Organometallic pyridylnaphthalimide complexes as protein kinase inhibitors

Article abstract of DOI:10.1021/om200366r

A new metal-containing scaffold for the design of protein kinase inhibitors is introduced. The key feature is a 3-(2-pyridyl)-1,8-naphthalimide "pharmacophore chelate ligand", which is designed to form two hydrogen bonds with the hinge region of the ATP-b

Full text of DOI:10.1021/om200366r

ARTICLE
pubs.acs.org/Organometallics
Organometallic Pyridylnaphthalimide Complexes as Protein Kinase
Inhibitors
Sebastian Blanck,† Thomas Cruchter,† Adina Vultur,‡ Radostan Riedel,† Klaus Harms,† Meenhard Herlyn,‡
and Eric Meggers*,†
†Fachbereich Chemie, Philipps-Universit€at Marburg, Hans-Meerwein-Strasse, 35043 Marburg, Germany
‡The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, United States
S Supporting Information
b
ABSTRACT: A new metal-containing scaffold for the design of protein kinase inhibitors
is introduced. The key feature is a 3-(2-pyridyl)-1,8-naphthalimide “pharmacophore
chelate ligand”, which is designed to form two hydrogen bonds with the hinge region of
the ATP-binding site and is at the same time capable of serving as a stable bidentate
ligand through CÀH activation at the 4-position of the electron-deficient naphthalene
moiety. This CÀH activation leads to a reduced demand for coordinating heteroatoms
and thus sets the basis for a very efficient three-step synthesis starting from 1,8-naphthalic
anhydride. The versatility of this ligand is demonstrated with the discovery of a
ruthenium complex that functions as a nanomolar inhibitor for myosin light-chain
kinase (MYLK or MLCK).
’ INTRODUCTION
of the ATP-binding site of protein kinases, whereas the remain-
ing coordination sphere can then be established through combi-
natorial chemistry, structureÀactivity relationships, structure-
based design, and combinations thereof. Although this approach
turned out to be highly successful, we recently became interested
in designing new pharmacophore chelate ligands for metal-
containing protein kinase inhibitors for two reasons: First, the
synthesis of the pyridocarbazole ligand is lengthy and includes
one photochemical step, thus complicating derivatizations in the
course of structureÀactivity relationships and inhibitor scale-up
reactions for in vivo studies.10 Second, we envisioned that placing
the metal center at a different position within the ATP-binding
site might allow us to discover metal-containing inhibitors with
very different binding profiles. In this respect it is noteworthy that
protein kinases are among the largest enzyme families with more
than 500 putative protein kinase genes,11 and we estimate that
our previous pyridocarbazole metal complex scaffold is probably
only suitable for a subset of them. Here we now introduce 3-(2-
pyridyl)-1,8-naphthalimide (1a; Scheme 1) as such a novel
pharmacophore ligand. It can be synthesized in just three steps
from readily available 1,8-naphthalic anhydride and coordinates
to ruthenium in a bidentate fashion by activating a CÀH bond
within the naphthalimide moiety, and as a proof of principle we
report an organoruthenium complex which serves as a selective
and nanomolar protein kinase inhibitor.
The last two decades have witnessed a steadily growing interest
for organometallic compounds in medicinal chemistry and chemical
biology.1 The unique properties of metal complexes such as
structural diversity, adjustable ligand exchange kinetics, fine-
tuned redox activities, photoreactivity, the availability of radio-
isotopes, and distinct spectroscopic signatures make them highly
versatile scaffolds for the modulation, sensing, and imaging of
biological processes.2
Several years ago we established a research program exploring
substitutionally inert metal-containing compounds as structural
scaffolds for the design of enzyme inhibitors, and we demonstrated
that ruthenium(II),3 platinum(II),4 osmium(II),5 and iridium(III)6
complexes, for example, can serve as highly potent and selective
inhibitors of protein kinases and lipid kinases.7 We believe that
especially octahedral coordination modes offer new opportunities
to design globular and well-defined molecular shapes that can fill
protein pockets such as enzyme active sites in unique ways.8
Our previous design was entirely based on the staurosporine-
inspired metallo-pyridocarbazole scaffold shown in Figure 1, in
which the maleimide moiety forms two key hydrogen bonds with
the hinge region of the ATP-binding site, the pyridocarbazole
heterocycle occupies the hydrophobic adenine binding cleft, and
the remaining globular space-demanding octahedral or half-
sandwich coordination sphere undergoes interactions in the region
of the ribose-triphosphate binding site.9 In this design strategy,
the pyridocarbazole heterocycle—called “pharmacophore chelate
ligand”—is rationally designed to interact with the hinge region
Received: May 3, 2011
Published: July 18, 2011
r
2011 American Chemical Society
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Scheme 2. Synthesis of Ruthenium Half-Sandwich
Complexes 5a,b as Racemic Mixturesa
a Only one enantiomer is shown.
Figure 1. Previous and new design for metal complexes as protein
kinase inhibitors. Shown are the intended interactions of the so-called
“pharmacophore chelate ligand” with the hinge region of the ATP-
binding site of protein kinases.
Scheme 1. Synthesis of 3-(2-Pyridyl)-1,8-naphthalimide (1a)
and N-Benzyl-3-(2-pyridyl)-1,8-naphthalimide (1b)
Figure 2. ORTEP drawing with 50% probability thermal ellipsoids,
giving the crystal structure of half-sandwich complex 5b. A disordered
CH2Cl2 solvent molecule is omitted. Selected bond distances (Å) and
angles (deg): Ru1ÀC1 = 2.073(8), Ru1ÀN15 = 2.094(9), Ru1ÀC27 =
2.230(10), Ru1ÀC30 = 2.274(7), Ru1ÀC111 = 1.838(12); C1ÀRuÀ
N15 = 78.8(4), C111ÀRu1ÀC1 = 87.2(4), C111ÀRu1ÀN15 =
97.0(4).
’ RESULTS AND DISCUSSION
Synthesis of the 3-(2-Pyridyl)-1,8-naphthalimide Ligand.
