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European Journal of Medicinal Chemistry 46 (2011) 1131e1139

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Carbaboranes as pharmacophores: Similarities and differences betweenaspirin and asborin

Matthias Scholz a, Goran N. KaluCerovi�c b,c, Harish Kommera b, Reinhard Paschke b, Joanna Will d,William S. Sheldrick d, Evamarie Hey-Hawkins a,*

a Institut für Anorganische Chemie der Universität Leipzig, Johannisallee 29, 04103 Leipzig, GermanybBiozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germanyc Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, 06120 Halle, Germanyd Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany

a r t i c l e i n f o

Article history:Received 25 November 2010Received in revised form5 January 2011Accepted 17 January 2011Available online 28 January 2011

Keywords:AspirinAsborinCarbaboraneCarboranePharmacophoreCytotoxicity

* Corresponding author. Tel.: þ49 341 9736151; faxE-mail address: hey@rz.uni-leipzig.de (E. Hey-Haw

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.01.030

a b s t r a c t

In medicinal chemistry carbaboranes have been used almost exclusively as boron carriers for boronneutron capture therapy (BNCT). Recent developments extended the carrier approach and use carba-boranes as scaffolds for radiodiagnostic or therapeutic agents. Most recent studies, however, focus oncarbaboranes as modern hydrophobic pharmacophores. This research employs preferably meta- andpara-carbaborane, because these isomers are extremely hydrophobic and very stable. In this paper wetherefore investigated the pharmacophoric behavior of the ortho isomer as putative phenyl mimetic bycomparing aspirin to asborin, its ortho-carbaborane analogue. Special emphasis is placed on the impactof the cluster properties on the pharmacological profile. Subjects under study are the mode of cyclo-oxygenase (COX) inhibition, stability, and toxicity. The straightforward syntheses of the correspondingnido compounds as well as their contribution to the pharmacology of the closo precursors will behighlighted. Finally, proof will be given that the ortho-carbaborane core of asborin merits the designation“pharmacophore” by definition and is a multifunctional group rather than just a hydrophobic, bulkyspectator.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Dicarba-closo-dodecaboranes(12), or carbaboranes, were firstreported in both the USA and USSR at the end of 1963 [1e3]. Theicosahedral clusters exist as three isomeric forms: 1,2-, 1,7-, and1,12-dicarba-closo-dodecaborane(12), usually referred to as ortho-,meta- and para-carbaborane. The neutral carbon-containing clus-ters are theoretically derived from the B12H12

2� dianion by replacingtwo BH� units by two isolobal CH groups [4]. ortho-Carbaborane isobtained by the reaction of acetylene with B10H14 in the presence ofLewis bases such as acetonitrile, alkyl amines, and alkyl sulfides andis the common precursor for the other isomers. Under inertconditions ortho-carbaborane rearranges to meta-carbaboranebetween 400 and 500 �C, and meta-carbaborane to para-carba-borane between 600 and 700 �C [3,5].

All isomers show similar but individually accented properties.The clusters are extremely hydrophobic and regarded stable under

: þ49 341 9739319.kins).

son SAS. All rights reserved.

various conditions [3]. Since the skeletal electrons are delocalizedwithin the cluster core, carbaboranes are considered to be three-dimensional aromatics [6]. Furthermore, all isomers are electronwithdrawing and weak Brønsted acids. The electron deficiencydecreases in the order ortho-,meta-, to para-carbaborane, while thestability increases in this order [7].

Exploration of carbaborane chemistry has revealed a wealth ofreactions to obtain modified cluster compounds. The orthogonalityof nucleophilic substitution reactions at the carbon atoms to elec-trophilic substitution reactions at the boron atoms allows for high-yield standard procedures without laborious requirements forprotecting groups [8]. The choice of carbaborane isomer allows fora desired two-dimensional orientation of the carbon-attachedsubstituents. The inclusion of boron substituents renders a three-dimensional orientation possible. Deboronation reactions generatequantitatively nido cluster anions and fine-tune solubility.Deboronation is observed when carbaboranes are exposed tostrong nucleophiles such as amines or alcohols at elevatedtemperatures [9]. Wet DMSO and CsF have been reported tosupport the formation of nido clusters [10,11]. Deboronation ratesdecrease from ortho- to para-carbaborane [12]. The latter requires

Fig. 1. Transparent ribbon presentation of COX monomers with active-site amino acids(green) and acetylated lysine (red) and acetylated serine (blue) due to asborin(generated using PyMOL). Left: ovine COX-1 data taken from RCSB protein data baseentry 1 eqg [31]. Right: human COX-2 data taken from RCSB protein data base entry1v0x. (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e11391132

very harsh conditions. Electron-withdrawing substituents at thecluster carbon atoms, however, facilitate deboronation intrinsically[13]. The resulting nido-carbaboranes also give access to metal-lacarbaboranes. These organometallic compounds feature a BHvertex replaced by an isolobal metal complex fragment andpotentially have new applications [14].

Since the discovery of carbaboranes in 1963 various applicationshave been found in catalysis [15], materials design [16], andmedicine [17]. In medicinal chemistry carbaboranes are preferablyused for the design of boron neutron capture therapy (BNCT)agents. BNCT is a cancer treatment based on the reaction 10B(n,a)7Liacting only locally on the dimensions of a cell diameter [17,18].

