On the Solubility and Lipophilicity of Metallacarborane

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On solubility and lipophilicity of metallacarborane pharmacophores Jakub Rak, Barbora Dejlova, Hana Lamplova, Robert Kaplánek, Pavel Matejicek, Petr Cigler, and Vladimir Kral Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300565z • Publication Date (Web): 18 Mar 2013 Downloaded from http://pubs.acs.org on April 1, 2013

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Molecular Pharmaceutics

On solubility and lipophilicity of metallacarborane pharmacophores Jakub Raka,b, Barbora Dejlováa, Hana Lampováa,b, Robert Kapláneka, Pavel Matějíčekc, Petr Cíglerd, *, and Vladimír Krála,b a) Department of Analytical Chemistry, Faculty of Chemical Engineering, Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic b) Zentiva R&D, part of Sanofi-Aventis, U Kabelovny 130, 102 37 Prague 10, Czech Republic c) Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague 2, Czech Republic d) Institute of Organic Chemistry and Biochemistry, AS CR, v. v. i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic

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TOC graphics

ABSTRACT

Metallacarborane moieties have been identified as promising pharmacophores. The pharmaceutical use of such compounds is, however, complicated by their low solubility and tendency to self-assemble in aqueous solution. In this work, we estimated the solubilities of a vast series of metallacarboranes [cobalt bis(dicarbollide) derivatives] in pure water, saline, and saline with human serum albumin as a model of blood plasma. In addition, we determined the octanol-water partition coefficients (Pow) as a lipophilicity descriptor. Pow weakly correlates with the water solubility of metallacarboranes, whereas the ability of HSA to increase the solubility of metallacarboranes correlates well with their Pow values. Because metallacarboranes are known inhibitors of HIV protease, the possible correlation between Pow and ability to inhibit HIV protease was investigated. Results from this study indicate that interaction of metallacarborane inhibitors with HIV protease is driven by specific binding rather than by promiscuous lipophilic interactions. The most promising candidates for further drug development were identified by ligand lipophilicity efficiency analysis.


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KEYWORDS metallacarborane, cobalt bis(dicarbollide), serum albumin, HIV protease, inhibitor, lipophilicity, Pow, solubility

1. Introduction The increasing number of pharmacological targets, along with the extensive synthetic efforts of medicinal chemists, has led to identification of numerous novel pharmacophores.1 Most of these are based on “traditional” organic frameworks; however, several inorganic and hybrid organic-inorganic structures have been identified as promising building blocks for drug design.2 One example is the use of boron clusters in medicine. In addition to their application as high boron-content agents for boron neutron capture therapy and boron neutron capture synovectomy3-6 or as radioimaging or magnetic resonance imaging probes,7 boron cluster moieties can serve as constituents of enzyme inhibitors. For example, boron-cluster-containing compounds have been shown to inhibit HIV-1 protease,8-12 cyclooxygenase,13,

14

serine

protease,15 and protein kinase C16, 17 (for review, see 18-22). Metallacarboranes, specifically cobalt bis(dicarbollide) derivatives substituted with exoskeletal organic groups, have been identified as promising HIV protease inhibitors.

8-12

In addition to

specifically inhibiting wild-type HIV protease, these substituted metallacarboranes efficiently inhibit drug-resistant protease variants. Metallacarborane HIV protease inhibitors show low toxicity in tissue culture and exhibit substantial chemical and biological stability.8, 9 Despite their structural similarity, these inhibitors show different mechanisms of HIV protease inhibition. However, it remains unclear which properties of metallacarboranes or assay conditions dictate the mechanism of inhibition.11


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Metallacarboranes are artificial structures with distinct physico-chemical properties, such as delocalization of cluster charge, high rigidity of the skeleton, lipophilicity,23-25 and the ability to form so-called dihydrogen bonds.26, 27 Dihydrogen bonds connect partially negatively charged hydrogen atoms bonded to electropositive boron atoms (i.e., hydrogen atoms in a B–H group with hydride-like character) with the partially positively charged hydrogen atoms in organic molecules (OH, NH, SH).28 The introduction of a cobalt bis(dicarbollide) moiety into a molecule most commonly results in the molecule’s sparing solubility in water and tendency to spontaneously self-assemble.12, 29-32 Both effects, combined with the impact of delocalized charge, result in limited permeation through biological membranes and unfavorable pharmacological properties. Surprisingly, systematic study focused on the physico-chemical properties of metallacarboranes lags behind the search for new structures and analysis of their potential use as pharmacophores. In our previous studies,33, 34 we showed that human serum albumin (HSA) and several biocompatible excipients can effectively increase the solubility of metallacarboranes and suppress their selfassembly (aggregation) via non-specific complexation. HSA is a well-known drug carrier that is able to bind negatively charged and (hetero)aromatic compounds and transfer them to target tissues. HSA can solubilize poorly soluble drugs in the circulatory system via its ligand-binding ability and can also delay the metabolic clearance of therapeutic agents. From this point of view, the interaction of metallacarboranes with HSA is expected to play a key role in the potential use of metallacarborane derivatives as drugs.35 Another issue to be addressed is the influence of salts on metallacarborane solubility and behavior in aqueous solution. For example, the spontaneous self-assembly of metallacarboranes (number and size of self-assemblies) is very sensitive to the presence of sodium chloride.12


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Generally, the presence of salts can lead to various salt-related effects. In the case of metallacarborane sodium salts, which contain the same ion present in saline (a 0.9% solution of NaCl), the common-ion effect (influence on solubility product, Ks) should play a major role. However, the solubility could also be affected by interactions of hydrophobic solute with ions (salting-in/salting-out) that depend on the concentration and nature of the salt added.34,

