 [MꢀHCl]+; found: 505.1289.
[C12H14N6RuClꢀHCl]+: 343.0245 [MꢀHCl]+; found: 343.0232.
[(h6-cymene)RuCl
N
(5c):
[{(h6-cymene)RuCl2}2]
(10 mg,
ACTHNGUTERNNUG
[(h6-triisopropylbenzene)RuCl(k2-4a)]·Cl (7a): Complex [{(h6-triisopro-
16 mmol) and 4c (2.1 equiv, 34 mmol, 9.7 mg) were used. The solution
turned from red to yellow–brown after stirring for 2 h. The product was
obtained as a brown solid (92.2%, 17.7 mg) with HPLC purity (99.8%,
eluent: 80% ACN/H2O, Rt: 2.17 min). 1H NMR (300 MHz, [D6]DMSO):
d=1.14 (d, J=6.9 Hz, 3H; Me2CH), 1.22 (d, J=6.3 Hz, 3H; Me2CH),
2.03 (m, 2H; CH2), 2.15 (s, 3H; Me), 2.58 (m, 2H; CH2), 2.83 (m, 1H;
CH), 4.14 (m, 2H; CH2), 5.45 (d, J=5.4 Hz, 1H; CH-cymene), 5.67 (s,
2H; CH-cymene), 5.86 (d, J=5.7 Hz, 1H; CH-cymene), 7.15–7.25 (m,
5H; Ph), 7.79 (d, J=8.7 Hz, 1H; NH), 8.10 (s, 1H; CH), 9.80 (d, J=
9.0 Hz, 1H; NH), 10.12 (s, 1H; NHCO), 12.72 ppm (s, 1H; NH); IR
(KBr): n˜ =3612, 3340, 3042, 2720, 1712, 1624, 1229, 1156, 792 cmꢀ1; ESI-
MS (MeOH): m/z: 554.8 [M]+, 518.8 [MꢀHCl]+; HRMS (ESI): m/z
calcd for [C24H30N6ORuClꢀHCl]+: 519.1446 [MꢀHCl]+; found:
519.1449.
pylbenzene)RuCl2}2] (10 mg, 13 mmol) and 4a (2.1 equiv, 27 mmol, 4.9 mg)
were used. The solution turned from red to deep orange after stirring for
2 h. The product was obtained as an orange solid (89.2%, 13.2 mg) with
HPLC purity (99.3%, eluent: 80% ACN/H2O, Rt: 1.75 min). 1H NMR
(400 MHz, [D6]DMSO): d=1.21 (d, J=6.6 Hz, 9H; Me2CH), 1.26 (d, J=
6.6 Hz, 9H; Me2CH), 2.90 (sep, J=6.9 Hz, 3H; CH), 3.75 (s, 3H; NMe),
5.59 (s, 3H; CH-isopropyl benzene), 7.85 (d, J=8.0 Hz, 1H; NH), 8.09 (s,
1H; CH), 9.64 (d, J=8.4 Hz, 1H; NH), 10.0 (s, 1H; NHCO), 12.77 ppm
(br, 1H; NH); IR (KBr): n˜ =3529, 3258, 2872, 1726, 1655, 1588, 1421,
1246, 881, 786 cmꢀ1; ESI-MS (MeOH): m/z: 521.0 [M]+, 484.7 [MꢀHCl]+
;
HRMS (ESI): m/z calcd for [C21H32N6ORuClꢀHCl]+: 485.1602
[MꢀHCl]+; found: 485.1611.
