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ACCOUNT 1177

Exploring Chemical Space with Organometallics: Ruthenium Complexes as Protein Kinase InhibitorsRuthenium Complexes as Protein Kinase InhibitorsEric Meggers,* G. Ekin Atilla-Gokcumen, Howard Bregman, Jasna Maksimoska, Seann P. Mulcahy, Nicholas Pagano, Douglas S. WilliamsDepartment of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USAFax +1(215)7460348; E-mail: [email protected] 28 August 2006

SYNLETT 2007, No. 8, pp 1177–118916.05.2007Advanced online publication: 03.04.2007DOI: 10.1055/s-2007-973893; Art ID: A44006ST© Georg Thieme Verlag Stuttgart · New York

Abstract: Complementing organic elements with a metal centerprovides new opportunities for building three-dimensional struc-tures with unique and defined shapes. Such access to unexploredchemical space may lead to the discovery of molecules with unprec-edented properties. Along these lines, this account article describesour successful design of highly potent and selective ruthenium-based inhibitors for the protein kinases GSK-3 and Pim-1 by usingthe class of indolocarbazole alkaloids as a lead structure. The de-scribed ruthenium complexes are kinetically inert scaffolds inwhich the ruthenium has the function to organize the orientation ofthe organic ligands in the three-dimensional space.

1 Introduction1.1 Biologically Relevant Chemical Space1.2 Organic vs. Organometallic Compounds1.3 Ruthenium as a Virtual Octahedral Carbon2 Mimicking the Natural Product Staurosporine with Simple

Ruthenium Complexes3 Synthesis of Cycloruthenated Pyrido[2,3-a]pyrrolo[3,4-

c]carbazole-5,7(6H)-diones4 Discovery of Ruthenium Complexes as Protein Kinase

Inhibitors4.1 Octahedral Complexes4.2 Half-Sandwich Complexes5 Structures of Ruthenium Half-Sandwich Complexes Bound

to Protein Kinase Pim-16 GSK-3 Inhibition in Mammalian Cells, Frog Embryos, and

Zebrafish Embryos7 Summary and Outlook

Key words: bioorganometallic chemistry, protein kinases, inhibi-tors, ruthenium, mammalian cells

1 Introduction

1.1 Biologically Relevant Chemical Space

Chemical substances are extensively used as drugs andtools for modulating biological processes.1 Thus, the iden-tification of compounds with novel and defined biologicalfunctions is of high importance for research in medicinalchemistry and chemical biology.

Per definition, the total number of possible compoundswith biological activity span the ‘biologically relevantchemical space’.2 Charting this subset of the chemicalspace is focused predominately on small organic mole-

cules. But are organic-based scaffolds capable of coveringall areas of the biologically relevant chemical space? Werecently started a research program to address this ques-tion by designing chemically inert organometallic sub-stances with unique structures and investigating theirbiological functions.3–13

1.2 Organic vs. Organometallic Compounds

In order to access unexplored and novel chemical struc-tures it is tempting to include elements from the periodictable of elements which adopt coordination geometriesthat differ from carbon. In fact, carbon only forms stablecoordination geometries that are either linear (sp-hybrid-ization), trigonal planar (sp2-hybridization), or tetrahedral(sp3-hybridization) and they are the basis of all bioactiveorganic scaffolds.14 Clearly, additional pentavalent orhexavalent coordination geometries would be highly de-sirable for the design of molecules with novel shapes.15

Intriguingly, an octahedral center with 6 different substit-uents is capable of forming 30 different stereoisomerscompared to just 2 for an asymmetric tetrahedral carbon.Thus, by just increasing the number of substituents from4 (tetrahedral center) to 6 (octahedral center), the abilityof the center to organize substituents in the three dimen-sional space increases by more than an order of magnitude(Figure 1). It can therefore be concluded that an octa-hedral center has potential to be extremely powerful inbuilding molecules with novel shapes and would con-stitute an important tool in the quest for accessingunexplored chemical space.

Figure 1 Structural opportunities with an octahedral metal center.

1.3 Ruthenium as a Virtual Octahedral Carbon

Many transition metals prefer octahedral coordinationspheres and they therefore are the primary focus of oursearch for an imaginary ‘octahedral carbon’. Current ef-forts in our laboratory are concentrating predominately on

A

CD C

BM

DE B

C

A

F

tetrahedral carbonmax. 2 stereoisomers

octahedral metalup to 30 stereoisomers

increased structuralcomplexity

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1178 E. Meggers et al. ACCOUNT

Synlett 2007, No. 8, 1177–1189 © Thieme Stuttgart · New York

Eric Meggers was born inBonn, Germany in 1968. He re-ceived his Diploma in chemis-try from the University of Bonnin 1995 and a PhD from theUniversity of Basel in 1999,working under the guidance ofProfessor Bernd Giese on thetransport of charge in DNA. Hethen spent three years as a post-doctoral researcher with Pro-

fessor Peter G. Schultz at theScripps Research Institute inLa Jolla, USA, investigatingmetal-mediated base pairing inDNA. Since 2002, EricMeggers is an Assistant Pro-fessor in the Chemistry Depart-ment at the University ofPennsylvania. His research in-terests broadly revolve aroundthe combination of synthetic

organic and inorganic chemis-try to create compounds withnovel biological, physicochem-ical, and materials properties.He was recently awarded aCamille and Henry DreyfusNew Faculty Award, a CamilleDreyfus Teacher-ScholarAward, and an Alfred P. SloanResearch Fellowship.

