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Halogen Atoms in the Modern Medicinal

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 Current Drug Targets, 2010, 11, 00-00 1

Halogen Atoms in the Modern Medicinal Chemistry: Hints for the Drug
Design
Marcelo Zaldini Hernandes*,1, Suellen Melo T. Cavalcanti1, Diogo Rodrigo M. Moreira 1,2,
Walter Filgueira de Azevedo Junior 3 and Ana Cristina Lima Leite1,*

1
Departamento 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.
2
Programa de Ps-graduao em Qumica, Departamento de Qumica Fundamental, CCEN, UFPE, Rua Prof. Artur S
S/N, Cidade Universitria, 50740-540, Recife, PE, Brazil.
3
Laboratrio 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]. In
most cases, elucidating the intermolecular interactions in
these drug-target complexes was a pre-requisite to reach
success [3-5]. Although the premise that structurally similar
molecules have similar biological targets seems to be one
starting point of reasoning, there are violations on this
statement [6, 7]. Modern biochemical and analytical tools are
nowadays available to elucidate drug-target interactions, but
this process remains complex. In the light of this, studying
the earlier cases of drug discovery is an essential exercise for
those who work in the field of medicinal chemistry. In this
review 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 years
marine animals have demonstrated to be rich sources of
halogenated metabolites [8, 9], the occurrence of halogena-
ted NPs in plants is rare [10]. Thus, it would be expected that
halogenated drugs would have little importance in the drugs
*Address correspondence to this author at the Departamento de Cincias
Farmacuticas, UFPE, Rua Prof. Artur S S/N, Cidade Universitria, ZIP
CODE 50740-520, Recife, PE, Brazil; Phone: +055-81-2126-8511; Fax:
+055-81-2126-8510. Email: acllb2003@yahoo.com.br; zaldini@ufpe.br
scenario, but this is not the case, since halogenated drugs
have a prominent position. Of course they are, in their
majority, of synthetic origin. The paradoxical occurrence of
these halogenated drugs is the result from the usefulness of
halogen atoms.
One of the most popular hypotheses in the field of
medicinal chemistry is that the appropriate attachment of
bulk groups (bulky flanking groups) in BioNCEs, such as
halogen atoms, could lead to volumetric and conformational
changes. In other words, bulk groups tend to occupy all the
active site of molecular targets, including the deeper pockets
[11-15]. Thus, in diverse cases, inserting bulk groups into
BioNCEs can induce antagonistic or agonist effects in
comparison 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 conver-
sions [16-18]. Besides, the incorporation of halogen atoms
into BioNCE is commonly used to increase membrane per-
meability and, therefore, improve the oral absorption [19].
Likewise, halogenation also enhances the BBB permeability
and this is a pre-requisite for the drugs that need to reach the
CNS [20]. It is estimated that one quarter of the total number
of papers and patents closely related to medicinal chemistry
involve the insertion of halogens during the synthesis of final
compounds [21]. However, the applicability of these atoms
in drug design is still far from been completely exploited.
That is, most of SAR discussions of halogenated compounds