[(h6-triisopropylbenzene)RuCl(k2-4b)]·Cl (7b): Complex [{(h6-triisopro-
ACTHNUGTRNENUG
pylbenzene)RuCl2}2] (10 mg, 13 mmol) and 4b (2.1 equiv, 27 mmol,
7.3 mg) were used. The solution turned from red to deep orange after
stirring of 2 h. The product was obtained as a pale-yellow solid (86.4%,
14.8 mg) with HPLC purity (98.3%, eluent: 80% ACN/H2O, Rt:
2.14 min). 1H NMR (400 MHz, CD3OD): d=1.20 (d, J=6.4 Hz, 9H;
Me2CH), 1.25 (d, J=7.2 Hz, 9H; Me2CH), 2.89 (m, 3H; CH), 3.00 (t, J=
7.2 Hz, 2H; CH2), 4.22 (br, 2H; CH2), 5.57 (s, 3H; CH-isopropyl ben-
zene), 7.05 (s, 1H; NH), 7.13–7.22 (m, 5H; Ph), 7.26 (s, 1H; NH), 7.28 (s,
1H; CH), 7.91 (s, 1H; NHCO), 9.51 ppm (d, J=8.8 Hz, 1H; NH); IR
ACHTUNGTRENNUNG
[(h6-cymene)RuCl(k2-4d)]·Cl (5d): Complex [{(h6-cymene)RuCl2}2]
(10 mg, 16 mmol) and 4d (2.1 equiv, 34 mmol, 8.7 mg) were used. The sol-
ution turned from red to deep violet after stirring for 2 h. The product
was obtained as a black solid (87.4%, 16.0 mg) with HPLC purity
(98.5%, eluent: 80% ACN/H2O, Rt: 2.23 min). 1H NMR (300 MHz,
CD3OD): d=1.23 (d, J=7.2 Hz, 3H; Me2CH), 1.30 (br, 3H; Me2CH),
2.23 (s, 3H; Me), 2.46–2.51 (m, 1H; CH), 4.03 (s, 3H; NMe), 6.01 (d, J=
5.7 Hz, 1H; CH-cymene), 6.16 (d, J=6.0 Hz, 1H; CH-cymene), 6.23 (d,
J=5.7 Hz, 1H; CH-cymene), 6.61 (d, J=6.3 Hz, 1H; CH-cymene), 7.09
(s, 1H; CH), 7.77–7.81 (m, 2H; CH), 7.89–7.92 (m, 1H; CH), 8.21 (d, J=
6.6 Hz, 2H; CH), 8.35 ppm (s, 1H; NHCO); IR (KBr): n˜ =3631, 3427,
3022, 2942, 1653, 1597, 1446, 1034, 878, 758 cmꢀ1; ESI-MS (MeOH): m/z:
527.4 [M]+, 491.4 [MꢀHCl]+; HRMS (ESI): m/z calcd for
[C22H26N6ORuClꢀHCl]+: 491.1133 [MꢀHCl]+; found: 491.1154.
(KBr): n˜ =3526, 3359, 2928, 1712, 1656, 1524, 1462, 1185, 857, 787 cmꢀ1
;
ESI-MS (MeOH): m/z: 611.1 [M]+, 574.9 [MꢀHCl]+; HRMS (ESI): m/z
calcd for [C28H38N6ORuClꢀHCl]+: 575.2072 [MꢀHCl]+; found:
575.2077.
ACTHNUTRGNEUNG
[(h6-triisopropylbenzene)RuCl(k2-4 f)]·Cl (7 f): Complex [{(h6-triisopro-
pylbenzene)RuCl2}2] (10 mg, 13 mmol) and 4 f (2.1 equiv, 27 mmol, 4.5 mg)
were used. The solution turned from red to deep orange after stirring of
2 h. The product was obtained as a yellow solid (82.6%, 11.9 mg) with
HPLC purity (99.8%, eluent: 80% ACN/H2O, Rt: 2.02 min). 1H NMR
(400 MHz, [D6]DMSO): d=1.21 (d, J=6.8 Hz, 9H; Me2CH), 1.27 (d, J=
6.8 Hz, 9H; Me2CH), 2.92 (sep, J=6.4 Hz, 3H; CH), 4.04 (s, 3H; NMe),
5.61 (s, 1H; CH-triisopropyl benzene), 7.98 (s, 1H; CH), 8.52 (s, 1H;
CH), 8.76 (d, J=8.0 Hz, 1H; NH), 9.93 (s, 1H; NH), 10.81 ppm (d, J=
8.0 Hz, 1H; NH); IR (KBr): n˜ =3514, 3250, 2964, 1663, 1587, 1425, 1246,