G. Ekin Atilla-Gokcumen wasborn in Ankara, Turkey in1980. She obtained her BSc inchemistry in 2003 from KocUniversity, Istanbul, Turkey

where she worked at the Poly-mer Research Laboratories. Shestarted her graduate studies in2003 at the University of Penn-sylvania and currently pursuing

a PhD degree in Chemistry un-der the supervision of ProfessorEric Meggers.

Howard Bregman was born inNew York in 1980. He receivedhis BSc degree in biochemistryfrom the University of Bing-hamton in 2002 during whichtime his research focused on the

total synthesis of heterocyclicnatural products. In 2002 hebegan his graduate studies atthe University of Pennsylvaniawhere his he has since workedin the group of Professor Eric

Meggers studying the synthesisand evaluation of metal-organiccomplexes as structural scaf-folds for protein kinase inhibi-tion.

Jasna Maksimoska was bornin Jagodina, Serbia. She gradu-ated from University of Bel-grade, Serbia in 2002 and

obtained a BSc degree in biolo-gy. In 2004 she commencedgraduate studies in chemistry atthe University of Pennsylvania.

She joined the group of Pro-fessor Eric Meggers and iscurrently pursuing a PhD inchemistry.

Seann P. Mulcahy was bornand raised in upstate NewYork. He received his BSc de-gree in chemistry from the Uni-versity of Richmond in 2004and performed undergraduate

research on new methodologyfor the synthesis of hetero-cycles. He then moved toPhiladelphia, Pennsylvania fordoctoral research at the Univer-sity of Pennsylvania and is

currently working in the labora-tory of Professor Eric Meggersstudying the biological,physicochemical, and reactiveproperties of transition-metalcomplexes.

Nicholas Pagano was born inMarlton, New Jersey in 1982and graduated with a BSc inchemistry in 2004 from Al-bright College in Reading,Pennsylvania. While an under-graduate, his research was cen-

tered on the synthesis of aminoacid derivatives under the guid-ance of Professor ChristianHamann. Currently, he is agraduate student studying or-ganic chemistry at the Univer-sity of Pennsylvania under the

supervision of Professor EricMeggers. His research interestsfocus on the synthesis and de-sign of organometallic com-pounds as protein kinaseinhibitors.

Douglas S. Williams was bornin Millville, New Jersey in1981. He received his BA de-gree in chemistry in 2003 fromFranklin and Marshall Collegein Lancaster, Pennsylvania,

where he conducted undergrad-uate research with ProfessorScott Van Arman. He thenmoved on to the labs of Profes-sor Eric Meggers at the Univer-sity of Pennsylvania where he is

pursuing a doctoral degree. Hiscurrent research is focused onexpanding the scope of the de-veloped organometallic inhibi-tors by incorporating differentmetals.

Biographical Sketches

ACCOUNT Ruthenium Complexes as Protein Kinase Inhibitors 1179


ruthenium. In our opinion, ruthenium possesses a combi-nation of properties that render it ideal for the design ofbioactive organometallic species with novel shapes assummarized in Figure 2 and outlined below.

Figure 2 Reasons for selecting ruthenium in chemically inert bioactive organometallics.

1.) Ruthenium is able to form substitutionally inert coor-dinative bonds.16

The power of organic chemistry lies in the ability of car-bon to form stable bonds to itself as well as to many otherelements. Thus, metal complexes can only serve as reli-able structural scaffolds if the coordinative bonds canwithstand the replacement by biological ligands, in partic-ular the millimolar concentrations of thiols in cells.17

2.) Ruthenium has a low general toxicity.

A significant concern in using metal-containing com-pounds is the potential toxicity of the metal. In this re-spect, it is our opinion that a metal complex that has asubstitutionally inert coordination sphere should not dis-play any ‘metal-specific’ toxicity itself and the biologicalproperties of the metal complex should be instead deter-mined by the entire entity. This has already been postulat-ed by Francis P. Dwyer almost half a century ago. In onepublication he and his coauthors make the following state-ment about the use of ruthenium tris(phenanthroline)complexes in vivo: ‘Such doubly charged preformedRu(II) chelates are stable in boiling concentrated acids oralkali and in animal tissues. Hence their biological effectsdepend solely on the physicochemical properties of theRu(II) complex cation as a whole since no ruthenium ionor ligand is liberated.’18 In his pioneering work, Dwyerfound that such ruthenium complexes can inhibit tumorgrowth and exert bacteriostatic effects.19

However, toxic effects due to metabolic conversion of co-ordinated ligands followed by the exposure of the metal tobiological ligands cannot be excluded in individual cases.It is therefore desirable that the metal itself or derivedmetal salts have a low toxicity. In this respect, data fromphase I clinical trials of two (substitutionally labile) ruthe-nium anticancer drugs, KP101920 and NAMI-A,21 are en-couraging since these compounds have proven to be muchless toxic compared to the anticancer drug cisplatin.

