Ruthenium Complexes as Protein Kinase Inhibitors

Article abstract of DOI:10.1021/ol036283s

(Equation presented) Replacing complex natural products with simple metal complexes could lead to a new class of metallopharmaceuticals in which the metal center plays mainly a structural role. A strategy is introduced for the creation of ruthenium comple

Full text of DOI:10.1021/ol036283s

ORGANIC
LETTERS
2004
Vol. 6, No. 4
521-523
Ruthenium Complexes as Protein
Kinase Inhibitors
Lilu Zhang, Patrick Carroll, and Eric Meggers*
UniVersity of PennsylVania, Department of Chemistry, 231 S. 34th Street,
Philadelphia, PennsylVania 19104
meggers@sas.upenn.edu
Received November 22, 2003
ABSTRACT
Replacing complex natural products with simple metal complexes could lead to a new class of metallopharmaceuticals in which the metal
center plays mainly a structural role. A strategy is introduced for the creation of ruthenium complex-based protein kinase inhibitors 1 (X )
CO or CH ), morphed out of the class of indolocarbazole inhibitors with the alkaloid staurosporine as its most prominent member.
2
The development of small molecules that perturb specific
protein functions is of great importance for probing biological
processes and ultimately for the generation of potent and
safe drugs. Medicinal chemistry is predominately focused
on the design of organic molecules, whereas the incorporation
of inorganic components into drugs is much less investi-
gated.1 We are interested in exploring organometallic and
inorganic compounds as structural scaffolds for enzyme
inhibition.2,3 Such metal-ligand assemblies allow convergent
synthetic approaches and give access to structural motifs that
differ from purely organic molecules. Our efforts are focused
on ruthenium complex scaffolds because ruthenium offers a
hexavalent coordination sphere that cannot be easily obtained
by any organic element. In addition, ruthenium tends to form
kinetically very inert coordinative bonds, making it possible
to obtain compounds that display stabilities that are com-
parable to purely organic molecules. We here introduce a
strategy that uses a class of natural products as a lead for a
ruthenium complex scaffold.
Protein kinases regulate most aspects of cellular life and
are one of the main drug targets.4-6 The microbial alkaloid
staurosporine is a very potent but relatively nonspecific
inhibitor of many protein kinases (for the structure, see
Abstract).7 Many staurosporine derivatives and related
organic compounds with modulated specificities have been
(1) Metal-based drugs: (a) Orvig, C.; Abrams, M. J. Chem. ReV. 1999,
99, 2201-2842. (b) Guo, Z.; Sadler, P. J. Angew. Chem., Int. Ed. 1999,
38, 1512-1531. (c) Farrell, N. Coord. Chem. ReV. 2002, 232, 1-230.
(2) Metal ions and metal complexes as enzyme inhibitors: Louie, A.
Y.; Meade, T. J. Chem. ReV. 1999, 99, 2711-2734.
(3) (a) Sakai, S.; Shigemasa, Y.; Sasaki, T. Tetrahedron Lett. 1997, 38,
8145-8148. (b) Takeuchi, T.; Bo¨ttcher, A.; Quezada, C. M.; Simon, M. I.;
Meade, T. J.; Gray, H. B. J. Am. Chem. Soc. 1998, 120, 8555-8556. (c)
Lebon, F.; deRosny, E.; Reboud-Ravauz, M.; Durant, F. Eur. J. Med. Chem.
1998, 33, 733-737. (d) Goral, V.; Nelen, M. I.; Eliseev, A. V.; Lehn, J.-
M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1347-1352. (e) Liang, X.;
Parkinson, J. A.; Weisha¨upl, M.; Gould, R. O.; Paisey, S. J.; Park, H.;
Hunter, T. M.; Blindauer, C. A.; Parsons, S.; Sadler, P. J. J. Am. Chem.
Soc. 2002, 124, 9105-9112.
(4) Role of protein kinases in disease: (a) Hunter, T. Cell 2000, 100,
113-127. (b) Blume-Jensen, P.; Hunter, T. Nature 2001, 411, 355-365.
(5) Protein kinase inhibitors: (a) Garc´ıa-Echeverr´ıa, C.; Traxler, P.;
Evans, D. B. Med. Res. ReV. 2000, 20, 28-57. (b) Cohen, P. Nature ReV.
Drug DiscoV. 2002, 1, 309-315. (c) Cole, P. A.; Courtney, A. D.; Shen,
K.; Zhang, Z.; Qiao, Y.; Lu, W.; Williams, D. M. Acc. Chem. Res. 2003,
36, 444-452.
(6) See also: (a) Gray, N. S.; Wodicka, L.; Thunnissen, A.-M. W. H.;
Norman, T. C.; Kwon, S.; Hernan Espinoza, F.; Morgan, D. O.; Barnes,
G.; LeClerc, S.; Meijer, L.; Kim, S.-H.; Lockhart, D. J.; Schultz, P. G.
Science 2000, 281, 533-538. (b) Maly, D. J.; Choong, I. C.; Ellman, J. A.
Proc. Natl. Acad. Sci 2000, 97, 2419-2424. (c) Bishop, A. C.; Buzko, O.;
Shokat, K. M. Trends Cell Biol. 2001, 11, 167-172.
10.1021/ol036283s CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/31/2004