The first BNCT agents were reported some time ago [19], but theinvestigation of carbaboranes as pharmacophores is more recent[20]. The most promising approach to studying carbaboranemoieties in pharmaceuticals is integration of the cluster into a drugwith known pharmacological profile. Since the aromatic cluster hasa diameter of about 5.5 Å it is just a little larger than a rotatingphenyl group (ca. 4.7 Å) [17]. The phenyl-mimetic geometry andaromaticity were already applied in the synthesis of carbaboraneanalogues of tamoxifen [21] and trimethoprim (TMP) [22]. Carba-borane-modified compounds, inspired by the cyclooxygenase(COX) inhibitors flufenamic acid and diflunisal [23], and finally theaspirin analogue, asborin (Scheme 1), were also reported [24].

Preliminary studies showed that asborin inhibits both enzymevariants, COX-1 and COX-2, which are pharmaceutically interestingenzyme targets [24e26].

2. Results

2.1. COX acetylation studies

The detailed mechanism of COX inhibition by aspirin is well-investigated [27e29]. Aspirin inhibits COX by acetylating an active-site serine residue. In the case of COX-1 Ser530 is acetylated, and inthe case of COX-2 Ser516. Assuming that asborin’s mode of action issimilar to that of aspirin we investigated the acetylation behaviorfirst.

LCeESI tandem mass spectrometry provides a useful tool tostudy covalent modification sites of enzymes [30]. Therefore, both

Scheme 1. Synthesis of asborin [24].

COX variants were incubated with asborin, and the peptide frag-ments obtained after trypsin digestion were examined by ESI MS/MS following reverse phase LC separation.

Surprisingly, the significant active-site serine residues (Ser530and Ser516), which were modified by aspirin, were not acetylatedby asborin [32]. By contrast, COX-1 was acetylated at Ser521 and ata further six lysine residues (Lys169, 211, 222, 253, 473, 560). COX-2showed no serine acetylation, but five lysine acetylation sites(Lys166, 346, 355, 432, 598) (Fig. 1). When incubated with aspirin,COX-1 and COX-2 showed exclusive acetylation of the active-siteserine residues S530 and S516, respectively. This mode of COX-2acetylation has already been reported for Co-ASS, a hexa-carbonyldicobalt-modified aspirin analogue, which showed thesame acetylation pattern [33]. Unlike Co-ASS, asborin showed oneextra acetylation at Lys355. All acetylated lysine residues arelocated on the surface of each monomer and not in the active-siteregion.

2.2. Stability studies by NMR measurements

2.2.1. 1H NMR studiesTo investigate the stability and acetylation potential of asborin

in comparison to aspirin we used 1H NMR spectroscopy to followhydrolysis of the acetyl group. As simple experimental setup,aspirin and asborin were dissolved in deuterated water, with wateracting both as natural solvent and as nucleophile. The samples werestored at room temperature and time-dependent spectra wererecorded.

Aspirin is hydrolyzed to salicylic acid and acetic acid underaqueous conditions (Scheme 2).

After one day, aspirin showed almost no signs of hydrolysis(Fig. 2). After 10 days more than 80% remained unhydrolyzed, andeven after 150 days aspirin was not completely deacetylated. In

Scheme 2. Hydrolysis of aspirin.

Fig. 2. Time-dependent 1H NMR signals of the methyl protons of aspirin (2.36 ppm)and the growing signal of acetic acid (2.08 ppm) in D2O.

Fig. 3. Time-dependent 1H NMR signals of the methyl protons of asborin (1)(2.13 ppm), the growing signal of acetic acid (2.08 ppm, *), and nido-asborin (3)(2.04 ppm) in D2O.

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e1139 1133

more complex serum samples the half-life is, of course, muchshorter [34,35].

The hydrolysis of asborinwas expected to bemore complex thanthat of aspirin (Scheme 3). Deacetylation of asborin 1 to salborin 2,the salicylic acid analogue, corresponded to the hydrolysis of aspirin(reaction A). The ortho-cluster, however, can additionally undergodeboronation with nucleophiles in aqueous solution. The resultinganionic nido-carbaboranes have one boron atom less than the closoclusters and a bridging hydrogen atom. Nucleophilic attack takesplace at one of the two boron atoms adjacent to both carbaboranecarbon atoms giving a planar-chiral product [36]. Since there is nochiral induction present, we expected the formation of a racemicnido-carbaborane mixture. In solution the bridging hydrogen atomis delocalized over the boron atoms of the open pentagonal face andexchanges with deuterium [37].

The deboronation reaction B can therefore compete withasborin saponification reaction A creating nido-asborin (3).Compound 2 is also susceptible to deboronation as shown inreaction C, and nido-asborin 3 can also undergo saponification(reaction D).

With the aim of confirming Scheme 3 we dissolved asborin indeuterated water and recorded the corresponding time-dependent1H NMR spectra of the methyl protons (Fig. 3).

We found that hydrolysis of asborin, in contrast to aspirin,started immediately. The first 1H NMR spectrum was thereforerecorded very quickly, even if the compound was not yetcompletely dissolved. Thus, the first spectrum directly after theaddition of water showed a low signal-to-noise ratio. The peak ofthe methyl group of asborin at 2.13 ppm was clearly seen togetherwith signals indicating the beginning of hydrolysis. After 1.5 h theacetic acid signal at 2.08 ppm has already grown noticeably. A

Scheme 3. Hydrolysis of asborin: reactions A and D: deacetylation, reactions B and C

signal at 2.04 ppm also developed indicating formation of 3. Afterone day, asborin has been completely converted to acetic acid and3. Integration of the 1H NMR signals indicated that the reactionrates of A and B were approximately the same. Reaction D pro-ceeded very slowly in comparison to reactions A and B. Even afterone month, compound 3was still detected. Reaction C could not befollowed by 1H NMR.