3.) The synthetic chemistry of ruthenium compounds ispredictable.

Synthetic routes to ruthenium-containing compounds canbe planned and predicted similarly to purely organic mol-ecules.22 Due to the kinetic inertness of coordinativebonds to ruthenium, compounds along the synthetic routecan generally be purified in the same fashion as purely or-ganic compounds by standard silica gel chromatography.

2 Mimicking the Natural Product Staurospo-rine with Simple Ruthenium Complexes

We initiated our research program by using structural dataof organic enzyme inhibitors as a lead for the design ofbioactive organoruthenium compounds. Our overall strat-egy can be summarized in three steps:

Step 1: Selection of a target active site and analysis of ex-isting co-crystal structures with bound organic molecules.

Step 2: Identification of a main pharmacophore and incor-poration into a chelating ligand.

Step 3: Targeting ruthenium complexes to the active siteby incorporation of the pharmacophore ligand. Additionalligands in the ruthenium coordination sphere can nowform contacts to other parts of the active site.

Following this outlined approach we started to design or-ganometallic protein kinase inhibitors by using the naturalproduct staurosporine as lead structure.23 Staurosporine(Figure 3 and Figure 4) is a member of the class of indolo-carbazole alkaloids, many of which are potent ATP-com-petitive protein kinase inhibitors.24 This family ofinhibitors shares the indolo[2,3-a]carbazole moieties 2(lactam form) or 3 (imide form, arcyriaflavin A) whichbind to the adenine binding site by establishing two hy-drogen bonds to the backbone of the hinge between the N-terminal and C-terminal kinase domain. Staurosporineadopts a defined globular structure with the carbohydratemoiety being oriented perpendicular to the plane of theindolocarbazole heterocycle. The indolo[2,3-a]carbazolemoiety occupies the hydrophobic adenine binding cleftwith the lactam group mimicking the hydrogen bondingpattern of the adenine base, and the carbohydrate moietyforming hydrophobic contacts and hydrogen bonds withinthe globular ribose binding site.25,26 Thus, staurosporineclosely matches the shape of the ATP-binding sitewhich makes it a highly potent inhibitor for many proteinkinases.

In order to match the overall shape of the ATP-bindingsite of protein kinases in a fashion similar to staurospo-

Figure 3 Binding of ATP (left) and staurosporine (right) to theATP-binding site of cyclin dependent kinase 2 (CDK2). Both ATPand staurosporine form hydrogen bonds with the backbone amidegroups of glutamate 81 and leucine 83. The green area indicates apatch of high hydrophobicity. Adapted from ref. 26.



rine, but with less synthetic effort and more extendedstructural options, we replaced the indolocarbazole alka-loid structure with simple metal complexes in which themain features of the indolocarbazole heterocycle 3 are re-tained in a metal-chelating pyridocarbazole 4 (Figure 4).This ligand 4 can serve as a bidentate ligand for rutheniumcomplexes of type 1. All of the ruthenium compounds de-scribed in this Account article include this pyridocarba-zole moiety as a key pharmacophore ligand. Additionalligands in the coordination sphere of the metal can nowsubstitute for the carbohydrate moiety of staurosporine,with the metal center serving as a ‘glue’ to unite all of theparts. This approach has resulted in the successful designof nanomolar and even picomolar protein kinase inhi-bitors.8–13

3 Synthesis of Cycloruthenated Pyrido[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-diones

The tert-butyldimethylsilyl (TBS)-protected pyridocarba-zole 5, and not the highly insoluble compound 4, is typi-cally our starting ligand for the synthesis of metalatedpyridocarbazole complexes (Scheme 1). We developed ashort route to 5 and its derivatives as shown in Scheme 1.Accordingly, pyridoindole 6, accessible through Fischerindole synthesis or Suzuki coupling, is lithiated withLiHMDS, and reacted with TBS-protected dibromomale-imide 7 affording the monobromide 8 in 68% yield. Thekey step is the following anaerobic photocyclization intoluene to pyridocarbazole 5 upon release of HBr(80%).10,27 This synthetic route is general and has alreadybeen applied to more than 20 substituted pyridocarbazolederivatives in our laboratory.

Pyridocarbazole 5 reacts with metal fragments that beartwo labile or semilabile coordinated ligands (leavinggroups). The reactions are usually performed in thepresence of a weak base in order to capture the proton that

is released from the indole upon coordination to themetal. For example, the reaction of 5 with [(h5-C5H5)Ru(CO)(MeCN)2]

+PF6– (9) in the presence of one

equivalent of potassium carbonate affords the half-sand-wich compound 10 (87%), and after TBAF deprotectionthe ruthenium half-sandwich complex 11 (96%,Scheme 2).

This neutral half-sandwich ruthenium complex 11 is sta-ble under air, in water, and can even withstand the pres-ence of millimolar concentrations of thiols as determinedby 1H NMR spectroscopy. Compound 11 is a nanomolarinhibitor for the protein kinases GSK-3 and Pim-1 as dis-cussed in later chapters.