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

2 Current Drug Targets, 2010, Vol. 11, No. ?
have only considered the classical steric parameters, ignoring
other contributions and effects of such atoms. It was only in
recent years that the role of halogen atoms in proteinligand
complexes 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-1 pocket 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 fashion
that resembles the H-bonds. The core definitions of halogen
bonds have been reviewed by Metrangolo and co-workers
[22, 23] and will be summarized in this review. Now, it is
worth to say that in the case of self-assembly of organic
crystals, it is estimated that the stabilizing potential of typical
halogen bonds is of about half of an average H-bond [24].
Regarding the binary protein-ligand complexes, there are
many examples suggesting that formation of halogen bonds
are favorable electrostatic interactions. For instance, due to
the involvement of halogen bonds, Auffinger and co-workers
showed that halogenated ligands bind to the cyclin-depen-
dent kinase 2 (CDK2), leading to more stable complexes
than non-halogenated ligands [25]. In light of these and other
findings, identifying halogen bonds could be of great
relevance to explain the molecular recognition (pharmaco-
dynamics), as well as in other steps of the medicinal
chemistry, 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 the
prevalence of halogenated drugs, listing some particular
properties of the halogen atoms. Features related to identi-
fication of halogen bonds in proteinligand complexes, how
to calculate (prediction) and how these atoms should be
exploited are discussed. Recent results of this approach
applied to the design of optimized lead-compounds will also
be summarized.
PREVALENCE OF HALOGENATED DRUGS
Despite the substantial investigations in the field by
medicinal chemists, the number of halogenated drugs that
have really reached the market at the last twenty years is
slightly smaller than the expected [26-28]. There are some
hypotheses to explain it. The first reason may be that the
drug discovery is, in many cases, serendipitous. The second
reason is related to the NPs. NPs remain working as key
templates for the discovery of new BioNCEs and as source
of bio-inspiration for the planning of synthetic and
Hernandes et al.
semisynthetic drug candidates [29]. However, as mentioned
before, halogenated NPs from plants are really scarce. Syn-
thetic considerations have also limited the number of halo-
genated BioNCE. The halogenation of BioNCE is mostly
performed in aromatic rings, while the insertion of halogens
in aliphatic carbons is less abundant. This statement can be
confirmed through the scarce numbers of halogenated
BioNCEs from peptides, carbohydrates, steroids, and
polyether macrolides. This occurs, in part, because of the
synthesis to achieve the selective halogenation on these
scaffolds is relatively difficulty.
By analyzing (Fig. 2), we can see that the majority of
halogenated drugs are fluorine drugs, followed by chlorine
ones, while bromine is rare and the only iodine drug is the
thyroid hormone thyroxine. In fact, with the exception of the
iodine compounds, which are relative unstable (because C
I
bonds are highly polarizable) and of expansive synthesis
(and obviously avoided by the pharmaceutical industry), the
chemical processes for the synthesis of fluorine, chlorine,
and bromine compounds are well known in industrial scale
and of desirable stability and cost. Regarding the fluorine
drugs, there are some reasons that justify their predo-
minance. Fluorine is the most electronegative atom in the
periodic table and has a small atomic radius, along with low
polarizability [30]. The highly electronegative nature of fluo-
rine renders it a poor halogen bond acceptor character, but
enables it to receive hydrogen bonds from H-bond donors
[31]. These chemical characteristics imply that the fluorina-
tion of BioNCE alters physical, chemical, electronic, and
conformational parameters, which eventually could result to
drugs with optimized pharmacological properties. In a more
detailed view, Biffinger and co-workers have adopted the
term polar hydrophobicity to describe the phenomenon in
which fluorinated fragments are less able to engage in
dispersion-based interactions with aqueous solvent than alkyl
or 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 clinical
use were excluded during this search.
In the medicinal chemistry, fluorine is generally viewed
as classic bioisosters of hydrogen and methyl [31]. One
illustration of this statement is the classical exchanging of
Halogen Atoms in the Modern Medicinal Chemistry Current Drug Targets, 2010, Vol. 11, No. ? 3

OH
O O O
biological OPO32-
H bioisosteric F H F
N exchange N metabolisation N O

N O N O N O
H H H 5-fluoro-dUMP
Uracile (Inhibitor of the
5-fluorouracil (5-FU)
thymidylate synthase)