36-39

Partial formation of ion-pairs instead of dissolved separated ions is also a possibility.40 Metallacarboranes bearing an oligo(ethylene glycol) spacer can form complexes with sodium cations via their oxygen atoms,41,

42

further supporting the proposed tendency of an alkaline

cation to interact with a metallacarborane cluster (B-H⋅⋅⋅M+ interaction).43 These various effects could contribute to an overall salting-out effect, resulting in the decreased solubility of metallacarboranes.44, 45 A metallacarborane pharmacophore present in blood will be exposed to two antagonistic effects – the overall effect of salts vs. the effect of deaggregation and solubilization by serum albumin. In the present work, we examine the sensitivity of metallacarboranes to these rival effects and evaluate their pharmacological impact. Using a large series of metallacarborane conjugates, we evaluate the overall molecular charge (zwitterions vs. anions), the presence of salt and HSA in solution, and the octanol-water partition coefficient (Pow), a lipophilicity descriptor. The interplay of these factors can result in changes in solubility and behavior of metallacarboranes in aqueous solutions. Using ligand lipophilicity efficiency (LLE) analysis, we attempt to identify methods for structural optimization of metallacarborane HIV protease inhibitors as targets for further drug development. Our efforts also contribute to answering an important question: whether the mechanisms of inhibition of HIV protease and/or the inhibition


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activities of metallacarborane inhibitors are driven preferentially by specific protein recognition or by non-specific lipophilic interactions.

2. Experimental Section 2.1 Chemicals and synthesis n-Octanol (spectrophotometric grade, 99%), sodium chloride, human serum albumin (HSA, containing fatty acid), sodium chloride (p.a.), and all anhydrous solvents were purchased from Sigma-Aldrich (Prague, Czech Republic). Cesium cobalt bis(dicarbollide) (1) and 8-dioxane3,3'-cobalt bis(dicarbollide) (7) were purchased from KatChem Ltd. (Czech Republic). The detailed synthetic procedures and spectral characterization of compounds are described in Supporting information. These procedures employ a nucleophilic dioxane-ring opening reaction of 8-dioxane-3,3'-cobalt bis(dicarbollide) (7) with O-, N-, or S- nucleophiles in a suitable anhydrous solvent (1,2-dimethoxyethane, acetonitrile, acetone, or a THF-toluene mixture).8, 10, 33, 46-48

HIV-1 protease inhibitors (26-39) were kindly provided by the authors of ref.10

2.2 Octanol-water partition coefficient (Pow) Pow is defined as the ratio of the equilibrium concentrations of a dissolved substance in a twophase system consisting of octanol and water: Pow = co/cw, where co is the equilibrium concentration in octanol and cw is the equilibrium concentration in water Water and octanol are not completely immiscible; the solubility of water in octanol is 3.8 g/100 g and the solubility of octanol in water is 0.032 g/100 g at 25°C.49 To determine Pow values, several milligrams of each compound were dissolved in 0.5 mL octanol. Various amounts of this stock solution were added to mixtures consisting of 0.5 mL


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octanol and 10 mL water. At least four samples containing different amounts of each compound were prepared. The difference between the highest and the lowest amount was at least two orders of magnitude. The system was left to shake overnight at 25 °C to reach equilibrium. Both phases were then carefully separated by centrifugation (11000g, 10 min). Absorbance of the octanol phase, Ao, was directly determined by UV/Vis spectrophotometry on a Biochrom Libra S22 spectrophotometer (Biochrom Ltd.). The maximum absorbance was recorded at ë ≈ 300 nm. If necessary, the octanol phase was appropriately diluted with water-saturated octanol. The water phase of a known weight (mw) was evaporated in a vacuum oven at 50 °C. The dry compound originating from the water phase was dissolved in a known amount of water-saturated octanol (mo). The absorbance of this solution at ë ≈ 300 nm, Aow, was recorded. Then, the partition coefficient was calculated according to the equation Pow = (Aomwño)/(Aowmoñw), where ρo and ρw are the densities of water-saturated octanol and octanol-saturated water at 25 °C, respectively.50

2.3 Solubility determinations The solubilities of the compounds studied were determined by preparing saturated solutions of metallacarboranes in deionized water, saline (0.9% NaCl), and saline supplemented with human serum albumin (0.9% NaCl containing 50 g/L of HSA, a model simulating human blood plasma). The samples were prepared as follows: solid metallacarborane (approximately 2 mg) was dissolved by shaking and sonication in an ultrasonic bath for 24 h, dissolved substances were centrifuged (11000g, 10 min), and the metallacarborane solutions (after appropriate dilution if necessary) were measured by UV/Vis spectrophotometry. Extinction coefficients were determined for each solution separately. We observed that extinction coefficients per metallacarborane moiety of all derivatives bearing an oligo(ethylene


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glycol) linker (i.e., all derivatives except 1 and 7) display very similar values (maximum difference ± 3%). This observation is in agreement with expectations that a substituent connected through an oligo(ethylene glycol) spacer does not significantly influence the electronic density of a metallacarborane moiety.

2.4 Quasi-elastic light scattering (QELS) The light scattering setup (ALV, Langen, Germany) consisted of a 633 nm He-Ne laser, an ALV CGS/8F goniometer, an ALV High QE APD detector, and an ALV 5000/EPP multibit, multitau autocorrelator. DLS data analysis was performed by fitting the measured normalized intensity autocorrelation function g2(t) = 1 + â|g1(t)|2, where g1(t) is the electric field correlation function, t is the lag-time, and â is a factor accounting for deviation from the ideal correlation. An inverse Laplace transform of g1(t) with the aid of a constrained regularization algorithm (CONTIN) provides the distribution of relaxation times, ôA(ô). Effective angle- and concentration-dependent hydrodynamic radii, RH(q,c), were obtained from the mean values of relaxation times, ôm(q,c), of individual diffusive modes using the Stokes-Einstein equation.