2.2.2. 11B NMR studiesTo follow reaction C and to support the proposedmechanismwe

additionally recorded the corresponding 11B NMR spectra (Fig. 4).The closo isomers 1 and 2 showed signals from �5 to �14 ppm,

whereas the nido compounds 3 and 4 covered a larger range from�8 to �38 ppm. The rising signals in the high-field region (after6 h) indicated formation of the nido derivatives. The signal of boricacid at þ19.5 ppm further confirmed deboronation. Due to thequadrupole moment of the boron nucleus, the 11B NMR signalswere broad and overlapping. Thus, neither could closo isomer 1 bedistinguished from closo isomer 2, nor could nido isomer 3 bedistinguished from nido isomer 4 in the aqueous mixture. Thesignal at �5.7 ppm, however, unambiguously corresponds to thecloso compounds 1 or 2. Observation of this signal finally allowed usto follow the termination of reaction C. The 1H NMR spectra showedthat asborin disappeared completely after one day. The 11B NMRspectra indicated that formation of the nido cluster was completedafter approximately four days. Long-term measurements revealedthat complete decomposition finally takes place, as proved by the

: nido cluster formation. 3 and 4 are representative enantiomers and tautomers.

Fig. 4. Time-dependent 11B NMR spectra of asborin (1) dissolved in D2O. The closoisomers show signals from �5 to �14 ppm, whereas the nido compounds cover therange from �8 to �38 ppm. The signal at þ19.5 ppm corresponds to boric acid. Theasterisks (�5.7 ppm) highlight a 11B NMR signal of the closo isomers.

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e11391134

final and single signal of boric acid at þ19.5 ppm. Major interme-diates were not observed during this process. Total degradation toboric acid, however, proceeded far more slowly than the initialformation of the nido clusters.

2.3. Hydrolysis products

For fast in vitro studies, such as the COX inhibition assay,hydrolysis products (compounds 2e5) were expected to playa minor role. For long-term investigations of the general pharma-cological profile of asborin, however, these products will be ofmajor relevance. Boric acid, the final degradation product, iscommercially available. Compound 2 was already obtained duringthe synthesis of asborin [24]. Therefore, a targeted synthesis of thenido clusters 3 and 4 remained. The electron-withdrawingsubstituents at the cluster carbon atoms rendered deboronationpossiblewithout addition of nucleophiles andwithout heating. Thiswas crucial to keep the reactive acetyl group of asborin attached tothe cluster. Asborinwas quantitatively converted to nido compound3 in methanol within 2 h. Salborin (2) had to be heated to reflux inwater to force the reaction to completeness within 15 min. Theresulting nido cluster salts were very strong acids, due to the H3Oþ

countercation. Replacementwith appropriate cationswas thereforepivotal to suppress further degradation upon concentration andallow isolation. nido-Asborin (3) was almost quantitatively isolatedas its sodium salt, which was obtained after treatment with sodiumexchanger material. nido-Salborin (4) was quantitatively

Table 1IC50 [mM] determined by sulforhodamine-B microculture colorimetric assay.

Compound

518A2 FaDu

Asborin (1) 139.1� 13.6 123.7� 17.2nido-Asborin (3) 247.2� 16.8 270.3� 19.7Aspirin >1000 >1000Salborin (2) 100.1� 15.6 100.3� 12.7nido-Salborin (4) 169.0� 2.8 218.1� 3.0Salicylic acid >1000 >1000Boric acid (5) >1000 >1000Cisplatin 1.52� 0.19 1.21� 0.14

IC50 values after 96 h incubation. nido-Asborin (3) was applied as sodium salt, nido-salb

precipitated as tetraethylammonium salt from aqueous solution.All hydrolysis products were isolated as solids and recrystallized forX-ray structure determination (Fig. 5).

Each of the chiral compounds 3 and 4 crystallized as a racemicmixture. The bridging hydrogen atoms (Hm) were located adjacentto the carboxyl group and refined freely. Details of the crystalstructures are given in the SI.

2.4. Cytotoxicity studies

2.4.1. Sulforhodamine-B microculture colorimetric assayToxicity is an important criterion of pharmacophores. Since the

COX-2 acetylation pattern induced by asborin was similar to that ofCo-ASS, which is discussed as a potential anticancer agent, wefocused on cancer cell lines first [33]. Human tumor cell lines(518A2 melanoma, FaDu head and neck tumor, HT-29 colon carci-noma, MCF-7 breast carcinoma, and SW1736 anaplastic thyroidcancer cells) were selected to determine the IC50 values usinga sulforhodamine-B microculture colorimetric assay (Table 1) [38].

The phenyl compounds aspirin and salicylic acid showed notoxicity up to 1000 mM concentration. The closo-carbaboraneanalogues, however, inhibited cells growth with IC50 valuesaround 100 mM. Salborin (2) and asborin (1) both showed thelowest IC50 against MCF-7 cells and the highest IC50 against thehealthy human fibroblast WWO70327 cell line, which we addi-tionally tested for asborin (1; IC50¼174.4�12.2 mM) and salborin(2; IC50¼148.5�7.2 mM). Deacetylation of asborin (1) did notinfluence the IC50 value significantly. Boric acid, the final degra-dation product, was found to be nontoxic in concentrations lowerthan 1000 mM. The nido compounds were less toxic than the closocompounds, the nido-asborin salt (3) was less toxic than nido-salborin salt (4). All IC50 values of the boron compounds are,however, much higher than those of cisplatin.

