Halogenated Antimicrobial Agents To Combat Drug

Pharmrev Fast Forward. Published on 16 October 2023 as DOI 10.1124/pharmrev.123.
000863 This
article has not been copyedited and formatted. The final version may differ from this version.
Halogenated Antimicrobial Agents to Combat Drug-Resistant Pathogens
Olajide Sunday Faleye§,1, Bharath Reddy Boya§,1, Jin-Hyung Lee1, Inho Choi2, and Jintae
Lee1,*
1
School of Chemical Engineering and 2Department of Medical Biotechnology, Yeungnam
University, 280 Daehak-Ro,
Gyeongsan, 38541, Republic of Korea (O.S.F., B.R.B., J-H.L., I.C., and J.L.)
§
These authors contributed equally to this work as first authors
*Corresponding Author
Running title: Halogenated Antimicrobial Agents
Address correspondence to
Jintae Lee, School of Chemical Engineering, Yeungnam University
Downloaded from pharmrev.aspetjournals.org at ASPET Journals on January 9, 2024

280 Daehak-Ro, Gyeongsan, Gyeongbuk, 38541, Republic of Korea
E-mail: [email protected]. Tel.: +82-53-810-2533. Fax: +82-53-810-4631
Number of text pages: 90
Number of tables: 1
Number of figures: 21
Number of references: 388
Number of words in the Abstract: 149
1
Pharmrev Fast Forward. Published on 16 October 2023 as DOI 10.1124/pharmrev.123.000863 This
Contents
Abstract
Significance Statement
I. Introduction
II. Halogenated Antibiotic Classes
A. Cell Wall Synthesis Inhibitors (Penicillins, Lipopeptides, Cephalosporins, and
Glycopeptides)
B. Cell Proliferation Inhibitors (Sulfonamides, Trimethoprim, and Quinolones)
C. Protein Synthesis Inhibitors (Aminoglycosides, Tetracyclines, Macrolides,
Lincosamides, and Oxazolidinones)
III. Halogenated Antimicrobial Scaffolds
A. Polyphenolic Compounds
1. Catechols
2. Magnolols

3. Resveratrols
4. Flavonoids
B. Essential Oil Compounds
1. Thymols
2. Cinnamaldehydes
C. Alkaloids
1. Indoles
2. Streptochlorins
3. Quinones and Quinolones
D. Benzopyrones and Phenazines
2
1. Coumarins
2. Phenazines
E. Azoles
F. Repurposed scaffolds
1. Isoniazids
2. Azo-aspirins
3. Haloperidols
4. Pyrrolopyrimidines
5. Phenothiazines
6. Salicylanilides
G. Other Halogenated Bioactive Compounds
1. β-Nitrostyrene and Nitrovinylfurans
2. 2-Aminothiopenes
3. Isophthalonitriles

4. β-Amino amides and Alboflavusins
IV. Halogenated polymerics
V. Halogenated Biomolecules
A. Peptides
B. Carbohydrates
C. Lipids
VI. Effects of Halogenation and Factors Affecting the Antimicrobial Activities
A. Halogen Bonding, Pharmacokinetics and Pharmacodynamics
B. Halogenated Agents and Gut Microbiota
C. Factors Affecting Antimicrobial Activities.
1. Sizes and Substitution Pattern

3
2. Hydrophobicity and Lipophilicity
3. Physicochemical and Biophysical Properties
4. Bacterial Cell Types
VII. Halogenation for Antibiotic Rejuvenation and Resistance Minimization
VIII. Current Practices and Prospects
IX. Conclusions
X. References
XI. Acknowledgements
Abbreviations
ADME- Adsorption, distribution, metabolism, and elimination
AHM- 2-Amino-5-(2-hydroxyethyl)-6-methyl pyrimidine-4-one
AMP- Antimicrobial peptide

AMR- Antimicrobial resistance
ATP- Adenosine triphosphate
CNMA- Cinnamaldehyde
DNA- Deoxyribo nucleic acid
ESKAPE- Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
FA- Fatty acids
FDA- Food and Drug Administration
HIV- Human immunodeficiency virus

4
HTCC- N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride
MDR- Multidrug resistant
MIC- Minimum inhibitory concentration
MOA- Mode of action
MRSA- Methicillin-resistant Staphylococcus aureus
MRSE- Methicillin-resistant Staphylococcus epidermis
NADH- Nicotinamide adenine dinucleotide hydrogen
NDH-2- Type II NADH dehydrogenase
PABA- Para-aminobenzoic acid
PBP- Penicillin binding proteins
PVP-I- Povidone iodine