The synthesis of the 3-(2-pyridyl)-1,8-naphthalimide ligand 1a
(R = H) and its benzylated derivative 1b (R = Bn), which merely
served as a crystallization handle, is outlined in Scheme 1. Accord-
ingly, a bromination of 1,8-naphthalic anhydride 2 in concen-
trated sulfuric acid using silver(I) sulfate as catalyst afforded
exclusively 3-bromo-1,8-naphthalic anhydride (3; 91%), which
was further converted to the free imide 4a (93%) or the benzyl-
protected imide 4b (71%) by treatment with concentrated ammo-
nia or benzylamine, respectively. A subsequent Pd-mediated
Stille cross coupling with 2-(trimethylstannyl)pyridine provided
the final ligands 1a (67%) and 1b (99%) in three steps overall and
total yields of 57% and 64%, respectively.
Synthesis of Half-Sandwich Compounds. To investigate the
coordination properties of the new pyridylnaphthalimide ligands,
we started with the synthesis of ruthenium half-sandwich com-
plexes. For example, the reaction of the ligands 1a,b with [Ru(η5-
C5H5)(CO)(MeCN)2]PF612 in the presence of Et3N at 60 °C
provided the complexes 5a (51%) and 5b (70%), which could be
purified by standard silica gel flash chromatography (Scheme 2).
As anticipated, the CÀH activation occurred solely at the
4-position of the naphthalimide moiety, while no CÀH activa-
tion at the sterically more hindered 2-position was observed. A
crystal structure of complex 5b is displayed in Figure 2 and
confirms the bidentate binding of the ruthenium to the pyridine
nitrogen (RuÀN = 2.09 Å) and the C-4 atom of the naphthalene
moiety (RuÀC = 2.07 Å). It is worth noting that the facile CÀH
activation and robustness of the formed RuÀC bond can be
attributed to the highly electron deficient nature of the naphtha-
lene moiety, being functionalized with electron-withdrawing
pyridyl and maleimide substituents.13
Synthesis of Octahedral Complexes. In order to evaluate the
synthesis of octahedral ruthenium complexes, ligand 1a was
14
reacted with [Ru(MeCN)3([9]aneS3)](CF3SO3)2 ([9]aneS3
= 1,4,7-trithiacylononane) and triethylamine in DMF at 60 °C to
obtain the monoacetonitrile complex 6 in 77% yield (Scheme 3).
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Scheme 3. Synthesis of the Octahedral 1,4,7-Trithiacyclononane Ruthenium Complexes 7À9 by Two Different Routesa
ARTICLE
a Only one enantiomer of the formed racemic mixtures 6À9 is shown.
This complex offers a quick access to different octahedral complexes
by exchanging the remaining semilabile acetonitrile with other
monodentate ligands. For example, heating the acetonitrile complex
6 under an atmosphere of CO for 3 h provided the CO complex 7
in 87% yield. The synthesis of the thiocyanate complex 8 and
the selenocyanate complex 9 were accomplished by reacting com-
plex 6 with NaSCN (23% yield) and KSeCN (30% yield),
respectively. All three complexes were purified by regular silica gel
flash chromatography. As an example, complex 7 was tested for its
stability under biologically relevant conditions and was found to be
stable in aqueous solution under air in the presence of millimolar
concentrations of 2-mercaptoethanol for several days without any
sign of decomposition, as determined by 1H NMR spectroscopy.
The crystal structure of complex 7 displayed in Figure 3 reveals
that, in contrast to the half-sandwich complex 5b, the pyridyl-
naphthalimide ligand in the octahedral complex is twisted, which
is apparently due to a steric repulsion between the CÀH group at
the 5-position of the naphthalimide moiety and the sterically
demanding 1,4,7-trithiacyclononane ligand. This steric repulsion
also results in a significant distortion of the entire octahedral
ruthenium coordination geometry. For example, the CO ligand is
not oriented perpendicular to the plane of the naphthalimide
moiety but instead is bent backward. Interestingly, the crystal
structure furthermore demonstrates the desired ability of the
imide moiety to form hydrogen bonds.
Synthesis of a Reactive “Precursor Complex”. For future
combinatorial chemistry it would be desirable to have access to a
complex in which the ruthenium is coordinated to the pyridyl-
naphthalimide pharmacophore ligand in addition to labile mono-
dentate coordinating ligands that can be replaced in a combinatorial
fashion against other mono-, bi-, tri-, or even tetradentate ligands,
thus providing rapid access to a diversity of novel structures just
by straightforward ligand substitution chemistry.15 In fact, the
reaction of pyridylnaphthalimide 1a with [Ru(η6-C6H6)Cl2]2 in
the presence of K2CO3 and KPF6 in refluxing acetonitrile afforded
in one step the tetraacetonitrile complex 10 in a yield of 40%
(Scheme 3).16 The crystal structure of complex 10 is shown in
Figure 4. Despite the apparent lability of the four acetonitrile
ligands, complex 10 is surprisingly robust and can be purified by
regular silica gel flash chromatography. As expected, the acetoni-
trile ligands of 10 are prone to substitution at elevated tempera-
ture. For example, the reaction of 10 with 1,4,7-trithiacyclononane
in DMF provided in a good yield of 73% the complex 6, in which
three acetonitrile ligands have been replaced by 1,4,7-trithiacyclononane.
Protein Kinase Inhibition. To quickly gain insight into the
protein kinase inhibition properties of pyridylnaphthalimide
metal complexes, we tested complex 7 as a representative member
of this class of compounds for its protein kinase binding affinity
profile against the majority of the human protein kinases encoded in
the human genome (human kinome).11 This was accomplished
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Figure 3. ORTEP drawing with 50% probability thermal ellipsoids, giving the crystal structure of the 1,4,7-trithiacyclononane complex 7. Solvent
molecules (MeCN and diethyl ether) and the hexafluorophosphate counterions are omitted for clarity. Selected bond distances (Å) and angles (deg):
Ru1ÀC1 = 2.075(4), Ru1ÀN15 = 2.095(4), Ru1ÀC26 = 1.864(5), Ru1ÀS1 = 2.3419(11), Ru1ÀS2 = 2.4169(11), Ru1ÀS3 = 2.4077(12);
C1ÀRu1ÀN15 = 78.72(15), C1ÀRu1ÀS3 = 87.53(12), C1ÀRu1ÀC26 = 92.29(17), N15ÀRu1ÀS3 = 84.01(10), N15ÀRu1ÀC26 = 94.91(16).
