Current Drug Targets, 2010, 11, 00-00

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Current Drug Targets, 2010, 11, 00-00

1389-4501/10 $55.00+.00 2010 Bentham Science Publishers Ltd.

Halogen Atoms in the Modern Medicinal Chemistry: Hints for the DrugDesign

Marcelo Zaldini Hernandes*,1, Suellen Melo T. Cavalcanti1, Diogo Rodrigo M. Moreira 1,2,Walter Filgueira de Azevedo Junior

3and Ana Cristina Lima Leite

1,*

1Departamento de Cincias Farmacuticas, Centro de Cincias da Sade, Universidade Federal de Pernambuco

(UFPE), Rua Prof. Artur S S /N, Cidade Universitria, 50740-520, Recife, PE, Brazil.

2Programa de Ps-graduao em Qumica, Departamento de Qumica Fundamental, CCEN, UFPE, Rua Prof. Artur S

S/N, Cidade Universitria, 50740-540, Recife, PE, Brazil.

3Laboratrio de Bioqumica Estrutural, Faculdade de Biocincias, Pontifcia Universidade Catlica do Rio Grande do

Sul, CEP 90619-900, Porto Alegre RS, Brazil

Abstract: A significant number of drugs and drug candidates in clinical development are halogenated structures. For a

long time, insertion of halogen atoms on hit or lead compounds was predominantly performed to exploit their steric

effects, through the ability of these bulk atoms to occupy the binding site of molecular targets. However, halogens in drug

target complexes influence several processes rather than steric aspects alone. For example, the formation of halogen

bonds in ligand-target complexes is now recognized as a kind of intermolecular interaction that favorably contributes to

the stability of protein-ligand complexes. This paper is aimed at introducing the fascinating versatility of halogen atoms. It

starts summarizing the prevalence of halogenated drugs and their structural and pharmacological features. Next, we

discuss the identification and prediction of halogen bonds in proteinligand complexes, and how these bonds should be

exploited. Interesting results of halogen insertions during the processes of hit-to-lead or lead-to-drug conversions are also

detailed. Polyhalogenated anesthetics and protein kinase inhibitors that bear halogens are analyzed as cases studies.

Thereby, this review serves as a guide for the virtual screening of libraries containing halogenated compounds and may be

a source of inspiration for the medicinal chemists.

Keywords: Halogen bond, virtual screening, molecular docking, anesthetics, protein kinase, flavopiridol, isoflurane.

INTRODUCTION

A large number of drugs launched in the market have, as

molecular targets, enzymes or membrane receptors [1, 2]. Inmost cases, elucidating the intermolecular interactions inthese drug-target complexes was a pre-requisite to reachsuccess [3-5]. Although the premise that structurally similarmolecules have similar biological targets seems to be onestarting point of reasoning, there are violations on thisstatement [6, 7]. Modern biochemical and analytical tools arenowadays available to elucidate drug-target interactions, butthis process remains complex. In the light of this, studyingthe earlier cases of drug discovery is an essential exercise forthose who work in the field of medicinal chemistry. In thisreview we analyze drugs that bear halogen atoms.

Due to their molecular complexity and diversity,

secondary metabolites from natural sources, mainly plants,still inspire the design of drugs. Although in recent yearsmarine animals have demonstrated to be rich sources ofhalogenated metabolites [8, 9], the occurrence of halogena-ted NPs in plants is rare [10]. Thus, it would be expected thathalogenated drugs would have little importance in the drugs

*Address correspondence to this author at the Departamento de CinciasFarmacuticas, UFPE, Rua Prof. Artur S S/N, Cidade Universitria, ZIPCODE 50740-520, Recife, PE, Brazil; Phone: +055-81-2126-8511; Fax:+055-81-2126-8510. Email: [email protected]; [email protected]

scenario, but this is not the case, since halogenated drugshave a prominent position. Of course they are, in theirmajority, of synthetic origin. The paradoxical occurrence ofthese halogenated drugs is the result from the usefulness ohalogen atoms.

One of the most popular hypotheses in the field ofmedicinal chemistry is that the appropriate attachment obulk groups (bulky flanking groups) in BioNCEs, such ahalogen atoms, could lead to volumetric and conformationachanges. In other words, bulk groups tend to occupy all theactive site of molecular targets, including the deeper pockets[11-15]. Thus, in diverse cases, inserting bulk groups intoBioNCEs can induce antagonistic or agonist effects incomparison with the original BioNCE (Fig. 1).

Over the years, insertion of halogen atoms has been used

in innumerous cases of hit-to-lead or lead-to-drug conversions [16-18]. Besides, the incorporation of halogen atominto BioNCE is commonly used to increase membrane permeability and, therefore, improve the oral absorption [19]Likewise, halogenation also enhances the BBB permeabilityand this is a pre-requisite for the drugs that need to reach theCNS [20]. It is estimated that one quarter of the total numbeof papers and patents closely related to medicinal chemistryinvolve the insertion of halogens during the synthesis of finacompounds [21]. However, the applicability of these atomin drug design is still far from been completely exploitedThat is, most of SAR discussions of halogenated compounds


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2 Current Drug Targets, 2010, Vol. 11, No. ? Hernandes et a

have only considered the classical steric parameters, ignoringother contributions and effects of such atoms. It was only inrecent years that the role of halogen atoms in proteinligandcomplexes has come to light.

