Challenges and Opportunities for the Application of Boron Clusters in

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Challenges and opportunities for the application of boron clusters in drug design Zbigniew Jan Lesnikowski J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01932 • Publication Date (Web): 28 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Challenges and opportunities for the application of boron clusters in drug design

Zbigniew J. Leśnikowski*

Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, 106 Lodowa St., Lodz 93-232, Poland.

KEYWORDS Boron clusters, carboranes, metallacarboranes, drug design.

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ABSTRACT There are two branches in boron medicinal chemistry: the first focuses on single boron atom compounds, and the second utilizes boron clusters. Boron clusters and their heteroatom counterparts belong to the family of cage compounds. A subset of this extensive class of compounds includes dicarbadodecaboranes, which have the general formula C2B10H12, and their metal biscarboranyl complexes, metallacarboranes, with the formula [M(C2B10H12)2-2]. The unique properties of boron clusters have resulted in their utilization in applications such as in pharmacophores, as scaffolds in molecular construction and as modulators of bioactive compounds. This perspective presents an overview of the properties of boron clusters that are pertinent for drug discovery, recent applications in the design of various classes of drugs and the potential use of boron clusters in the construction of new pharmaceuticals.

INTRODUCTION Boron has a wide range of applications in chemistry, materials science, energy research, electronics, and in the life sciences. The chemistry of boron has many facets. One of the reasons for this is its location in the periodic table of elements and, consequently, its electronic structure. Boron lies next to carbon, which causes boron to share some similarities with carbon but also possesses important differences. It is the combination of these similarities and differences that give boron its unique potential in medicinal applications. In contrast to single boron atom compounds, boron atoms in boron clusters act in concert to create new qualities such as threedimensional aromaticity, hydrophobicity, and the formation of non-classical proton-hydride bonds. The following sections include a discussion on some of the properties of boron clusters

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that are pertinent to their application in the design of bioactive molecules and recent developments in the use of boron clusters in medicinal chemistry.

ELEMENTS IN DRUGS Most clinically used drugs are pure organic compounds. Traditional chemotherapeutic agents comprise less than 10 elements and usually consist of carbon, hydrogen, nitrogen and oxygen; occasionally, these agents contain sulfur and phosphorus and are sometimes supplemented with chlorine and fluorine. Interestingly the sextet C, H, N, O, S, P represents the elements that are most abundant in the human body. Clearly, they play important roles in physiology; therefore, it is not surprising that most pharmaceutical drugs are composed of this small set of elements.1 Nevertheless, there are several exceptions in which one can find unusual components, which often include metals or semi-metals.2 As examples of clinically used drugs that contain atypical elements, one can mention Polaprezinc (contains zinc), which is used for the treatment of peptic ulcers, Auranofin (gold complex), which is used as an antirheumatic agent, Oxaliplatin, which is a platinum-based drug that is used as an antineoplastic agent in anticancer therapy, Melarsoprol (contains arsenium), which is a prodrug used for the treatment of trypanosome-based diseases or Bortezomib (contains boron), which is used in the anticancer treatment of multiple myeloma (Figure 1). Several other agents containing single boron atoms are currently in different phases of clinical trials.3-10 Boron is an element that is generally not observed in living bodies; however, it possesses considerable potential for the facilitation of new biological activity and for use in pharmaceutical drug design. Following advances in boron-based drugs, this review focuses on boron in the form of boron clusters as a component of bioactive molecules.

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Figure 1. Examples of single boron atom-containing drugs that are clinically used (1,2) or are in clinical trials (3,4): bortezomib (Velcade®), anticancer, proteasome inhibitor (1) (Bioorg. Med. Chem. Lett. 1998, 8, 333–338); tavaborole (Kerydin™), antifungal, aminoacyl-tRNA synthetase inhibitor (2) (Science 2007, 316, 1759–1761); crisaborole, topical anti-inflammatory, nonsteroidal PDE-4 inhibitor (3) (Cell. Signal., doi:10.1016/j.cellsig.2015.08.003); SCYX-7158, anti-African trypanosomiasis agent (4) (Parasitology 2014, 141, 104–118).

This account is not meant to be comprehensive but is rather centered on new developments and ideas. It focuses on studies conducted over the last decade, although not all of them could be included due to space limitations. Readers interested in previous investigations on the medicinal chemistry of boron clusters can refer to one of the several excellent reviews and books on the subject.11-19 The large number of studies on the medicinal chemistry of single boron atom drugs are beyond the designed scope of this perspective and have thus been omitted.

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BORON IN MEDICINE – A HISTORICAL OUTLINE The use of boron-containing compounds as medication has a long history.20-22 It can be divided into three chronological stages. The first is based on the use of inorganic, naturally occurring boric acid compounds, such as borax and borax-containing minerals. Its use began in the darkness of the past though some preparations are still used in modern wrapping techniques as mild antiseptic, antibacterial and antifungal treatment methods. The second stage took advantage of the development of boron-based organic chemistry, which started in the beginning of the 20 century. After several decades of studies numerous bioactive molecules and molecular tools containing single boron atoms have been developed.3-6 Several such boron compounds are in different stages of clinical studies; one of these compounds, bortezomib,7 was approved as an anticancer drug in 2003, and tavaborole was approved as a topical antifungal agent in 2014 (Figure 1).8 Polyhedral boron compounds have added a new dimension to the medicinal chemistry of boron-based drugs, facilitating the present quest for biologically active molecules containing boron clusters rather than those containing one boron atom per molecule as observed in derivatives of boric or boronic acid-based molecules.

BORON CLUSTERS The discovery of polyhedral boranes23,24 and the subsequent syntheses of carboranes25,26 created a foundation for a new and exciting field of medicinal chemistry that takes advantage of the properties of boron cage compounds that contain not one but several boron atoms. This emerging field of drug discovery is the topic of this overview. Several experts have already summarized the chemistry24,27-29 and properties of boron clusters that are relevant for the syntheses of

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bioactive molecules;13,16-18,30,31 therefore, only subjects pertinent to the focus of this perspective will be discussed.

Geometry Polyhedral boranes constitute a family of cage compounds with the general formula BnHn2-. Not all members of this family are stable under standard conditions, and stability is limited to boron hydride anions where n = 6–12. The molecular geometries of these clusters are deltahedra (polyhedra with only triangular faces), that are designated by the prefix closo (“clusus”, from Latin for “closed”), or deltahedra, that have one missing vertex and are labeled nido (“nidus”, from Latin for nest). Analogous families of carboranes exist in which one or more BH units is replaced by isoelectronic CH+ units.28 Carboranes have the extraordinary ability to accommodate nonmetals, giving rise to heteroboranes, and metals, giving rise to an entire branch of coordination compounds called metallacarboranes (Figure 2). Among heteroboranes with twelve vertices, neutral dicarba-closo-dodecaborane (C2B10H12) exists in the form of three isomers with carbon atoms in ortho-, meta-, or para-positions in the cage (5, 6 and 7); the negatively charged dodecaborate (2-) ion (B12H122-) (10) and 3-cobalt-bis(1,2-dicarbollide)ate, [Co(C2B9H11)22-] (12), are the most commonly used boron cluster compounds in medicinal chemistry. This list is complemented with neutral, ten-vertex carboranes, such as dicarba-closo-decaborane (C2B8H10)32 (8), negatively charged ionic 7,8-dicarba-nido-undecaborate (1-) (C2B9H111-) (9), an open cage form of 5 that forms due to its closo- to nido- transformation, and ionic carba-closo-dodecaborate (1-) (CB11H121-)33,34 (11). Studies on the bioorganic chemistry of compounds 8 and 11 are still in their infancy.

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Figure 2. Examples of boron clusters and metallacarboranes (boron cluster-based coordination compound) used in medicinal chemistry: 1,2-dicarba-closo-dodecaborane (5), ortho-isomer; 1,7dicarba-closo-dodecaborane (6), meta-isomer; 1,12-dicarba-closo-dodecaborane (7), para-isomer (C2B10H12); dicarba-closo-decaborane (C2B8H10) (8); 7,8-dicarba-nido-undecaborate (1-) ion (C2B9H111-) (9); closo-dodecaborate ion (B12H122-) (10); carba-closo-dodecaborate ion (CB11H121) (11) and 3-cobalt-bis(1,2-dicarbollide)ate (12).

The electroneutral dicarba-closo-dodecaboranes (5-7) are rigid, slightly distorted icosahedral clusters with 20 sides, 12 vertices, have nearly spherical shapes and are derived from the B12H122dianion (10). The space occupied by dicarbadodecaborane is approximately 50% larger than that of a rotating phenyl group (van der Waals volumes of 141-148 Å3 vs 102 Å3, respectively16). The dodecaborate (10) dianion, more specifically its B12 core, is a regular icosahedron and has the highest molecular symmetry of all the known boron clusters.29 In terms of geometry, the

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globular-shaped clusters of 5-11 are complemented with ellipsoidal-shaped metallacarboranes, such as 3-cobalt-bis(1,2-dicarbollide)ate (12) (Figure 2) and a range of double-cage boranes.18

Figure 3. Schematic representation of the directions for 1,2-dicarba-closo-dodecaborane (5) core functionalization.