Figure 4. ORTEP drawing with 50% probability thermal ellipsoids,
giving the crystal structure of the tetraacetonitrile precursor complex 10.
Selected bond distances (Å) and angles (deg): Ru1ÀC1 = 2.041(4),
Ru1ÀN14 = 2.043(4), Ru1ÀN100 = 2.018(4), Ru1ÀN200 = 2.165(4),
Ru1ÀN300 = 2.049(4), Ru1ÀN400 = 2.018(4); C1ÀRu1ÀN14 =
80.35(15), C1ÀRu1ÀN100 = 92.06(15), C1ÀRu1ÀN200 = 175.11(16),
C1ÀRu1ÀN300 = 102.31(15), C1ÀRu1ÀN400 = 90.49(15), C100À
Figure 5. Protein kinase selectivity of complex 7 as determined by an
N100ÀRu1 = 179.4(4), C200ÀN200ÀRu1 = 169.0(4), C300ÀN300À
active-site-directed affinity screening (KINOMEscan, DiscoveRx)
Ru1 = 168.4(3), C400ÀN400ÀRu1 = 174.5(3).
against 442 human protein kinases: representation of the main hits
(<0.1% of control; 0% = highest affinity, 100% = no affinity) within the
by using an active-site-directed competition binding assay with
442 different protein kinases (KINOMEscan, DiscoveRx) which
provides primary data (%ctrl = percent of control: 0% = highest
affinity, 100% = no affinity) that correlate with binding constants
(Kd).17,18 Interestingly, out of the 442 enzymes tested, 8 protein
kinases, namely CLK2, DMPK, HIPK2, MAP3K8, MYLK,
PKCθ, RSK2, and RSK4, were identified as the main hits with
%ctrl values below 0.1%. The human kinase dendrogram shown
in Figure 5 reveals that these eight kinases are distributed among
four protein kinase families. We selected myosin light-chain
kinase (MYLK or MLCK) for further studies because MYLK
plays a very important role in smooth muscle contraction.19
Upon stimulation by agonists, the intracellular concentration of
Ca2+ of smooth muscle is elevated, causing Ca2+ to bind to
calmodulin. The complex of Ca2+ and calmodulin then activates
MYLK. We determined complex 7 to be a nanomolar inhibitor of
human kinase dendrogram, which displays the protein kinase families
and the evolutionary relationships between the individual kinases. See
the Supporting Information for additional data.
MYLK with an IC50 value (concentration of inhibitor at which
the enzyme activity is reduced to 50%) of 75 ( 20 nM (100 μM
ATP), which according to the ChengÀPrusoff equation
(Km(ATP) = 50 μM) would reflect an inhibition constant
(Kd) of approximately 25 nM.20,21 Surprisingly, the pyridyl-
naphthalimide ligand 1a itself does not show any sign of MYLK
inhibition even at a concentration of 1 mM. Thus, the Ru(1,4,7-
trithiacyclononane)(CO) fragment increases the binding affinity
by more than 4 orders of magnitude. To evaluate if complex 7
binds as designed to the hinge region of the ATP-binding site, we
synthesized a derivative of 7 in which the imide hydrogen is
replaced by a methyl group (7Me). As shown in Figure 6, this
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3.5 ( 0.9 μM) or selenocyanate 9 (IC50 = 4.2 ( 1.3 μM) display
any significant affinity for MYLK, indicating that it is the bent
position of the coordinated CO that is crucial for MYLK binding,
most likely through a specific interaction with a region of the
flexible glycine-rich loop.
Acitivities in Human Cells. Finally, because of the recent
clinical success of kinase inhibitors for the treatment of metastatic
melanoma,22,23 we investigated complexes 5a and 7 as represen-
tative members of the new class of metallo-naphthalimides in an
in vitro MTS proliferation assay24 with two human-derived
melanoma cell lines (451Lu and WM3918), as well as human
melanocytes (FOM102010) and human fibroblast cells (FF2508)
as controls (Figure 7). Unfortunately, even at a concentration as
high as 20 μM, complex 7 did not show any significant effects on
the melanoma or normal human cell lines (Figure 7a). It can be
assumed that this is due to the cationic nature of complex 7,
which may prevent an efficient cellular uptake. In fact, in contrast
to complex 7, the neutral complex 5a inhibited the proliferation
of the melanoma cell lines 451Lu and WM3918 in a dose-
dependent fashion to 53% and 40% at 20 μM, respectively, while
fibroblasts were only slightly affected and melanocytes not at all
(Figure 7b). Thus, due to this promising therapeutic window,
complex 5a and derivatives thereof should be further evaluated
for their antimelanoma activities.
Figure 6. IC50 curves of complex 7 and the methylated control com-
plex 7Me against MYLK at an ATP concentration of 100 μM. The
pyridylnaphthalimide ligand 1a does not show any inhibition of MYLK
even at a concentration of 1 mM.
’ CONCLUSIONS
In summary, we have introduced 3-(2-pyridyl)-1,8-napththa-
limide as a novel pharmacophore chelate ligand for metal-based
protein kinase inhibitors and demonstrated its versatility by pre-
senting a ruthenium complex as a nanomolar inhibitor of myosin
light-chain kinase. 3-(2-Pyridyl)-1,8-naphthalimide is designed
to interact with the hinge region of the ATP-binding site by
forming two hydrogen bonds with the imide moiety. A key feature is
its capability of serving as a stable bidentate ligand through CÀH
activation at the 4-position of the electron-deficient naphthalene
moiety, which simplifies the structure by reducing the number of
required heteroatoms for transition-metal binding.