3. Results and Discussion

The widely used reaction of the synthetic precursor dioxane-cobalt(III) bis(dicarbollide) zwitterion with nucleophiles [for review see

51

] gives rise to either negatively charged salts or

electroneutral, zwitterionic compounds. Similarly, introduction of two metallacarborane moieties into a molecule using this reaction enables formation of dianionic, zwitterionic-anionic, or biszwitterionic salts. For evaluation of solubility and lipophilicity trends, we synthesized a series


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of compounds containing the cobalt(III) bis(dicarbollide) pharmacophore modified with either neutral or cationic substituents. The diversity of compounds is shown in Fig. 1: 1-4 are simple alkaline metal salts, and 5-6 are double salts with two cobalt bis(dicarbollide) moieties and two alkaline cations. Compounds 7-25 behave as zwitterions with the negative charge located on the cobalt bis(dicarbollide) cluster and with the positive charge on the periphery (protonated amino group or quaternary ammonium, guanidinium, aminoguanidinium, or thiouronium group).

3.1 Octanol-water partition coefficient (Pow) In the mid-1970s, extraction of cations (especially 137Cs+) with metallacarboranes was used as a method for work-up of nuclear wastes (for review, see

52

). Later, Chevrot and Wipff et al.

performed several studies investigating the interfacial behavior of dicarbollides in various aqueous-organic systems.53-55 They used molecular dynamics simulations to observe freezeframe dissected kinetics of the transfer between the aqueous and organic phases and the processes adjacent to the interface layer. The authors see dicarbollide anion as an "ellipsoid," and they connect its surface activity to its adsorption at the interface and to its ability to extract cations.54 Adsorption of dicarbollides at the aqueous-organic interface has also been demonstrated experimentally.56 Compared with extraction using an excess of hydrophobic anion, the study performed here – partitioning of pure salts between aqueous and organic phases – is different: the molar ratios of cations to anions are always determined by the stoichiometry of a salt, i.e., they are fixed. Several lipophilicity descriptors can be used to characterize equilibria between two immiscible liquid phases. Among these, the most commonly used descriptor in medicinal chemistry is the octanol-water partition coefficient, Pow.57


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We determined Pow for all metallacarboranes studied (logarithmic values are shown in Table 1 for 1-25 and in Table 2 for 26-39). The presence of a metallacarborane moiety in a molecule results in relatively strong lipophilic behavior. The values of log Pow found in this study for compounds containing one metallacarborane moiety (1–25) lie in the following ranges: 2.1–2.2 for dianionic compounds, 2.1–2.7 for anionic compounds, and 2.5–3.9 for zwitterionic compounds. The compounds bearing two metallacarborane moieties (25–38) cover a wider range (1.5–3.9) and show similar trends (lower values for dianionic molecules, higher for monoanionic). Apparently, the overall charge of the molecule is the driving force for Pow. Predictions of Pow values based on the chemical nature of molecular substituents would not sort the molecules according to the experimental results. For instance, the acridinium (23) and phenazinium (24) derivatives behave like most polar compounds, even though they contain relatively lipophilic substituents. Similarly, ester derivatives (12) are typically less polar than hydroxyethyl derivatives (10), but their predicted log Pow values are reversed. The only previously available data on Pow values of metallacarborane derivatives were measured for nucleoside analogues of metallacarboranes.29 Previous work showed that Pow can be used for qualitative prediction of the aggregation behavior of this class of compounds in aqueous solutions. Metallacarboranes with log Pow > 0 tend to form aggregates in aqueous solutions, while no aggregation is observed at lower log Pow.29 The behavior of compounds 1–38 corresponds with these earlier findings (log Pow values in the 1.5–3.9 range). The difficulty of predicting Pow in carborane or metallacarborane compounds was established by in silico modeling studies.28 The dual solvation properties of octanol [its polar OH heads preferably solvate moderately hard ions (e. g., alkaline metals), while its alkyl chains solvate soft hydrophobic anions like metallacarboranes15] play a role in an individual compound’s affinity to


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the octanol phase. Based on theoretical modeling, the association of a hexachlorinated derivative of cobalt bis(dicarbollide) occurs in an octanol microphase present in octanol-saturated water.54 To examine the aggregation behavior of metallacarboranes we performed QELS measurements of compounds 1-25 in saline, water, octanol, and both water and octanol phases. We measured the light scattering intensity and, in cases where the intensity was high enough, we determined hydrodynamic radii, RH (data are summarized in Figure S1 in Supporting Information). In line with our previous observations,11 the self-assembling was most pronounced in pure water and saline and aggregates were somehow more monodisperse and “well-behaving” in salted solutions than those in pure water. The presence of octanol in water phase substantially suppressed the aggregation, but in some samples it was still detectable on the limit of the method. For the most of studied samples, we did not observe presence of aggregates in octanol phase and octanol (RH could not be therefore calculated and the corresponding columns in the graphs are missing). However, these data are not precluding the possible existence of self-assemblies smaller than the detection limit of QELS which were predicted by molecular dynamics calculations in the octanol phase.54 In general, RH of the aggregates extremely vary from 100 nm to 1000 nm, which is also typical for metallacarboranes.11

3.2 Solubility in water In general, salts (1-6) display relatively high solubility in pure water compared with zwitterions. Surprisingly, compounds with two lipophilic cobalt bis(dicarbollide) moieties (5 and 6) display similar solubility, probably due to their double salt character. In most cases, the solubility of zwitterions is roughly two orders of magnitude lower than that of salts. The aqueous solubility of metallacarboranes weakly correlates with log Pow (see Fig. 2).