Fig. (1). Generically view of how insertion of bulk groups

(represented as R) could enhance the bidding affinity. A)

Molecular target bound to its natural substrate. B) Molecular target

bound to an analogue of its natural substrate. The occupation of the

P-1pocket by bulk group implies in a different accommodation of

the ligand by the target, leading to a distinct biological response.

This strategy is valid to either receptor antagonists or agonists.

In spite of being less pronounced, halogens are endowed

with the ability to establish intermolecular bonds in a fashionthat resembles the H-bonds. The core definitions of halogenbonds have been reviewed by Metrangolo and co-workers[22, 23] and will be summarized in this review. Now, it isworth to say that in the case of self-assembly of organiccrystals, it is estimated that the stabilizing potential of typicalhalogen bonds is of about half of an average H-bond [24].Regarding the binary protein-ligand complexes, there aremany examples suggesting that formation of halogen bondsare favorable electrostatic interactions. For instance, due tothe involvement of halogen bonds, Auffinger and co-workersshowed that halogenated ligands bind to the cyclin-depen-dent kinase 2 (CDK2), leading to more stable complexesthan non-halogenated ligands [25]. In light of these and other

findings, identifying halogen bonds could be of greatrelevance to explain the molecular recognition (pharmaco-dynamics), as well as in other steps of the medicinalchemistry, such as structural planning and molecular docking(virtual screening).

Thereby, this paper is aimed at introducing the fascina-ting versatility of halogen atoms. It starts by describing theprevalence of halogenated drugs, listing some particularproperties of the halogen atoms. Features related to identi-fication of halogen bonds in proteinligand complexes, howto calculate (prediction) and how these atoms should beexploited are discussed. Recent results of this approachapplied to the design of optimized lead-compounds will alsobe summarized.

PREVALENCE OF HALOGENATED DRUGS

Despite the substantial investigations in the field bymedicinal chemists, the number of halogenated drugs thathave really reached the market at the last twenty years isslightly smaller than the expected [26-28]. There are somehypotheses to explain it. The first reason may be that thedrug discovery is, in many cases, serendipitous. The secondreason is related to the NPs. NPs remain working as keytemplates for the discovery of new BioNCEs and as sourceof bio-inspiration for the planning of synthetic and

semisynthetic drug candidates [29]. However, as mentionedbefore, halogenated NPs from plants are really scarce. Synthetic considerations have also limited the number of halogenated BioNCE. The halogenation of BioNCE is mostlyperformed in aromatic rings, while the insertion of halogenin aliphatic carbons is less abundant. This statement can beconfirmed through the scarce numbers of halogenatedBioNCEs from peptides, carbohydrates, steroids, and

polyether macrolides. This occurs, in part, because of thesynthesis to achieve the selective halogenation on thesescaffolds is relatively difficulty.

By analyzing (Fig. 2), we can see that the majority ohalogenated drugs are fluorine drugs, followed by chlorineones, while bromine is rare and the only iodine drug is thethyroidhormone thyroxine. In fact, with the exception of theiodine compounds, which are relative unstable (because Cbonds are highly polarizable) and of expansive synthesi(and obviously avoided by the pharmaceutical industry), thechemical processes for the synthesis of fluorine, chlorineand bromine compounds are well known in industrial scaleand of desirable stability and cost. Regarding the fluorinedrugs, there are some reasons that justify their predo-minance. Fluorine is the most electronegative atom in theperiodic table and has a small atomic radius, along with lowpolarizability [30]. The highly electronegative nature of fluorine renders it a poor halogen bond acceptor character, buenables it to receive hydrogen bonds from H-bond donor[31]. These chemical characteristics imply that the fluorination of BioNCE alters physical, chemical, electronic, andconformational parameters, which eventually could result todrugs with optimized pharmacological properties. In a moredetailed view, Biffinger and co-workers have adopted theterm polar hydrophobicity to describe the phenomenon inwhich fluorinated fragments are less able to engage indispersion-based interactions with aqueous solvent than alkyor aryl groups [32].

Fig. (2). Classification of the halogenated drugs according with the

kind of halogen. This estimative considered the drugs approved by

FDA from 1988 to 2006. Salts and metallic complexes in clinica

use were excluded during this search.