obtained by condensing deprotonated bisbenzimidazole 5
with N-benzyl-2,3-dibromomaleimide 7, yielding 9, followed
by a reduction and deprotection sequence as shown in
Scheme 1. Accordingly, one carbonyl group of 9 was first
reduced to the alcohol 10 with NaBH4, followed by acety-
lation of the alcohol with acetic anhydride and reductive
elimination to 11 after addition of zinc dust.9 Finally,
deprotection of the benzyl group under acidic conditions
yielded the lactam 4.10
Scheme 1. Synthesis of Ligands 3 and 4a
Despite their unique geometry, having nitrogen donor
ligands from two five-membered rings annulated to a central
six-membered ring, 3 and 4 are able to serve as bidentate
ligands. For example, refluxing the benzyl derivative 9 with
cis-RuCl2(DMSO)4 in toluene yielded diastereoselectively the
cis(Cl),cis(DMSO) complex 12a, which isomerized to the
cis(Cl),trans(DMSO) isomer upon crystallization from chlo-
roform (Figure 1). The structure reveals that the two
a Reagents and conditions: (a) Deprotonation of 5 with 2.1 equiv
of NaH in DMF, followed by addition 6 or 7 (8, 33%; 9, 35%). (b)
TBAF, CH2Cl2 (71%). (c) NaBH4, EtOH (90%). (d) First reflux in
Ac2O, then addition of Zn and reflux (89%). (e) TFA, H2SO4,
anisole, reflux (76%).
developed, and several are in clinical trials as anticancer
drugs.5 They all share the indolo[2,3-a]carbazole aglycon 2,
which binds to the ATP binding site and can hydrogen bond
with two conserved amino acids.8 For this class of inhibitors,
specificity for a particular protein kinase can be achieved
by the moiety that is attached to the indole nitrogen atoms.
We envisioned that by replacing the indolocarbazole
alkaloid scaffold with metal complex 1, elaborate structures
could be assembled in an efficient manner by variation of
the ligands. Key components of our design are ligands 3 and
4, derived from the indolocarbazole aglycon 2 by just
replacing two carbon against two nitrogen atoms. This
transformation does not change the shape of the ligand but
generates two benzimidazole moieties (bold in the structure
in Abstract) that can function as coordination sites for the
ruthenium center. The remaining four coordination sites at
the ruthenium can become filled by ligands L1-L4 and
substitute for the carbohydrate moiety, with the metal center
serving as a “glue” for holding all parts together.
Figure 1. ORTEP drawing with 35% probability thermal elipsoids
of the X-ray structure obtained upon crystallization of complex 12a.
The complex isomerized to the cis(Cl),trans(DMSO) isomer.
benzimidazoles can indeed serve as coordination sites for
the ruthenium and that the ligand 9 is, as expected, almost
superimposable to the indolocarbazole 2. The ruthenium-
nitrogen bonds are remarkably long with distances of 2.15
and 2.16 Å, respectively, supposedly a consequence of both
Bisbenzimidazolomaleimide 3 was synthesized in an
economical fashion from readily available precursors in two
steps by condensing deprotonated bisbenzimidazole 5 with
N-TBDMS-2,3-dibromomaleimide 6 followed by deprotec-
tion with TBAF to afford 3 (Scheme 1). Lactam 4 was
(9) This method was originally reported for the reductive cleavage of
an R-keto acetate: Woodward, R. B.; Sondheimer, F.; Taub, D.; Heusler,
K.; McLamore, W. M. J. Am. Chem. Soc. 1952, 74, 4223-4251.
(10) Hargrave, K. D.; Proudfoot, H. R.; Grozinger, K. G.; Cullen, E.;
Kapadia, S. R.; Patel, U. R.; Fuchs, V. U.; Mauldin, S. C.; Vitous, J.;
Behnke, M. L.; Klunder, J. M.; Pal, K.; Skiles, J. W.; McNeil, D. W.; Rose,
J. M.; Chow, G. C.; Skoog, M. T.; Wu, J. C.; Schmidt, G.; Engel, W. W.;
Eberlein, W. G.; Saboe, T. D.; Campbell, S. J.; Rosenthal, A. S.; Adams,
J. J. Med. Chem. 1991, 34, 2231-2241.
(7) (a) Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kato, Y.; Morimoto,
M.; Tomita, F. Biochem. Biophys. Res. Commun. 1986, 135, 397-402. (b)
Caravatti, G.; Meyer, T.; Fredenhagen, A.; Trinks, U.; Mett, H.; Fabbro,
D. Bioorg. Med. Chem. Lett. 1994, 4, 399-404.
(8) Toledo, L. M.; Lydon, N. B. Structure 1997, 5, 1551-1556.
522
Org. Lett., Vol. 6, No. 4, 2004