Icosahedral clusters such as dicarba-closo-dodecaboranes (5-7) and dodecaborate (10) theoretically allow the attachment of several different substituents at the carbon and boron atoms in strictly defined ways, allowing the 3D spatial organization of the ligands. This, in turn, provides unique potential for the construction of molecules that are best adapted to their biological targets. 1,2-Dicarba-closo-dodecaborane (5) allows the attachment of two substituents at the carbon atoms at angles of approximately 60º, 1,7-dicarba-closo-dodecaborane (6) allows the attachment of these substituents at approximately 120º and para-carbaborane (7) allows the same attachment at an angle of 180º. Attachment of substituents to boron atoms offers a similar amount of diversity. Introduction of three or more substituents at both carbon and boron atoms makes the 3D arrangement of substituents possible (Figure 3). This synthetic versatility makes carbaboranes unique and very useful scaffolds for the design of drugs in a way that is not possible in the case of limited to 2D benzene systems. The vast majority of carborane derivatives described to date are monosubstituted or disubstituted compounds, and methods for oligofunctionalization (attachment of three or more substituents) of carborane cages remain

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rare;35-38 however, the need for such molecules due to their use in medicinal and material chemistry applications has resulted in a growing interest in these compounds. Dodecaborate (10) offers similar possibilities in principle, but its high symmetry and the chemical identity of the BH vertices makes selective substitution of different boron atoms difficult. So far, mono-, oligo- or persubstituted compounds with the same ligands have mainly been synthesized.39-42 Recently, dodecaborates with two different types of substituents were described.43-45 The ellipsoidal shapes of metallacarboranes offer another platform for the 3D organization of attached ligands. Attempts to create methods for the oligofunctionalization of metallacarboranes are underway but are in the early stages of development.46-48

Electronic properties Boron has an atomic number of 5 and electron configuration of 1s22s22p1. It has a vacant p orbital that readily accepts electrons from a donor molecules and interconverts with ease between neutral sp2 and anionic sp3 hybridization states. Boron forms stable three-center two electron (3c2e) boron–hydrogen–boron bonds that produce a wide variety of stable boron clusters.24 The closo-dodecaborate

anion

(B12H122-)

and

isoelectronic,

electroneutral

dicarba-closo-

dodecaboranes (C2B10H12) feature 2n + 2 skeletal electrons (e_), that is, 26 e_ for 12 vertices and 20 triangular faces.28 The clusters characteristically reveal nonclassical bonding interactions resulting in complex overall electronic structures in which carbon and boron atoms are hexacoordinated. The high connectivity of the atoms in the clusters compensates for the relatively low-electron densities of the skeletal bonds. Consequently, the structures of boron clusters cannot be described in terms of usual two-center two electron (2c2e) organic bonds that

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are marked as a line between two atoms. Instead, a line connecting atoms in a cluster only illustrates the polyhedral geometry and does not represent an electron pair (Figure 4).

Figure 4. Electron configuration of boron (A) and three-center two electron (3c2e) boron– hydrogen–boron bond formation (B).

Because the cluster electrons are three-dimensionally delocalized, boron clusters can be described as 3D aromatic compounds.49,50 This spherical aromaticity is strongly reflected in the reaction chemistry of boron clusters, where substituent effects are transmitted through 3D cage skeletons in a manner reminiscent of 2D organic arene chemistry. It is worthy to stress that this property also extends to open-cage clusters, although in such cases lower electron-delocalization results in somewhat lower extents of aromaticity. The electronegativities of the components of major boron clusters increases in the following order: boron (2.051) < hydrogen (2.300) < carbon (3.066). The C-H units in carboranes give a part of their electron density to the common fund of 26 skeleton electrons; the C-H vertices have partial positive charges (δ+), which are compensated by a sum of partial negative charges (δ-) on particular B-H vertices.49,51 The double negative charge of a dodecaborate anion B12H122- is spread throughout the whole cage and participates in the charge of hydridic B-H hydrogen atoms. It is worth noticing that in contrast to the negatively charged B-H vertices, the C-H

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vertices in benzene (C6H6), a two dimensional aromatic organic system, have a partial positive charge similar to the C-H vertices in dicarbadodecaborane clusters (C2B10H12). One of the most important consequences of the electronic structures of clusters as a whole and the electronegative characteristics of their elementary components is the fact that hydrogen atoms carry a partial negative charge and are thus considered to be hydridic. In contrast to B-H bonds in carboranes, which have low polarity and exhibit virtually no acidic characteristics, C-H bonds in clusters are more polar and exhibit clear acidic properties. Furthermore, because carbon atoms are more electronegative than boron atoms, the electronic effects of boron atoms in carborane cages change depending on the mutual location of carbon and boron atoms in a cluster: boron atoms that are more remote from carbon atoms have stronger electron-donating effects. This affects the electron density of various vertex positions in different ways, giving rise to different hydricities of B-H hydrogens and chemical reactivities. Moreover, substitution at one or another boron or carbon atom changes the electron density pattern of the whole cluster. This allows the further tuning of its chemical properties and makes the chemistry of boron clusters a real game of chess.51,52

Hydrophobicity and lipophilicity Hydrophobicity is the physical property of a molecule that repels it from a mass of water. This repulsion is due to the inability of hydrophobic molecules to form standard hydrogen bonds with water molecules. This leads to the disruption and reorganization of a network of water molecules and the tendency of hydrophobic compounds to interact with each other and aggregate (Figure 5). Lipophilicity refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents. The terms “hydrophobicity” and “lipophilicity” can be used to describe the

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same tendency and are often used interchangeably. However, the terms "lipophilic" and "hydrophobic" are not synonymous because there are molecules that are hydrophobic but not lipophilic (e.g., silicones and fluorocarbons). Compounds that include both hydrophobic and hydrophilic regions are amphiphilic and have surfactant properties. The increasing interest in lipophilicity can be ascribed to its proven fundamental relevance in absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies of drug candidates.53

Figure 5. Hydrophobicity of boron clusters and their tendency to aggregate: A) water molecules next to a solute (5 is used as an example) cannot move freely. They are ordered and have less entropy; B) the system changes so that fewer water molecules are on the surface layer and the hydrophobic solutes aggregate (a hypothetical hydration and aggregation pattern).

The presence of hydride-like hydrogens next to the boron atoms contributes to the unique properties of boron clusters and affects their behavior in biological environments. Their impact is twofold: First, the partial negative charge of the hydrogens prevents them from participating in classic proton donor-proton acceptor hydrogen bonding with water, the most abundant substance in biological systems, which gives them hydrophobic characteristics. Second, it promotes hydrophobic interactions with biological macromolecules, which are possible drug targets. In addition, the electronegativity of the hydrogen atoms allows boranes to form unconventional hydrogen bonds, namely dihydrogen bonds. These bonds, also called proton–hydride bonds,

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generally occur between a positively charged hydrogen atom of a classical proton donor and a hydridic proton acceptor.54 However, dihydrogen bonds are weaker than classic hydrogen bonds; therefore, the repulsive effects toward the surrounding water molecules prevail, and the hydrophobic nature of many boron clusters is observed. In boranes, NH…HB, CH…HB and SH…HB dihydrogen bonds have been found. Another type of interaction was found for CH moieties in carborane hydrogen-bonded complexes. These complexes were, however, much less stable.55,56 Dynamic simulation of the hydration of dodecaborate revealed the formation of the first solvation shell, which resulted from the formation of dihydrogen bonds between the hydridic hydrogens of the cage and the hydrogen atoms of the surrounding water molecules, although the water molecules were very weakly bound.57 The hydrophobicity of the boron clusters resulting from the hydride character of the B-H hydrogens is modulated by the fact that depending on the structures of the clusters, they can carry a negative charge, a double negative charge as seen in parent compound 10 (B12H122-), a double or single negative charge as seen in biscarbollide metal complexes (i.e., M(C2B10H122-)2) such as 12 or a single negative charge as seen in 11 (CB11H121-). Base-degraded dicarbadodecaboranes 5-7, which are truncated icosahedrons (nido-carborane), carry a single negative charge, as seen in 9 (C2B9H121-). The lipophilicity of neutral dicarbadodecaborane (C2B10H12) is further differentiated by existing in three isomeric forms 5-7 that differ in polarity (Figure 2).

It is necessary to stress that boron clusters do not function as drugs per se but are used as structures functionalized with organic substituents or as components of conjugates bearing both

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an inorganic boron cluster and an organic component. The hydrophobicity of such conjugates stems from the properties of their constituents.58 However, low water solubility of the biomolecules conjugated with highly hydrophobic dicarba-closo-dodecaboranes (5-7) or metallacarboranes, such as metal-bis(1,2-dicarbollide)ate (12), is often observed. Substituting a more hydrophobic boron cluster for a charged cluster (9 or 10 anions), attaching hydrophilic hydroxyl, amine or carboxylic groups or conjugate phosphorylation59 decreases the total hydrophobicity of these conjugates and improves their water solubility.

Stereochemistry The effect of the regioisomerism (positional isomerism) of boron clusters on the biological activities of boron cluster conjugates has only been occasionally studied until recently. Now, its importance is generally recognized, but the value for tuning the biological properties of boron cluster compounds has not been fully explored. Examples of the influence of positional isomerism of boron clusters, such as dicarba-closo-dodecaboranes 5-7, on drug activity are described in the following sections. Stereoisomerism is a type of isomerism where the isomers differ only in their spatial arrangements and not in their connectivity. It is a widespread phenomenon in organic chemistry and is a characteristic feature of almost all natural compounds; the best-known examples include L-amino acids in proteins and D-sugars in nucleic acids. Protein receptors and enzymes, which are standard drug targets, usually have stereospecific interactions with their ligands and substrates. The stereochemistry of deltahedral borane derivatives could be potentially as extensive as organic chemistry60 but still awaits systematic exploration.

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Figure 6. Example chiral derivatives of boron clusters: A) enantiomers of 2-methyl-9dimethylthio-7,8-dicarba-nido-undecaborate zwitterion (13a) and (13b) (Inorg. Chim. Acta., 1999,

289,

45-50);

B)

asymmetric,

chiral

8,8’-µ-chlorophosphate[(1,2-dicarba-closo-

undecaborane)-3,3’-cobalt(_1)(1’,2’-dicarba-closo-undecaborane)]ate

(14)

(J.

Organomet.