An X-ray structure of the N-benzylated derivative of 11(Figure 5) verifies that the pyridocarbazole can in factserve as a bidentate ligand for ruthenium, having one clas-sical coordinative bond with the pyridine moiety (Ru–N = 2.13 Å) in addition to one covalent s bond with theindole nitrogen (Ru–N = 2.11 Å).8,28 The coordinationgeometry of the ruthenium is pseudo-octahedral with theCO being oriented perpendicular to the plane of thepyridocarbazole and the cyclopentadienyl (Cp) moietyoccupying the remaining three binding sites.

In a different synthetic strategy we were seeking a ruthe-nium precursor complex that would enable us to introducenew ligands in the final synthetic step, thus acceleratingthe synthesis of new coordination spheres, eventuallyeven in a combinatorial fashion. We developed the syn-thesis of such a ruthenium complex 12, bearing four leav-ing groups (three acetonitriles and one chloride) inaddition to the pyridocarbazole ligand (Scheme 3).12 Forthe synthesis of 12, the TBS-protected pyridocarbazole 5is cyclometallated with [(benzene)RuCl2]2 at room tem-perature and in the presence of one equivalent of K2CO3,affording the half-sandwich complex 13 in 69% yield.The benzene can subsequently be replaced by three aceto-nitrile molecules upon photolysis in acetonitrile using a

Figure 4 Designing ruthenium complexes 1 as protein kinase inhibitors by using the ATP-competitive natural product staurosporine as a leadstructure. The chelating ligand 4 contains the main pharmacophore of the indolocarbazole heterocycles 2 and 3.

N

NNO

H

O

O

HHN

staurosporine as a lead structure

NH

NH

NX O

H

NH

N O

H

N

O

2 (X = CH2), 3 (X = CO) 4

NNO

HO

NRu

A

D

C

B

ruthenium complexes 1



medium-pressure mercury lamp yielding complex 12 dia-stereoselectively after TBAF deprotection (54% over twosteps). A crystal structure of the N-benzylated derivativeof 12 (Figure 6) reveals a trans stereochemistry betweenthe chloride and indole.

This compound 12 allows a rapid access to a diversity ofnovel structures 14 by simple ligand replacement chemis-try in the final steps. For example, compounds 15–17(Figure 7) have been synthesized through 12.

4 Discovery of Ruthenium Complexes as Protein Kinase Inhibitors

4.1 Octahedral Complexes

Simple ligand substitution chemistry with rutheniumcomplex 12 opened an economic avenue for the rapidscanning of ligands around the ruthenium center, search-ing for three-dimensional structures that are complemen-

tary in shape and functional group presentation to theactive site of individual protein kinases.12 Surprisingly,even the synthesis of just a small library of rutheniumcomplexes (around 100 members) by reacting 12 with dif-ferent combinations of ligands, followed by a screeningagainst a small panel of kinases, led already to the identi-fication of nanomolar ruthenium kinase inhibitors(Figure 7, 15–17).

Scheme 1 Synthesis of TBS-protected pyridocarbazole 5.

8

7

NH

NNH N

N OO

TBS

BrBr

N OO

TBS

Br

NH

N OO

TBS

N

56

hν

LiHMDS – HBr

68% 80%

Scheme 2 Synthesis of the cycloruthenated pyridocarbazole 11. Only one enantiomer of the racemic mixture is displayed.

5

Ru PF6–

NN C

OC

C+

N

N O

TBS

N

O

Ru

CO

N

N O

H

N

O

Ru

CO

TBAF

10 11

K2CO3

9

87%

96%

Figure 5 Crystal structure of the N-benzylated derivative of 11.ORTEP drawing with 35% probability thermal elipsoids.

Scheme 3 Synthesis of ruthenated pyridocarbazoles by ligand sub-stitution chemistry starting from the precursor complex 12.

N

N O

TBS

O

N

Ru

Cl

N

N O

H

O

N

Ru

N Cl

NN

5

12

13

[Ru(C6H6)Cl2]2

MeCN, K2CO3

69%

1) hν, MeCN2) TBAF

54%

N

N O

H

O

N

Ru

L3 L2

L1L4

14

L1–L4

heat



Figure 7 Some protein kinase inhibitors with octahedral rutheniumcoordination sphere. Note that all these compounds are racemicmixtures. IC50 values were determined with an ATP concentration of100 mM.

Figure 8 Inhibitory activities of the organoruthenium compounds15–18 at a concentration of 100 nM against the protein kinases GSK-3a, Pim-1, MSK1, and CDK2/CyclinA. ATP concentration was 100mM. Compound 18 is an inactive reference complex derived from 16by replacing the CO ligand by P(OMe)3.