2.4.2. Apoptosis studiesCell death can be induced by necrosis or apoptosis. To determine

which pathway is triggered by the most toxic closo-carbaboranecompounds, we performed dye exclusion test, DNA laddering, andcaspase activity assays using the MCF-7 breast carcinoma cell linerepresentatively.

2.4.2.1. Dye exclusion test and DNA laddering. Apoptosis and cell-mediated cytotoxicity are characterized by fragmentation of thegenomic DNA. The resulting DNA fragments have a length ofabout 180 base pairs or multiples of this number, which is thecharacteristic DNA length of a nucleosome (DNAehistonecomplex). These DNA fragments are resolved to a distinctiveladder pattern in agarose gel electrophoresis. This pattern wasfound for the MCF-7 cell line treated with asborin and salborin(Fig. 6).

IC50 [mM]

HT-29 MCF-7 SW1736

132.5� 16.9 96.8� 8.0 162.3� 14.4247.8� 8.6 269.0� 4.7 451.0 � 11.1>1000 >1000 >1000121.7� 13.9 91.9� 3.4 142.4� 14.0232.7� 4.3 182.1� 6.9 156.8� 10.2>1000 >1000 >1000>1000 >1000 >10000.63� 0.03 2.03� 0.11 3.20� 0.24

orin (4) was tested as tetraethylammonium salt, as obtained from the syntheses.

Fig. 5. ORTEP of decomposition products salborin (2), (R)-nido-asborin (3), (S)-nido-salborin (4); thermal ellipsoids are drawn at 50% probability.

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e1139 1135

Dye exclusion verified the apoptotic pathway triggered bycompounds 1 and 2 (Fig. 6).

2.4.2.2. Caspase 2, 3, 8, and 9 activities. In order to gain deeperinsight into the induced apoptosis mechanism, we analyzedwhether and which caspases are involved as upstream anddownstream effectors. Caspases are cysteineeaspartatic proteases,which play a major role in inducing programmed cell death.Apoptotic caspases are divided into initiator or upstream caspasesand executor or downstream caspases [39]. Therefore, the levels ofupstream caspases 2, 8, and 9, and of downstream caspase 3 wereexamined after treatment with the most toxic closo-carbaboranecompounds and cisplatin (Fig. 7). Asborin (1) induced accumulationof all caspases rapidly. Salborin (2) had no effect after 2 h. After 6 hincubation, 2 increased the levels of initiator caspases 2 and 9similarly to asborin (1). The level of downstream caspase 3,however, together with caspase 8 was noticeably higher after 6 h ofincubation when cisplatin was applied. Asborin (1) and cisplatingenerally triggered both extrinsic and intrinsic caspase pathways;salborin (2) is less effectivewhich is reflected as slower induction ofcaspase accumulation in the cells.

3. Discussion

This study was performed with the aim of investigating thepotential of ortho-carbaborane as phenyl-mimetic pharmacophore.This isomer has attracted less attention in this field than the meta-and para-carbaborane derivatives, which are obtained from theortho-cluster by thermal isomerization [40,41]. Aspirin wasselected as lead structure for two reasons. First, the carbaboraneanalogue is a very small molecule, so that the influence of the

Fig. 6. Dye exclusion test and DNA laddering (MCF-7 cell line treated for 24

cluster should come to the fore. Second, aspirin is an inhibitor of thetarget enzymes COX-1 and COX-2 with a well-investigated mode ofaction and a plethora of data reported for comparison. COX inhi-bition was already observed for asborin, which would support thephenyl analogy [24]. However, the efficiency of COX inhibition wasnoticeably lower compared to aspirin. Aspirin is activated inside theCOX active site and thereby acetylates an active-site serine residue[27,28]. In the case of COX-1, Ser530 is acetylated, and this preventsconversion of the substrate arachidonic acid (AA) into prosta-glandin H2 (PGH2). COX-2 treated with aspirin reveals acetylatedSer516 [28]. Unfortunately, asborin did not imitate this ingeniousmode of COX inhibition. Mass spectrometry investigations revealedthat asborin irreversibly acetylated different lysine residues on theprotein surface instead of the serine residue in the active site. Themodification of the protein periphery influenced the general COXactivity less than active-site serine acetylation and explains thelower COX inhibition potency of asborin compared to aspirin.Interestingly, in contrast to aspirin, asborin is capable of trans-ferring the acetyl group without external activation. We hold thehigh electron deficiency of the ortho-cluster responsible for causingthis increase in activity. The electron-withdrawing character makesthe hydroxyl-ortho-carbaborane unit a very good leaving group bystabilizing the negative charge of the alcohol oxygen atom afteracetyl transfer. Therefore, the ortho-carbaborane cluster renderedasborin too reactive to enter the complex COX active site with theacetyl group still attached. This illustrates clearly that ortho-car-baborane can activate functional groups in contrast to the phenylgroup. An impression of the degree of activation was revealed bythe stability studies in water followed by NMR spectroscopy.Hydrolysis of aspirin to acetic acid in water proceeds very slowly,while asborin is hydrolyzed within one day. The hydrolysis of

h with IC90 concentration of asborin (1) (left) and salborin (2) (right)).