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3.3 Solubility in saline All the substances studied show lower solubility in saline than in pure water. This could be explained by a combination of multiple salt effects such as the common-ion effect, salting-out, possible partial formation of ion-pairs, self-assembly, and complexation of alkaline cations by oligo(ethylene glycol) spacers. The solubility of the cesium salt of cobalt bis(dicarbollide) (1) is not significantly affected by the presence of sodium ions. This could be explained by the fact that the sodium salt of cobalt bis(dicarbollide) is several orders of magnitude more soluble than the cesium salt; thus, in practice, only the concentration of cesium cations plays a role. The small decrease in solubility could be caused by the salting-out effect of sodium cations. The solubility of metallacarboranes bearing an oligo(ethylene glycol) moiety with no further substitution (2 and 5) is relatively high; strikingly, the presence of sodium cations decreases solubility by up to two orders of magnitude. This unusually strong effect of salt on solubility is probably due to chelation of sodium cation by the penta(ethylene glycol) spacer and possible coordination with partially negatively charged hydrogens bonded to electropositive boron atoms (B-H ⋅⋅⋅ M+ interaction).43 The cationic charge of the metal ion could be compensated by the negative charge of the nearby cobalt bis(dicarbollide) anion.47 For example, the complexation of sodium cation by poly(ethylene glycol) in the presence of cobalt bis(dicarbollide) in aqueous solutions leads to formation of insoluble composite from otherwise water soluble compounds.41, 42

The di(ethylene glycol) spacers in 3, 4, and 6 do not contain enough oxygen atoms to

coordinate sodium cations. Correspondingly, the effect of sodium on 2 and 5 was not detectable. These results suggest an important consideration for future design of metallacarborane-based


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drugs: the potential interaction of sodium with an oligo(ethylene glycol) spacer should be carefully checked before further structural optimization. The solubility of formally non-charged zwitterionic compounds 7-25 is slightly decreased by the presence of saline, most likely due to the salting-out effect.

3.4 Solubility in saline with HSA HSA can bind a broad range of small lipophilic compounds in its hydrophobic cavities. The protein principally serves as a solubilizer and transporter for these compounds and. Our previous studies33,

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showed that HSA forms unspecific complexes with metallacarboranes, thus

increasing their water solubility. In the case of HSA in saline, two opposing effects – complexation and general salt-effect – occur. The solubility of a metallacarborane with a penta(ethylene glycol) spacer and no further substitution (2 and 5) greatly decreases in the presence of sodium ions due to chelation of sodium cations by the spacer (see Section 3.3). However, the addition of HSA to saline increases the solubility of 2 and 5. The solubilities of these derivatives in saline with HSA are comparable to their solubilities in pure water. The solubilities of zwitterions and salts (except 2 and 5) usually increase by one to two orders of magnitude in the presence of HSA (compared with their solubilities in saline or pure water). This relatively high increase is likely due to the high binding capacity of HSA and a relatively weak salting-out effect. The ratio of a metallacarborane’s solubility in saline with HSA to its solubility in water weakly correlates with its log Pow (Fig. 3). The correlation of this relative effect (strength of binding)


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with lipophilicity (log Pow) is in agreement with the fact that HSA binds small lipophilic compounds due to hydrophobic interactions.

3.5 Lipophilicity and inhibition of HIV protease The ligand lipophilicity is an important parameter to consider in drug discovery projects. It helps in assessment of toxicity, promiscuity, solubility, target affinity, in vivo distribution, intestinal absorption, plasma protein binding, and metabolism.58 Lipophilicity is a complex physical property dependent on molecular size, polarity, and hydrogen bonding characteristics.59 For evaluation of the optimization process in drug discovery, ligand lipophilicity efficiency, or LLE, (LLE = –log IC50 – log Pow) has been introduced as a descriptor of drug potency per unit of lipophilicity. Increasing LLE values in a series of similar derivatives can indicate a pathway for further structural optimization.60 Inhibition of HIV protease (IC50 and mechanism of action) by various metallacarboranes was recently described.10,

11

We determined the LLE values for these inhibitors, and an LLE

correlation graph is shown in Fig. 4. An optimized value of LLE > 5 (i.e., IC50 < 10 nM and log Pow < 3), has been proposed.61 An LLE value > 5 often correlates with low toxicity and high bioavailability. The present study marks the first time that lipophilicity optimization of metallacarboranes has been taken into account, and we show that even the most effective known metallacarborane inhibitors of HIV protease have LLE values far from the optimum. Although eight compounds from the series studied fulfill the criterion log Pow < 3, none of them shows sufficient inhibitory efficacy. Nevertheless, we observed a structural trend leading to higher LLE values in this series of metallacarboranes: a higher negative charge positively influences the


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LLE. Based on the LLE analysis, the inhibitors with no positively charged group (i. e., amino group) can be considered the most promising structures for future drug development. One might also expect that lipophilicity plays a role in the binding of inhibitor to the hydrophobic cavity of the target protein. However, we did not find any correlation between the Pow values of derivatives 26-39 and either IC50 or mechanism of action (see Table 2). Our findings support the fact that inhibition of HIV protease by metallacarboranes in either a competitive or non-competitive mode is driven more by specific interactions than by the promiscuous lipophilic behavior of metallacarboranes.