In the medicinal chemistry, fluorine is generally viewedas classic bioisosters of hydrogen and methyl [31]. Oneillustration of this statement is the classical exchanging of


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Halogen Atoms in the Modern Medicinal Chemistry Current Drug Targets, 2010, Vol. 11, No. ?

hydrogen by fluorine on the purine base uracile, resulting tothe anticancer drug 5-fluorouracil (5-FU) (Fig. 3). This drugis not incorporated into DNA, but is converted to the bioac-tive metabolite 5-fluoro-monophosphate (5-fluoro-dUMP),which inhibits the thymidylate synthase that is of pivotal rolefor the pyrimidine biosynthesis [33, 34]. Thereby, the fluo-rine chemistry provides good opportunities for enhancing thebinding affinity of potential drug candidates. These featureshave made the trifluoromethyl (CF3) [35] and pentafluoro-sulfanyl (SF5) [36] useful chemical groups in the contem-

porary drug design. Another interesting issue is that incorpo-rating fluorinated functionality into endogenous substrates orligands through

19F-markers is a powerful technique to probe

protein functions and to study their catalytic cycle, consti-tuting an essential tool for the chemical biology [37].

Chlorine is also of noticeable prevalence and deservesmore discussion. Chlorine occupies an intermediate positionon the halogen series. Different from fluorine, chlorine is amoderate halogen bond acceptor, besides being larger in sizethan fluorine. The CCl bond is enough stable, allowing itsinsertion on diverse heterocyclics of pharmacological value.Another feature of chlorine drugs regards to its bindingaffinity. Replacing hydrogen by chlorine also provides asubstantial alteration on the volumetric and shape issues. In

the light of these features, chlorination of BioNCEs couldeventually result to BioNCEs that are best accommodated onthe active site of targets in comparison with the non-chlorinated BioNCEs. It was also described that subunitsbearing chlorine can be accommodated in tight and deepcavities, as well as in hydrophobic pockets of the biologicaltargets [38].

The following examples illustrate a somewhat more res-trictive aspect of how chlorine could crucial during molecu-lar recognition. Nemonapride was found to be a potent dopa-minergic receptor ligand. Although Nemonapride has notshowed affinity to other targets (muscarinic, adrenergic,histaminic), a lack of selectivity among the subtypes (D1,

D2, D3, D4) was observed [39]. To overcome this low selec-tivity over the D4 receptor, a number of molecular modi-fications were performed in the Nemonapride. During thestep of molecular simplification, it was observed that the 1-chloro-2-methoxylbenzamide constitutes a ubiquitous func-tion-determining domain, i.e., the minimal central scaffold.The removal of the chlorine was deleterious for theselectivity among the dopaminergic receptors. Thereby,preserving this central scaffold, a number of molecularmodifications were performed, which allowed the discoveryof novel and selective dopaminergic receptor ligands (Fig.4), being YM50001 a selective ligand for D4 versus D2 and

of insignificant affinity for 1-adrenergic, adrenergicserotonergic, muscarinic, and histaminic receptors [40, 41].

Cl

H2N O

NH

NO

Cl

NH

O

NH

ON

Cl

NH

O

NH

O

N

Cl

NH

O

NH

O

N

O

O

O

Clebopride

YM-50001

YM-43611

Nemonapride

Fig. (4). Chlorobenzamides as dopaminergic antagonists. Dashed

lines are delineating the molecular motifs in common. From these

cases, removing the chlorine is deleterious for the selectivity in the

DOPA-receptors.

PERMEABILITY, TOXICITY AND METABOLISMOF HALOGENATED DRUGS

According to Kubinyis studies, inappropriate ADME isnot the major reason for failure in drug development, beinganimal toxicity and inefficacy the major problems (Fig. 5[42]. Apart from the pharmacological efficacy, which depends on the therapeutically application, ADME and toxicitycan be measured independently of the pharmaceutical classThat is, theoretical predictions of oral permeability and BBBpenetration are fast achieved and of good accuracy. Analysisof Lipinksis rule of five [43], the polar surface area [44]molecular flexibility [45] and VolSurf parameters [46] areexamples of useful tools to predict the oral permeability.

N

N

O

O

H

H

N

N

O

O

H

HF

N

N

O

O

H

FO

OPO32-

OH

Uracile 5-fluorouracil (5-FU)

bioisostericexchange

5-fluoro-dUMP(Inhibitor of the

thymidylate synthase)

biologicalmetabolisation

Fig. (3). Conception of the anticancer prodrug 5-fluorouracil (5-FU), exploring the bioisosteric exchange of hydrogen by fluorine atom.


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Fig. (5). Diagram showing the reasons for failure in drug

development. These data were gathered from the analysis of 121

BioNCEs in the period of 1964-1985. Adapted from reference [42].

Experimentally, octanol / saline partition coefficientshave been usually employed for rapid and accurate assessingof lipid solubility, while the drug toxicity potential ismeasurable using in vitro cellbased assays, albeit with somelimitations [47]. In the chemical point of view, there aremany molecular modifications that could eventually increasethe intestinal permeability of BioNCE. In particular, it wasreported that inserting halogens in BioNCE increases thelipophilicity, with subsequently improvement of oralpermeability and CNS penetration [19, 20]. This logic wasinitially confirmed by Gentry and co-workers [20] during theconception of new halogenated enkephalin analogs, proto-types of opioid analgesic agents. Through the appropriateattachment of chlorine, bromine, fluorine, or iodine at thepara-position of the Phe-4 residue on the prototype Tyr-D-Pen-Gly-Phe-L-Pen-Phe, it was possible to investigate theeffects of those halogenations on the in vitro BBB permea-

bility. Except for the fluorine, enhancements of in vitro permeability in bovine brain microvessel endothelial cells wereobserved in all the other halogenated peptides, and thistendency was also accompanied by improvement in theoctanol/saline partition coefficient. Later on, more accuratestudies also corroborated these statements, noticing that thisstrategy is also valid for BioNCEs of nonpeptidic nature[48].