Figure 8 shows the inhibitory activities of these com-pounds at a concentration of 100 nM against a small panelof the protein kinases CDK2/CyclinA, MSK-1, Pim-1,and GSK-3a. For example, the racemic compound 16 is asubnanomolar inhibitor for the protein kinase Pim-1 withan IC50 value (concentration of compound at which 50%of the enzyme is inhibited) of 0.45 nM at 100 mM ATP.With this, 16 is at least two orders of magnitude more po-tent against Pim-1 compared to the unspecific inhibitorstaurosporine. Because of the cyclic nature of the triden-tate ligand and the sp3-hybridization at the coordinatingsulfur atoms, 1,4,7-trithiacyclononane has to occupy twocoordination sites within the plane of the pyridocarbazoleligand, thus leaving one position perpendicular to thepyridocarbazole plane for the monodentate ligand. In-triguingly, the nature of this monodentate ligand has adramatic effect on kinase inhibition. For example, substi-tuting the CO for P(OMe)3 (18) renders the compoundcompletely inactive (Figure 8). Other ligands such asDMSO, cyanide, and ammonia also result in a significantdecrease in potency for Pim-1. Interestingly, substitutingthe CO for an azide (15) yields a fairly potent MSK-1 in-hibitor (IC50 = 70 nM).

The bar diagram in Figure 8 further demonstrates that theCO compound 16 is most potent against Pim-1 but also in-hibits GSK-3a to a significant extent at 100 nM. Appar-ently, the CO ligand in the plane perpendicular to thepyridocarbazole chelate is an important pharmacophoreboth for Pim-1 and GSK-3, but not for most other kinases.The rest of the ligand sphere can then be used to render thecomplex either selective for Pim-1 or GSK-3. This is wellillustrated by the compounds 16 and 17. Whereascomplex 16 significantly prefers Pim-1, the complex 17 isinstead selective for GSK-3 with an IC50 of 8 nM forGSK-3a at 100 mM ATP compared to 95 nM for Pim-1.

It is also noteworthy that the entire coordination sphere of17 renders the complex around 19,000 fold more potentfor GSK-3a compared to the plain pyridocarbazole ligand4 (Figure 9), thus demonstrating the power of using theentire coordination sphere of the ruthenium for the designof enzyme inhibitors.

Figure 9 IC50 curves of pyridocarbazole ligand 4 and the rutheniumcomplex 17 at an ATP concentration of 100 mM.

Figure 6 Crystal structure of the N-benzylated derivative of pre-cursor complex 12. ORTEP drawing with 40% probability thermalelipsoids.

N

N O

H

O

N

Ru

S S

NSN

N

N

N O

H

O

N

Ru

Cl

CH2N O

N

MSK1: IC50 = 70 nM

GSK-3α: IC50 = 8 nM

N

N O

H

O

N

Ru

S S

CS

Pim-1: IC50 = 0.45 nM

O+

PF6–

15

16

17



4.2 Half-Sandwich Complexes

A structurally simplified class of GSK-3 and Pim-1 inhib-itors is constituted by the half-sandwich scaffold 11(Scheme 2 and Figure 10), bearing a h5-coordinatedcyclopentadienyl moiety and CO ligand in addition to thepyridocarbazole moiety.8–11,13 This scaffold is pseudo-octahedral because the Cp ring occupies three sites of anoctahedral coordination sphere. This has the practicaladvantage of reducing the number of possible stereo-isomers down to two for compound 11.

Screening complex 11 against a panel of 57 kinases re-vealed that 11 is a quite selective inhibitor for the proteinkinases GSK-3 and Pim-1.13 The IC50 curve of 11 againstthe a-isoform of GSK-3 (GSK-3a) and a comparison withthe corresponding pyridocarbazole ligand 4 is shown inFigure 10 at an ATP concentration of 10 mM (red andgreen curves, respectively). The pyridocarbazole ligand 4itself is a very weak inhibitor for GSK-3a with an IC50 ofonly 50 mM at 10 mM ATP. This means that upon forma-tion of the metal complex 11 the potency increases by afactor of more than 15,000. Consequently, the activity ofcomplex 11 requires the entire assembly, kept together bythe central ruthenium atom. In order to test if 11 does, asdesigned, bind to the ATP site, we synthesized a deriva-tive of 11 with the imide hydrogen replaced by a methylgroup (11Me). This methylation abolishes the activitycompletely (IC50 > 300 mM, see pink curve in Figure 10),consistent with the assumption that the imide hydrogenis involved in hydrogen bonding with the adenine bindingcleft. Additionally, a Lineweaver–Burk analysis(Figure 11) of relative initial velocities of GSK-3a atdifferent concentrations of ATP and 11 reaffirms ATPcompetitive binding and yields an inhibition constant of0.98 nM.

Derivatizations as well as the absolute configuration canbias this scaffold towards the binding to either GSK-3 orPim-1. For example, the S-isomer of compound 19, (S)-19(Figure 12), having an additional hydroxyl group at the in-

dole, is 30-fold selective for Pim-1 with an IC50 of 220 pMfor Pim-1 (100 mM ATP). In contrast, the R-isomer ofcompound 20, (R)-20, having a hydroxyl group and bro-mine at the indole and methoxycarbonyl group at the Cp,is by more than one order of magnitude selective for GSK-3a (IC50 = 0.35 nM, 100 mM ATP) and GSK-3b(IC50 = 0.55 nM) vs. Pim-1.