4. Conclusions In this work, we analyzed the solubilities of metallacarboranes in water, saline (0.9% NaCl), and saline with HSA (a model of blood plasma) and calculated the corresponding Pow values as a descriptor of lipophilicity. We found that, in general, the solubility of metallacarboranes in water weakly correlates with their Pow values. Some experimental values deviated from this trend, but these deviations could be accounted for by the structural peculiarities of individual molecules. For example, complexation of sodium cations by peripheral oligo(ethylene glycol) units and consequent charge neutralization likely plays a significant role in solubility and aggregation of particles and we recommend to focus in future on this phenomenon. We also identified a correlation between Pow and the effect of HSA on metallacarborane solubility. This indicates that the interaction of metallacarboranes with HSA is driven mainly by their lipophilic character. In contrast, we did not observe any relationship between Pow and inhibition of HIV protease, which, like HSA, contains a hydrophobic cavity. The inhibition of HIV protease is most likely driven by specific interactions and not by the general lipophilic behavior of metallacarboranes. From the


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available series of known HIV protease inhibitors, we identified several promising candidates for further development by LLE analysis. These molecules can serve as platforms for structural development of pharmacologically active HIV protease inhibitors based on metallacarborane clusters.

AUTHOR INFORMATION Corresponding Author * Petr Cígler, Institute of Organic Chemistry and Biochemistry, AS CR, v. v. i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic; email: [email protected]; tel.: +420-220-183-429.

ACKNOWLEDGMENT The authors thank Dr. Bohumír Grüner and Dr. Václav Šícha (IIC AS CR, v.v.i., Rez near Prague, Czech Republic) for kind provision of HIV protease inhibitors, Dr. Jan Konvalinka and Dr. Martin Lepšík (IOCB AS CR, v.v.i., Prague, Czech Republic) for valuable discussions, and Dr. Hillary Hoffman for for critical proofreading of the manuscript. This work was supported by the Ministry of Education, Youth, and Sports of the Czech Republic (projects MSM0021620857 and LH11027); the Grant Agency of the Czech Republic (GAP303/11/1291); the Technology Agency of the Czech Republic (project TE01020028); the Grant Agency of the Academy of Sciences of the Czech Republic (project IAAX00320901); and Biomedreg project (CZ.1.05/2.1.00/01.0030).

ABBREVIATIONS


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HIV, human immunodeficiency virus; HSA, human serum albumin; IC50, half maximal inhibitory concentration; LLE, ligand lipophilicity efficiency; Pow, octanol-water partition coefficient; QELS, quasi-elastic light scattering.

SUPPORTING INFORMATION AVAILABLE Chemical synthesis and structural characterization of compounds, additional results from light scattering study. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F. G.; Barth, R. F.; Codogni, I. M.;

Wilson, J. G., The chemistry of neutron capture therapy. Chem. Rev. 1998, 98 (4), 1515-1562. 7.

Hawthorne, M. F.; Maderna, A., Applications of radiolabeled boron clusters to the

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Cigler, P.; Kozisek, M.; Rezacova, P.; Brynda, J.; Otwinowski, Z.; Pokorna, J.; Plesek, J.;

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11. Řezáčová, P.; Cígler, P.; Matějíček, P.; Lepšík, M.; Pokorná, J.; Grüner, B.; Konvalinka, J., Medicinal Application of Carboranes: Inhibition of HIV Protease. In Boron Science: New Technologies and Applications, Hosmane, N. S., Ed. CRC Press: New York, 2011; pp 41-70. 12. Matejicek, P.; Cigler, P.; Prochazka, K.; Kral, V., Molecular assembly of metallacarboranes in water: Light scattering and microscopy study. Langmuir 2006, 22 (2), 575581. 13. Scholz, M.; Bensdorf, K.; Gust, R.; Hey-Hawkins, E., Asborin: The Carbaborane Analogue of Aspirin. ChemMedChem 2009, 4 (5), 746-748. 14. Scholz, M.; Steinhagen, M.; Heiker, J. T.; Beck-Sickinger, A. G.; Hey-Hawkins, E., Asborin Inhibits Aldo/Keto Reductase 1A1. ChemMedChem 2011, 6 (1), 89-93. 15. Page, M. F. Z.; Jalisatgi, S. S.; Maderna, A.; Hawthorne, M. F., Design and synthesis of a candidate alpha-human thrombin irreversible inhibitor containing a hydrophobic carborane pharmacophore. Synthesis 2008, (4), 555-563. 16. Endo, Y.; Yoshimi, T.; Kimura, K.; Itai, A., Protein kinase C modulators bearing dicarbacloso-dodecaborane as a hydrophobic pharmacophore. Bioorg. Med. Chem. Lett. 1999, 9 (17), 2561-2564. 17. Tsuji, M.; Koiso, Y.; Takahashi, H.; Hashimoto, Y.; Endo, Y., Modulators of tumor necrosis factor alpha production bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore. Biol. Pharm. Bull. 2000, 23 (4), 513-516. 18. Lesnikowski, Z. J., Boron units as pharmacophores - New applications and opportunities of boron cluster chemistry. Collect. Czech. Chem. Commun. 2007, 72 (12), 1646-1658.