’ EXPERIMENTAL SECTION
General Methods. All reactions were carried out using oven-dried
glassware and conducted under a positive pressure of nitrogen unless
otherwise specified. 1,8-Naphthalic anhydride, silver(I) sulfate, and other
chemicals were used as received from standard suppliers unless otherwise
specified. 3-Bromo-1,8-naphthalic anhydride,25 2-(trimethylstannyl)pyridine,26
tetrakis(triphenylphosphine)palladium(0),27 [Ru(η5-C5H5)(CO)-
(MeCN)2]PF6,12 [Ru(MeCN)3([9]aneS3)](CF3SO3)2,14 and [RuCl2-
(η6-C6H6)]2 were prepared according to literature procedures. All
28
Figure 7. Effects of the pyridylnapthalimide complexes 7 and 5a on the
proliferation of human-derived cells. Cellular proliferations of melanoma
cell lines (451Lu and WM3918), human melanocytes (FOM102010), and
human fibroblasts (FF2508) were assessed with the MTS assay. Data
show the mean value of three independent experiments, including the
standard deviations. The proliferation is given as the percent of DMSO
control experiments.
solvents were distilled prior to use. Acetonitrile and DMF were dried by
common methods and freshly distilled prior to use. NMR spectra were
recorded on an Avance 300 (300 MHz), DRX-400 (400 MHz), Avance
500 (500 MHz), or DRX-500 (500 MHz) spectrometer. Infrared spectra
were recorded on a Bruker Alpha FTIR instrument . High-resolution
mass spectra were obtained with a Finnigan LTQ-FT instrument using
either APCI or ESI. Complex 7Me was synthesized analogously to 7 (see
the Supporting Information).
methylation dramatically reduces the activity by almost 2 orders
of magnitude (IC50 = 5.2 ( 1.0 μM), consistent with the
expectation that the imide hydrogen forms a hydrogen bond
with a carbonyl oxygen of the hinge region, as indicated in
Figure 1. It is also worth noting that neither the half-sandwich
complex 5a (IC50 = 83 ( 36 μM) nor the thiocyanate 8 (IC50 of
3-Bromo-1,8-naphthalimide (4a). 3-Bromo-1,8-naphthalic an-
hydride (3; 5.07 g, 18.3 mmol) was added to 255 mL of concentrated
ammonia (28%) and heated to 70 °C for 90 min. The solution was
cooled to ambient temperature, and the resulting precipitate was filtered
off, washed with ethanol twice, and dried under vacuum to provide
3-bromo-1,8-naphthalimide (4a) as an off-white solid (4.71 g, 93%). 1H
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NMR (300 MHz, DMSO-d6): δ (ppm) 11.82 (br s, 1H), 8.70 (d, J = 2.1
Hz, 1H), 8.34À8.43 (m, 3H), 7.87 (t, J = 7.8 Hz, 1H). 13C NMR (75
MHz, DMSO-d6): δ (ppm) 163.7, 162.9, 135.7, 133.3, 132.9, 131.7,
130.2, 128.2, 127.2, 124.5, 122.7, 119.9. IR (film): ν (cmÀ1) 3180, 3067,
2827, 1693, 1617, 1589, 1408, 1326, 1262, 883, 821, 783, 745, 726, 515,
477, 429. HRMS: m/z calcd for C12H7BrNO2 (M + H)+ 277.9634,
found 277.9624.
1701, 1662, 1626, 1591, 1476, 1420, 1331, 1200, 1180, 782, 736, 701.
HRMS: m/z calcd for C24H17N2O2 (M + H)+ 365.1285, found 365.1281.
Complex 5a. 3-(2-Pyridyl)-1,8-naphthalimide (4a; 30 mg, 108
μmol), and [Ru(η5-C5H5)(CO)(MeCN)2]PF6 (71 mg, 165 μmol)
were dissolved in 5 mL of DMF and the reaction mixture was purged
with nitrogen for 10 min. Following the addition of triethylamine
(35 μL, 249 μmol), the mixture was heated to 60 °C for 18 h. The
reaction mixture was cooled to ambient temperature, diluted with 50 mL
of CH2Cl2, and washed with saturated NH4Cl solution. The aqueous
phase was washed with 2 Â 25 mL of CH2Cl2, the combined organic
phases were dried using MgSO4, and the crude product was subjected to
silica gel chromatography with CH2Cl2/MeOH (40/1). The combined
product eluents were dried in vacuo, and the final complex 5a was
obtained as a yellow solid (27 mg, 51%). 1H NMR (300 MHz, DMSO-
d6): δ (ppm) 11.56 (s, 1H), 9.11 (d, J = 5.7 Hz, 1H), 8.76 (s, 1H), 8.53
(d, J = 8.4 Hz, 1H), 8.44À8.47 (m, 2H), 8.01 (t, J = 7.5 Hz, 1H), 7.80 (t,
J = 7.8 Hz, 1H), 7.23 (t, J = 6.6 Hz, 1H), 5.29 (s, 5H). 13C NMR (75
MHz, DMSO-d6): δ (ppm) 201.0, 200.8, 165.9, 164.8, 164.1, 157.7,
144.7, 143.9, 142.8, 142.2, 137.9, 130.3, 125.7, 123.3, 122.7, 121.6, 120.4,
117.3, 85.1. IR (film): ν (cmÀ1) 2922, 2851, 1934, 1670, 1577, 1557,
1471, 1381, 1318, 1257, 1244, 999, 965, 791, 777, 741. HRMS: m/z
calcd for C23H15N2O3Ru (M + H)+ 469.0121, found 469.0123.