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

hydrogen by fluorine on the purine base uracile, resulting to of insignificant affinity for 1-adrenergic,
adrenergic,
the anticancer drug 5-fluorouracil (5-FU) (Fig. 3). This drug serotonergic, muscarinic, and histaminic receptors [40, 41].
is 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 role O N
for the pyrimidine biosynthesis [33, 34]. Thereby, the fluo-
rine chemistry provides good opportunities for enhancing the Cl
N
binding affinity of potential drug candidates. These features H
have made the trifluoromethyl (
CF3) [35] and pentafluoro- Clebopride
H2N O
sulfanyl (
SF5) [36] useful chemical groups in the contem-
porary drug design. Another interesting issue is that incorpo-
O
rating fluorinated functionality into endogenous substrates or N
ligands through 19F-markers is a powerful technique to probe Cl
N
protein functions and to study their catalytic cycle, consti- H
tuting an essential tool for the chemical biology [37]. N O
H Nemonapride
Chlorine is also of noticeable prevalence and deserves
more discussion. Chlorine occupies an intermediate position O
on the halogen series. Different from fluorine, chlorine is a N
Cl
moderate halogen bond acceptor, besides being larger in size O N
than fluorine. The C
Cl bond is enough stable, allowing its H
YM-43611
insertion on diverse heterocyclics of pharmacological value. N O
Another feature of chlorine drugs regards to its binding H
O
affinity. Replacing hydrogen by chlorine also provides a O
substantial alteration on the volumetric and shape issues. In N
the light of these features, chlorination of BioNCEs could Cl
O N
eventually result to BioNCEs that are best accommodated on H
the active site of targets in comparison with the non- YM-50001
N O
chlorinated BioNCEs. It was also described that subunits H
bearing chlorine can be accommodated in tight and deep
cavities, as well as in hydrophobic pockets of the biological Fig. (4). Chlorobenzamides as dopaminergic antagonists. Dashed
targets [38]. lines are delineating the molecular motifs in common. From these
The following examples illustrate a somewhat more res- cases, removing the chlorine is deleterious for the selectivity in the
trictive aspect of how chlorine could crucial during molecu- DOPA-receptors.
lar recognition. Nemonapride was found to be a potent dopa-
minergic receptor ligand. Although Nemonapride has not PERMEABILITY, TOXICITY AND METABOLISM
showed affinity to other targets (muscarinic,
adrenergic, OF HALOGENATED DRUGS
histaminic), a lack of selectivity among the subtypes (D1,
D2, D3, D4) was observed [39]. To overcome this low selec- According to Kubinyis studies, inappropriate ADME is
tivity over the D4 receptor, a number of molecular modi- not the major reason for failure in drug development, being
fications were performed in the Nemonapride. During the animal toxicity and inefficacy the major problems (Fig. 5)
step of molecular simplification, it was observed that the 1- [42]. Apart from the pharmacological efficacy, which dep-
chloro-2-methoxylbenzamide constitutes a ubiquitous func- ends on the therapeutically application, ADME and toxicity
tion-determining domain, i.e., the minimal central scaffold. can be measured independently of the pharmaceutical class.
The removal of the chlorine was deleterious for the That is, theoretical predictions of oral permeability and BBB
selectivity among the dopaminergic receptors. Thereby, penetration are fast achieved and of good accuracy. Analysis
preserving this central scaffold, a number of molecular of Lipinksis rule of five [43], the polar surface area [44],
modifications were performed, which allowed the discovery molecular flexibility [45] and VolSurf parameters [46] are
of novel and selective dopaminergic receptor ligands (Fig. examples of useful tools to predict the oral permeability.
4), being YM50001 a selective ligand for D4 versus D2 and
4 Current Drug Targets, 2010, Vol. 11, No. ?
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 coefficients
have been usually employed for rapid and accurate assessing
of lipid solubility, while the drug toxicity potential is
measurable using in vitro cellbased assays, albeit with some
limitations [47]. In the chemical point of view, there are
many molecular modifications that could eventually increase
the intestinal permeability of BioNCE. In particular, it was
reported that inserting halogens in BioNCE increases the
lipophilicity, with subsequently improvement of oral
permeability and CNS penetration [19, 20]. This logic was
initially confirmed by Gentry and co-workers [20] during the
conception of new halogenated enkephalin analogs, proto-
types of
opioid analgesic agents. Through the appropriate
attachment of chlorine, bromine, fluorine, or iodine at the
para-position of the Phe-4 residue on the prototype Tyr-D-
Pen-Gly-Phe-L-Pen-Phe, it was possible to investigate the
effects of those halogenations on the in vitro BBB permea-
Blocking the
metabolism
CF3
1,1-dimethyl-2,2,2-trifluoroethyl
TRVR1 antagonist ligands
N Assay of 45Ca2+ influx
Cl
Fig. (6). Bioisosteric replacement of tert-butyl (3i) by 1,1-dimethyl-2,2,2-trifluoroethyl (2) towards the design of more stable and equipotent
analogue.
Hernandes et al.
bility. Except for the fluorine, enhancements of in vitro per-
meability in bovine brain microvessel endothelial cells were
observed in all the other halogenated peptides, and this
tendency was also accompanied by improvement in the
octanol/saline partition coefficient. Later on, more accurate
studies also corroborated these statements, noticing that this
strategy is also valid for BioNCEs of nonpeptidic nature
[48].
The task of rationally designing a chemistry strategy for
circumventing a limiting toxicity can be frustrating. Because
of this status and with the great advent of HTS and
combinatorial chemistry, in silico filtering techniques (the
so-called garbage filters) to eliminate undesirable atoms or
fragments endowed of intrinsic toxicity in potential are hot
topics that have called much attention of the medicinal
chemists [49-51]. However, the toxic potential of a drug
depends on various factors that are very far from being fitted
in just a few predictive models. Nitro group is an example of
fragment that is frequently considered as undesirable frag-
ment by these garbage filters. Actually, nitrated BioNCEs
frequently display more pronounced toxicity than nonnitra-
ted BioNCEs, because the nitro group undergoes biological
reduction, leading to the production of toxic metabolites [52,
53]. While this statement is certainly true, at least for some
nitro-drugs, sweeping generalizations such this can also be
misleading. As stated by Kubinyi, if such prediction tools
are nevertheless applied, they should only be used to score
groups of compounds, not to decide the fate of individual
candidates [42].
In light of these observations, we have tried to summa-
rize information regarding the toxicity of halogenated drugs.
Proudfoot has provided an excellent analysis of the launched
drugs in recent years, listing each drug and its respective lead
compound [54]. Taking this work as guide, we analyzed,
point-by-point, all the cases in which halogenated lead com-
pounds were explored as potential candidates during the step
of lead-to-drug conversion. In only two cases, halogenated
lead compounds were excluded during this process because
Rapid
conversion into
non-selective O
metabolit
OH
tert-butyl product of oxidative metabolism
CF3
N Analogue 2
H
Prototype 3i Antagonist (IC50=42nM)
Assay of 45Ca2+ influx Clearance: 48mL\min.\kg
Antagonist (IC50=37nM)
Clearance: 168mL\min.\kg
Halogen Atoms in the Modern Medicinal Chemistry Current Drug Targets, 2010, Vol. 11, No. ? 5