Chem., 2002, 657, 59-70); C) diastereomeric oligothymidylates d(T)l2 containing enantiomeric 7,8-dicarba-nido-undecaborate-7-yl groups (15a) and (15b) (J. Am. Chem. Soc., 1994, 116, 7494-7501); J. Organomet. Chem. 1999, 581, 156-169).

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Boron clusters of the BnHn2- series are highly symmetric molecules, where the B12H122- ion is the most symmetric species. However, relatively minor changes, such as replacing B-H hydrogens with substituents, replacing skeletal boron atoms with heteroatoms, or combining these changes, make the original clusters prochiral or chiral. Tethering such clusters with a chiral organic component adds an additional chirality factor that may make the resulting conjugate a complex mixture of diastereomers. The chirality of a prospective drug molecule can be an advantage due to the expected higher activity and selectivity of a specific stereoisomer, although it 61-63

simultaneously makes drug synthesis more difficult.

In the case of future boron cluster

drugs, the problem of cluster chirality can be mitigated, if necessary, via the use of singlesubstituted clusters, symmetrically bis-substituted clusters and clusters that do not easily undergo transformation from the more symmetric closo-form to the less symmetric nido-form, such as 1,12-dicarba-closo-dodecaborane. Chirality in a single boron cluster species is due to molecular asymmetry induced by marking appropriate positions in an otherwise very symmetric parent framework (13a,b and 15a,b).60,64,65 The cause of asymmetry in bridged metallacarboranes, such as metal-bis(1,2-dicarbollide)ate (14), is more complex (Figure 6). In all compounds of this type, the pentagonal planes of 7,8dicarba-nido-undecacarborate ligands are mutually arranged prismatically; in one projection they are eclipsed, but the carbon atoms never eclipse in prochiral species. The pentagonal ligand planes are mutually inclined, where the inclination angle is dependent on the structure of the bridging substituent. Therefore, species of this type resemble more or less bent bows and give rise to asymmetry in the entire structure. Nearly two-thirds of the drugs currently on the market are chiral drugs. Chirality greatly influences the biological and pharmacological properties of drugs. Requirements that chiral

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medicines must be as pure as possible places great demands on drug synthesis, purification, analysis, and testing. This is also the case for future drugs that contain boron clusters.66

Chemical stability and metabolism of boron cluster conjugates To discuss the chemical stability and metabolism of boron cluster derivatives and conjugates, one must distinguish the two parts of the conjugate molecules: 1) the boron cluster and 2) the organic component. The parent boron clusters that are pertinent to medicinal chemistry (Figure 2) are chemically and thermally stable. In addition, because they are abiotic and are unfamiliar to living species and because they were synthesized only few decades ago, it can be safely assumed that no specific enzymatic systems that are able to metabolize these molecules have evolved. Consequently, boron clusters are bioorthogonal and are stable in biological environments, according to the present state of knowledge. Though, it should be stressed that metabolism and elimination of these boron clusters have not been well studied yet. The organic components of conjugates can undergo chemical/biological transformations similar to purely organic species. However, it can be judicially expected that the type, extent and kinetics of the metabolic transformations of the organic parts of the boron cluster conjugates will be affected by the presence of boron cluster modification. 1,2-Dicarba-closo-dodecaborane (5) is resistant to strong acids and oxidants, although methods for its oxidative hydroxylation are known. It is also stable up to 400 °C, above 425 °C it rearranges to the isomer 1,7-dicarba-closo-dodecaborane (6) and above 600 °C it further rearranges to generate a 1,12-dicarba-closo-dodecaborane (7) isomer that can survive temperatures in excess of 700 °C.28 The calcium salt of closo-dodecaborate (10) is stable upon heating up to 400 °C67 and the metallacarborane 3-cobalt-bis(1,2-dicarbollide)ate (12) is resistant

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to 2 M HNO3 for several days, high temperatures and ionizing radiation. It decomposes slowly upon treatment with concentrated solutions of NaOH or KOH in protic solvents, conditions that are far from biological circumstances.68 Chemical degradation of the cluster moieties in biological systems is most likely only relevant for 1,2-dicarba-closo dodecaborane (5) and its derivatives, which generate ionic 7,8-dicarbanido-undecacarborates. In general, the most stable dicarbadodecaborane isomers are those that have the greatest separation between the carbon atoms in the cage. The rational driving force for this phenomenon is the mutual repulsion between the relatively electropositive carbon nuclei in the cluster skeletons. The stability of dicarbadodecaboranes increases in the order of 5 < 6 < 7, and the order of susceptibility to deboronation and consequent closo/nido transformation follows the same pattern.28 1,12-Dicarba-closo dodecaborane (7) requires very harsh conditions to enforce the formation of the nido-form, and as a result, it is completely stable in vivo. Deboronation is initiated by nucleophilic attack at one of the most positive boron vertices followed by a second nucleophilic attack at the same boron atom. The rate of deboronation generally depends on various parameters such as the parent isomer, substituents, temperature, and solvent, as well as the presence, ratio, and types of nucleophiles. Carboranes substituted with strongly electron-withdrawing elements, such as oxygen, are deboronated even in water and at ambient temperatures. In contrast, the presence of electron-donating groups on the cages hinders the deboronation process. The corresponding nido- clusters can eventually hydrolyze to boric acid. An illustrative example of chemically induced in vitro deboronation of 1,2-dicarba-closo dodecaborane (5) in a bioorganic conjugate is depicted through the closo/nido transformation of the cluster in a DNA-oligonucleotide conjugate,69 phenylalanine derivatives70 or asborin71 (Figure 12B). The total degradation of the 1,2-dicarba-closo-dodecaborane cluster to boric acid

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proceeds far more slowly than the initial formation of the nido-clusters, and in the case of asborin takes more than 28 days. Despite this, for asborin, substantial closo-to-nido transformation of the cluster can be observed after 1.5 h, and after 24 h, all of the clusters are in the nido-form. 71

Figure 7. Examples for the metabolic transformation of boron cluster compounds: A) oxidation of sodium borocaptate (16) in patients with brain tumors (Drug Metabol, Disp., 2001, 29, 1588– 1598); B) phosphorylation of 5-(1,2-dicarba-closo-dodecaboran-1-yl)-2’-deoxyuridine (CDU) (22) in human peripheral blood mononuclear (PBM) cell culture (Intl. J. Radiation Oncol. Biol. Phys., 1994, 28, 1113-1120); C) dephosphorylation of 2’-O-{[(1,12-dicarba-closo-dodecaboran1-yl)propoxy]methyl} adenosine 5’-monophosphate (24) in human blood plasma (Coll. Czech. Chem. Commun., 2008, 73, 175-186).

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The enzymatic metabolism of boron cluster derivatives is even less recognized. The only study conducted to investigate the stability of boron cluster drugs in vivo was performed for sodium undecahydromercapto-closo-dodecaborate (BSH) (Na2B12H11SH) (16), a thiol derivative of closo-dodecaborate (10) used clinically in boron neutron capture therapy (BNCT) of tumors as a boron carrier. BSH was administered to brain tumor patients, and the products of its transmutation were observed in urine using electrospray ionization mass spectrometry (ESI-MS). Signals corresponding to several BSH derivatives were detected: BSH sulfenic acid [B12H11SOH]-2 (17), BSH sulfinic acid, [B12H11SO2H]-2 (18), BSH disulfide, [B12H11SSB12H11]-4 (19), BSH thiosulfinate, [B12H11SOSB12H11]-4 (20), and a BSH-S-cysteine conjugate, [B12H11SSC3H5NO2]-3 (21) (Scheme 7A). The remainder of the parent compound (between 73 and 89%) was unchanged BSH.72 Although oxidation of thiol groups and the transformation of further oxidized products can be catalyzed in vivo by a number of enzymes, the available data do not permit conclusions on whether the formation of the observed products was a spontaneous chemical reaction or an enzymatically mediated process.72,73 A metabolic process involving the enzymatic transformation of boron cluster-containing molecules that has been studied in more detail is the phosphorylation of nucleoside/boron cluster conjugates by nucleoside kinases. These studies were stimulated by the importance of nucleoside kinase activity toward nucleoside/boron cluster conjugates used as boron donors for BNCT74 and as potential antiviral75 and anticancer agents.76 Libraries of pyrimidine nucleoside/boron cluster conjugates were evaluated in in vitro phosphoryl transfer assays with TK1, TK2 and dCK. Their substrate specificities

and the relationship between structure and susceptibility to

phosphorylation were studied. In addition, kinetic parameters were established for several enzyme/conjugate pairs.77-80 For some nucleoside/boron cluster conjugates, e.g., 5-(1,2-dicarba-

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closo-dodecaboran-1-yl)-2’-deoxyuridine (22), enzymatic phosphorylation in cell cultures and animal models was studied (Figure 7B).81,82 The opposite process, dephosphorylation of phosphorylated nucleoside/boron cluster conjugates by human plasma phosphatases was studied for 2’-O-{[(1,12-dicarba-closo-dodecaboran-1-yl)propoxy]methyl} adenosine phosphates (AMP, cAMP, ATP) (Figure 7C)59. The effect of boron cluster modification on the resistance of DNA against phosphodiesterase degradation was also evaluated, and an increase in stability for the modified DNA-oligomers in the presence of the nuclease was observed.83 Analogously, an increase in stability of boron cluster-modified peptides towards degradation by proteolytic enzymes can be expected.84 The above considerations illustrate the incipient and fragmentary status of research progress on the metabolism of boron cluster derivatives and enzymology, and are obviously terra incognita that await exploration. One may expect that the emergence of drug candidates consisting of boron clusters will expedite developments in this field.