Complex 5b. N-Benzyl-3-(2-pyridyl)-1,8-naphthalimide (4b; 16
mg, 44 μmol) and [Ru(η6-C5H5)(CO)(MeCN)2]PF6 (28 mg, 66
μmol) were dissolved in 4 mL of DMF, and the reaction mixture was
purged with nitrogen for 10 min. Following the addition of triethylamine
(7.3 μL, 53 μmol), the mixture was heated to 60 °C for 18 h. The solvent
was removed and the crude product subjected to silica gel chromatog-
raphy with CH2Cl2. The combined product eluents were dried in vacuo,
and the complex 5b was obtained as an orange solid (18 mg, 70%). 1H
NMR (400 MHz, CDCl3): δ (ppm) 8.92 (d, J = 5.2 Hz, 1H), 8.83 (s,
1H), 8.65 (dd, J = 7.6, 1.2 Hz, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 8.09 (d,
J = 8.0 Hz, 1H), 7.81 (td, J = 8.4, 1.6 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H),
7.55À7.57 (m, 2H), 7.28À7.32 (m, 2H), 7.20À7.24 (m, 1H), 7.00 (td,
J = 6.6, 1.2 Hz, 1H), 5.44 (s, 2H), 5.08 (s, 5H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 202.6, 199.8, 167.2, 165.4, 164.4, 157.1, 144.1, 142.8,
142.5, 137.8, 137.0, 131.9, 128.7, 128.4, 127.2, 125.5, 124.6, 122.9, 120.6,
119.9, 117.3, 84.5, 43.4. IR (film): ν (cmÀ1) 2925, 2854, 1930, 1690,
1649, 1581, 1561, 1474, 1390, 1319, 1229, 1180, 779, 731, 701. HRMS:
m/z calcd for C30H21N2O3Ru (M + H)+ 559.0598, found 559.0608.
Complex 6. Method a. 3-(2-Pyridyl)-1,8-naphthalimide (1a;
70 mg, 255 μmol) and [Ru(MeCN)3([9]aneS3)](CF3SO3)2 (269 mg,
383 μmol) were dissolved in 5 mL of DMF, triethylamine (92 μL, 663
μmol) was added, and the reaction mixture was heated to 60 °C for 14 h.
The solution was cooled to ambient temperature, the solvent was
removed, and the crude product was subjected to silica gel chromatog-
raphy, first with acetonitrile, followed by acetonitrile/water/saturated
KNO3 solution (50/3/1). The combined product eluents were dried in
vacuo, the residue was dissolved in a minimal amount of acetonitrile/
water, and NH4PF6 was added. The solution was centrifuged, and the
resulting orange filter cake was washed three times with water and dried
in vacuo to provide the monoacetonitrile 6 complex as an orange solid
(145 mg, 77%).
3-(2-Pyridyl)-1,8-naphthalimide (1a). 3-Bromo-1,8-naphthali-
mide (3; 0.95 g, 3.44 mmol) and 2-(trimethylstannyl)pyridine (0.84 g,
3.47 mmol) were dissolved in 15 mL of m-xylene, and the reaction
mixture was purged with nitrogen for 15 min. Tetrakis(triphenylphosphine)-
palladium(0) (197 mg, 0.17 mmol) was added and the mixture was
heated to reflux for 22 h. After the mixture was cooled to ambient
temperature, the solvent was removed and the crude product adsorbed
onto silica gel, and subjected to silica gel chromatography with CH2Cl2/
MeOH (75/1). The combined product eluents were dried in vacuo, and
3-(2-pyridyl)-1,8-naphtalimide (1a) was obtained as a pale yellow solid
(0.63 g, 67%). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 11.77 (s, 1H),
9.11À9.13 (m, 2H), 8.76À8.78 (m, 1H), 8.52 (dd, J = 8.1, 0.9 Hz, 1H),
8.42 (dd, J = 7.2, 0.9 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.99 (td, J = 7.8,
1.8 Hz, 1H), 7.87 (dd, J = 8.1, 7.4 Hz, 1H), 7.47 (ddd, J = 7.5, 4.8, 0.9 Hz,
1H). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 164.0, 154.2, 149.8,
137.6, 137.0, 134.9, 132.0, 131.4, 130.2, 128.7, 127.9, 127.5, 123.5, 123.1,
122.5, 120.8. IR (film): ν (cmÀ1) 3174, 3064, 2846, 1672, 1595, 1586,
1414, 1381, 1360, 1337, 1259, 1203, 843, 833, 780, 748, 737, 513, 500,
475, 439, 401. HRMS: m/z calcd for C17H10N2O2Na (M + Na)+
297.0634, found 297.0633.
N-Benzyl-3-bromo-1,8-naphthalimide (4b). 3-Bromo-1,8-
naphthalic anhydride (3; 3.00 g, 10.8 mmol) was suspended in 75 mL
of glacial acetic acid, benzylamine (1.78 mL, 16.2 mmol) was added, and
the reaction mixture was heated to reflux for 18 h. The reaction mixture
was carefully poured into ice-cold water, and the resulting beige
precipitate was filtered off and washed with water. The crude product
was dissolved in CH2Cl2, adsorbed onto silica gel, and subjected to silica
gel chromatography with CH2Cl2/hexane (3/2). The combined pro-
duct eluents were dried in vacuo to provide N-benzyl-3-bromo-1,8-
1
naphtalimide (4b) as an off-white solid (2.82 g, 71%). H NMR (300
MHz, CDCl3): δ (ppm) 8.66 (d, J = 2.1 Hz, 1H), 8.59 (dd, J = 7.8,
1.2 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H), 8.01 (dd, J = 8.4, 0.9 Hz, 1H), 7.76
(dd, J = 8.4, 7.5 Hz, 1H), 7.53À7.56 (m, 2H), 7.24À7.34 (m, 3H), 5.37
(s, 2H). 13C NMR (75 MHz, CDCl3): δ (ppm) 163.6, 163.0, 137.0,
135.5, 134.1, 132.82, 132.75, 131.5, 129.1, 128.5, 128.0, 127.6, 126.5,
124.1, 122.8, 121.1, 43.7. IR (film): ν (cmÀ1) 3065, 3033, 1702, 1662,
1621, 1587, 1432, 1407, 1358, 1322, 1232, 1177, 1154, 1074, 827, 732,
701. HRMS: m/z calcd for C19H13BrNO2 (M + H)+ 368.0106, found
368.0102.