of toxicity problems, being inefficacy the real reason of
failure. From these analyses, there is not evidence that
halogen 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 specific
drug candidate during clinical test. Conversely, hepatocytes
have a central role in the metabolism of xenobiotics, being
drug-induced liver injury the most frequent reason for the
withdrawal of an approved drug from the market [47].
Regarding this issue, it is believed that carbon-halogen
bonds are not easily metabolized by the cytochrome p450
system and, therefore, this is a feasible strategy to block the
metabolically labile positions of a particular BioNCE.
Following this line of reason, an interesting initiative was
done by researchers from Pfizer Global Research &
Development during the molecular optimization of ionic
channel 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, prototype
3i was thought to undergo oxidative metabolism, which
eventually could lead to non-selective or non-sedative meta-
bolites. Aiming to avoid this, replacement of tert-butyl (3i)
by 1,1-dimethyl-2,2,2-trifluoroethyl (2) was performed (Fig.
6). This replacement was rationalized assuming that fluorine
atoms could block the metabolism of the tert-butyl chain. On
the basis of the pharmacological studies, the 1,1-dimethyl-
2,2,2-trifluoroethyl derivative (2) was equipotent as antago-
nistic agent of TRPV1 and more stable in human liver micro-
somes than its prototype (3i) [55]. The synthetic method to
achieve this kind of molecular modification has been
recently improved, enabling its use on drug design [56].
Consequently, 1,1-dimethyl-2,2,2-trifluoroethyl has assumed
the position of more stable bioisoster of tert-butyl groups.
THERMODYNAMIC VIEW OF HALOGENATED
LIGANDS
The understanding of biomolecular interactions in binary
complexes of protein-ligand in current use are based on the