Interestingly, the stereochemistry of the ruthenium frag-ment plays a crucial role as demonstrated with the IC50

curves in Figure 13. Whereas (R)-20 binds more tightly toGSK-3, the mirror-image isomer (S)-20, significantly pre-fers Pim-1 (IC50 = 3 nM, 100 mM ATP) and thus leadingto a complete switch in kinase selectivity just by invertingthe absolute configuration at the ruthenium.

Figure 10 IC50 curves of the ruthenium complex 11, a methylatedderivative of 11 (11Me), pyridocarbazole ligand 4, and staurosporineagainst GSK-3a at an ATP concentration of 10 mM.

Figure 11 Double-reciprocal plots of relative initial velocities (Vrel)against varying ATP concentrations in the presence of 11. The plotsintersect at the 1/Vrel axis, confirming that 11 binds competitivelywith respect to ATP.

Figure 12 The ruthenium half-sandwich compounds 11, 19, and 20have metal-centered chirality. The absolute configuration at the ruthe-nium center has been assigned according to the priority order of theligands being h5-C5H5 > pyridine [N(C, C, C)] > indole [N(C, C, lonepair)] > CO.

N

N O

H

N

O

R1

Ru

CO

N

NO

H

N

O

R1

Ru

CO

s

R-isomer S-isomer

R2 R2R3 R3

11 R1 = R2 = R3 = H19 R1 = OH, R2 = R3 = H20 R1 = OH, R2 = Br, R3 = CO2Me

N

N O

H

N

O

HO

Ru

CO

N

NO

H

N

O

OH

Ru

CO

(R)-20GSK-3 selective

(S)-19Pim-1 selective

Br CO2CH3



5 Structures of Ruthenium Half-Sandwich Complexes Bound to Protein Kinase Pim-1

To investigate the binding mode of our ruthenium com-pounds, we co-crystallized full-length Pim-1 with enan-tiomerically pure organoruthenium compound (S)-19 andsolved the structure to 1.9 Å resolution (PDB code 2BZI)in collaboration with the group of Stefan Knapp (OxfordUniversity).11,29

The structure shows the typical two-lobe protein kinasearchitecture, connected by a hinge region, with the cata-lytic domain positioned in a deep intervening cleft. The N-terminal lobe is comprised primarily of anti-parallel b-sheets and the C-terminal lobe mainly of a-helices.11

Figure 14 demonstrates that (S)-19 occupies the ATP-binding site, in accordance with the initial design.

Superimposing cocrystallized (S)-19 and staurosporinewith Pim-1 (PDB code 1YHS)25d reveals how closely thehalf-sandwich complex mimics the binding mode of stau-rosporine (Figure 15). The pyridocarbazole moiety of (S)-19 is nicely placed inside of hydrophobic pocket formedbetween residues from N-terminal and C-terminal domain(Figure 16, b), mimicking the binding position of the in-dolocarbazole moiety of staurosporine, while the rest ofthe ruthenium complex occupies the binding site of thecarbohydrate moiety of staurosporine (Figure 15).

In agreement with the initial design, (S)-19 retains the hy-drogen bonding pattern of ATP by forming one character-istic hydrogen bond between the maleimide NH group ofthe pyridocarbazole moiety and the backbone carbonyloxygen atom of Glu121 within the hinge region. Pro123does not allow the formation of the second hydrogen bondwhich is analoguous to the binding of ATP and staurospo-rine to Pim-1. Additional hydrogen bonds are formed be-tween the hydroxyl group of (S)-19 and two ordered watermolecules (Figure 16, a).

The carbonyl ligand does not form any hydrogen bonds.However, the surprisingly short distances between thecarbonyl oxygen and Gly45 (3.0 Å to the methylenegroup, 3.0 Å to the nitrogen, and 3.2 Å to the carbonylcarbon) are strong evidence for dipolar interactions ofthe CO ligand and positively polarized methylene andcarbonyl group of Gly45 (Figure 16, c).30,31

Since both enantiomers of the half-sandwich rutheniumcomplexes 11 and 19 bind with nanomolar affinities toPim-1, we were interested in comparing their bindingmodes and therefore co-crystallized also (R)-11 with Pim-1 and solved the structure to a 1.9 Å resolution (PDB code2BZH).11 The structure reveals that the maleimide group,the carbonyl ligand, and the cyclopentadienyl moiety of(R)-11 occupy almost identical binding positions com-pared to (S)-19. This is accomplished by a 180° flippedpyridocarbazole moiety (Figure 16, d).

These co-crystal structures clearly demonstrate that theruthenium complexes (R)-11 and (S)-19 bind to their tar-get in a fashion typical for organic enzyme inhibitors. The

Figure 13 Influence of the absolute configuration at the rutheniumon kinase selectivity. IC50 curves (100 mM ATP) of the half-sandwichorganometallics (R)-20 and (S)-20 against GSK-3b (solid lines) andPim-1 (dotted line).

Figure 14 Overview of the crystal structure of Pim-1 with the enan-tiomerically pure ruthenium complex (S)-19 bound to the ATP-bin-ding site. Insert: Electron density map at 1 s of (S)-19 plus onecoordinated water molecule (red sphere).