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19. Satapathy, R.; Dash, B. P.; Maguire, J. A.; Hosmane, N. S., New Developments in the Medicinal Chemistry of Carboranes. Collect. Czech. Chem. Commun. 2010, 75 (9), 995-1022. 20. Issa, F.; Kassiou, M.; Rendina, L. M., Boron in Drug Discovery: Carboranes as Unique Pharmacophores in Biologically Active Compounds. Chem. Rev. 2011, 111 (9), 5701-5722. 21. Scholz, M.; Hey-Hawkins, E., Carbaboranes as Pharmacophores: Properties, Synthesis, and Application Strategies. Chem. Rev. 2011, 111 (11), 7035-7062. 22. Grimes, R. N., Carboranes in medicine. In Carboranes, Academic Press: 2011; pp 10531082. 23. Sivaev, I. B.; Bregadze, V. V., Polyhedral Boranes for Medical Applications: Current Status and Perspectives. Eur. J. Inorg. Chem. 2009, (11), 1433-1450. 24. Chen, Z. F.; King, R. B., Spherical aromaticity: Recent work on fullerenes, polyhedral boranes, and related structures. Chem. Rev. 2005, 105 (10), 3613-3642. 25. King, R. B., Three-dimensional aromaticity in polyhedral boranes and related molecules. Chem. Rev. 2001, 101 (5), 1119-1152. 26. Fanfrlik, J.; Lepsik, M.; Horinek, D.; Havlas, Z.; Hobza, P., Interaction of carboranes with biomolecules: Formation of dihydrogen bonds. Chemphyschem 2006, 7 (5), 1100-1105. 27. Fanfrlik, J.; Hnyk, D.; Lepsik, M.; Hobza, P., Interaction of heteroboranes with biomolecules - Part 2. The effect of various metal vertices and exo-substitutions. Phys. Chem. Chem. Phys. 2007, 9 (17), 2085-2093.


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28. Farras, P.; Juarez-Perez, E. J.; Lepsik, M.; Luque, R.; Nunez, R.; Teixidor, F., Metallacarboranes and their interactions: theoretical insights and their applicability. Chem. Soc. Rev. 2012, 41 (9), 3445-3463. 29. Matejicek, P.; Cigler, P.; Olejniczak, A. B.; Andrysiak, A.; Wojtczak, B.; Prochazka, K.; Lesnikowski, Z. J., Aggregation behavior of nucleoside-boron cluster conjugates in aqueous solutions. Langmuir 2008, 24 (6), 2625-2630. 30. Kubat, P.; Lang, K.; Cigler, P.; Kozisek, M.; Matejicek, P.; Janda, P.; Zelinger, Z.; Prochazka, K.; Kral, V., Tetraphenylporphyrin-cobalt(III) bis(1,2-dicarbollide) conjugates: From the solution characteristics to inhibition of HIV protease. J. Phys. Chem. B 2007, 111 (17), 45394546. 31. Hao, E.; Sibrian-Vazquez, M.; Serem, W.; Garno, J. C.; Fronczek, F. R.; Vicente, M. G. H., Synthesis, aggregation and cellular investigations of porphyrin-cobaltacarborane conjugates. Chem. Eur. J. 2007, 13 (32), 9035-9042. 32. Bauduin, P.; Prevost, S.; Farras, P.; Teixidor, F.; Diat, O.; Zemb, T., A Theta-Shaped Amphiphilic Cobaltabisdicarbollide Anion: Transition From Monolayer Vesicles to Micelles. Angew. Chem. Int. Edit. 2011, 50 (23), 5298-5300. 33. Rak, J.; Kaplanek, R.; Kral, V., Solubilization and deaggregation of cobalt bis(dicarbollide) derivatives in water by biocompatible excipients. Bioorg. Med. Chem. Lett. 2010, 20 (3), 1045-1048.


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34. Rak, J.; Jakubek, M.; Kaplanek, R.; Matejicek, P.; Kral, V., Cobalt bis(dicarbollide) derivatives: Solubilization and self-assembly suppression. Eur. J. Med. Chem. 2011, 46 (4), 1140-1146. 35. Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P., The extraordinary ligand binding properties of human serum albumin. Iubmb Life 2005, 57 (12), 787-796. 36. Kalra, A.; Tugcu, N.; Cramer, S. M.; Garde, S., Salting-in and salting-out of hydrophobic solutes in aqueous salt solutions. J. Phys. Chem. B 2001, 105 (27), 6380-6386. 37. Lee, T. B.; Mckee, M. L., Dissolution Thermochemistry of Alkali Metal Dianion Salts (M2X1, M = Li+, Na+, and K+ with X = CO32-, SO42-, C8H82-, and B12H122-). Inorg. Chem. 2011, 50 (22), 11412-11422. 38. Bockris, J. O.; Bowlerreed, J.; Kitchener, J. A., The Salting-in Effect. Trans. Faraday Soc. 1951, 47 (2), 184-192. 39. Tasleem, S.; Durani, S.; Anwar, M.; Niaz, M.; Muzaffar, K., Salting-out coefficients and activity coefficients of alkali and alkaline earth metals in aqueous and aqueous organic mixed solvents. J. Chem. Soc. Pak. 2004, 26 (1), 39-43. 40. Ivanov, I. M.; Volkov, V. V.; Kalish, N. K., Solvent-Extraction and Solvatation of Alkali Cations in Form of Salts Cobalt(Iii) Dicarbollide Complexes. Sibirskii Khim. Zh. 1991, (4), 7881.