THE USE OF BORON CLUSTERS IN THE DESIGN OF BIOACTIVE MOLECULES Over the last 10 years, the number of new drugs introduced to the market has declined. The causes of this trend are complex, and the remedies are not obvious, although the weighty impact of the economy on this trend cannot be overlooked. Moving beyond the most popular drug targets and toward the exploration of new areas of chemical space may contribute to a reduction in the falling output of new drugs. Additionally, introducing more intellectual diversity to that pure synthetic organic chemistry or molecular biology alone can do may help.85 The use of boron clusters in the design of biologically active molecules is a step in this direction.

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The fact that boron compounds, especially those based on polyhedral boron hydrides, will be unfamiliar to life has potential advantages. This is because the active substances are less likely to be prone to the development of resistance and are expected to be more stable in biological systems compared to carbon-based molecules. Additionally, the types of interactions would be somehow different from key-lock systems that have been built up in living cell lines in nature for billions of years. This is an obvious benefit for future boron cluster drugs. While pathogens, such as bacteria and viruses, are eventually capable of evolving resistance against almost any molecule that attacks them, one could hypothesize that this process would take longer for boronbased compounds. Therefore, this would make it easier for humans to remain one-step ahead rather than struggling to keep pace as it is at present. The properties of boron clusters that are useful in drug design include the following: 1) hydridic character of H atoms – the types of interactions with biological targets may differ from this based on classic hydrogen bond formation, 2) spherical or ellipsoidal geometry and rigid 3D arrangement – these offer versatile platforms for 3D molecular construction, 3) lipophilicity, amphiphilicity or hydrophilicity – these qualities depend on the type of boron cluster used that allows the tuning of pharmacokinetics and bioavailability, 4) chemical stability and simultaneous susceptibility to functionalization, 5) bioorthogonality – stability in biological environments and a decreased susceptibility to metabolism, 6) abiotic origin – foreignness to existing enzymes and a decreased likelihood of triggering the development of drug resistance, 7) high boron contents – important for BNCT, and 8) resistance to ionizing radiation – a feature that is important for the design of radiopharmaceutical agents. At the present stage of medicinal chemistry, the development of boron cluster drugs faces several remaining obstacles, which include the following: 1) boron clusters cannot be categorized in the

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same way as organic moieties due to their unique electronic properties, different hydrogen bonding patterns, hexacoordinate carbon and boron atoms, and different properties for ionic forms, 2) despite the tremendous progress made in recent years, the chemistry used to functionalize boron clusters requires advancement focused on the needs of applications in medicinal chemistry, 3) in silico drug design and evaluation of boron cluster drugs is difficult or impossible due to the lack of appropriate descriptors for the interaction potentials of boron and the attached hydrogen atoms, and 4) the lack of libraries of boron cluster compounds for high throughput screening (HTS). Despite the gaps in the knowledge of the chemistry, pharmacology and biology of boron clusters, they have been successfully used for the syntheses of a variety bioactive molecules and have been tested against diverse biological targets. Bioorganic boron cluster derivatives can be divided into two major classes. One class includes physiologically inert compounds such as boron carriers used for BNCT, and diagnostics (e.g., radiodiagnostics, redox active molecular probes, IR and Raman labels). The second class consists of physiologically active molecules and includes compounds that affect cellular metabolism, such as drugs, drug candidates and molecular tools. The present review focuses on the second class of boron cluster derivatives. In these molecules, boron clusters have a central function as pharmacophores or scaffolds, or are used in the periphery of pharmaceutically relevant compounds as modulators of physicochemical and/or biological properties of already bioactive compounds to fine-tune drug activity.

Technically, two major avenues in the search for new biologically active molecules containing boron clusters have been exploited. The first is based on the modification of natural products, previously identified molecular tools or clinically used drugs with the hope of finding improved

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biological or pharmacokinetic properties. The second focuses on screening of available compound collections in search for novel structures with desired activities and properties. A third approach, based on rational drug design supported with in silico methods, still needs to be developed to match the level of methods that are available for the discovery of purely organic compounds. Due to the complexity of bonding within carborane cages and because current commercial software packages do not provide built-in default empirical potential energy functions for boron atoms, molecular modeling of boron cluster-containing molecules is difficult. This leads to forced, though necessary, simplifications that result in approximate and often inaccurate calculations with limited usefulness in the design of new molecules. However, computational methods are often useful for rationalizing and explaining experimentally observed results.86-91

Boron clusters were first exploited to modify amino acids and peptides. 1,12Dicarbadodecarboranylalanine and several other carborane-modified amino acids were synthesized in the late seventies. Subsequently, several analogs of biologically active peptides, such as enkephalin, angiotensin, bradykinin, substance P, and insect neuropeptide pyrokinin in which the Phe and/or Tyr residue was replaced with carborane-bearing amino-acid analogs, were synthesized.13,17 One of the recent focus areas has been on neuropeptide Y. Due to overexpression of its receptor (hY1 receptor) in most breast cancer tissues and in all breastcancer-derived metastases, it is considered to be a valuable tumor-targeted boron carrier for BNCT.92,93 Several carborane-containing antibodies for BNCT and radio imaging studies have also been prepared.94-100 Because the subject of the perspective is physiologically active compounds, discussions on BNCT agents have been omitted.

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The revival of interest in the application of boron clusters for drug design was initiated by extensive studies conducted by Endo and associates at the turn of the century. These authors conducted experiments on a range of receptor modulators containing carboranes as parts of their steroid frameworks, such as analogs of retinoic acid, estradiol, or androgene.101 Among the many low molecular weight compounds synthesized for BNCT are carborane-containing amino acids, lipids, carbohydrates, porphyrins, nucleic acid bases, nucleosides, and DNA groove binders.11,14 A new generation of radiosensitizers for BNCT includes biopolymers bearing one or more carboranyl residues. This class of boron trailers includes carboranyl peptides and proteins,95-97 carboranyl oligophosphates, and nucleic acids (DNA-oligonucleotides).83,102 However, because this review is focused on the developments made over the last decade, these studies will not be discussed. For discussions on earlier studies, interested readers may consult several excellent reviews and overviews that were published earlier.11-18

HIV protease inhibitors based on boron cluster metal complexes HIV protease (PR) inhibitors represent a substantial portion of the antiviral drugs used in clinics to treat AIDS. All these drugs, similar to other antiviral and anticancer agents, suffer from limited bioavailability, side effects, and the development of resistant strains. The tetrakis-(1,2dicarba-closo-dodecacarboranyl)carboxylate

ester

of

2,4-bis-(α,β-

dihydroxyethyl)deuteroporphyrin was identified as an HIV PR inhibitor more than two decades ago.103 It is of interest that similar porphyrin derivatives bearing 3-cobalt bis(1,2dicarbollide)]diate modifications instead of dicarbadodecaborane were later found to also exhibit anti-HIV PR activity, proving the importance of the hydrophobic characteristics of boron clusters for interactions with the enzyme.104

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Figure 8. Examples of HIV protease inhibitors based on boron cluster structures: sodium dihydrogenimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]diate (26), IC50 =140 nM; sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]diate (27), IC50 =100 nM; sodium hydrogen butylimino bis-10,10-[5-(3-oxa-pentoxy)-7,8-dicarbanido-undecaborate] (28), IC50 = 8.5 µM (IC50 established in in vitro enzyme assay) (Boron Sciences. New technologies and Applications, Hosmane, N.S., Ed.; CRC Press: Boca Raton, 2011; pp 41-70).

More recently, another class of 3-cobalt-bis(1,2-dicarbollide)ate (12) derivatives was discovered to be potent, specific, and selective competitive inhibitors of HIV PR. The most active compound,

sodium

hydrogen

butylimino

bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt

bis(1,2-

dicarbollide)]diate (27) (Figure 8), exhibited high anti-HIV activity and no toxicity in cell cultures. Resolution by protein crystallography of the PR and the parent compound, 3-cobalt-

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bis(1,2-dicarbollide)ate (12) (Figure 2), showed that two molecules of 12 bound to the hydrophobic pockets of the subsites of PR, similar to conventional protease inhibitors, suggesting that compound 26 and its homologues had similar modes of binding.105 In a search for more potent inhibitors of HIV PR based on two cobalt bis(dicarbollide) clusters connected via linkers of different types and length, several derivatives were synthesized and evaluated for their anti-HIV PR activities.106,107 Unfortunately, despite the promising characteristics, which included greater tolerance of metallacarborane drugs to mutations within the HIV PR binding pocket than clinical agents (lower susceptibility to drug resistance),86 the development of these compounds into clinically useful agents has proceeded slowly.108 One obstacles has been the difficulty in controlling the tendencies of metallacarborane derivatives to aggregate in aqueous media.107

Flufenamic acid and diflunisal analogs containing carborane pharmacophores Flufenamic acid is an anthranilic acid derivative and belongs to the class of NSAID drugs. Its clinical use as an analgesic and anti-inflammatory agent is limited due to high rates of gastrointestinal side effects characteristic for nonspecific COX-1/COX-2 activity. In contrast, diflunisal is a salicylic acid derivative similar to aspirin that also belongs to the NSAID family. Due to its favorable therapeutic profile, it is widely available and is used as an aspirin alternative. Both flufenamic acid and diflunisal, as well as several of theirs analogues, have been shown to also be active against transthyretin-related hereditary amyloidosis. Transthyretin (TTR) is a thyroxine-transport protein found in blood that has been implicated in a variety of amyloidrelated diseases. TTR dissociative fragmentation and subsequent aggregation to form putative toxic amyloid fibrils is considered to be a possible molecular mechanism for the role of TTR in

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amyloidosis. However, the COX activity associated with these pharmaceuticals has limited their potential as long-term therapeutic agents.

Figure 9. Selected examples of dicarba-closo-dodecaborane analogues of flufenamic acid (29) and diflunisal (33) shown in decreasing orders of inhibitory activity of amyloid fibril formation in a TTR assay; inhibitor concentration [µM] and percent fibril formation [%] are given in sequence (Proc. Nat. Acad. Sci. USA, 2007, 104, 4808–4813).