N-Benzyl-3-(2-pyridyl)-1,8-naphthalimide (1b). N-Benzyl-
3-bromo-1,8-naphthalimide (4b; 417 mg, 1.14 mmol) and 2-
(trimethylstannyl)pyridine (250 mg, 1.04 mmol) were dissolved in
25 mL of m-xylene, and the reaction mixture was purged with nitrogen
for 15 min. Tetrakis(triphenylphosphine)palladium(0) (120 mg, 0.10
mmol) was added and the mixture heated to reflux for 22 h. After cooling
to ambient temperature the solvent was removed, the crude product
adsorbed onto silica gel, and subjected to silica gel chromatography with
CH2Cl2/MeOH (75/1 to 35/1). The combined product eluents were
dried in vacuo, and N-benzyl-3-(2-pyridyl)-1,8-naphthalimide (1b) was
Method b. Tetraacetonitrile complex 10 (15 mg, 22 μmol) was
dissolved in 3 mL of DMF, 1,4,7-trithiacyclonane was added, and the
mixture was heated to 90 °C for 24 h. The solution was cooled to
ambient temperature, the solvent was removed, and the crude product
was subjected to silica gel chromatography, first with acetonitrile
followed by acetonitrile/water/saturated KNO3 solution (50/3/1).
The combined product eluents were dried in vacuo, the residue was
dissolved in a minimal amount of acetonitrile/water, and NH4PF6 was
added. The solution was centrifuged, and the resulting orange filter cake
was washed three times with water and dried in vacuo to provide
monoacetonitrile complex 6 as an orange solid (12 mg, 73%). 1H NMR
1
obtained as a pale yellow solid (381 mg, 99%). H NMR (300 MHz,
CDCl3): δ (ppm) 9.17 (d, J = 1.8 Hz, 1H), 8.95 (d, J = 1.8 Hz, 1H),
8.76À8.78 (m, 1H), 8.59 (dd, J = 7.2, 1.2 Hz, 1H), 8.28 (dd, J = 8.1, 0.9
Hz, 1H), 7.96 (dt, J = 8.1, 0.9 Hz, 1H), 7.85 (td, J = 7.5, 1.8 Hz, 1H), 7.75
(dd, J = 8.1, 7.5 Hz, 1H), 7.56À7.59 (m, 2H), 7.24À7.36 (m, 4H), 5.41
(s, 2H). 13C NMR (75 MHz, CDCl3): δ (ppm) 164.2, 164.1, 155.2,
150.1, 138.0, 137.3, 137.2, 134.8, 132.1, 131.9, 131.7, 129.8, 129.0, 128.4,
128.2, 127.5, 127.4, 123.1, 122.6, 120.8, 43.6. IR (film): ν (cmÀ1) 3062,
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Table 1. Crystallographic Data for Ruthenium Complexes 5b, 7, and 10a
5b 1.3CH2Cl2
7 Et2O CH3CN
10
3
3
3
À
formula
fw
C
30H20N2O3Ru, 0.65CH2Cl2
[C24H21N2O3RuS2]+, PF6À, Et2O, CH3CN
[C25H21N6O2Ru]+, PF6
612.88
883.88
683.52
a( Å)
23.7464(13)
7.0753(5)
29.415(2)
12.6950(4)
14.1048(4)
21.4127(7)
102.734(2)
95.437(3)
99.004(3)
3660.4(2)
4
8.3493(6)
11.6436(8)
14.5174(8)
102.461(5)
101.109(5)
101.134(5)
1311.06(15)
2
b (Å)
c (Å)
R (deg)
β (deg)
γ (deg)
V (Å3)
Z
4942.2(6)
8
space group
dcalcd (Mg/m3)
Pbca
P1
P1
1.647
0.814
1.38À25
3082
1.604
1.731
μ (mmÀ1
)
0.716
0.739
θ range (deg)
no. of indep rflns
no. of params
wR2 (all data)b
R1 (I > 2σ(I))b
CCDC no.c
4.65À25
12 812
4.71À25
4476
381
960
391
0.121
0.053
812716
0.136
0.059
0.051
0.040
812717
812718
a Mo KR radiation (λ = 0.71073 Å). b R1 = ∑||Fo| À |Fc||/∑|Fo|; wR2 = [∑w(Fo À Fc2)2/∑w(Fo2)2]1/2. Crystallographic data (excluding struc-
ture factors) have been deposited with the Cambridge Crystallographic Data Center. CIF files can be obtained from the CCDC free of charge via
http://www.ccdc.cam.ac.uk/data_request/cif.
2
c
(300 MHz, CD3CN): δ (ppm) 9.39 (s, 1H), 8.95 (d, J = 8.4 Hz, 1H),
8.86 (s, 1H), 8.77 (d, J = 5.7 Hz, 1H), 8.55 (d, J = 7.2 Hz, 1H), 8.37 (d, J =
6.9 Hz, 1H), 8.08 (t, J = 7.5 Hz, 1H), 7.85 (t, J = 7.8 Hz, 1H), 7.38 (t, J =
6.9 Hz, 1H), 3.15À3.31 (m, 2H), 2.76À3.00 (m, 6H), 2.08À2.46 (m,
7H). 13C NMR (75 MHz, CD3CN): δ (ppm) 194.4, 189.9, 165.4, 164.5,
163.9, 153.1, 144.9, 141.5, 139.4, 139.0, 130.8, 129.5, 126.7, 124.0, 123.7,
chromatography with CH2Cl2/MeOH (gradient 20/1 to 10/1). The
combined product eluents were dried in vacuo, and the thiocyanate
complex 8 was obtained as a red solid (3 mg, 23%). The ruthenium
coordination of the ambidente NCS ligand through nitrogen was
verified by 15N NMR and the crystal structure of a benzylated derivative
(see the Supporting Information). 1H NMR (300 MHz, DMSO-d6): δ
(ppm) 11.42 (s, 1H), 9.22 (dd, J = 8.4, 1.1 Hz, 1H), 8.96 (d, J = 4.8 Hz,
1H), 8.64 (s, 1H), 8.40 (dd, J = 7.2, 0.9 Hz, 1H), 8.31 (d, J = 8.1 Hz, 1H),
7.90 (dt, J = 8.4, 1.5 Hz, 1H), 7.74 (dd, J = 7.6, 6.6 Hz, 1H), 7.31À7.36
(m, 1H), 3.14À3.20 (m, 1H), 2.87À2.97 (m, 2H), 2.64À2.76 (m, 5H),
2.07À2.19 (m, 1H), 1.83À2.01 (m, 3H). 13C NMR (125 MHz, DMSO-
d6): δ (ppm) 215.2, 165.6, 165.4, 164.6, 152.6, 145.2, 143.1, 140.4,
137.0, 130.3, 129.1, 125.2, 124.3, 123.4, 122.8, 122.3, 119.9, 115.3, 36.6,
35.5, 35.2, 31.5, 30.3, 29.9. IR (film): ν (cmÀ1) 3159, 3002, 2920, 2854,
2096, 1673, 1574, 1379, 1312, 1248, 1016, 951, 841, 768, 739. HRMS:
m/z calcd for C24H21N3O2RuS4Na (M + Na)+ 635.9453, found
635.9467.