Fig. 7. Caspase 2, 3, 8, and 9 activity relative to an untreated control after 2 and 6 htreatment with asborin (1), salborin (2), and cisplatin on MCF-7 cell line.

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e11391136

asborin additionally consists of two competing reactions, which aredeacetylation and deboronation with almost the same reactionrate. The nido compounds were more stable than the parent closoprecursors, probably due to electronic repulsion between theanionic cluster and nucleophiles. In solution complete deborona-tion to boric acid was observed after long times. In aqueous bufferand cell medium solutions similar degradation is anticipated. Thepresence of electron-withdrawing groups at the carbaboranecarbon atoms made deboronation possible even at room temper-ature and without additional nucleophiles. We took advantage ofthis intrinsic activation while synthesizing the nido deboronationproducts. nido-Asborin (3) formed readily and quantitatively inmethanol, and salborin (2) in water. The lack of stronger nucleo-philes in the reaction mixture made the formation of nido-asborin(3) possible, with the reactive acetyl group still directly attached tothe cluster alcohol oxygen atom. The choice of cation was crucial toprevent self-destruction and to facilitate purification. The applica-tion of appropriate counterions made nearly quantitative isolationof the nido salts possible, as well as the characterization by X-raycrystallography. This shows that not only the closo compounds canbe prepared by high-yield procedures, but also the correspondingnido analogues.

The presence of a cluster had an even greater impact on thetoxicity of the compounds. While the phenyl compounds werenontoxic, moderate cytotoxicity was observed for the closo-carba-borane analogues. As salborin (2) and asborin (1) were almostequally toxic (IC50 z 100e150 mM), the influence of the acetylgroup is negligible. Thus, the cytotoxic behavior must be attributedsolely to the ortho-closo-carbaborane moiety, which then meritsthe designation “pharmacophore” by definition [42]. A furtherstudy on the inhibition of the aldo/keto reductase 1A1 (AKR1A1)additionally proved this claim [43]. Aspirin and salicylic acid wereinactive against AKR1A1 whereas the integration of the cluster

made them active inhibitors. The toxicity studies showed alsoa slight selectivity trend toward cancer cells. The lowest IC50 valuesare obtained for the MCF-7 breast carcinoma cell line. More inter-esting is the detoxification upon decomposition. In all cell linesinvestigated, the nido compounds were less toxic than the closoclusters and the final boric acid was completely nontoxic. This trendwaspreviouslyobserved for the trimethoprimanalogues [22].ortho-Carbaborane induced cell death via apoptosis. Asborin (1) actedrapidly and triggered both intrinsic and extrinsic apoptotic path-ways. Salborin (2) induced only slowly accumulation of initiatorcaspases. Paradoxically, the acetyl group had no influence on theoverall IC50 value, but on the velocity and kind of caspases activated.

4. Summary and conclusion

The results of this study show the validity of regarding ortho-closo-carbaborane as an independent pharmacophore rather thanjust as a phenyl mimetic. Although size and aromaticity support thephenyl analogy, the cluster properties make it a multifunctionalgroup rather than a bulky spectator. The unique electron-with-drawing character of the ortho isomer was found to dominate theproperties. It influences not only the cluster itself, but also adjacentgroups. This is clearlydemonstratedbyacetylationof COXbyasborin.In this particular case, theunspecific COXmodification is unfavorableand results in lower enzyme inhibition.With respect to other targets,such as AKR1A1, this mode of acetylation is advantageous.

The toxicity of the closo compounds is solely caused by thecluster core, which thereby fulfills the criterion of being designateda pharmacophore. The overall toxic profile of asborin (1) is worthinvestigation in more depth as it shows a promising trend. It actsfast and temporally limits its toxicity by degrading to boric acid. Inrecent years, NSAIDS, especially aspirin, have emerged as cancer-protective or anticancer drugs, with both COX-dependent and-independent action [44,45]. The detailed mode of this behavior isstill unknown, but indications arose that those derivatives will beuseful in combination with classical cytostatics [46e48]. In thiscontext we hope to promote consideration of the versatile andmultifunctional ortho-carbaborane pharmacophore outside theniche of simple BNCT boron carriers.

5. Experimental section

5.1. LCeESI tandem mass spectrometry

COX-1 and COX-2 (both 10 mg) were incubated in 380 mL proteinbuffer solutions at a 1:10 ratio for 24 h at 310 K with asborin.Following trypsin digestion (1.6 mg) of the individual reactionsolutions at 310 K for 12 h, 2 mL 98% formic acid was added to theresulting peptide mixtures to reduce the pH to about 2.3. For theMUDPIT analyses (see Ref. [30] of the main text), a dual-gradientsystem HPLC pump (Dionex, Amsterdam), including a Famos autosampler and Switchos, was connected to a Finnigan LTQ ion trapmass spectrometer (Thermo Electron Corp., San Jose, USA). Amonophasic 100 mm microcapillary column packed with 12 cm ofEclipse XDB C18 was employed to separate the tryptic peptides ata flow rate of 0.12 mL/min using a linear CH3CN/0.1% formic acidgradient. Full MS scans were acquired in the m/z range 350e2000and were followed by MS/MS scans of the three most intense ions.The following parameters were implemented for SEQUESTmatching [49] of experimental MS/MS spectra to theoretical COX-1or COX-2 peptide sequences: Del Mass <2.5, Sp (preliminary score)>500, RSp (preliminary score rank) <6, Xcorr >3.75 for a charge 3þand >2.50 for charges of 2þ and 1þ, Delta(CN) >0.10. Potentiallyacetylated residues were included in the search file as modifiedresidues with a mass gain of þ42.