In the search for TTR dissociation inhibitors that were unburdened by COX activities, several flufenamic acid and diflunisal analogs containing carborane pharmacophores have been synthesized.109 The replacement of a phenyl ring in the NSAIDs with a carborane moiety greatly decreased their COX activity with a retention of similar efficacy as an inhibitor of TTR dissociation. The most promising of these compounds, 1-carboxylic acid-7-[3-fluorophenyl]-1,7dicarba-closo-dodecaborane (34) (Scheme 9), showed effectively no COX-1 or COX-2 inhibition at a concentration more than an order of magnitude higher than the concentration at which TTR dissociation was almost completely inhibited in vitro.110 In the case of diflunisal the strong effect of boron cluster regioisomerism on the inhibition of TTR-related amyloid fibril formation by the

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boron cluster analogues needs to be emphasized. The boron cluster structure-function relationship is evocatively illustrated by the much higher activity of diflunisal analogue 34 bearing 1,7-dicarba-closo-dodecaborane (6) (7 ± 3% fibril formation at 7.2 µM of 34) compared to that of 1,7-dicarba-closo-dodecaborane (5)-bearing counterpart 35 (67 ± 4% fibril formation at 7.2 µM of 35).

New boron cluster-containing antifolates. Antifolates are drugs that block the actions of folic acid (vitamin B9). The primary function of folic acid is to act as a cofactor to various methyltransferases involved in serine, methionine, thymidine and purine biosyntheses. Consequently, antifolates inhibit cell division, DNA/RNA synthesis and repair and protein synthesis. The majority of antifolates work by inhibiting dihydrofolate reductase (DHFR).

N

N

H 2N

H2N

H 2N

N

H 2N

NH

N

NH H2N

H 2N

-1

> OCH3

H3CO OCH3

36, IC50 = 133 M

37, IC50 = 0.15 M

38, IC50 = 15 M

Trimethoprim = BH,

= C or CH

Figure 10. Boron cluster analogues of the antifolate drug trimethoprim (36). The inhibition activity of 36 against rat DHFR and of 37 and 38 against human enzyme is shown.

An example of an antifolate drug is the antibiotic trimethoprim (36) (Figure 10), which is mainly used to treat bacterial infections; another example of an antifolate is the broadly used anticancer

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drug methotrexate. In addition to the development of resistance, major adverse effects of treatment with trimethoprim include nausea, vomiting, diarrhea, rash and sun sensitivity. In an attempt to obtain antifolates with novel structural features, two trimethoprim (36) carborane analogues were synthesized: one contained 1,2-dicarba-closo-dodecaborane (5) and the other contained negatively charged 7,8-dicarba-nido-undecaborane (9) instead of a parachlorophenyl group. The compounds were screened for activity against DHFR from six sources and showed good to modest activities against these enzymes. The compounds were also tested for their antibacterial activities against Lactobacillus casei, Mycobacterium tuberculosis H37Ra, and three Mycobacterium avium strains and for their cytotoxic activities against seven different human tumor cell lines. The closo-analogue was a more potent inhibitor than the nidocounterpart in all the DHFRs except Toxoplasma gondii. The closo-compound also exhibited up to a 10-fold greater inhibitory growth activity than the nido-analogue in all seven of the human tumor cell lines. Interestingly, X-ray analyses revealed that, despite the presence of a nidocarborane group, the resulting zwitterionic biomolecule was able to maintain hydrophobic characteristics for interaction with the hydrophobic cavity of the DHFR receptor.111

[5,5]-trans-Lactone thrombin inhibitor-containing carborane pharmacophores α-Human thrombin, a serine protease, is a potent platelet agonist involved in the blood coagulation cascade. As part of its activity, thrombin also promotes platelet aggregation via activation of protease-activated receptors on the cell membrane of platelets. The coagulation cascade is regulated by natural anticoagulants, such as antithrombin. Loss of control over hemostasis, the coagulation process and platelet aggregation leads to arterial thrombosis-related cardiovascular diseases.

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Figure 11. Naturally occurring [5,5]-trans-lactam α-human thrombin inhibitor (39) and its analogue (40) bearing a permethylated 1,12-dicarba-closo-dodecaborane group (SynthesisStuttgart 2008, 555-563).

There is an array of antithrombotic drugs used in clinics, but all of them are not free of adverse effects. Recently identified naturally occurring bicyclic trans-lactones type of 39 gave a rise to new class of thrombin inhibitors. The disadvantage of these agents is the lactone moiety’s susceptibility to hydrolysis in plasma. In one of approaches to solve this problem, a novel [5,5]trans-lactam inhibitor with a boron cluster cage (40) was rationally designed using a computational docking approach (Figure 11). In designing the molecule, it was assumed that a ligand containing a hydrophobic, methyl persubstituted 1,12-dicarba-closo-dodecaborane unit would have an increased affinity for binding the protein based on the ability of a boron cluster to lodge itself in the hydrophobic pocket of the enzyme. An innovative approach used for the design and synthesis of the above potential thrombin inhibitor deserves attention. First, instead of modifying the bioactive lead structures via simple use of a boron cluster as a phenyl mimetic, the whole molecule of the potential antithrombotic agent was designed using molecular modeling and computational docking approaches. Computer-aided drug design based on boron clustercontaining structures has been applied only in a few cases so far. Second, the fully methylated

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carborane structure was used as a modification instead of the dicarbadodecaborane structure, which is most often utilized as a pharmacophore. Third, 1,12-dicarba-closo-dodecaborane was used as a boron cluster scaffold instead of the more common 1,2-isomer. Unfortunately, no biological evaluations of the obtained agents have been reported to date and have limited the impact of this original work.87

Asborin, the boron cluster analogue of aspirin. Aspirin (acetylsalicylic acid) (41) belongs to the class of nonsteroidal anti-inflammatory agents (NSAIDs). Synthesized in 1897, it is one of the most successful and widely used medications in the world. Its mechanism of action is based on the irreversible inhibition of cyclooxygenase (COX), one of the key enzymes involved in the metabolism of prostaglandins. COX exists in the form of two isoenzymes: COX-1 and COX-2. These forms differ slightly in their amino acid sequences, molecular weights, and most importantly, the presence of isoleucine at position 523 in COX-1 and valine in COX-1. Because of these differences, aspirin affects the COX-1 variant more than the COX-2 variant of the enzyme, although effective inhibition of COX-2 would be more desirable due to the decrease in gastric irritation associated with COX-2 inhibitors. This and other side effects of aspirin (as well as other NSAIDs) have prompted a continuous quest for better drugs. As a result, several new COX inhibitors have been designed (some are discussed in the following sections), though aspirin still maintains its unrivaled position.

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O O

A

OH

OH O

41

O

O

O

42, 500 µM: COX-1 = 49 ± 5 COX-2 = 28 ± 11 Asborin

Aspirin B OH

O

H3O+ -1 B(6) O

O

O

OH

O

H3O+ -1

H

O

O

H

+ B(OH)3 - CH3COOH

B(3)

OH OH B(OH)3

42 = BH,

=C

Figure 12. A) Aspirin (41) and asborin (42), its 1,2-dicarba-closo-dodecaborane mimetic. Asborin

concentration [µM] and percent of COX inhibition [%] are given in sequence

(ChemMedChem. 2009, 4, 746-748); B) Chemical hydrolysis and deboronation of asborin (Eur. J. Med. Chem. 2011, 46, 1131-1139).

Inspired by the structure of aspirin, “asborin” (42), a 1,2-dicarba-closo-dodecaborane analogue of the parent compound, was synthesized (Figure 12).112 It is a model example for the use of boron clusters as pharmacophores to modulate properties of known drugs. It is also an example of a case in which judicially designed modifications of bioactive molecules did not necessarily lead to the expected results. Due to the different electronic properties of the phenyl and 1,2dicarba-closo-dodecaborane (5) cores and the strong electron-withdrawing nature of the latter, the acetoxy group attached to the boron cluster became too reactive. As a result, instead of acetylating the Ser residues of the interior active site of COX, asborin has been shown to acetylate Lys residues on the surface of the protein, leading to lower COX inhibition activity than aspirin.

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The pronounced effect of a boron cluster on the attached functional group is illustrated by the pKa values of benzoic acid (C6H5COOH) and 1-carboxy-(1,2-dicarba-closo-dodecaborane) (C2B10H11COOH), which are 5.76 and 2.61, respectively.16 A similar trend for thiophenol (C6H5SH) and 1-mercapto-(1,2-dicarba-closo-dodecaborane) (C2B10H11SH) is observed, yielding pKa values of 7.50 and 3.30, respectively.51 However, further studies revealed the high and selective inhibition of aldo/keto reductase (AKR) 1A1, which belongs to a family of NADPHdependent oxidoreductases. AKR are enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Some enzymes of the AKR family are associated with epilepsy, depressive disorders, diabetic neuropathy and the development of cancer, forming a promising group of targets for adjusted and optimized asborin and its analogues as potential modulators of AKR activity.71,113

Inhibitors of hypoxia-inducible factors (HIF). The discovery of hypoxia-inducible factor-1 has led to a rapid increase in the understanding of the molecular mechanism of tumor hypoxia over the past two decades. Today, it is generally accepted that HIF-1 plays a pivotal role in the cellular response to tumor hypoxia, which represents a major obstacle for the success of radiotherapy and chemotherapy. Targeting HIF-1 has become a novel and efficient strategy for cancer therapy, and a number of agents have been developed aiming to suppress HIF-1. Several low-molecular-weight compounds with anti-HIF activity have been identified, including phenoxyacetanilide derivatives, polyamides, quinols, naphthoquinone spiroketal analogues and two representative drugs: echinomycin (bis-intercalator peptide antibiotic) and bortezomib (proteasome inhibitor anticancer drug) (1) (Figure 1).114

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Figure 13. A) Selected examples of phenoxyacetanilides shown in decreasing order of inhibitory activity against hypoxia-inducible factor (HIF)-1α (Bioorg. Med. Chem. Lett. 2010, 20, 1453– 1456); B) Manassantin A (46) and combretastatin A4 (49) anti-HIF-1 boron cluster mimics and the effects of boron clusters 1,2- vs 1,7-regioisomerism on anti-HIF- 1 activity (ChemMedChem., 2013, 8, 265–271).