A
NH2 CO2H R
O O
4 2
H H
N N NH CO2H
H2N N N
H H
hexapeptides O 4
O O
NH2 OH

F Cl Br I

2 4 5
1 3
Cl Cl F
Cl Cl Cl Cl F

6 7 8 10
9
B
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.
6 Current Drug Targets, 2010, Vol. 11, No. ?
model where binding affinity can be decomposed in terms
that reflect the various contributions to the binding [57].
Most prominently, the binding affinity in such complexes
can be estimated by building scoring functions to the Gibbs
free energy (
G) of binding. As such, the mechanism by
which halogen substituents contribute to the proteinligand
binding, in a thermodynamic view (
G), remained unknown
for 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
energy
G and enthalpy
H) that are closely related to the
binding affinity (dissociation constant, Kd), Memic and
Spaller explored a congener series of hexapeptides mono- or
bi-halogenated (Fig. 7) that bind to the PDZ3 domain of the
mammalian neuronal protein (PSD-95) (PDB access code:
1TP5). In comparison with the nonhalogenated hexapeptide,
halogenations altered the values of
G in a significant order
of magnitude, confirming at first moment, which
thermodynamic is a relevant contributor to the binding
affinity. Apart from a few outliers, it was observed that, in
general, the halogenated hexapeptides with lowest
G scores
(or conversely, most negative scores) also displayed highest
protein affinity (or conversely, lowest Kd values). The order
of Kd values for the mono-halogenated and para-substituted
hexapeptides was: I >> Br > Cl > F and this order also was
followed for the
G values. As expected, variations of the
halogen atom positions also influenced the Kd and
G
values, but in these cases, steric hindrance effects was more
pronounced than thermodynamic contribution. Most exciting
were the findings with the di-halogenated peptides. Although
sensible for the position of the halogen, di-halogenated
peptides 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 biomolecular
interactions does not occur because of steric differences
alone, but also as a result of modification of thermodynamic
parameters, being the
G the best descriptor to correlate with
protein 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 the
nature or position of the halogens.
From these results, it is evident that thermodynamic
considerations are closely correlated with the kind of halo-
gen. However, in cases where halogens are attached in
aliphatic carbons, there is the possibility of some kind of
covalent reaction between the target and halogenated ligand.
For example, nitrogen mustards are halogenated anticancer
drugs that bind to DNA, reacting covalently through of
nucleophilic substitution reactions [59]. Thus, in the absence
of crystal structure information of the binary protein-ligand
complexes, thermodynamics (
G) correlates with target
affinity if the halogen atom is attached in aromatic rings.
HALOGEN BONDS: STRUCTURAL AND ELECTRO-
NIC ASPECTS
In a recent review, Auffinger and coworkers [25], have
discussed that the short contacts involving halogen atoms,
Hernandes et al.
originally called charge-transfer bonds, were attributed to the
negative charge transfer from oxygen, nitrogen or sulfur
atoms (Lewis base) to a polarizable halogen atom (Lewis
acid). Nowadays, these interactions are named halogen
bonds, because of the similarity with typical H-bonds (Fig.
8). A number of structural surveys [60, 61], in addition to ab
initio molecular orbital calculations, have established that
this interaction is essentially electrostatic, with contributions
from polarization, dispersion, and charge transfer. This was
estimated in organic crystals, which the contribution of the
halogen bonds to the stabilization can vary from about half
until slightly greater than a typical H-bond, depending on the
environment [62].
Fig. (8). General representation of the halogen (X) bonds with
several functional groups containing oxygen atoms, where Y can be
carbon, phosphorous or sulfur. The geometrical aspects of this
interaction are defined by the distance dx-o, the
1 angle of the
oxygen relative to the C-X bond, and the
2 angle of the halogen
relative to the O-Y bond. In the left side, the lone pair of the oxygen
atom 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 equal
or less than the sum of respective van der Waals radii (3.27
for ClO, 3.37 for BrO and 3.50 for IO). The
strength of the interaction typically decreases in the follo-
wing order I > Br > Cl. With respect to the
1 and
2 angles
(Fig. 8), various structural surveys have statistically iden-
tified that the
1 angle is ideally near 180, typically greater
than 140. For the
2 angle, there are two main possibilities,