The task of rationally designing a chemistry strategy focircumventing a limiting toxicity can be frustrating. Becauseof this status and with the great advent of HTS andcombinatorial chemistry, in silico filtering techniques (theso-called garbage filters) to eliminate undesirable atoms orfragments endowed of intrinsic toxicity in potential are hotopics that have called much attention of the medicinachemists [49-51]. However, the toxic potential of a drugdepends on various factors that are very far from being fittedin just a few predictive models. Nitro group is an example ofragment that is frequently considered as undesirable fragment by these garbage filters. Actually, nitrated BioNCEsfrequently display more pronounced toxicity than nonnitrated BioNCEs, because the nitro group undergoes biologicareduction, leading to the production of toxic metabolites [5253]. While this statement is certainly true, at least for somenitro-drugs, sweeping generalizations such this can also bemisleading. As stated by Kubinyi, if such prediction toolsare nevertheless applied, they should only be used to scoregroups of compounds, not to decide the fate of individuacandidates [42].

In light of these observations, we have tried to summarize information regarding the toxicity of halogenated drugsProudfoot has provided an excellent analysis of the launcheddrugs in recent years, listing each drug and its respective leadcompound [54]. Taking this work as guide, we analyzedpoint-by-point, all the cases in which halogenated lead com

pounds were explored as potential candidates during the stepof lead-to-drug conversion. In only two cases, halogenatedlead compounds were excluded during this process because

CF3

N

NH

O

Cl

Prototype 3i

Assay of45Ca2+ influx

Antagonist (IC50=37nM)

Clearance: 168mL\min.\kg

CF3

Analogue 2

Assay of45Ca2+ influx

Antagonist (IC50=42nM)

Clearance: 48mL\min.\kg

Blocking the

metabolism

Rapid

conversion into

non-selective

metabolit

TRVR1 antagonist ligands

1,1-dimethyl-2,2,2-trifluoroethyl

OH

O

product of oxidative metabolismtert-butyl

Fig. (6). Bioisosteric replacement oftert-butyl (3i) by 1,1-dimethyl-2,2,2-trifluoroethyl (2) towards the design of more stable and equipoten

analogue.


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of toxicity problems, being inefficacy the real reason offailure. From these analyses, there is not evidence thathalogen insertion itself induces the toxic potential of a drug.

Drug metabolism is, on the one hand, a crucial deter-minant of drug clearance and inter-individual pharmaco-kinetic differences, leading to efficacy or failure of a specificdrug candidate during clinical test. Conversely, hepatocyteshave a central role in the metabolism of xenobiotics, being

drug-induced liver injury the most frequent reason for thewithdrawal of an approved drug from the market [47].Regarding this issue, it is believed that carbon-halogenbonds are not easily metabolized by the cytochrome p450system and, therefore, this is a feasible strategy to block themetabolically labile positions of a particular BioNCE.Following this line of reason, an interesting initiative wasdone by researchers from Pfizer Global Research &Development during the molecular optimization of ionicchannel vanilloid receptor 1 (TRPV1) antagonists, candi-dates to analgesic drugs. After the pharmacological screen-ing of seventeen congener sets, biarylcarboxybenzamide (3i)was disclosed to be a potent and selective antagonist of

TRPV1. However, because of the tert-butyl chain, prototype3i was thought to undergo oxidative metabolism, whicheventually could lead to non-selective or non-sedative metabolites. Aiming to avoid this, replacement of tert-butyl (3iby 1,1-dimethyl-2,2,2-trifluoroethyl (2) was performed (Fig6). This replacement was rationalized assuming that fluorineatoms could block the metabolism of the tert-butyl chain. Onthe basis of the pharmacological studies, the 1,1-dimethyl-

2,2,2-trifluoroethyl derivative (2) was equipotent as antagonistic agent of TRPV1 and more stable in human liver micro-somes than its prototype (3i) [55]. The synthetic method toachieve this kind of molecular modification has beenrecently improved, enabling its use on drug design [56]Consequently, 1,1-dimethyl-2,2,2-trifluoroethyl has assumedthe position of more stable bioisoster oftert-butyl groups.

THERMODYNAMIC VIEW OF HALOGENATEDLIGANDS

The understanding of biomolecular interactions in binarycomplexes of protein-ligand in current use are based on the

A

B

H2N

HN

NH

HN

NH

NH CO2H

O

O

O

O

O

R

OH

CO2H

NH2

NH2

4

4

2

FCl Br I

Cl Cl FCl

Cl

ClCl F

hexapeptides

1 2 34 5

6 7 8 9 10

Fig. (7).A) Comparison between bidding affinity (kd, M) and Gibbs free energy (G, kcal.mol-1

) for the compounds 1-10 [58]. For clarity

compound 6 (outlier) was excluded and the (dashed) line of tendency was added. B) Full structures of the compounds 1-10.