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e1139 1137

5.2. Chemistry

5.2.1. GeneralCompounds 1 and 2were synthesized according to the literature

[24].Methanol and acetoneweredistilled before use. Distilledwaterwas applied. Amberlite� IR-120 sodium exchanger (Merck) and allother chemicals were used as purchased. The infrared spectra wererecorded on a PerkineElmer System 2000 FT-IR spectrometer usingKBr disks. The 1H, 13C, and 11B NMR spectra were recorded on anAVANCEDRX 400 spectrometer (Bruker). The chemical shifts for the1H, 13C, and 11B NMR spectra are reported in parts per million (ppm)at 400.13, 100.63, and 128.38 MHz, respectively, with tetrame-thylsilane as standard for the first two and BF3(OEt2) as externalstandard for last-named. The 1HNMRsignals of theBHprotons covera larger range containing both solvent residual signals and themethyl protons in the case of 2 and were therefore not integrated.The spectra were recorded directly after dissolving the compounds.The mass spectra were recorded on an FT-ICR-MS Bruker-DaltonicsESI mass spectrometer (APEX II, 7 T). The melting points weredetermined in capillaries (Gallenkamp) and are uncorrected.

5.2.2. NMR studiesAspirin and asborin (1) were dissolved in D2O with 3-(trime-

thylsilyl)propionic acid as an internal standard. The samples werekept at room temperature. Time-dependent NMR spectra wererecorded. The first 1H NMR spectrum was measured as fast aspossible, even if 1 was not yet completely dissolved.

5.2.3. Syntheses

5.2.3.1. Sodium 7-acetoxy-7,8-dicarba-nido-undecaborate-8-carbox-ylic acid (nido-asborin sodium salt) (3). A solution of 1 (50 mg,0.20 mmol) in methanol (1 mL) was stirred for 2 h in the presence ofAmberlite� IR-120 sodium exchanger at room temperature.Hydrogen evolution could be observed. The reaction mixture wasthen filtered and Amberlite� IR-120 was washed with methanol(3�1 mL). The combined methanol layers were concentrated invacuoandgave3asawhitehygroscopic solid.Yield:50 mg (95%).Mp:102e104 �C. 1H NMR (DMSO-d6, 25 �C, ppm): d¼ 11.44 (s, br, 1H,COOH), 2.90 to�0.70 (m, vbr, 9H, C2B9H9),1.84 (s, 3H, CH3),�2.37 (m,vbr, 1H, Hm) (the value for the acidic protons is concentration-dependent). 11B NMR (DMSO-d6, 25 �C, ppm): d¼�8.5 (d,1J(B,H)¼ 128 Hz, 1B, C2B9H10), �11.6 (vbr, 1B, C2B9H10), �13.3 (d,1J(B,H)¼ 128 Hz, 2B, C2B9H10),�20.3 (vbr,1B, C2B9H10),�23.7 (vbr,1B,C2B9H10), �25.7 (d, 1J(B,H)¼ 141 Hz, 1B, C2B9H10), �34.2 (vbr, 1B,C2B9H10), �37.1 (d, 1J(B,H)¼ 141 Hz, 1B, C2B9H10) (1J(B,H) could not beresolved for all signals; due to the broad signals the number of boronatoms could not be determined unambiguously). 13C{1H} NMR(DMSO-d6, 25 �C, ppm): d¼ 170.9 (COO),170.8 (COO), 21.2 (CH3) (thebroad signals of the cluster carbon atoms could not be resolved). IR(KBr): ~n¼ 3566 (s, n (OeH)), 3507 (m), 3447 (m), 2981 (m), 2898 (m),2547 (s, n (BeH)), 2367 (w), 1733 (s, n (COO)), 1691 (s, n (COO)), 1668(m), 1616 (w), 1559 (w), 1540 (w), 1521 (w), 1507 (w), 1430 (m), 1372(m),1263 (s, n (CclustereO)),1161 (m),1049 (m),1034 (m),1021 (m),998(m),915 (w),877 (w), 739(w),668(w),642 (w), 504 (w) cm�1.HR-ESI-MS (�) (DMSO/CH3CN): m/z calculated for C5B9H14O4

� 236.1772,found 236.1770. Larger quantities of water present in Amberlite� IR-120 result in the formationof boric acid. In this case3wasdissolved inacetone and filtered through silica (0.035e0.070 mm, 60 Å) and thesolutionwas concentrated again in vacuo.