Figure 15 Superimposed co-crystal structures of ruthenium com-pound (S)-19 and staurosporine with Pim-1.



metal center is not involved in any direct interactions withthe active site of Pim-1. Instead, the metal controls the ori-entation of the organic ligands in the receptor space, thusyielding three-dimensional structures that are comple-mentary in shape and functional group presentation to theactive site of Pim-1 (Figure 17).

Figure 16 Interactions of (S)-19 (a-c) and (R)-11 (d) within the ac-tive site of Pim-1. (a) Hydrogen bond of the maleimide NH of (S)-19with backbone amide carbonyl group of Glu121 and water-mediatedcontacts of the hydroxyl group. (b) The most important hydrophobicinteractions with (S)-19. (c) The close contact of the CO ligand of (S)-19 with Gly45. (d) Hydrogen bonding of (R)-11 within the active siteof Pim-1.

Figure 17 (S)-19 in the binding pocket of Pim-1 demonstrating theshape match.

6 GSK-3 Inhibition in Mammalian Cells, Frog Embryos, and Zebrafish Embryos

In order to investigate the cellular activity of our rutheni-um compounds we probed the wnt signaling pathway withthe selective GSK-3 inhibitor (R)-20.13 In this pathway,GSK-3b is a negative regulator by phosphorylating b-catenin.32 Phosphorylated b-catenin itself is unstable andis degraded rapidly by the proteasome. In the presence ofa wnt signal, GSK-3b is inactivated, resulting in an accu-mulation of b-catenin in the cytoplasm, followed by a sub-

sequent translocation into the nucleus where b-cateninserves as a transcriptional co-activator through its interac-tion with the T-cell factor (TCF) family of transcriptionfactors. Thus inhibition of GSK-3b by pharmacologicalinhibitors or by wnt signaling leads to increased b-cateninlevels and activation of wnt dependent transcription(Figure 18).

Figure 18 Wnt pathway: Inactivation of GSK-3b by a wnt signal orsmall molecule inhibitor results in the transcription of target genes.

In order to determine the activation of the wnt pathway asa response to intracellular inhibition of GSK-3b, we usedhuman embryonic kidney cells (HEK-293OT) that havestably incorporated a Tcf-luciferase transcription-reportergene (OT-Luc cells).33 This transcription reporter gener-ates luciferase in response to increased concentrations ofb-catenin. We incubated these OT-Luc cells with varyingconcentrations of (R)-20. An upregulation of luciferasewas then determined by the luminescence signal upon ad-dition of luciferin to the cell lysate. Intriguingly, (R)-20shows high activities down to nanomolar concentrations(Figure 19, a). For example, at 30 nM, (R)-20 displays al-most complete activity with a luminescence increase by afactor of almost 700. No other tested compound couldmatch this cellular activity. For example, the organic in-hibitors kenpaullone34 and 6-bromo-indirubin-3¢-oxime(BIO)35 need micromolar concentrations for inducing sig-nificant wnt signaling.

We also analyzed the cellular b-catenin concentration byWestern blotting after incubation with (R)-20. Figure 19(b) demonstrates a qualitative increase in b-catenin in thepresence of (R)-20 at concentrations down to 30 nM.

To further verify the activation of the wnt pathway result-ing in the translocation of b-catenin into the nucleus, weperformed b-catenin staining experiments in melanomacells (1205Lu) after exposure to (R)-20. For this, cellswere fixed and incubated first with a primary antibody



against b-catenin followed by a secondary Texas Red con-jugated antibody. Immunofluorescence microscopy thenrevealed that most of the b-catenin was indeed located inthe nuclei and that this effect was observed at low concen-trations of only 30 nM (R)-20 (Figure 19, c). Thus, theseexperiments unambiguously demonstrate that (R)-20activates the wnt pathway in mammalian cell culture atnanomolar concentrations.

In order to test the utility of the organometallic reagent(R)-20 for experiments in entire organisms, we performedphenotypic experiments in zebrafish embryos. Wnt sig-naling, and thus GSK-3b, plays a crucial role in the devel-opment of metazoan. For example, the exposure ofzebrafish embryos at the four-hour stage to LiCl, a knownGSK-3 inhibitor, promotes a perturbed development of

the head structure with a no-eye-phenotype, among oth-ers.36 Similarly, treatment with (R)-20 resulted in a de-creased head structure without eyes and a stunted andcrooked tail. In addition, the yolk is enlarged and mis-shaped (Figure 20, c). Intriguingly, under the same condi-tions, zebrafish embryos which were instead treated underidentical conditions with the mirror imaged compound(S)-20, develop completely normal (Figure 20, b). Appar-ently, it is the GSK-3b inhibition of (R)-20 that inducesthe observed phenotype.

Figure 20 Exposure of zebrafish embryos to, (a) DMSO (2%),(b) 1 mM (S)-20, and (c) 1 mM (R)-20. The embryos were collectedand maintained in E3 media at 28.5 °C, compounds added at 4 h postfertilization (hpf), and the phenotypes were compared at 25 hpf.