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Several derivatives of phenoxyacetanilide containing as well organic as 1,2-dicarba-closododecaborane substituents have been synthesized, based on the recent development of AC1-001 (43) (Figure 13). Interestingly, some of the obtained phenoxyacetanilides, such as 44, simultaneously contained both types of boron modifications, i.e. boron cluster and boronic acid residue. Among these compounds, some with highest anti-HIF activities have been recruited.115,116

The boron cluster effect on improving the inhibitory properties of the purely organic inhibitor 43 (IC50 = 3.1 ± 0.07 µM) was illustrated by the more efficient inhibition of HIF-1 by the counterpart (44), which contained 1,2-dicarba-closo-dodecaborane (6) instead of a lipophilic adamantyl group and a boronic acid modification instead of a carboxyl function group (IC50 = 0.74 ± 0.24 µM). Interestingly, an analogue of 45, where boronic acid replaced only the carboxyl group, was less active than the parent compound (IC50 = 4.6 ± 0.86 µM).

Other examples of the successful introduction of boron cluster modifications into anti-HIF agents are the modifications of manassantin A (46) and combretastatin A4 (49), natural products that have potent inhibitory activities against HIF-1. In the case of manassantin analogues, a carborane framework was used to replace the tetrahydrofuran scaffold in the structure. It was found that the compound containing the 1,7-dicarba-closo-dodecaborane framework (47) showed more potent inhibitory activity (IC50 = 2.2 ± 1.6 µM) than the 1,2-dicarba-closo-dodecaborane framework (48) (IC50 = 3.2 ± 1.1 µM); however, in both cases, the anti-HIF-1 activities of the boron cluster analogues were comparable with YC-1 (IC50 = 1.5 ± 0.7 µM), a potential anticancer drug targeting HIF-1.113 A similar dependence was observed for combretastatin A4 mimetics. A

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derivative containing 1,7-dicarba-closo-dodecaborane was used as the replacement for the cis geometry of the stilbene framework (50) (IC50 = 5.1 ± 0.6 µM) and was shown to be a more efficient inhibitor of hypoxia-induced HIF-1 transcriptional activity than the counterpart (51) bearing a 1,2-dicarba-closo-dodecaborane system (IC50 = 17 ±1.5 µM).117

The use of boron cluster derivatives of indomethacin and other indole derivatives as COX inhibitors. Indomethacin, an indole derivative discovered in 1963, is one of the most extensively studied NSAIDs. Intense studies established COX as indomethacin’s protein target. It shows a preference for COX-1, similar to aspirin, although inhibition of COX-2 is responsible for a significant portion of the therapeutic effects of the drug. The added inhibition of COX-1, as in the case of aspirin, contributes to its gastrointestinal side effects. Therefore, COX-2-selective inhibitors still remain attractive targets in the search for better NSAIDs. In the quest for a better indomethacin derivative, extensive studies on its boron cluster analogues have been performed. A series of indomethacin (52) esters was constructed with 1,2- and 1,7dicarba-closo-dodecaborane clusters and with phenyl and adamantanyl groups for comparison (Figure 14 A). All the derivatives were less active than the parent compound [52, IC50 = 0.05 µM (COX-1), 0.75 µM (COX-2)]. Derivative 53 was the most active of the studied modifications and contained a 1,2-dicarba-closo-dodecaborane group [53, IC50 = 2.6 µM (COX-1), 4.20 µM (COX2)]. Interestingly, the indomethacin derivative bearing isomer 1,7 was not active [54, IC50 > 25 µM (COX-1), > 25 µM (COX-2)], much like derivatives bearing phenyl and adamantanyl substituents. The strong effects of boron cluster regioisomerism illustrates the exceptional characteristics of boron cluster modification.118

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Figure 14. The use of boron cluster derivatives of indomethacin and other indole derivatives as COX inhibitors: A) (Bioorg. Med. Chem., 2011, 19, 3242–3248; B) (Bioorg. Med. Chem., 2012, 20, 4830–4837); C) ChemMedChem., 2013, 8, 329 – 335; D) ChemMedChem., 2015, DOI: 10.1002/cmdc.201500199.

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In a further search for indomethacin analogues with improved selectivity toward COX-2, a set of indomethacin and indomethacin alkyl esters bearing 1,2- (55), 1,7- (56) or 1,12- isomers (57) of dicarba-closo-dodecaborane or adamantanyl groups attached via amide bond to the nitrogen of indole systems instead of the ester linkage at position 7 were prepared (Figure 14 B).119 The results obtained using indomethacin indicated that its biochemical profile clearly depended not only on the boron cluster location but also on the carborane isomer.

The COX inhibition potentials of the obtained derivatives were lower than that of unmodified indomethacin,

although

all

the

alkyl

esters

bearing

1,2-dicarba-closo-dodecaborane

modifications expressed slightly higher inhibition potencies toward COX-2 than COX-1 (similar to the purely organic counterpart, indomethacin methyl ester). The 1,2-dicarba-closododecaborane indomethacin derivative 55 exhibited the highest activity [IC50 > 25 µM (COX-1), 3.68 µM (COX-2)] compared with the 1,7- and 1,12- isomers, which were inactive in concentrations as high as 25 µM.119 The transformation of 55 into a methyl ester 61 further improved its selectivity towards COX-2. Dicarba-closo-dodecaboranyl derivatives of 2,3-disubstituted indole,120 a core part of indomethacin, have also been synthesized (Figure 14 C). Evaluations of their biological activities confirmed the significance of boron cluster isomerism that was observed previously. A lack of COX inhibitory activity for the 1,7-dicarba-closo-dodecaborane-substituted derivative 59 was observed (IC50 >> 4 µM (COX-1 and (COX-2)], while the 1,2-dicarba-closo-dodecaborane analogue 58 was active; however, 58 showed a shift in selectivity toward COX-1 [IC50 = 1.6 µM (COX-1), > 4 µM (COX-2)].121 These observations are in agreement with earlier results and demonstrate that replacement of a phenyl ring in indomethacin esters117 and replacement of the

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chlorophenyl ring in indomethacin116 with a 1,7-dicarba-closo-dodecaboranyl moiety results in a loss in the potency of COX inhibition. The culmination of the research on selective COX inhibitors based on indole structures and boron cluster modifications has only been published recently, which includes exploitation of negatively charged 7,8-dicarba-nido-undecaborane (9) instead of the usual use of electroneutral 1,2-dicarba-closo-dodecaborane (5).88 To improve the stability of the previously obtained indomethacin analogues (55-57), which are labile due to the introduction of the boron clusters via amide bonds, to check the tendency of closo clusters to undergo uncontrolled transformations into nido forms and to increase water solubility, anionic 7,8-dicarba-nido-undecaborane (9) was introduced into the methyl ester of indomethacin in place of the 4-chlorophenyl ring. As expected, in the indomethacin derivative bearing a nido-carborane cluster (60) [IC50 > 4 µM (COX-1), 0.051 µM (COX-2)], the amide bond proved to be more stable toward hydrolysis than counterpart 61 that exhibited similar activity [IC50 > 4 µM (COX-1), 0.072 µM (COX-2)], which included electroneutral carborane 5. In addition to stabilizing the compound, the nido-cluster led to high water solubility. Moreover, the nido-form of indoborin 60 was found to be a more potent inhibitor of both the COX isoforms with much greater selectivity for COX-2 than purely organic indomethacin methyl ester [IC50 33 µM (COX-1), 0.25 µM (COX-2)]. Although the selectivities of 60 and 61 were in similar ranges as the methyl ester of indomethacin, their potencies were significantly enhanced by the introduction of the boron cluster modification. The above results show that, in contrast to previous paradigms, ionic nido-cages can be accommodated in hydrophobic receptor pockets and lead to highly active and selective compounds with better chemical characteristics and physicochemical properties.

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Purinergic receptor ligands bearing boron clusters Purinergic receptors are a family of plasma membrane molecules that are involved in several cellular functions, including proliferation, migration, differentiation, vascular reactivity, apoptosis, cytokine secretion, thrombosis and inflammation. There are three known distinct classes of purinergic receptors called P1, P2X, and P2Y with adenosine, nucleotides (ATP, ADP, UTP, UDP or UDP-glucose) and ATP, respectively, as naturally occurring ligands. There is growing evidence that these receptors could be promising therapeutic targets for a wide range of conditions, including cardiovascular diseases, neurological disorders, immune system diseases, inflammatory disorders and cancer.122-125 In addition to the natural ligands, a plethora of artificial agonists and antagonists based on the structure of adenosine or non-nucleoside designs have been identified for P1 and P2 receptors and are already being used as drugs or molecular tools.

Figure 15. Selected examples of adenosine, 2’-deoxy adenosine boron cluster conjugates: efficient inhibitors of blood platelet aggregation (62) (ChemMedChem., 2010, 5, 749-756) and species that inhibit the formation of reactive oxygen species (62 and 68) (Bioorg. Med. Chem., 2012, 20, 6621–6629).