ACHTUNGTRENNUNG
[(h6-cymene)RuCl(k2-4 f)]·Cl (5 f): Complex [{(h6-cymene)RuCl2}2]
(10 mg, 16 mmol) and 4 f (2.1 equiv, 34 mmol, 5.6 mg) were used. The solu-
tion turned from red to yellowish orange after stirring for 2 h. The prod-
uct was obtained as a deep-yellow solid (86.2%, 13.4 mg) with HPLC
purity (99.8%, eluent: 80% ACN/H2O, Rt: 1.99 min). 1H NMR
(400 MHz, [D6]DMSO): d=1.16 (d, J=6.8 Hz, 3H; Me2CH), 1.23 (d, J=
6.8 Hz, 3H; Me2CH), 2.18 (s, 3H; Me), 2.88 (sep, J=6.8 Hz, 1H; CH),
4.04 (s, 3H; NMe), 5.49 (d, J=5.6 Hz, 1H; CH-cymene), 5.72 (d, J=
&
8
&
www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Templated Assembly of Organoruthenium Antagonists of Adenosine Receptors
FULL PAPER
894, 788 cmꢀ1; ESI-MS (MeOH): m/z: 505.0 [M]+, 469.0 [MꢀHCl]+;
HRMS (ESI): m/z calcd for [C21H32N6RuClꢀHCl]+: 469.1653 [MꢀHCl]+;
Acknowledgements
found: 469.1633; single crystals suitable for X-ray diffraction studies were
obtained by vapor diffusion of diethyl ether into a saturated solution of
the compound in MeOH.
This work was supported by the National University of Singapore and
Singapore Ministry of Education (MOE2009-T2–2–011 and R-398–000–
068–112 to G.P. and R-143–000–411–133 and R-143–000–512–112 to
W.H.A.). The authors acknowledge support by A*STAR-SERC TSRP-
iNPB 102 152 0016. The authors thank Sonja Kachler for her assistance
and expertise in the pharmacological evaluation of the compounds.
[(h6-cymene)Ru (k2-4a)]·2Cl (P5a):
ACHTUNGTRENNUNG(PTA)ACHTUNGTRENNUNG A solution of 5a (10 mg,
20 mmol) and PTA (1.1 equiv, 22 mmol, 3.6 mg) in MeOH turned from
orange to green after stirring for 2 h. A green precipitate was obtained
upon addition of diethyl ether and collected by filtration (83.2%, 11 mg)
with HPLC purity (99.5%, eluent: 80% ACN/H2O, Rt: 1.63 min).
1H NMR (300 MHz, [D6]DMSO): d=1.16 (d, J=6.9 Hz, 3H; Me2CH),
1.20 (d, J=6.6 Hz, 3H; Me2CH), 2.19 (s, 3H; Me), 2.73–2.80 (m, 1H;
CH), 3.71 (s, 3H; NMe), 4.07 (d, Jgem =13.5 Hz, 3H; CH2), 4.30 (d, Jgem=
13.5 Hz, 3H; CH2), 4.43 (s, 6H; CH2), 5.71 (d, J=5.7 Hz, 1H; CH-p
cymene), 6.11 (d, J=6.3 Hz, 1H; CH-p cymene), 6.21 (d, J=6.3 Hz, 1H;
CH-p cymene), 6.37 (br, 1H; CH-p cymene), 8.28 (s, 1H; CH), 9.17 (s,
1H; NHCO), 9.81 ppm (s, 1H; NH); 31P{1H} NMR (400Mz, [D6]DMSO):
d=ꢀ34.5 ppm; IR (KBr): n˜ =3477, 3261, 3149, 2970, 1659, 1537, 1420,
1284, 1151, 897, 774 cmꢀ1; ESI-MS (MeOH): m/z: 286.3 [M]2+, 414.8
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ꢀ
[MꢀH-PTA] ; HRMS (ESI): m/z calcd for [C22H34N9OPRu H-PTA] :
415.0820 [MꢀH-PTA]+; found: 415.0831.