22

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41. Matejicek, P.; Zednik, J.; Uselova, K.; Plestil, J.; Fanfrlik, J.; Nykanen, A.; Ruokolainen, J.; Hobza, P.; Prochazka, K., Stimuli-Responsive Nanoparticles Based on Interaction of Metallacarborane with Poly(ethylene oxide). Macromolecules 2009, 42 (13), 4829-4837. 42. Matejicek, P.; Brus, J.; Jigounov, A.; Plestil, J.; Uchman, M.; Prochazka, K.; Gradzielski, M., On the Structure of Polymeric Composite of Metallacarborane with Poly(ethylene oxide). Macromolecules 2011, 44 (10), 3847-3855. 43. Llop, J.; Masalles, C.; Vinas, C.; Teixidor, F.; Sillanpaa, R.; Kivekas, R., The [3,3 'Co(1,2-C2B9H11)(2)](-) anion as a platform for new materials: synthesis of its functionalized monosubstituted derivatives incorporating synthons for conducting organic polymers. Dalton Trans. 2003, (4), 556-561. 44. Uchman, M.; Cigler, P.; Gruner, B.; Prochazka, K.; Matejicek, P., Micelle-like nanoparticles

of

block

copolymer

poly(ethylene

oxide)-block-poly(methacrylic

acid)

incorporating fluorescently substituted metallacarboranes designed as HIV protease inhibitor interaction probes. J. Colloid Interf. Sci. 2010, 348 (1), 129-136. 45. Uchman, M.; Jurkiewicz, P.; Cigler, P.; Gruner, B.; Hof, M.; Prochazka, K.; Matejicek, P., Interaction of Fluorescently Substituted Metallacarboranes with Cyclodextrins and Phospholipid Bilayers: Fluorescence and Light Scattering Study. Langmuir 2010, 26 (9), 62686275. 46. Gruner, B.; Plesek, J.; Baca, J.; Cisarova, I.; Dozol, J. F.; Rouquette, H.; Vinas, C.; Selucky, P.; Rais, J., Cobalt bis(dicarbollide) ions with covalently bonded CMPO groups as selective extraction agents for lanthanide and actinide cations from highly acidic nuclear waste solutions. New J. Chem. 2002, 26 (10), 1519-1527.


23


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Page 24 of 34

47. Plesek, J.; Gruner, B.; Hermanek, S.; Baca, J.; Marecek, V.; Janchenova, J.; Lhotsky, A.; Holub, K.; Selucky, P.; Rais, J.; Cisarova, I.; Caslavsky, J., Synthesis of functionalized cobaltacarboranes based on the closo-[(1,2-C2B9H11)(2)-3,3 '-Co](-) ion bearing polydentate ligands for separation of M3+ cations from nuclear waste solutions. Electrochemical and liquidliquid extraction study of selective transfer of M3+ metal cations to an organic phase. Molecular structure of the closo-[(8-(2-CH3O-C5H4-O)-(CH2CH2O)(2)-1,2-C2B9H10)-(1 ',2 '-C2B9H11)3,3 '-Co]Na determined by X-ray diffraction analysis. Polyhedron 2002, 21 (9-10), 975-986. 48. Sivaev, I. B.; Starikova, Z. A.; Sjoberg, S.; Bregadze, V. I., Synthesis of functional derivatives of the [3,3 '-Co(1,2-C2B2H11)(2)](-) anion. J. Organomet. Chem. 2002, 649 (1-2), 18. 49. Smallwood, I. M., Handbook of organic solvent properties. John Wiley & Sons Inc.: New York, 1996; pp 101-103. 50. Dissociation and association of the dissolved compounds could result in deviations from the partition law, especially in the case of partially dissociated compounds. Such deviation would be indicated by the fact that the Pow value becomes dependent upon the concentration. The potential surface activity of the compounds would also be indicated by the Pow value dependence upon the concentration. In accordance with OECD guidelines, in which Pow is defined as a descriptor for non-charged compounds without surface activity, our experimental values should be denoted as apparent Pow (Powapp); however, for simplicity, we will use the abbreviation Pow throughout the manuscript. In any case, we did not observe any concentration-dependence of Pow in this study.


24

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51. Semioshkin, A. A.; Sivaev, I. B.; Bregadze, V. I., Cyclic oxonium derivatives of polyhedral boron hydrides and their synthetic applications. Dalton Trans. 2008, (8), 977-992. 52. Grüner, B.; Rais, J.; Selucký, P.; Lučaníková, M., Recent Progress in Extraction Agents Based on Cobalt Bis(Dicarbollides) for Partitioning of Radionuclides from High-Level Nuclear Waste. In Boron Science: New Technologies and Applications, Hosmane, N. S., Ed. CRC Press: New York, 2011; pp 463-490. 53. Chevrot, G.; Schurhammer, R.; Wipff, G., Surfactant behavior of "ellipsoidal" dicarbollide anions: A molecular dynamics study. J. Phys. Chem. B 2006, 110 (19), 9488-9498. 54. Chevrot, G.; Schurhammer, R.; Wipff, G., Synergistic effect of dicarbollide anions in liquid-liquid extraction: a molecular dynamics study at the octanol-water interface. Phys. Chem. Chem. Phys. 2007, 9 (16), 1991-2003. 55. Chevrot, G.; Schurhammer, R.; Wipff, G., Molecular dynamics study of dicarbollide anions in nitrobenzene solution and at its aqueous interface. Synergistic effect in the Eu(III) assisted extraction. Phys. Chem. Chem. Phys. 2007, 9 (44), 5928-5938. 56. Popov, A.; Borisova, T., Adsorption of dicarbollylcobaltate(III) anion {(pi-(3)-1,2B9C2H11)(2)Co(III)(-)} at the water/1,2-dichloroethane interface. Influence of counterions' nature. J. Colloid Interf. Sci. 2001, 236 (1), 20-27. 57. Zhao, Y. H.; Abraham, M. H., Octanol/water partition of ionic species, including 544 cations. J. Org. Chem. 2005, 70 (7), 2633-2640. 58. Waring, M. J., Lipophilicity in drug discovery. Expert Opin. Drug Dis. 2010, 5 (3), 235248.