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In a search for new types of P1 receptor modulators, a series of adenosine derivatives bearing electroneutral 1,12-dicarba-closo-dodecaborane (7), ionic 7,8-dicarba-nido-undecaborane (9) and a metallacarborane complex with 3-cobalt-bis(1,2-dicarbollide)ate (12) were synthesized. Modification of the nucleobase at the 6-N, 2-C or 8-C positions, or the sugar residue at the 2’-C position with suitable boron clusters tethered via different types of linkers (Figure 15) were prepared. The obtained library of adenosine derivatives was tested for the effects of these modifications on the functions of stimulated platelets126,127 and on the production of reactive oxygen species (ROS) by human neutrophils124. The modification of adenosine at the 2’-C position with 1,12-dicarba-closo-dodecaborane (68) was shown to result in efficient inhibition of platelet aggregation induced by thrombin or ADP (68, IC50 = 0.05 µM; adenosine, IC50 = 1.27 µM).126 Remarkably, inhibition of ROS production in activated neutrophils by adenosine modified at the 6-N position (62) with 7 was also observed (62, IC50 = 0.72 ± 0.22 µM; adenosine, IC50 = 1.81 ± 0.31 µM).128,129 The high affinities of the selected compounds for adenosine receptor A2A were established,128 which may suggest the possible involvement of the A2A receptor in the observed biological activities of the adenosine/boron cluster conjugates. Compounds 62 and 68 displaced a specific receptor A2A agonist [3H]CGS 21680 in a radioligand binding assay with inhibitory constants (Ki) of 9.9 nM and 1.04 nM, respectively, and displayed 3- and 26-fold higher affinities for the A2A receptor than CGS 21680 did, respectively.128 These preliminary findings, and the new chemistry proposed, form the basis for the development of a new class of adenosine analogs that can modulate human blood platelet activities and extend the range of innovative molecules available for testing as agents that affect inflammatory processes.

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Figure 16. Boron cluster analogues of purinergic receptor P2X7 antagonists with antidepressant activity (ACS Chem. Neurosci. 2014, 5, 335−339).

Another example of purinergic receptor targeting with boron cluster containing ligands are novel P2X7 receptor antagonists with antidepressant activities (72, 73). The receptor is found in the central (CNS) and peripheral nervous systems (PNS) in microglia, macrophages, uterine endometrium, and the retina. Its natural ligand is ATP, and P2X7 is a cation-selective ion channel. However, under specific conditions, it forms a nonselective pore. Formation of this pore results in apoptosis and the subsequent production and release of interleukin 1β (IL-1β). High levels of IL-1β in the brain have been implicated in depression, hyperalgesia and neurodegeneration. The development of a P2X7 antagonist is thus presumed to have antidepressant, analgesic and/or neuroprotective properties. One of the potent P2X7 receptor antagonists that has been identified is 1-[(5-methoxy-2-chlorobenzamido)methyl]adamantane (71).130 To obtain boron cluster analogues of the lead compound, the adamantanyl residue was replaced with electroneutral 1,12-dicarba-closo-dodecaborane (7) and ionic 7,8-dicarba-nidoundecaborane (9). For comparison, analogues of 71 bearing cubanyl or trishomocubanyl modifications instead of an adamantanyl group have also been synthesized.

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All the obtained compounds exhibited the ability to inhibit human P2X7 receptor pore formation in an in vitro assay (71, pIC50 = 7.98 ± 0.15 µM; 72, pIC50 = 6.43 ± 0.10 µM; 73, pIC50 = 8.07 ± 0.19 µM). They also showed significant antidepressant activity in an in vivo mouse model, and the derivative bearing 7,8-dicarba-nido-undecaborane (72) was found to be the most active compound.131 This study represents the first account of a carborane molecule possessing CNSmodifying activity. A consistent comparison of the biological properties of the boron cluster derivatives with counterparts bearing lipophilic, organic modifications such as adamantane, trishomocubane and cubane, deserves further attention.

Boron cluster-based carbonic anhydrase inhibitors Human carbonic anhydrases (CAs) are a family of enzymes that catalyze the interconversion of carbon dioxide and water to bicarbonate and protons. The active sites of most carbonic anhydrases contain a zinc ion; they are therefore classified as metalloenzymes. One of the functions of the enzyme in animals is to maintain an acid-base balance in blood and other tissues and to help transport carbon dioxide out of tissues. To date, 15 human CA isozymes with different subcellular localization and tissue expression profiles have been identified. Approximately 30 CA inhibitors, such as acetazolamide, methazolamide, ethoxzolamide or dichlorphenamide (diclofenamide) (74), are used clinically as anti-glaucoma drugs, anticonvulsants, diuretics, and anti-obesity agents, which target different CA isoenzymes. Most of the clinically used CA inhibitors that contain a sulfonamide or sulfamide moiety that coordinates to the zinc cation located in the CA catalytic site lack selectivity, causing numerous side effects. In an attempt to develop new types of CA inhibitors, a series of electroneutral 1,2-dicarba-closododecaborane (5), 1,7-dicarba-closo-dodecaborane (6), negatively charged 7,8-dicarba-nido-

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undecaborane (9) and carba-closo-dodecaborate (11) derivatives, which contain a sulfamide group connected through a linker to the carborane cluster, has been synthesized and tested against CA isozymes CA II and CA IX (Figure 17).

Figure 17. Selected examples of carborane-based carbonic anhydrase (CA) inhibitors in order of decreasing inhibitory activity (Angew. Chem. Int. Ed. 2013, 52, 13760 –13763).

Depending on the type of carborane cluster that is used as a scaffold (5-7), the closo-/nido statuses of the carborane cages and the substitution on the cluster, different inhibitory constants were obtained. The highest inhibitory activity was observed for compound 75 [Ki = 0.38 ± 0.14 (CA II), 0.23 ± 0.04 (CA IX), SI (Ki CA II/Ki CA IX = 1.7], which resembles the clinically used drug diclofenamide (74). In one case, a derivative of 77 bearing a phenyl substituent attached to a carbon atom of a carborane cage exhibited high activity [Ki = 2.78 ± 0.47 (CA II), 0.15 ± 0.06 (CA IX)] and selective inhibition of CA IX [SI (Ki CA II/Ki CA IX = 18.1).132 To investigate the observed CA II vs CA IX selectivity of some boron cluster inhibitors, crystallographic analysis of CA II complexed with 1-methylenesulfamide-1,2-dicarbaclosododecaborane (76), a parent compound of a reported series of CA inhibitors, was performed. The crystal structure analysis then served as a base for the construction of the CA IX-76

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computational model. Structural and computational analyses revealed the contributions of the individual residues to the binding energies of 1-methylenesulfamide-1,2-dicarba-closododecaborane to CAII and CAIX, substantiating the differences in the affinity of 76 towards CA II and CA IX. 89,90

Modification of selected anti-viral drugs with boron clusters and novel conjugates with antiviral activities. Nucleoside-derived drugs consist of a substantial part of the current armamentarium for the chemotherapy of viral infections and cancers. However, despite the undoubted benefits of nucleoside drugs, they have several drawbacks that restrict their clinical utility. Some compounds have limited oral bioavailability and must be administered intravenously. In addition, most of the drugs exhibit significant toxicity and result in serious adverse effects. The emergence of drug-resistant viral strains and drug-resistant cancer cells also poses an increasing problem for disease management. Anti-infectious disease drugs bearing essential boron components form an area of medicinal chemistry that still awaits exploration. To take advantage of the unique properties of boron clusters and to recognize their utility in the construction of antiviral agents, the synthesis of a clinically used anti-HCM and anti-influenza virus pro-drug modified with boron cluster was performed (Figure 18). The major characteristic of the obtained acyclovir (ACV) (78), ganciclovir (GCV) (80), and cidofovir (CDV) (82) derivatives 79, 81, 83 was the presence of a phosphoric or phosphonate acid residue and lipophilic modification in the form of a 1,12dicarba-closo-dodecaborane cluster (7) (Figure 18).133 This fulfilled two major requirements for the potential nucleoside pro-drugs: 1) the presence of a phosphate residue helped bypass the limiting first phosphorylation step which is a prerequisite for nucleoside antiviral activity, and 2)

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the presence of a lipophilic structural element that facilitated permeation through biological membranes of the otherwise lipophobic nucleoside phosphates and phosphonates, potentially increasing the bioavailability of the molecules. O

O N

N

NH

N

NH2

N

HO

O

O

* P

N

O O

H

O

O

N NH

N

HO

NH2

N

79, HCMV, IC50 = 80 M HSV-1, IC50 = 0.008 M

78, HCMV, IC50 = 15 M HSV-1, IC50 = 0.004 M Aciclovir N

NH

O

N

HO

NH2 O

HO

P

N

NH2

O

*

O

O

NH

O

ONa

81, HCMV, IC50 = 0.41 M HSV-1, IC50 = 75 M

80, HCMV, IC50 = 0.33 M HSV-1, IC50 = 20 M Ganciclovir NH2

NH 2

N

N N

O

O

O

P

N OH

O O O

*

OH OH

P

O

*

O

O

O

83, HCMV, IC50 = 2.8 M

82, HCMV, IC50 = 0.4 M Cidofovir

O

O O

O

O

O HN

HN NH2

NH2

O

O

85, IAV, IC50 = 0.5 g/mL

84, IAV, IC50 < 0.05 g/mL Oseltamivir = BH,

= C or CH

Figure 18. Selected anti-HCMV, anti-HSV-1 and anti-IAV and IBV boron cluster pro-drugs. Center of chirality is marked with star (Acta Pol. Pharm. Drug Res., 2012, 69, 1218-1223; ibid. 2013, 70, 489-504).