the open biting angle of the two nitrogens and their large
distance apart. Interestingly, a comparison with the crystal
structure of the free ligand 9 reveals that, upon complexation,
the distance between the two coordinating nitrogen atoms
decreases from 3.05 to 2.80 Å, a remarkable change in length
of 8.2%.
The synthesis of ruthenium complexes with unprotected
maleimide nitrogens was accomplished by using the TB-
DMS-modified ligand 8. Accordingly, reaction of 8 with cis-
RuCl2(DMSO)4 yielded diastereoselectively the unprotected
ruthenium complex 12b in one step (Figure 2). Reaction of
Table 1. Inhibition of Some Protein Kinases by Ligands 3 and
4 and the Ruthenium Complexes 12b, 13, and 14a
compound
Abl
RSK1
Src
PKCR
ZAP70
staurosporine
3
4
12b
13
14
2
25
20
10
2
<1
30
25
8
8
8
<1
>100
60
<1
>100
>100
>100
>100
50
<1
>100
50
30
40
30
40
30
40
5
a Concentrations required for 50% inhibition (IC50) in µM. Determined
by phosphorylation of peptide or protein substrates with [γ-32P]ATP in the
presence of varying concentrations of inhibitors.
for the Abelson tyrosine kinase (Abl) with an IC50 of 2 µM,
which is more than 10 times lower than the IC50 of the
corresponding free ligand 3. Additionally, the precursor Ru-
(COD)(CH3CN)2Cl2 does not show any signs of inhibition
against Abl even at 100 µM. Consequently, the activity of
compound 13 requires the entire assembly, kept together by
the central ruthenium ion. To test if 13 does, as designed,
bind to the ATP site, we synthesized a derivative of 13 with
the imide hydrogen replaced by a benzyl group. This
derivative shows a potency that is strongly reduced by a
factor of 25, consistent with the assumption that the imide
hydrogen is involved in hydrogen bonding within the adenine
binding cleft. Additionally, a Lineweaver-Burk analysis of
the initial velocities with Abl at different ATP concentrations
and fixed concentrations of ruthenium compound 14 reaf-
firms ATP competitive binding (see Supporting Information
for details). It is also noteworthy that the specificities of the
COD complex 13 and bipyridine complex 14 are different
from that of staurosporine. In our assays, staurosporine is a
nanomolar inhibitor for all tested kinases, except for Abl,
against which it has an IC50 of 2 µM.
Figure 2. Synthesized ruthenium complexes 12-14. See Support-
ing Information for experimental details of the synthesis.
8 with Ru(COD)(CH3CN)2Cl2 followed by treatment with
TBAF yielded 13. Refluxing the lactam 4 in ethanol with
Ru(bpy)2(EtOH)22+, generated from Ru(bpy)2Cl2 and AgOSO2-
CF3 in situ, yielded the ionic complex 14.
All three metal complex scaffolds are air-stable and can
be stored on the bench for weeks without any signs of
decomposition. Bipyridine complex 14 is completely stable
in a 1:1 water/DMSO solution for 12 h and can even
withstand a 1 mM methanolic solution of 2-mercaptoethanol
for 3 h without any decomposition. Time-dependent 1H NMR
measurements show that the compounds 12b and 13 slowly
release ligand 3 in 1:1 water/DMSO mixtures, with half-
lifes of 8 and 3 h, respectively.11 However, their stability
was sufficient for examining their potential as protein kinase
inhibitors.
We examined the potency of ruthenium complexes 12b,
13, and 14 against a small panel of protein kinases and
compared the results to the potency of the free ligands 3
and 4. Table 1 gives the concentrations of compounds
required for 50% inhibition (IC50). As expected, 3 and 4 have
some affinity against most of the tested kinases. The
important observation is that upon formation of the ruthenium
complexes, affinity and specificity become modulated. For
example, the bipyridine complex 14 is the only compound
that inhibits protein kinase C R (PKCR) below 100 µM. On
the other hand, the COD complex 13 is the best inhibitor
To our knowledge, this is the first report of ruthenium
complexes as protein kinase inhibitors.12 The scaffold 14 is
chemically very robust and conformationally rigid and thus
may be a promising lead structure for the development of
potent and specific inhibitors of Abl by derivatizing the
bipyridine ligands.13
Acknowledgment. We thank the University of Pennsyl-
vania and the Camille and Henry Dreyfus Foundation for
financial support of this research.
Supporting Information Available: Experimental de-
tails, spectroscopic data, and crystallographic data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL036283S
(12) Noncompetitive inhibition of PKC with respect to ATP has been
reported for adriamycin-Fe(III) and anthracycline-Cu(II) complexes: (a)
Hannun, Y. A.; Foglesong, R. J.; Bell, R. M. J. Biol. Chem. 1989, 264,
9960-9966. (b) Monti, E.; Monzini, F.; Morazzoni, F.; Perletti, G.; Piccinini,
F. Inorg. Chim. Acta 1993, 205, 181-184.
(13) Abl as a drug target: Nimmanapalli, R.; Bhalla, K. Oncogene 2002,
21, 8584-8590.
(11) Stability of ruthenium complexes with 3 and 4 is coupled to the
electronic nature of the other ligands. Ligand 3 seems to prefer an electron-
rich center, whereas 4 prefers a more electron-poor ruthenium center.
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