[(h6-cymene)Ru (k2-4 f)]·2Cl (P5 f):
ACHTUNGTRENNUNG(PTA)ACHTUNGTRENNUNG A solution of 5 f (10 mg,
20 mmol) and PTA (1.1 equiv, 22 mmol, 3.6 mg) in MeOH turned from
orange to green after stirring for 2 h. A green precipitate was obtained
upon addition of diethyl ether and collected by filtration (86.4%,
11.5 mg) with HPLC purity (88.3%, eluent: 80% ACN/H2O, Rt:
2.08 min). 1H NMR (300 MHz, [D6]DMSO): d=1.18 (d, J=6.0 Hz, 3H;
Me2CH), 1.22 (d, J=6.6 Hz, 3H; Me2CH), 2.20 (s, 3H; Me), 2.78–2.84
(m, 1H; CH), 4.06 (s, 3H; NMe), 4.34–4.45 (m, 12H; CH2), 5.80 (br, 1H;
CH-p cymene), 6.20 (br, 1H; CH-p cymene), 6.42 (br, 2H; CH-p
cymene), 8.17 (s, 1H; CH), 8.78 (s, 1H; CH), 9.56 (br, 1H; NH), 10.82
(br, 1H; NH), 11.22 ppm (br, 1H; NH); 31P{1H} NMR (400Mz,
[D6]DMSO): d=ꢀ35.4 ppm; IR (KBr): n˜ =3466, 3266, 3149, 2906, 1659,
1544, 1240, 781. cmꢀ1; ESI-MS (MeOH): m/z: 278.3 [M]2+, 399.0 [MꢀH-
PTA]+; HRMS (ESI): m/z calcd for [C22H34N9PRuꢀH-PTA]+: 399.0871
[MꢀH-PTA]+; found: 399.0861.
X-ray diffraction studies: X-ray data were collected with a Bruker AXS
SMART APEX diffractometer using MoKa radiation at 223(2) K with the
SMART suite of Programs.[30] Data were processed and corrected for
Lorentz and polarization effects by means of SAINT software,[31] and for
absorption effects using the SADABS software.[32] Structural solution and
refinement were carried out by using the SHELXTL suite of programs.[33]
The structure was solved by using Direct Methods. Non-hydrogen atoms
were located by using difference maps and were given anisotropic dis-
placement parameters in the final refinement. All the H atoms were put
at calculated positions by means of the riding model. CCDC-900858 and
CCDC-900859 contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Binding studies: Radio
ands were carried out at room temperature as described previously.[34]
Competition-binding studies were carried out at a concentration of 1 nm,
in saturation experiments a radioligand concentration of 0.2–10 nm was
ACHTUNGTRENNUNGliACHTUNGTRENNUNGgand-binding experiments with tritiated radiolig-
ACHTUNGTRENNUNG
A
A
used. Nonspecific binding was determined in the presence of 1 mm theo-
phylline for A1AR and 100 mm (R)-N6-phenylisopropyladenosine (R-
PIA) for A2AAR and A3AR. For the incubation and separation of bound
from free ligand, a 96-well microplate filtration system (Millipore Multi-
screen MAFC) was used. The Ki values were calculated from competition
curves by nonlinear curve fitting with the program SCTFIT.[35] In the case
of hA2BAR, adenylyl cyclase experiments were carried out as described
previously with minor modifications.[34,36] Membranes transfected with
hA2B receptor were incubated with 100 nm NECA and 150000 cpm of
[a-32P]ATP and tested compounds in different concentrations for 20 min
in the incubation mixture without ethylene glycol tetraacetic acid
(EGTA) and NaCl. The Ki values for the concentration-dependent inhib-
ition of NECA-mediated adenylyl cyclase activity caused by tested antag-
onists were calculated accordingly.
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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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ÞÞ
These are not the final page numbers!

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Received: September 14, 2012
Revised: January 15, 2013
Published online: && &&, 0000
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10
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www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Templated Assembly of Organoruthenium Antagonists of Adenosine Receptors
FULL PAPER
Scaffold design: A novel class of ruthe-
nium(II)–arene complexes containing
chelating N,N-pyrazolo–pyrimidine
ligands was rationally developed to be
selective antagonists of human A3
adenACHTUNGTRENNUNGosine receptors based on the
proven pyrazolo–triazolo–pyrimidine
design (see figure).
Inhibitors
P. Paira, M. J. Chow, G. Venkatesan,
V. K. Kosaraju, S.-L. Cheong,
K.-N. Klotz, W. H. Ang,*
G. Pastorin* ................... &&&& — &&&&
Organoruthenium Antagonists of
Human A3 Adenosine Receptors
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
11
&
ÞÞ
These are not the final page numbers!