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The toxicities of the new derivatives were evaluated in adherent cells; no cytotoxicity was observed in five different cell lines (MRC-5, A549, LLC-MK2, Vero and L-929) at concentrations up to 1000 µM. The compounds were active against HCMV or HSV-1 through cytopathic effects or plaque reduction assays; the anti-HCMV activity of the GCV pro-drug was comparable to the unmodified GCV, the activity of CDV was slightly lower than that of the parent compound (81, IC50 = 0.41 µM; GCV, IC50 = 0.33 µM; 83, IC50 = 2.8 µM; CDV, IC50 = 0.4 µM), and the anti-HSV-1 activity of modified ACV was similar to that of the unmodified ACV (79, IC50 = 0.008 µM; ACV, IC50 = 0.004 µM). None of these tested compounds had exhibited activity against HPIV-3 or VSV. It should be noted that due to the presence of the centers of chirality at the carbon atoms of the GCV, ACV and CDV derivatives, they were used in antiviral assays as a mixture of enantiomers (GCV, ACV) or diastereomers (CDV) without separation into individual species. One can presume that the activity of one or the other stereoisomer alone was higher. Another example of antiviral drug modification with boron clusters is oseltamivir (Tamiflu ®) (84) (Figure 18). Oseltamivir is an antiviral medication used to treat IAV and IBV (flu). To obtain a boron cluster pro-drug, an ethyl group in the carborane ester of oseltamivir carboxylic acid was replaced with a 1,12-dicarba-closo-dodecaborane-1-prop-3-yl group. Antiviral tests of the boron cluster pro-drug 85 in MDCK cell monolayers against IAV revealed the antiviral activity of 85 (IC50 = 0.5 µg/mL), though the efficacy of 85 had decreased in comparison to the original drug.134

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Figure 19. Selected examples of a library of boron cluster-modified uridines in order of increasing anti-HCMV activity (J. Organomet. Chem., 2015, 798, 99–105).

An example of new molecular entities with antiviral properties containing boron clusters include uridine bearing 1,12-dicarba-closo-dodecaborane (7) at position 5 of uracil75 (Figure 19). The leading compounds 86, 87 have been identified via screening of a small library of novel nucleoside conjugates. Three types of C-5-modified uridines and 2’-deoxyuridines were generated: 1) derivatives bearing 1,12-dicarba-closo-dodecaborane attached to the nucleobase through a short and rigid ethynyl linker (86, 87), 2) derivatives containing a boron cluster modification tethered via a flexible pentyn-1-yl linker (88) and 3) bicyclic furo[2,3-d]pyrimidin2(3H)-one 2’-deoxynucleosides (89) bearing a boron cluster attached directly to the furan ring. The designed compounds demonstrated low to moderate cytotoxicities in several cell lines. The antiviral activities of the agents were measured against a panel of DNA (HCMV, HSV-1) and RNA (HPIV-3, EMCV) viruses. The most potent compound was 5-[(1,12-dicarba-closododecaboran-2-yl)ethyn-1-yl]uridine (86) (IC50 = 3.8 ± 1.1 µM). However, its safety margin was smaller than that of its 2’-deoxyuridine counterpart (87) (IC50 = 5.5 ± 0.7 µM), which possessed a much higher selectivity index (SI > 180) because its toxicity was lower (CC50 > 1000 µM in

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the MRC-5 cell line) than that of 86 (CC50 = 66.2 ± 1.7 in MRC-5 cells). Compounds 86 and 87 are unusual in that they exhibit specific antiviral activities against HCMV and are inactive not only against HPIV-3 or EMCV but also against HSV-1.

Boronicaine, a boron cluster analogue of the local anesthetic lidocaine Clinically, there is a need for local anesthetics with greater specificity of action on target cells and fewer unwanted side effects that act over longer durations. Lidocaine (90, Figure 20) belongs to the class of amide local anesthetics. It has a dimethyl-substituted phenyl ring attached to its amide side chain. The dependence of anesthetic activity on the presence of a hydrophobic aromatic moiety, such as a phenyl ring, is well recognized.

Figure 20. Lidocaine boron cluster analogs bearing 1,2- (91), 1,7- (92, 94) or 1,12-dicarbacloso-dodecaboranes (93) in decreasing order of analgesic activity (ChemMedChem., 2015, 10, 62–67).

In the search for better local anesthetics, the aromatic dimethylphenyl group of lidocaine (90) was replaced with the following isomeric carborane clusters: 1,2-dicarba-closo-dodecaborane (5), 1,7-dicarba-closo-dodecaborane (6), 1,12-dicarba-closo-dodecaborane (7) and 1,7-dimethyl-

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1,7-dicarba-closo-dodecaborane. Additionally, adamantane lidocaine, in which the phenyl group of lidocaine was replaced with a nonaromatic, hydrophobic, adamantanyl group, was synthesized.135 The analgesic properties of boronicaines 91-94 were tested and compared with lidocaine in vivo using the hot plate test in a mouse model. The compounds differed in their analgesic efficacies in the following order: boronicaine bearing 1,2-dicarba-closo-dodecaborane (91) = 1,7-dimethyl1,7-dicarba-closo-dodecaborane (92) > 1,12-dicarba-closo-dodecaborane (93) > lidocaine (90) > 1,7-dicarba-closo-dodecaborane (94). The adamantanyl analogue of lidocaine had the lowest analgesic activity. In addition, boronicaine bearing 1,2-dicarba-closo-dodecaborane (91) had longer lasting analgesic effects than the parent molecule, lidocaine. The significant effects observed as a result of altering carborane stereochemistry on the biological activities of carborane-based compounds are in agreement with earlier observations made for COX118-121 ligands and demonstrates the potential of boron clusters in tuning the properties of prospective medicinal compounds. Molecular calculations and docking experiments revealed that the analgesic activities of boronicaines were affected by 1) the presence of delocalized electron density in the hydrophobic part of the molecule; 2) exposure of hydrophobic B-H vertices to the protein; and 3) the presence or absence of acidic C-H groups in the vicinity of the substituent.

CONCLUSIONS The physical and chemical properties of boron clusters make the design of boron clustercontaining molecules with new biological characteristics possible and offer medicinal chemists a rare opportunity to explore and pioneer new areas of molecular design and medicinal applications. Single boron atom-containing drugs have already reached clinics. Boron cluster-

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containing bioactive molecules can be considered to be second-generation boron drugs. As decades of research begin to bear fruit, the field of bioorganic and medicinal chemistry of boron clusters is on the verge of providing a new generation of drugs and therapeutics. Although several gaps in our knowledge must still be filled and extensive basic research on boron cluster medicinal chemistry, pharmacology and biology is required, new bioactive molecules bearing boron cluster are already synthesized and studied. Thus far, these molecules have usually been boron cluster analogues of known drugs (“me too” compounds), but the development of new molecular entities, mainly antivirals, has also been proposed. Together with developments in the medicinal chemistry and biochemistry of boron clusters, the appearance of boron cluster compound libraries for HTS and progress in the computational chemistry of boron clusters are expected to result in the syntheses of growing number of original boron cluster bioactive molecules. As a result, one may anticipate that this class of compounds is likely to grow over the next decade and that boron could become widely accepted as a useful element in the development of future medicinal compounds.

CORRESPONDING AUTHOR * Zbigniew J. Lesnikowski, Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, 106 Lodowa St., Lodz 93-232, Poland. E-mail: [email protected], Phone: 48-42-2723629, Fax: 48-42-2723630

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ACKNOWLEDGMENT This work was supported in part by the National Science Center, Poland, grant 2014/13/B/NZ1/03989, contributions from the Statutory Fund of IMB PAS is also gratefully acknowledged.

ABBREVIATIONS ACV

-

aciclovir

AIDS

-

Acquired Immune Deficiency Syndrome

AKR

-

aldo/keto reductase

AMP

-

adenoasine 5’-monophosphate

ATP

-

adenosine 5’-triphosphate

cAMP

-

adenosine 3’,5’-cyclic monophosphate

ADMET

-

absorption, distribution, metabolism, excretion, and toxicity

BNCT

-

Boron Neutron Capture Therapy

BSH

-

undecahydromercapto-closo-dodecaborate

CDU

-

5-(1,2-dicarba-closo-dodecaboran-1-yl)-2’-deoxyuridine

CDV

-

cidofovir

CNS

-

central nervous system

dCK

-

2’-deoxycitidine kinase

COX

-

cyclooxygenase

DHFR

-

dihydrofolate reductase

3D

-

tridimensional

CA

-

carbonic anhydrase

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EMCV

-

encephalomyocarditis virus

ESI-MS

-

electrospray ionization mass spectrometry

GCV

-

ganciclovir

HCMV

-

human cytomegalovirus

HIF

-

hypoxia-inducible factors

HIV

-

human immunodeficiency virus

HPIV

-

human parainfluenza virus

HSV

-

herpes simplex virus

HTS

-

High Throughput Screening

IR

-

infrared

IAV

-

influenza A virus

IBV

-

infectious bronchitis virus

NSAID

-

nonsteroidal anti-inflammatory agents

PNS

-

peripheral nervous systems

PR

-

protease

TK1

-

thymidine kinase 1

TK2

-

thymidine kinase 2

TTR

-

transthyretin

UDP

-

uridine 5’-diphosphate

UTP

-

uridine 5’-triphosphate

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BIOGRAPHY Zbigniew J. Lesnikowski received Ph.D. in organic chemistry at the CMMS PAS in 1981, D.Sc. degree at the Technical University of Lodz in 1992, and prof. of chemistry title in 2004. After a postdoctoral study at the University of Virginia (1981-1983) he continued his affiliation with CMMS till 1994. He was a Fulbright Scholar at Emory University in 1992, he worked at Emory University and VAMC in Atlanta from 1993 till 1996. In 1997 he joined the Center of Microbiology and Virology (CMV) PAS in Lodz (at present Institute of Medical Biology PAS) where he directs the Laboratory of Molecular Virology and Biological Chemistry.

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