Complex 9. The monoacetonitrile complex 6 (16 mg, 22 μmol) was
dissolved in 2 mL of DMF, KSeCN (5 mg, 32 μmol) in water (100 μL)
was added, and the solution was heated to 95 °C for 12 h. The solvent
was removed and the crude product subjected to silica gel chromatog-
raphy with CH2Cl2/MeOH (gradient 20/1 to 10/1). The combined
product eluents were dried in vacuo, and the selenocyanate complex 9
was obtained as a red solid (4 mg, 30%). The ruthenium coordination of
the ambidente SeCN ligand through selenium was verified by the crystal
structure of a benzylated derivative (see the Supporting Information).
1H NMR (500 MHz, DMSO-d6): δ (ppm) 11.39 (s, 1H), 9.22 (dd, J =
8.0, 1.0 Hz, 1H), 8.94À8.95 (m, 1H), 8.64 (s, 1H), 8.39 (dd, J = 7.0, 1.0
Hz, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.90 (ddd, J = 8.5, 7.5, 1.5 Hz, 1H),
7.73 (dd, J = 8.5, 7.0 Hz, 1H), 7.32 (ddd, J = 7.0, 5.5, 1.5 Hz, 1H),
3.15À3.19 (m, 1H), 2.90À2.98 (m, 2H), 2.64À2.77 (m, 4H), 2.50À
2.54 (m, 1H), 2.06À2.13 (m, 1H), 1.87À1.99 (m, 3H). 13C NMR (125
MHz, DMSO-d6): δ (ppm) 165.6, 165.3, 164.4, 152.5, 145.4, 143.2,
140.7, 137.0, 136.9, 130.4, 129.4, 125.0, 123.5, 122.8, 122.3, 119.9, 115.3,
108.4, 36.5, 36.3, 35.3, 31.3, 31.1, 30.0. IR (film): ν (cmÀ1) 3344, 3224,
123.6, 121.7, 119.4, 37.0, 36.5, 34.5, 33.6, 31.8, 30.6. IR (film): ν (cmÀ1
)
2991, 1974, 1675, 1582, 1564, 1476, 1412, 1386, 1322, 1248, 837, 778,
748. HRMS: m/z calcd for C25H24N3O2RuS3 (M À PF6)+ 596.0069,
found 596.0078.
Complex 7. The monoacetonitrile complex 6 (15 mg, 20 μmol) was
dissolved in 4 mL of DMF and purged with CO gas for 30 s. The solution
was heated to 95 °C for 3 h. The solvent was removed and the crude
product subjected to silica gel chromatography, first with acetonitrile
followed by acetonitrile/water/saturated KNO3 solution (50/3/1). The
combined product eluents were dried in vacuo, the residue was dissolved
in a minimal amount of acetonitrile/water, and NH4PF6 was added. The
solution was centrifuged, and the resulting yellow filter cake was washed
three times with water and dried in vacuo to provide monoacetonitrile
complex 7 as a yellow solid (13 mg, 87%). 1H NMR (400 MHz,
CD3CN): δ (ppm) 9.36 (s, 1H), 8.95 (dd, J = 8.4, 1.2 Hz, 1H), 8.86 (s,
1H), 8.77 (dt, J = 5.2, 0.8 Hz, 1H), 8.55 (dd, J = 7.2, 1.2 Hz, 1H), 8.37 (d,
J = 8.0 Hz, 1H), 8.08 (td, J = 8.4, 0.8 Hz, 1H), 7.85 (dd, J = 8.4, 7.2 Hz,
1H), 7.38 (td, J = 5.6, 1.2 Hz, 1H), 3.17À3.30 (m, 2H), 2.86À3.00 (m,
4H), 2.77À2.85 (m, 2H), 2.28À2.46 (m, 3H), 2.19À2.25 (m, 1H). 13C
NMR (100 MHz, CD3CN): δ (ppm) 194.4, 189.9, 165.4, 164.5, 163.9,
153.1, 144.9, 141.5, 139.4, 138.9, 130.8, 129.4, 126.7, 124.0, 123.7, 123.6,
121.7, 119.4, 37.0, 36.5, 34.5, 33.6, 31.8, 30.6. IR (film): ν (cmÀ1) 3164,
3053, 2839, 1991, 1675, 1584, 1474, 1387, 1317, 1266, 834, 780, 745.
HRMS: m/z calcd for C24H21N2O3RuS3 (M À PF6)+ 582.9752, found
582.9752.