i.e., about 120 when the interaction of the halogen atoms
occurs with the lone pair of the oxygen atom, and about 90
which occurs when the interaction occurs with the  system
of the double bond between O and Y.
Since the halogen bond interaction is essentially elec-
trostatic, another important aspect is the electrostatic poten-
tial around the halogen atom. By using the methyl bromide
(CH3Br) as an exemplification model (Fig. 9), one can see a
small positive electrostatic potential cap at the end region of
the halogen atom along the CX axis. This anisotropic distri-
bution of the electron density, forming the positive cap,
was named by Politzer and coworkers [63] as a sigma-
hole. Lu and coworkers [64] have emphasized that halogen
atoms could exhibit both electrophilic character along the
axes of the CX bonds, and nucleophilic character along the
vectors that are perpendicular to these bonds. In this way, the
halogen atoms can form a halogen bond with nucleophiles
Halogen Atoms in the Modern Medicinal Chemistry
(electronegative atoms like oxygen, for example), displaying
roughly linear arrangement, but can also form a hydrogen
bond 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 to
accommodate the geometry (length, angles and alignments)
of halogen bonds. Regarding the drug-target complexes,
formation of halogen bonds is supposed to be more sensitive
to steric factors when compared with H-bonds. This
statement was recently ascertained, measuring the catalytic
activity for one series of semisynthetic isomerases that bears
halogens instead of hydroxyl group in Tyr16-residue from
the original isomerase. Having in hands these semisynthetic
isomerases, they were evaluated through their ability to
convert 5-androstene-3,17-dione in 4-androstene-3,17-dione.
In this model it was observed that all the halogenated
isomerases were of low capacity to promote the
isomerization of 5-androstene-3,17-dione [65]. Although
theoretically feasible, there was no formation of halogen
bond in these halogenated isomerases. Molecular modeling
with these halogenated isomerases-5-androstene-3,17-dione
complexes was performed and showed that the active site,
composed by a oxyanion hole, it was not enough flexible to
accommodate halogen bonds. This work may serve as basis
to explain why the number of ligand-target complexes con-
taining halogen bonds is relatively small. Obviously, the lack
of computational approaches available to precisely predict
Current Drug Targets, 2010, Vol. 11, No. ? 7
this kind of interaction within virtual screening is also
responsible, and will be further discussed.
Since the halogen bonds are quite well characterized both
by the structural and electronic points of view, it is worth to
analyze how accurate is the description of this specific
interaction, particularly during the application of the virtual
screening methods, such as molecular docking.
A recent study made by Lu and coworkers [66] conclu-
ded that the unique features of the halogen bonding,
including the anisotropic distribution of the charge density,
represents an important challenge for the current force fields
used in simulation (Molecular Dynamics, Monte Carlo and
Docking, for example), because they are not ready to take
this interactions into account in a accurate way. Auffinger
and coworkers [25] also concluded that current force fields
should be used with great caution when applied to halo-
genated compounds.
By focusing the molecular docking applications typically
used to investigate drug-target complexes in medicinal
chemistry area, one can identify that the typical force fields
used in molecular docking programs are, a priori, generally
neglecting the specific and unique characteristics of the
halogen bonding, when halogenated ligands (drugs) are
present. If analyzed the scoring functions and programs for
molecular docking [67, 68], it is clear that not a single one of
the most popular softwares for molecular docking has, on
their mathematical formalism, specific terms to take into
account the halogen bonds in an accurate fashion,
particularly with the strong directional feature caused by the
anisotropic effect (sigma-hole).
In a general way, disregarding the electrostatic term
based on typical Coulombs law for charged atoms, the
majority of the scoring functions used in molecular docking,
particularly those based on force fields, are considering the
interaction between the non-bonded halogen (located in the
ligand 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 (see
Equation 1), where Aij and Bij are specific parameters that
depend on what atoms are interacting, and dij is the
geometrical distance between these atoms.
A Bij 
 ij
EvdW =    d 12