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model where binding affinity can be decomposed in termsthat reflect the various contributions to the binding [57].Most prominently, the binding affinity in such complexescan be estimated by building scoring functions to the Gibbsfree energy (G) of binding. As such, the mechanism bywhich halogen substituents contribute to the proteinligandbinding, in a thermodynamic view (G), remained unknownfor a long time. More recently, with the studies disclosed by

Memic and Spaller, this question started to be clarified [58].By measuring thermodynamic parameters (Gibbs free

energyG and enthalpyH) that are closely related to thebinding affinity (dissociation constant, Kd), Memic andSpaller explored a congener series of hexapeptides mono- orbi-halogenated (Fig. 7) that bind to the PDZ3 domain of themammalian neuronal protein (PSD-95) (PDB access code:1TP5). In comparison with the nonhalogenated hexapeptide,halogenations altered the values ofG in a significant orderof magnitude, confirming at first moment, whichthermodynamic is a relevant contributor to the bindingaffinity. Apart from a few outliers, it was observed that, ingeneral, the halogenated hexapeptides with lowest G scores(or conversely, most negative scores) also displayed highestprotein affinity (or conversely, lowest Kd values). The orderofKd values for the mono-halogenated and para-substitutedhexapeptides was: I >> Br > Cl > F and this order also wasfollowed for the G values. As expected, variations of thehalogen atom positions also influenced the Kd and Gvalues, but in these cases, steric hindrance effects was morepronounced than thermodynamic contribution. Most excitingwere the findings with the di-halogenated peptides. Althoughsensible for the position of the halogen, di-halogenatedpeptides led to more stable complexes (lowest G values)with subsequently improvement in the affinity, being the di-fluorinated peptides the most potent of them.

Although there were not all-or-nothing roles defined, two

interesting conclusions can be drawn based on these results.First, the influence of halogen substituents on biomolecularinteractions does not occur because of steric differencesalone, but also as a result of modification of thermodynamicparameters, being the G the best descriptor to correlate withprotein affinity. Second, in the course of a standard hit-to-lead optimization campaign, di-halogenation of hit com-pounds demands investigation, instead of just varying thenature or position of the halogens.

From these results, it is evident that thermodynamicconsiderations are closely correlated with the kind of halo-gen. However, in cases where halogens are attached inaliphatic carbons, there is the possibility of some kind ofcovalent reaction between the target and halogenated ligand.For example, nitrogen mustards are halogenated anticancerdrugs that bind to DNA, reacting covalently through ofnucleophilic substitution reactions [59]. Thus, in the absenceof crystal structure information of the binary protein-ligandcomplexes, thermodynamics (G) correlates with targetaffinity if the halogen atom is attached in aromatic rings.

HALOGEN BONDS: STRUCTURAL AND ELECTRO-NIC ASPECTS

In a recent review, Auffinger and coworkers [25], havediscussed that the short contacts involving halogen atoms,

originally called charge-transfer bonds, were attributed to thenegative charge transfer from oxygen, nitrogen or sulfuatoms (Lewis base) to a polarizable halogen atom (Lewisacid). Nowadays, these interactions are named halogenbonds, because of the similarity with typical H-bonds (Fig8). A number of structural surveys [60, 61], in addition to abinitio molecular orbital calculations, have established thathis interaction is essentially electrostatic, with contribution

from polarization, dispersion, and charge transfer. This waestimated in organic crystals, which the contribution of thehalogen bonds to the stabilization can vary from about haluntil slightly greater than a typical H-bond, depending on theenvironment [62].

Fig. (8). General representation of the halogen (X) bonds withseveral functional groups containing oxygen atoms, where Y can becarbon, phosphorous or sulfur. The geometrical aspects of thisinteraction are defined by the distance dx-o, the 1 angle of theoxygen relative to the C-X bond, and the 2 angle of the halogenrelative to the O-Y bond. In the left side, the lone pair of the oxygenatom is interacting with the halogen, while in the right side, the system of the double bond between O and Y is interacting with the

halogen atom.It is important to notice that dx-o distance must be equa

or less than the sum of respective van der Waals radii (3.27for ClO, 3.37 for BrO and 3.50 for IO). Thestrength of the interaction typically decreases in the follo-wing order I > Br > Cl. With respect to the 1 and 2 angle(Fig. 8), various structural surveys have statistically identified that the 1 angle is ideally near 180, typically greaterthan 140. For the 2 angle, there are two main possibilitiesi.e., about 120 when the interaction of the halogen atomsoccurs with the lone pair of the oxygen atom, and about 90which occurs when the interaction occurs with the systemof the double bond between O and Y.