5.2.3.2. Tetraethylammonium 7-hydroxy-7,8-dicarba-nido-undeca-borate-8-carboxylic acid (nido-salborin tetraethylammonium salt)(4). 2 (105 mg, 0.51 mmol) was dissolved in water (2 mL) and the

solution heated to reflux for 15 min. While the solution cooledNEt4Cl (127 mg, 0.77 mmol) was added and the suspension stirredfor an additional 15 min at ambient temperature. The precipitatewas filtered off at room temperature and washed with ice-coldwater (3�1 mL). Evaporation in vacuo gave 4 as a white solid.Yield: 162 mg (97%). Mp: 295e297 �C. 1H NMR (acetone-d6, 25 �C,ppm): d¼ 11.08 (s, br, 1H, COOH), 6.79 (s, br, 1H, OH), 3.50 (q, 8H,CH2), 3.00 to �0.70 (m, vbr, 9H, C2B9H9), 1.41 (t, 12H, CH3), �2.72(m, vbr, 1H, Hm) (the values for the acidic protons are concentra-tion-dependent). 11B NMR (acetone-d6, 25 �C, ppm): d¼�9.8(d, 1J(B,H) ¼ 154 Hz, 1B, C2B9H10), �12.2 (d, 1J(B,H) ¼ 128 Hz,3B, C2B9H10), �19.5 (d, 1J(B,H) ¼ 154 Hz, 1B, C2B9H10), �22.2(d, 1J(B,H) ¼ 154 Hz, 1B, C2B9H10), �28.3 (d, 1J(B,H) ¼ 141 Hz,1B, C2B9H10), �32.8 (d, 1J(B,H)¼ 128 Hz, 1B, C2B9H10), �36.1 (d,1J(B,H)¼ 141 Hz, 1B, C2B9H10); 13C{1H} NMR (acetone-d6, 25 �C,ppm): d¼ 173.3 (COO), 52.0 (CH2), 6.7 (CH3); (the broad signals ofthe cluster carbon atoms could not be resolved). IR (KBr): ~n¼ 3432(m, n (OeH)), 3093 (m), 2994 (m), 2564 (s, n (BeH)), 2351 (w), 2316(w), 1681 (s, n(COO)), 1644 (m), 1486 (s), 1454 (m), 1439 (m), 1417(m), 1395 (m), 1366 (m), 1302 (m), 1264 (m, n (CclustereO)), 1192 (m),1174 (m), 1132 (w), 1070 (w), 1028 (m), 999 (m), 959 (w), 907 (w),877 (w), 785 (m), 747 (w), 709 (w), 682 (w), 669 (w), 652 (w), 441(w). HR-ESI-MS (�) (acetone): m/z calculated for C3B9H12O3

�

194.1666, found 194.1664.

5.2.4. Crystallography

5.2.4.1. General. The crystallographic data were collected ona CCD Oxford Xcalibur S diffractometer (l (Mo-Ka)¼ 0.71073 Å) inu scan mode. Semi-empirical from equivalents absorptioncorrections were carried out with SCALE3 ABSPACK [50] and thestructures were solved with direct methods [51]. Structurerefinement was carried out with SHELXL-97 [52]. All non-hydrogen atoms were refined anisotropically, and all H atoms,except those of disordered NEt4 in 4, were located on differenceFourier maps and refined freely. CCDC 792729 (2), 792730 (3) and792731 (4) contain the supplementary crystallographic data forthis paper. These data can be obtained free of charge from TheCambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

5.2.4.2. Structural data for 2 obtained from dichloromethane/pentane at 4 �C as colorless prisms in a commensurable modulatedstructure: C3H12B10O3, Mr¼ 204.23, monoclinic, space group P21/c,a¼ 1351.84(3), b¼ 1275.62(2), c¼ 1410.88(3) pm, b¼ 116.970(3)�,T¼ 110 K, V¼ 2.16837(7) nm3, Z¼ 8, rcalcd¼ 1.251 Mg/m3, m¼ 0.076mm�1, 2.90< q< 30.51�, R¼ 0.0416, wR¼ 0.0929, GOF¼ 0.941.

5.2.4.3. The sodium salt of 3 was dissolved in water and 1.5 equiv-alents tetraethylammonium chloride were added to precipitate thewater-insoluble ammonium salt. The precipitate was washed withwater and dried in vacuo. The white solid was recrystallized fromwater/acetone. Evaporation of acetone at room temperature gavecolorless crystals of the NEt4 salt of nido-asborin (3) together withcrystals of nido-salborin (4) as anion. Structural data for 3:C13H34B9NO4, Mr¼ 365.70, monoclinic, space group P21/n, a¼872.5(1), b¼ 1096.1(1), c¼ 2256.8(3) pm, b¼ 96.64(1), T¼ 130 K,V¼ 2.1438(4) nm3, Z¼ 4, rcalcd¼ 1.133 Mg/m3, m¼ 0.071 mm�1,3.00<q< 30.51�, R¼ 0.0473, wR¼ 0.1187, GOF¼ 0.918.

5.2.4.4. Structural data for the crystals of 4 obtained as described for3 as colorless prisms: C11H32B9NO3, Mr¼ 323.67, triclinic, spacegroup P1, a¼ 1015.36(4), b¼ 1161.54(4), c¼ 1797.34(7) pm,a¼ 83.969(3), b¼ 74.576(3), g¼ 66.396(4)�, T¼ 130 K, V¼ 1.8724

M. Scholz et al. / European Journal of Medicinal Chemistry 46 (2011) 1131e11391138

(1) nm3, Z¼ 4, rcalcd¼ 1.148 Mg/m3, m¼ 0.069 mm�1, 3.03<q

< 28.28�, R¼ 0.0605, wR¼ 0.1698, GOF¼ 1.046.

5.3. In vitro studies

5.3.1. General

5.3.1.1. Preparation of drug solutions. Stock solutions of investi-gated compounds were freshly prepared in phosphate bufferedsaline (PBS) at a concentration of 20 mM and filtered througha Millipore 0.22 mm filter before use. Working concentrations wereobtained by dilution with nutrient medium RPMI-1640 (PAALaboratories) supplemented with 10% fetal bovine serum (Bio-chrom AG) and penicillin/streptomycin (PAA Laboratories).