dij6 
(1)
protein drug
i j
 ij
Some force fields (Autodock, for example) consider a
slight different term to compute hydrogen bonding, as
represented in Equation 2, where the power of the attractive
(negative) term is increased from d6 to d10, besides the
addition of an angular weight factor (Et), that is important to
take into account the directional character of the hydrogen
bonding interactions.
C Dij 
 ij
E HB =   E  d t 12


dij10 
(2)
protein drug
i j
 ij
Therefore, it seems important to develop new and more
accurate force fields capable to consider the halogen bonding
effects in a very precise way, taking into account all the
specific features like the strong directional dependence of
8 Current Drug Targets, 2010, Vol. 11, No. ?
F
F
F F F F
F F
O F O F
F F F F
desflurane sevoflurane
lower clogP
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).
such interaction caused by the anisotropic distribution of the
charge density.
CASE STUDIES
The next topics describe in detail recent observations of
polar interactions involving halogens in drug-target comp-
lexes and how these informations could be valuable for
further design of drug candidates.
ANESTHETIC DRUGS
Prominent among volatile anesthetics are the class known
collectively as polyhalogenated ethers (Fig. 10). The history
of their early development has been well summarized [69].
The key mechanisms of actions of these polyhalogenated
anesthetics involve the functional modulation of ionic
channels and proteins on CNS implicated in the anesthetic
response. That is, they act through perturbations in the
existent allosteric regulatory sites of the ion channels [70,
71]. As the ionic channels have transmembrane domains,
crystallization of ionic channels in complex with anesthetics
is hard to be performed. Consequently, studying proteins
involved 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 is
available in the market as racemate because the difference of
minimum alveolar anesthetic concentration values (MAC,
the best descriptor of anesthetic potency) for it enantiomers,
is minimal [72]. Although isoflurane reached the market
about twenty years ago and has inspired the design of other
potential anesthetics, its mechanism of action was only
understood in recent years. The initial experiments disclosed
that isoflurane does not interact neither with glycine 1 nor
aminobutyric acid (GABA) receptors in a significant way
[73]. Later on, studies have shown that isoflurane modulates
the immune responses, through interactions with leukocyte
targets [74].
Taking the previous knowledge that some volatile anes-
thetics are endowed with activity to suppress leukocyte
accumulation 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 the
first high-resolution crystals of LFA-1 in uncomplexed form
and 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,
Hernandes et al.
F Cl F
F F Cl F
F F F
O F O F
H Cl F F Cl O
enflurane isoflurane methoxyflurane
higher clogP
blocking the activation-dependent conformational conver-
sion of LFA-1 at clinically relevant concentrations. Crystal
structure of ligand-free LFA-1 is composed by three