Since the halogen bond interaction is essentially elec-trostatic, another important aspect is the electrostatic potential around the halogen atom. By using the methyl bromide(CH3Br) as an exemplification model (Fig. 9), one can see asmall positive electrostatic potential cap at the end region ofthe halogen atom along the CX axis. This anisotropic distribution of the electron density, forming the positive capwas named by Politzer and coworkers [63] as a sigma-hole. Lu and coworkers [64] have emphasized that halogenatoms could exhibit both electrophilic character along theaxes of the CX bonds, and nucleophilic character along thevectors that are perpendicular to these bonds. In this way, thehalogen atoms can form a halogen bond with nucleophiles


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(electronegative atoms like oxygen, for example), displayingroughly linear arrangement, but can also form a hydrogenbond with electrophiles (H-bond donors), occurring laterally.

Fig. (9). Ab initio electrostatic potential surfaces of methyl bromide

(b and d) in order to compare the induced negative (red), neutral

(green), and positive (blue) electrostatic potentials around the

halogen surface. The potential energies (a) are presented in the

89.9 to +105.9 kJ/mol range. A schematic representation (c) of the

positive cap was also included. The geometry was optimized at

the HF/6-31G(d) level and the electrostatic potential surface was

generated by mapping this property onto vdW surface by using the

program Spartan08 v1.0.0 (Wavefunction, Irvine, CA).

HALOGEN BONDS IN DRUG-TARGET COMPLEXES

As previously described, there are many requirements toaccommodate the geometry (length, angles and alignments)of halogen bonds. Regarding the drug-target complexes,formation of halogen bonds is supposed to be more sensitiveto steric factors when compared with H-bonds. Thisstatement was recently ascertained, measuring the catalyticactivity for one series of semisynthetic isomerases that bearshalogens instead of hydroxyl group in Tyr16-residue fromthe original isomerase. Having in hands these semisyntheticisomerases, they were evaluated through their ability toconvert 5-androstene-3,17-dione in 4-androstene-3,17-dione.In this model it was observed that all the halogenatedisomerases were of low capacity to promote theisomerization of 5-androstene-3,17-dione [65]. Althoughtheoretically feasible, there was no formation of halogenbond in these halogenated isomerases. Molecular modelingwith these halogenated isomerases-5-androstene-3,17-dionecomplexes was performed and showed that the active site,composed by a oxyanion hole, it was not enough flexible toaccommodate halogen bonds. This work may serve as basisto explain why the number of ligand-target complexes con-taining halogen bonds is relatively small. Obviously, the lackof computational approaches available to precisely predict

this kind of interaction within virtual screening is alsoresponsible, and will be further discussed.

Since the halogen bonds are quite well characterized bothby the structural and electronic points of view, it is worth toanalyze how accurate is the description of this specificinteraction, particularly during the application of the virtuascreening methods, such as molecular docking.

A recent study made by Lu and coworkers [66] conclu-ded that the unique features of the halogen bondingincluding the anisotropic distribution of the charge densityrepresents an important challenge for the current force fieldused in simulation (Molecular Dynamics, Monte Carlo andDocking, for example), because they are not ready to takethis interactions into account in a accurate way. Auffingerand coworkers [25] also concluded that current force fieldsshould be used with great caution when applied to halogenated compounds.

By focusing the molecular docking applications typicallyused to investigate drug-target complexes in medicinachemistry area, one can identify that the typical force fieldsused in molecular docking programs are, a priori, generally

neglecting the specific and unique characteristics of thehalogen bonding, when halogenated ligands (drugs) arepresent. If analyzed the scoring functions and programs formolecular docking [67, 68], it is clear that not a single one ofthe most popular softwares for molecular docking has, ontheir mathematical formalism, specific terms to take intoaccount the halogen bonds in an accurate fashionparticularly with the strong directional feature caused by theanisotropic effect (sigma-hole).

In a general way, disregarding the electrostatic termbased on typical Coulombs law for charged atoms, themajority of the scoring functions used in molecular dockingparticularly those based on force fields, are considering the

interaction between the non-bonded halogen (located in theligand or drug) and oxygen (located in the protein or target)atoms, for example, using van der Waals terms (EvdW)usually represented by a Lennard-Jones equation (seeEquation 1), where Aij and Bij are specific parameters thadepend on what atoms are interacting, and dij is thegeometrical distance between these atoms.

EvdW=

Aij

dij

12B

ij

dij

6

drugj

protein

i

(1)

Some force fields (Autodock, for example) consider aslight different term to compute hydrogen bonding, arepresented in Equation 2, where the power of the attractive(negative) term is increased from d6 to d10, besides theaddition of an angular weight factor (Et), that is important totake into account the directional character of the hydrogenbonding interactions.

EHB= E

t

Cij

dij

12D

ij

dij

10

drugj

protein

i

(2)

Therefore, it seems important to develop new and moreaccurate force fields capable to consider the halogen bondingeffects in a very precise way, taking into account all thespecific features like the strong directional dependence o


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such interaction caused by the anisotropic distribution of thecharge density.