Complex 8. The monoacetonitrile complex 6 (16 mg, 22 μmol)
was dissolved in 2 mL of DMF, NaSCN (3 mg, 32 μmol) in water
(100 μL) was added, and the solution was heated to 95 °C for 12 h. The
solvent was removed and the crude product subjected to silica gel
4604
dx.doi.org/10.1021/om200366r |Organometallics 2011, 30, 4598–4606

Organometallics
ARTICLE
3003, 2921, 2851, 2098, 1674, 1575, 1507, 1432, 1406, 1378, 1321, 1256,
1019, 827, 778. HRMS: m/z calcd for C24H21N3O2RuS3SeNa (M +
Na)+ 683.8904, found 683.8903.
included at calculated positions, N-bonded H atoms in 10 were located
and refined isotropically. The crystal of 5b was twinned. Only the
reflections of one twin domain were used for refinement. In 5b two
positions of a disordered CH2Cl2 molecule were refined with a total
occupation of 0.65 in the asymmetric unit. In 7 one hexafluorophosphate
anion was disordered. High displacement factors indicate non esolved
disorder of the solvent ether molecules. Crystallographic data for 5b, 7,
and 10 are given in Table 1.
Complex 10. 3-(2-Pyridyl)-1,8-naphthalimide (1a; 78 mg, 0.28 mmol),
[RuCl2(η6-C6H6)]2 (71 mg, 0.14 mmol), KPF6 (199 mg, 1.08 mmol), and
K2CO3 (110 mg, 0.80 mmol) were dissolved in acetonitrile and heated to
reflux for 16 h. The solvent was removed and the crude product subjected to
silica gel chromatography with CH2Cl2/MeOH (gradient 35/1 to 20/1).
The combined product eluents were dried in vacuo, and the tetraacetonitrile
complex 10 was obtained as a red solid (77 mg, 40%). 1H NMR (300 MHz,
CD3CN): δ(ppm) 9.21 (br s, 1H), 9.14 (dd, J=8.4, 1.2Hz, 1H), 9.04À9.06
(m, 1H), 8.65(s, 1H), 8.52(dd, J= 7.2, 1.2 Hz, 1H), 8.15 (d, J= 8.4 Hz, 1H),
7.85À7.91 (m, 1H), 7.78 (dd, J = 8.4, 7.2 Hz, 1H), 7.28 (ddd, J = 7.2, 5.7 Hz,
1.2 Hz, 1H), 2.55 (s, 3H), 2.14 (s, 6H), 1.99 (s, 3H). 13C NMR (125 MHz,
CD3CN): δ (ppm) 215.8, 167.2, 165.0, 164.1, 152.7, 145.8, 143.4, 140.6,
136.8, 130.3, 128.8, 126.2, 124.8, 123.4, 122.2, 121.7, 121.5, 118.9, 115.6, 2.8.
IR (film): ν (cmÀ1) 3363, 2924, 2851, 2271, 1681, 1578, 1561, 1473, 1316,
1242, 830, 776, 757, 557. HRMS: m/z calcd for C23H18N5O2Ru (M À
CH3CN À PF6)+ 498.0505, found 498.0487.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures, text, and tables giving
b
screening data of compound 7 against a panel of 442 protein
kinases, stability test with 7, investigation of the binding mode of
the ambidente thiocyanate and selenocyanate ligands in com-
plexes 8 and 9, synthesis and analytical data of the complex 7Me,
and the complete references 17, 18, and 22, and CIF files giving
crystallographic data for 5b, 7, and 10. This material is available
free of charge via the Internet at http://pubs.acs.org.
Protein Kinase Assays. The protein kinase selectivity profile of
complex 7 at an assay concentration of 10 μM was derived from an
active-site-directed affinity screening against 442 human protein kinases
(KINOMEscan, DiscoveRx). See the Supporting Information for addi-
tional data. For the determination of IC50 values, various concentrations
of the ruthenium complexes or the ligand 1a were incubated at room
temperature in 20 mM MOPS, 30 mM Mg(OAc)2, 0.8 μg/μL BSA, 5%
DMSO (7À9, 7Me), or 10% DMSO (5a and 1a) (resulting from the
inhibitor stock solution) at pH 7.0 in the presence of CaCl2 (500 μM),
calmodulin (1 μM), substrate ZIPtide (62.5 μM), and human MYLK
(purchased from Millipore as MLCK) (6.9 nM). After 30 min, the
reaction was initiated by adding ATP to a final concentration of 100 μM
and approximately 0.1 μCi/μL [γ-33P]ATP. Reactions were performed
with a total volume of 25 μL. After 45 min, the reaction was terminated
by spotting 15 μL on a circular P81 phosphocellulose paper (2.1 cm
diameter, Whatman), followed by washing three times with 0.75%
phosphoric acid and once with acetone. The dried P81 papers were
transferred to a scintillation vial, 5 mL of scintillation cocktail was added,
the counts per minute (CPM) were measured with a Beckmann
CoulterTM LS6500 Multi-Purpose Scintillation Counter, and the IC50
values were defined to be the concentration of inhibitor at which the
counts per minute (CPM) were 50% of the control sample, corrected by
the background CPM.
Cell Viability Assays. Human melanoma cell lines (451Lu,
WM3918) were isolated as previously described29 and were further
cultured in Dulbecco’s modified Eagle’s medium supplemented with 5%
fetal bovine serum. Normal human primary melanocytes (FOM102010)
and fibroblasts (FF2508) were isolated from the human epidermis of
neonatal foreskins and cultured as described.30,31 Cells (5000/well)
were seeded in 96-well plates and allowed to adhere overnight. The
following day, complex 5a, 7, or DMSO vehicle control was added to the
cells at different concentrations and the cells were incubated for another
96 h. Cells were then directly incubated with MTS substrate (CellTiter-
96 Aqueous One Solution Cell Proliferation Assay, Promega).24 The
absorbance was measured at 490 nm as per the supplier’s instructions,
and the percent proliferation was normalized to the absorbance of
DMSO-treated cells. For each cell line and treatment, the absorbance
values of at least 4 wells were used to analyze the data.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: meggers@chemie.uni-marburg.de.
’ ACKNOWLEDGMENT
We thank the US National Institutes of Health (Grant No.
CA114046) for support of this research. S.B. acknowledges a
stipend from the Fonds der Chemischen Industrie.
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