domains, where two of them contain Mg2+ ions. The cavity
of Mg2+ ion is characterized by a typical architecture called
of Metal Ion-Dependent Adhesion Site (MIDAS). LFA-1
isoflurane complex was crystallized without Mg2+ and it was
composed by three
domains, where isoflurane was
observed to bind in two binding sites of these domains. As
expected, isoflurane was found to bind the allosteric cavity
of LFA-1, acting therefore as an allosteric antagonist. Ana-
lysis of binding mode has given rise to the observation of
interesting interactions (Fig. 11). The CF3 subunit was
A B
Fig. (11). A) Ribbon representation of the integrin lymphocyte
function-associated antigen-1 (LFA-1,
domain, PDB, access code
3F74). B) Crystal structure of the LFA-1 bound to the S-
stereoisomer isoflurane (stick model, colored by atom) (PDB,
access code 3F78). Dashed lines represent the polar interactions
between the residue Y307 (Tyr) and the fluorine atoms of the
isoflurane.
accommodated on hydrophobic pocket of the LFA-1, while
another pocket, located close to the E301 (Glu) residue,
accommodates the chlorine. The chlorine and 1,1-difluoro-
methoxy establish polar interactions with the residues E301
(Glu) and Y307 (Tyr), with respective measurements of 3.4
and 3.2 . Based on the length that was apparently longer
than expected to halogen bonds, these authors hesitated to
state if these polar interactions are or not halogen bonds. But
apart from this, it is well plausible that the intermolecular
bond between the difluoromethoxy moiety of the inhibitor
and the hydroxyl of Y307 (Tyr), which was the strongest
Halogen Atoms in the Modern Medicinal Chemistry
OH O OH
hit-to-lead
conversion
HO O HO
HO
N N
L86-8276
Fig. (12). Hit-to-lead conversion resulting to flavopiridol, a potent CDK2 inhibitor which is under clinical trials.
polar interaction observed in this complex, is pivotal for the
coordination of isoflurane on LFA-1.
Moreover, Shimaoka and coworkers have noticed that
allosteric binding by isoflurane governs the conformational
states of the MIDAS present in the LFA-1 structure, as it is
typically expected for allosteric small molecules [76]. Assu-
ming that the class of polyhalogenated ethers share similar
structures, these authors proposed that the allosteric binding
observed with isoflurane could be extended to other
members of this class and, in fact, this generalization sounds
well pertinent.
In the light of these interesting findings, it is conspicuous
that halogens play a pivotal role in the binding affinity of
anesthetic drugs rather than providing a simple contribution
to increase the lipid solubility. If in the past century, struc-
tural basis of anesthetic drugs was primarily based on the
MeyerOverton correlation [77], in this century, significant
insights of the structural basis of volatile anesthetic, such as
the above disclosed example, will advance our capacity to
drive this subject, from serendipity to prediction.
CYCLIN-DEPENDENT KINASE 2 INHIBITORS
Cyclin-dependent kinase 2 (CDK2) plays critical roles in
important 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 its
inhibitors, a large number of structural features important for
halogen binding could be inferred [81]. (Fig. 12) presents the
structure of flavopiridol (L86-8275; NSC 649890), a semi-
synthetic inhibitor of CDK2 which is in clinical trials for
cancer chemotherapy [82-85]. Although the crystal structure
for CDK2-flavopiridol is not available, the crystallographic
structure of human CDK2 in complex with a flavopiridol
analog, L86-8276 (called dechloroflavopiridol), provides the
structural basis for understanding the specificity and potency
of this inhibitor compared with its chlorinated form
(flavopiridol). Especially interesting is the region of CDK2
taken by the phenyl ring of the L86-8276 molecule that is
pointing away from the ATP-binding pocket. This region is
not occupied by any part of the ATP molecule in the ATP
complex but generates 10 van der Waals contacts to the
phenyl ring of the inhibitor in the L86-8276 complex. The
main contact residues are Leu83, His84, and Asp86. The
position of the phenyl ring of L86-8276 is also accountable
for the different position of the side chain of residue Lys89,
which is moved away from the binding pocket in the L86-
Current Drug Targets, 2010, Vol. 11, No. ? 9
O
O
HO
Cl
flavopiridol (L86-8275)
8276 complex [86]. The flavopiridol molecule currently in
clinical trials has a chlorophenyl instead of the phenyl in the
L86-8276 molecule, and this change increases the kinase
inhibition by a factor of six [87-92]. This is most likely due
to the new potential contacts that the chlorine makes with
residues Leu10, Phe82, and Leu83, increasing the total
number of contacts between flavopiridol and CDK2 to 61.
Considering that there are many reports where either
halogen bonds or polar contacts involving halogens were
observed in binary complexes of CDK-ligand [81], the iden-
tification of such interactions seems to be of relevance,
aiding chemistry toward CDK inhibitors of second-genera-
tion that, eventually, could show marked improvement.
FINAL REMARKS
Apart from the cases above discussed, there are other
binary complexes with lysozyme [93], flavoprotein iodotyro-
sine deiodinase [94], taxane-tubulin binding pocket [95],
DNA [96], serine protease factor Xa [97], and tyrosine
kinases [98], where intermolecular interactions involving
halogens are viewed as important interactions to the binding
affinity, show us that the relevance of this subject is steadily
increasing.
Here, we have tried to provide a guide to some of the
effects that the introduction of halogens can have on drug
development. The capacity of halogen atoms to improve oral
absorption, BBB permeability, metabolic and chemical sta-
bility or improvements in potency exemplifies well the ver-
satility of these atoms. Apart from these aspects, there are
other ideas and applications that can emerge from the appro-
priated use of halogen atoms during the experience with
various discovery technologies and optimization techniques.
More conspicuously, the possibility to establish halogen
bonds or polar interactions may increase protein-ligand sta-
bility and subsequently, contributes to the binding affinity.
Thereby, the identification of halogen bonds in binary
complexes, as well as its in silico prediction, can provide not
only insights into the mechanism by which the ligand
achieves specificity but also a greater understanding of the
binding pocket that will enable more selective drug
candidates to be discovered.
ACKNOWLEDGEMENTS
Work in our laboratories was funded by Conselho
Nacional de Pesquisas (CNPq grant #479982/2008-2 to
10 Current Drug Targets, 2010, Vol. 11, No. ?
A.C.L.L. and #473699/2008-7 to M.Z.H), Programa de
Apoio aos Grupos de Excelncia (PRONEX grant #APQ-
FACEPE-0981-1.06/08), Institutos Nacionais de Cincias e
Tecnologia (INCT in Dengue, Tuberculosis and Inovao
Farmacutica), and Fundao de Cincias e Tecnologia do
Estado de Pernambuco (FACEPE grant APQ-0123-4.03/08
to A.C.L.L. and M.Z.H). W.F.A.Jr is senior researcher for
CNPq. D.R.M.M. acknowledges support from CNPq by
providing his PhD fellowship, while S.M.T.C. acknowledges
support from FACEPE by providing her MSc fellowship.
ABBREVIATIONS
ADME = Absorption, distribution, metabolism and
excretion
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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Accepted: October 20, 2009