CASE STUDIES

The next topics describe in detail recent observations ofpolar interactions involving halogens in drug-target comp-lexes and how these informations could be valuable forfurther design of drug candidates.

ANESTHETIC DRUGS

Prominent among volatile anesthetics are the class knowncollectively as polyhalogenated ethers (Fig. 10). The historyof their early development has been well summarized [69].The key mechanisms of actions of these polyhalogenatedanesthetics involve the functional modulation of ionicchannels and proteins on CNS implicated in the anestheticresponse. That is, they act through perturbations in theexistent allosteric regulatory sites of the ion channels [70,71]. As the ionic channels have transmembrane domains,crystallization of ionic channels in complex with anestheticsis hard to be performed. Consequently, studying proteinsinvolved in the anesthetic response have received an

immense amount of scientific attention.Regarding the polyhalogenated anesthetics, a curious

case is the polyhalogenated ether isoflurane. Isoflurane (Fig.10) is a chiral compound but used clinically as racemate. It isavailable in the market as racemate because the difference ofminimum alveolar anesthetic concentration values (MAC,the best descriptor of anesthetic potency) for it enantiomers,is minimal [72]. Although isoflurane reached the marketabout twenty years ago and has inspired the design of otherpotential anesthetics, its mechanism of action was onlyunderstood in recent years. The initial experiments disclosedthat isoflurane does not interact neither with glycine 1 noraminobutyric acid (GABA) receptors in a significant way[73]. Later on, studies have shown that isoflurane modulates

the immune responses, through interactions with leukocytetargets [74].

Taking the previous knowledge that some volatile anes-thetics are endowed with activity to suppress leukocyteaccumulation at sites of inflammation, Shimaoka and cowor-kers determined the inhibition kinetics of integrin lympho-cyte function-associated antigen-1 (LFA-1), the major leuko-cyte cell adhesion molecule, by isoflurane and presented thefirst high-resolution crystals of LFA-1 in uncomplexed formand LFA-1 co-crystallized in complex with isoflurane (S-enantiomer) [75, 76]. Nuclear magnetic resonance spectro-scopy revealed initially that isoflurane binds to LFA-1,

blocking the activation-dependent conformational conversion of LFA-1 at clinically relevant concentrations. Crystastructure of ligand-free LFA-1 is composed by threedomains, where two of them contain Mg2+ ions. The cavityof Mg2+ ion is characterized by a typical architecture calledof Metal Ion-Dependent Adhesion Site (MIDAS). LFA-1isoflurane complex was crystallized without Mg2+ and it wacomposed by three domains, where isoflurane waobserved to bind in two binding sites of these domains. Asexpected, isoflurane was found to bind the allosteric cavityof LFA-1, acting therefore as an allosteric antagonist. Ana

lysis of binding mode has given rise to the observation ointeresting interactions (Fig. 11). The CF3 subunit wa

A B

Fig. (11). A) Ribbon representation of the integrin lymphocytefunction-associated antigen-1(LFA-1, domain, PDB, access code3F74). B) Crystal structure of the LFA-1 bound to the Sstereoisomer isoflurane (stick model, colored by atom) (PDBaccess code 3F78). Dashed lines represent the polar interactionsbetween the residue Y307 (Tyr) and the fluorine atoms of the

isoflurane.accommodated on hydrophobic pocket of the LFA-1, whileanother pocket, located close to the E301 (Glu) residueaccommodates the chlorine. The chlorine and 1,1-difluoromethoxy establish polar interactions with the residues E301(Glu) and Y307 (Tyr), with respective measurements of 3.4and 3.2 . Based on the length that was apparently longerthan expected to halogen bonds, these authors hesitated tostate if these polar interactions are or not halogen bonds. Buapart from this, it is well plausible that the intermoleculabond between the difluoromethoxy moiety of the inhibitoand the hydroxyl of Y307 (Tyr), which was the strongest

Cl

FO F

F

F

F

F

O F

FClFF

FO

FF

F

FF

F

F

FO

FF

F

F

F

H

enflurane isofluranedesflurane sevoflurane

OCl

Cl FF

methoxyflurane

lower clogP higherclogP

Fig. (10). Structures of the most representative polyhalogenated ether anesthetics. The correlation of MeyerOverton indicates that potency(minimum alveolar anesthetic concentration, MIC) of a volatile anesthetic is directly proportional to it lipid solubility (clogP).


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polar interaction observed in this complex, is pivotal for thecoordination of isoflurane on LFA-1.

Moreover, Shimaoka and coworkers have noticed thatallosteric binding by isoflurane governs the conformationalstates of the MIDAS present in the LFA-1 structure, as it istypically expected for allosteric small molecules [76]. Assu-ming that the class of polyhalogenated ethers share similarstructures, these authors proposed that the allosteric binding

observed with isoflurane could be extended to othermembers of this class and, in fact, this generalization soundswell pertinent.