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59. Oliferenko, P. V.; Oliferenko, A. A.; Poda, G.; Palyulin, V. A.; Zefirov, N. S.; Katritzky, A. R., New Developments in Hydrogen Bonding Acidity and Basicity of Small Organic Molecules for the Prediction of Physical and ADMET Properties. Part 2. The Universal Solvation Equation. J. Chem. Inf. Model. 2009, 49 (3), 634-646. 60. Edwards, M. P.; Price, D. A., Role of Physicochemical Properties and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks. Annu. Rep. Med. Chem. 2010, 45, 381-391. 61. Leeson, P. D.; Springthorpe, B., The influence of drug-like concepts on decision-making in medicinal chemistry. Nat. Rev. Drug Discov. 2007, 6 (11), 881-890.


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TABLES Table 1. Solubilities of metallacarboranes (µmol/L) in water, saline, and saline with HSA and their corresponding octanol-water partition coefficients (expressed as log Pow values). The solubilities are highlighted in grey tones (darker shade indicates higher solubility). Compounds are divided into groups based on their charge and structural similarities. Type a Salt

Double salt Zwitterion

a

Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Water 1500 2300 3200 990 220 1500 72 14 32.6 150 46 1800 5.3 5.8 67 14 2.6 25 190 40 25 2700 4800 300 6.0

Saline 1400 27 1800 490 11 700 71 12 14 47 27 1400 3.7 3.3 29 5.1 0.1 6.9 42 4.7 5.3 1400 1200 46 0.9

Saline+HSA 19000 1400 17000 15000 970 14000 690 250 1600 5900 3500 16000 370 110 4400 1200 240 460 1600 1500 380 25000 21000 5700 28

log Pow 2.5 2.1 2.4 2.7 2.2 2.1 3.2 3.8 3.8 3.1 3.7 2.7 3.6 3.3 3.5 3.4 3.9 3.0 3.3 3.7 3.3 2.9 2.6 2.5 3.1

Type of compound – salt (Na+, K+), double charged salt (2Na+, 2K+), and zwitterions


27


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Page 28 of 34

Table 2. Octanol-water partition coefficient (expressed as log Pow values), IC50 (µM), and mechanism of HIV protease inhibition of compounds 26 - 39. Compound

log Pow

IC50 (µM)

Mechanism (ref 8, 10, 11)

26 27 28 29 30 31 32 33 34 35 36 37 38 39

3.1 3.8 2.7 3.9 1.7 3.5 2.9 1.5 1.6 2.8 2.8 2.6 3.0 1.7

140 100 140 130 190 140 110 110 58 50 70 160 250 8500

competitive competitive competitive competitive non-competitive concentration-dependent competitive non-competitive concentration-dependent non-competitive concentration-dependent concentration-dependent competitive not determined


28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURES Figure captions: Figure 1. Structures of a) metallacarboranes (1 – 25) and b) metallacarborane-based HIV 1 protease inhibitors (26 – 39) used in this study. Figure 2. Solubility of metallacarboranes 1-25 in water as a function of log Pow (R² = 0.632). Figure 3. Relative ratio of solubility of metallacarboranes 1-25 in saline with HSA to solubility in water as a function of log Pow (R² = 0.572). The data for compound 2 were excluded from the graph, because of additional effect of oligo(ethyleneglycol) arm on the solubility. Figure 4. Ligand lipophilicity efficiency (LLE) for a series of metallacarborane-containing HIV protease inhibitors (for structures corresponding to the numbers, see Fig. 1). The solid lines indicate regions of constant LLE value. The arrow shows the direction of LLE optimization leading leading toward the optimum LLE value of > 5.


29


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Page 30 of 34

OH

Zwitterions:

CB O

7

O

CB O = CB

CB O

O

O

18 N S

8

NH2

Co NH2 CB O

= BH or B (if substituted) = CH

CB O

O

O

9

CB O

NH2

10

OH

NH2

CB O

Salts:

CB H Cs O

CB O HO

1

NH

19

NH2

NH

NH2

O

NH2

OH

CB O

O

NH

NH

NH

2

O Na

CB O

O

20

NH2

NH2

O O

O

O

NH

NH2

11 CB O

O

12

O

NH2

NH2 CB O

O

21

O

S

22

NH2

OH CB O

O

O

3

K

CB O

O

13

NH

CB O

O

O

CB O O

K

CB O O

O

O

N

14

N

OH OH 15

23

4

Double salts: CB O

N N

O CB O

O

O

O

CB O

O

N

24

OH 2 Na

O CB O

O

O

O

F

5

CB O

O

NH

N

O CB O

O

O

O 2K

6 CB O

O

O

CB O

O

F F

N

16

N O

CB O

O

25 N

17

N S

Figure 1a.


30

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HIV-1 protease inhibitors: 2 O

O

NH2

Co

O

O Co

Na

O

O

26

O

O

NH

O

NH

O

O Co

Na

Co

O

O

NH

O

27

O

O

NH

O

2 Na

Co

Na

Co

Na

O

34

O

Co

Co

Na

O

O

NH

O

O

Co

28

OH

Co

33

SO3

Co

O

Co

35 2

O

O

NH

O

O

Co

Co

Na

Co

O

O

N O S O

O

O

NH

O

O

Co

Co

HO

Co

2 Na

Co

Na

36

29

O

O

Na

O

O

N

O

O

Co

37

OH OH

30 2

O

O

NH

O

O

Co

Co

Na

O

O

N

O

Co

Co

O

O

NH

O

O Co

Na

O

O

NH

2 Na

38

31

Co

O

Co

O

O

K

39

32 OOC

Figure 1b.


31


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Page 32 of 34

Figure 2.


32

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.


33


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Page 34 of 34

Figure 4.


34

On the Solubility and Lipophilicity of Metallacarborane

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