Figure 21 Effects of ruthenium compound 19 on the developmentof Xenopus embryos. Organometallic 19 (ca. 0.5 mM in 10% DMSO)was injected into a ventral blastomere of 32–64 cell stage Xenopusembryos (upper embryo, with twinned dorsal axis). Control injectionswith water or DMSO did not affect dorsal axis development (lowerembryo).

Finally, we investigated the effects of organorutheniumGSK-3 inhibitor racemic 19 on Xenopus laevis embryos incollaboration with Peter S. Klein (University of Pennsyl-vania).10 Injection of 19 into a ventral blastomere at the32–64 cell stage caused formation of a complete second-ary dorsal axis with the formation of a small second head(Figure 21), similar to effects observed with injection ofthe GSK-3 inhibitor LiCl.37

In summary, with these studies we demonstrated thatmetal-containing compounds are suitable as molecularprobes for chemical biology.10,13 Compound (R)-20 is su-perior as a molecular probe for the function of GSK-3compared to many reported organic GSK-3 inhibitorswith respect to binding affinity, cellular potency, andkinase selectivity.

Figure 19 Wnt activation with (R)-20. a) Cells transfected with a b-catenin-responsive luciferase reporter were treated with different con-centrations of (R)-20 for 24 h. Luminescence signals were measuredafter cell lysis and the addition of luciferin. b) Qualitative detection ofcellular concentrations of b-catenin as a function of incubation with(R)-20 for a period of 24 h. DMSO (0.5%) and LiCl (30 mM) wereused as negative and positive controls, respectively. The cells were in-cubated with the compounds for 24 h and the concentration of b-cate-nin verified by Western blotting. Each lane contains the same totalamount of protein. c) Cellular b-catenin staining in melanoma cells(1205Lu) as a function of the concentration of (R)-20. The cells wereseeded onto glass coverslips and incubated overnight in the presenceof, 0 (left) and 30 nM (right) (R)-20. Cells were then fixed in 4%formaldehyde solution, permeabilized with Triton X-100, incubatedwith a primary antibody raised against b-catenin, and subsequentlytreated with an anti-mouse Texas Red conjugated secondary antibo-dy. Coverslips were then analyzed using immunofluorescencemicroscopy.



7 Summary and Outlook

This Account makes an argument for exploring newchemical space with organoruthenium compounds. Theapproach is based on the hypothesis that complementingan organic scaffold with a metal center opens new oppor-tunities for the design of small molecules with novelthree-dimensional structures and thus provides the chanceto discover compounds with novel biological properties.

We applied this strategy to the design of organorutheniumprotein kinase inhibitors by mimicking the binding modeof staurosporine. Intriguingly, we discovered rutheniumcomplexes which display distinguished properties withhigh kinase selectivities, picomolar binding constants,and superior activities in mammalian cell cultures. Crystalstructures unambiguously verify that the metal center isnot involved in any direct interactions and that the metalinstead controls the orientation of the organic ligands inthe receptor space, thus yielding three-dimensional struc-tures that are complementary in shape and functional-group presentation to the active site. It is likely that someof our inhibitors cannot be easily mimicked by organicscaffolds and we believe that we are accessing an area ofthe chemical space that is mostly unexplored.

Future work will extend this methodology to other classesof biomolecular targets. Target sites, including protein–protein interactions, which are large and globular seem tobe especially advantageous because we believe that bybuilding the structure from a single center, our approachmay have an advantage in synthesizing small andmedium-sized globular molecules in a very economicalfashion.

Furthermore, it will be interesting to evaluate if these ru-thenium-based kinase inhibitors are potential scaffolds fortherapeutic use. Along these lines, we recently found thatsome of our ruthenium GSK-3 inhibitors display highcytotoxicities in mammalian cells. Mechanistic investiga-tions in collaboration with the Meenhard Herlyn laborato-ry (Wistar Institute, Philadelphia) revealed that thesecompounds induce p53-activated apoptosis38 and that theapoptotic properties are at least in part due to the rescue ofp53 from degradation in an MDM2 dependent mannerthrough the prevention of phosphorylation by GSK-3b.39,40 Similar results have been reported recently bypharmacological inhibition of GSK-3b in colorectal can-cer cells.41 This anticancer activity of ruthenium com-pounds is distinguished from the traditional mode ofaction of metal-based anticancer drugs such as cisplatin orPeter Sadler’s and Paul Dyson’s organoruthenium com-pounds,4k–m which trigger apoptosis by initially reactingwith guanine bases in DNA.

Last but not least, thinking into a different direction, it isalso appealing to complement the structural role of metalswith their physicochemical,42 reactive, and/or catalyticproperties in one molecule to yield compounds with un-precedented abilities to probe and modulate biologicalprocesses.43

Acknowledgment

Financial support was provided by the University of Pennsylvaniaand the National Institutes of Health (1R01 GM071695-01A1). Wethank our collaborators Scott L. Diamond (University of Pennsylva-nia), Peter S. Klein (University of Pennsylvania), Stefan Knapp(Oxford University), Keiran S. M. Smalley, Meenhard Herlyn (bothWistar Institute, Philadelphia), and Michael A. Pack (University ofPennsylvania) for their important contributions to this research pro-ject.

References and Notes

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