In the light of these interesting findings, it is conspicuousthat halogens play a pivotal role in the binding affinity ofanesthetic drugs rather than providing a simple contributionto increase the lipid solubility. If in the past century, struc-tural basis of anesthetic drugs was primarily based on theMeyerOverton correlation [77], in this century, significantinsights of the structural basis of volatile anesthetic, such asthe above disclosed example, will advance our capacity todrive this subject, from serendipity to prediction.

CYCLIN-DEPENDENT KINASE 2 INHIBITORS

Cyclin-dependent kinase 2 (CDK2) plays critical roles inimportant intracellular pathways and cell cycle progression,being thus an attractive biological target for drug develop-ment [78-80]. From the plethora of crystallographic struc-tures available for the complexes involving CDK2 and itsinhibitors, a large number of structural features important forhalogen binding could be inferred [81]. (Fig. 12) presents thestructure of flavopiridol (L86-8275; NSC 649890), a semi-synthetic inhibitor of CDK2 which is in clinical trials forcancer chemotherapy [82-85]. Although the crystal structurefor CDK2-flavopiridol is not available, the crystallographicstructure of human CDK2 in complex with a flavopiridol

analog, L86-8276 (called dechloroflavopiridol), provides thestructural basis for understanding the specificity and potencyof this inhibitor compared with its chlorinated form(flavopiridol). Especially interesting is the region of CDK2taken by the phenyl ring of the L86-8276 molecule that ispointing away from the ATP-binding pocket. This region isnot occupied by any part of the ATP molecule in the ATPcomplex but generates 10 van der Waals contacts to thephenyl ring of the inhibitor in the L86-8276 complex. Themain contact residues are Leu83, His84, and Asp86. Theposition of the phenyl ring of L86-8276 is also accountablefor the different position of the side chain of residue Lys89,which is moved away from the binding pocket in the L86-

8276 complex [86]. The flavopiridol molecule currently inclinical trials has a chlorophenyl instead of the phenyl in theL86-8276 molecule, and this change increases the kinaseinhibition by a factor of six [87-92]. This is most likely dueto the new potential contacts that the chlorine makes withresidues Leu10, Phe82, and Leu83, increasing the totanumber of contacts between flavopiridol and CDK2 to 61.

Considering that there are many reports where either

halogen bonds or polar contacts involving halogens wereobserved in binary complexes of CDK-ligand [81], the identification of such interactions seems to be of relevanceaiding chemistry toward CDK inhibitors of second-generation that, eventually, could show marked improvement.

FINAL REMARKS

Apart from the cases above discussed, there are otherbinary complexes with lysozyme [93], flavoprotein iodotyrosine deiodinase [94], taxane-tubulin binding pocket [95]DNA [96], serine protease factor Xa [97], and tyrosinekinases [98], where intermolecular interactions involvinghalogens are viewed as important interactions to the bindingaffinity, show us that the relevance of this subject is steadilyincreasing.

Here, we have tried to provide a guide to some of theeffects that the introduction of halogens can have on drugdevelopment. The capacity of halogen atoms to improve oraabsorption, BBB permeability, metabolic and chemical stability or improvements in potency exemplifies well the versatility of these atoms. Apart from these aspects, there areother ideas and applications that can emerge from the appropriated use of halogen atoms during the experience withvarious discovery technologies and optimization techniques.

More conspicuously, the possibility to establish halogenbonds or polar interactions may increase protein-ligand stability and subsequently, contributes to the binding affinityThereby, the identification of halogen bonds in binarycomplexes, as well as its in silico prediction, can provide noonly insights into the mechanism by which the ligandachieves specificity but also a greater understanding of thebinding pocket that will enable more selective drugcandidates to be discovered.

ACKNOWLEDGEMENTS

Work in our laboratories was funded by ConselhoNacional de Pesquisas (CNPq grant #479982/2008-2 to

O

N

OOH

HO

HO

O

N

OOH

HO

HOCl

hit-to-lead

conversion

L86-8276 flavopiridol (L86-8275)

Fig. (12). Hit-to-lead conversion resulting to flavopiridol, a potent CDK2 inhibitor which is under clinical trials.


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A.C.L.L. and #473699/2008-7 to M.Z.H), Programa deApoio aos Grupos de Excelncia (PRONEX grant #APQ-FACEPE-0981-1.06/08), Institutos Nacionais de Cincias eTecnologia (INCT in Dengue, Tuberculosis and InovaoFarmacutica), and Fundao de Cincias e Tecnologia doEstado de Pernambuco (FACEPE grant APQ-0123-4.03/08to A.C.L.L. and M.Z.H). W.F.A.Jr is senior researcher forCNPq. D.R.M.M. acknowledges support from CNPq by

providing his PhD fellowship, while S.M.T.C. acknowledgessupport from FACEPE by providing her MSc fellowship.

ABBREVIATIONS

ADME = Absorption, distribution, metabolism andexcretion

BioNCE = New bioactive chemical entity

BBB = Bloodbrain barrier

CNS = Central nervous system

H-bond = Hydrogen bond

HTS = High throughput screening

NP = Natural product

SAR = Structure-activity relationship

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Received: October 01, 2009 Revised: October 10, 2009 Accepted: October 20, 200

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