5.3.1.2. Cell lines and culture conditions. The cell lines 518A2melanoma, FaDu head and neck tumor, HT-29 colon carcinoma,MCF-7 breast carcinoma, SW1736 anaplastic thyroid cancer, andWWO70327 normal cell line (fibroblasts) were maintained asmonolayers in RPMI-1640 (PAA Laboratories, Pasching,Germany) supplemented with 10% heat-inactivated fetal bovineserum (Biochrom AG, Berlin, Germany) and penicillin/strepto-mycin (PAA Laboratories) at 37 �C in a humidified atmosphere of5% (v/v) CO2.

5.3.2. Sulforhodamine-B (SRB) microculture colorimetric assayExponentially growing cells were seeded into 96-well plates on

day 0 at the appropriate cell densities to prevent confluence of thecells during the period of the experiment. After 24 h, the cells weretreated with serial dilutions of the studied compounds to achievefinal concentrations in the range 0e1000 mM. The percentages ofsurviving cells relative to untreated controls were determined 96 hafter drug exposure. The supernatant medium from the 96-wellplates was discarded and the cells were fixed with 10% trichloro-acetic acid (TCA). For a thorough fixation plates were then allowedto stand at 4 �C. After fixation the cells were washed in a stripwasher four times with water using alternate dispensing andaspiration procedures. The plates were then dyed with 100 mL of0.4% SRB (Sigma-Aldrich) for about 45 min, washed again with 1%acetic acid to remove the dye and allowed to dry in air overnight.100 mL of 10 mM Tris base solution was added to each well of theplate, and absorbance was measured at 570 nm by using a 96-wellplate reader (Tecan Spectra, Crailsheim, Germany). The IC50 value,defined as the concentrations of the compound at which 50% cellinhibition was observed, was calculated from the dose-responsecurves.

5.3.3. Apoptosis tests

5.3.3.1. Trypan blue exclusion test. Apoptotic cell death wasanalyzed by trypan blue dye (SigmaeAldrich, Germany) on MCF-7cell line. The cell cultureflaskswith70e80%confluencewere treatedwith IC90 dose of the compounds for 24 h. The supernatant mediumwith floating cells was collected and centrifuged to collect the deadand apoptotic cells. The cell pellet was re-suspended in serum-freemedia. Equal amounts of cell suspension and trypan blue weremixed and analyzed under a microscope. The cells with preservedmembrane excluded the dye (colorless) while cells with destroyedmembrane were blue. If the proportion of colorless cells is higherthan the colored cells, the death can be characterized as apoptotic.

5.3.3.2. DNA fragmentation assay. MCF-7 cells were treated withthe respective IC90 dose of the compounds for 24 h. Floating cellsinduced by drug exposure were collected, washed with PBS and

lysed with lysis buffer (100 mM Tris-HCL, pH 8.0; 20 mM EDTA;0.8% sodium dodecyl sulfate (SDS); all from SigmaeAldrich). Thenthey were treated with RNAse A at 37 �C for 2 h and proteinase K at50 �C (both from Roche Diagnostics chemical company, Mannheim,Germany). DNA laddering was observed by running the samples on2% agarose gel followed by ethidium bromide (SigmaeAldrich)staining.

5.3.3.3. Caspases 2, 3, 8 and 9 enzyme activity assay. Activity ofcaspases 2, 3, 8, and 9 was measured by the caspase substratecleavage assay. After exposure of MCF-7 cells to equitoxic IC50concentrations of 1, 2, and cisplatin, cells were sampled after 2 and6 h. Adherent cells were washed with cold PBS, collected with a cellscraper and suspended in cell lysis buffer (50 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.4, 1% TritonX100, all from SigmaeAldrich). After incubation for 10 min on iceand centrifugation, protein concentrations of the supernatantswere measured according to the method of Bradford (Bio-RadLaboratories). Samples (50 mg protein extract respectively) wereincubated on a microplate at 37 �C overnight in reaction buffer(50 mM Hepes, pH 7.4, 0.1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA), 5% glycerol)containing 10 mM threo-1,4-dimercapto-2,3-butanediol (DTT) (allfrom SigmaeAldrich) and a specific substrate of caspases (2, Ac-VDVAD-pNA; 3, Ac-DEVD-pNA; 8, Ac-IETD-pNA; 9, Ac-LEHD-pNA,Axxora, Loerrach, Germany). Extinction of released p-nitroanilinewas measured at 405 nm (Tecan Spectra, Crailsheim, Germany) andactivity of caspases 2, 3, 8, and 9 was evaluated by optical density(OD) ratio of treated/untreated samples [53].

Acknowledgment

The authors thank Dr. T. Mueller for providing the cell lines,BioSolutionsHalle GmbH for cell culture facilities, andDr. P. Lönneckefor advice concerning the crystallographic data. M.S. thanks theStudienstiftungdesDeutschenVolkes for a Ph.D. grant. Thisworkwassupported by the Graduate School of Excellence “Building withMolecules and Nano-objects (BuildMoNa)”, funded by the DeutscheForschungsgemeinschaft. J.W. and W.S.S. thank the DeutscheForschungsgemeinschaft for financial support within the researchgroup FOR 630 ”Biological Function of Organometallic Compounds”.

Supplementary data

Supplementary data associated with this article can be found inthe online version at doi:10.1016/j.ejmech.2011.01.030.

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