QS- Quorum sensing
RHS- Reactive halogen species
RNA- Ribonucleic acid
ROS- Reactive oxygen species
SAR- Structure activity relationship
VRE- Vancomycin-resistant enterococci
5
Abstract
Antimicrobial resistance presents us with a potential global crisis as it undermines the
abilities of conventional antibiotics to combat pathogenic microbes. The history of
antimicrobial agents is replete with examples of scaffolds containing halogens. In this review,
we discuss the impacts of halogen atoms in various antibiotic types and antimicrobial
scaffolds and their modes of action, structure-activity relationships, and the contributions of
halogen atoms in antimicrobial activity and drug resistance. Other halogenated molecules,
including carbohydrates, peptides, lipids, and polymeric complexes, are also reviewed, and
the effects of halogenated scaffolds on pharmacokinetics, pharmacodynamics, and factors
affecting antimicrobial and antivirulence activities are presented. Furthermore, the potential
of halogenation to circumvent antimicrobial resistance and rejuvenate impotent antibiotics is
addressed. This review provides an overview of the significance of halogenation, the abilities
of halogens to interact in biomolecular settings and enhance pharmacological properties, and
their potential therapeutic usages in preventing a post-antibiotic era.

Significance Statement
Antimicrobial resistance and the increasing impotence of antibiotics are critical
threats to global health. The roles and importance of halogen atoms in antimicrobial drug
scaffolds have been established, but comparatively little is known of their pharmacological
impacts on drug resistance and antivirulence activities. This review is the first to extensively
evaluate the roles of halogen atoms in various antibiotic classes and pharmacological
scaffolds and to provide an overview of their abilities to overcome antimicrobial resistance.
Keywords
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Antibiotics; Multidrug resistance; Halogenated hydrocarbons; Structure-activity relationships;
Microbial chemotherapy
I. Introduction
Antimicrobial resistance (AMR) is a phenomenon whereby pathogenic microbes like
bacteria, fungi, viruses, and parasites can adapt and grow in the presence of antimicrobial
compounds to which they were once susceptible (Founou et al., 2017). AMR leads to
prolonged illness and hospital admissions, the use of more expensive second-line drugs, and
treatment failures. It also adversely affects health economies; for example, in 2019 estimates
of additional annual burdens were nine billion euros in Europe and 20 billion dollars in the
USA (Dadgostar, 2019). Microbes have several drug resistance mechanisms in their arsenals,
such as modifying drug targets, limiting drug uptake, enhancing drug efflux, or deactivating
drugs that are deployed based on antibiotics administered (Reygaert, 2018). Furthermore,
acquired resistance mechanisms based on drug modifying β-lactamases and carbapenemases,

multidrug efflux pumps, drug transport proteins, and conformation-modification are
components of pathogen resistomes and further enhance drug recalcitrance (Davies and
Davies, 2010).
Pathogen virulence factors also confer antimicrobial resistance. Biofilm formation is
a significant virulence factor that provides physical protection from antibiotics and
facilitates horizontal gene transfer of resistance genes among microbial communities (Molin
and Tolker-Nielsen, 2003). Regulation systems like the two-component systems and quorum
sensing also modulate resistance and virulence when pathogens are exposed to external
stresses (Haque et al., 2018). Other virulence factors like toxin production, adhesion, iron
metabolism, immune response evasion, and bacterial secretory systems further heighten
antimicrobial resistance (Beceiro et al., 2013). Thus, acquired resistance mechanisms and
7
virulence factors ensure the survival of pathogens in various ecological niches and
nosocomial settings. Countermeasures and alternate therapeutic strategies are warranted to
combat increasingly resistant strains and maintain beneficial commensal microbes, which
are increasingly resistant to common antibiotics.
Halogens are important in several classes of antibiotics and antimicrobial scaffolds
and are present in ~25% of licensed drugs and ~40% of actively tested lead compounds (Xu
et al., 2014) (Fig. 1). Notably, pharmaceuticals referred to as “blockbuster drugs” are usually
halogenated (Suárez-Castro et al., 2018). Furthermore, of the 50 compounds approved by
FDA in 2021, 14 contained halogens (Benedetto Tiz et al., 2022). Halogen bond formation is
one of the primary drivers underlying the inclusion of halogens in antimicrobial drugs.
Halogenated compounds are capable of forming multiple covalent interactions with ligands
and can act as both electrophiles and nucleophiles (Turunen and Erdélyi, 2020). Furthermore,
the introduction of a carbon-halogen (C-X) bond variously influences biological activities,
such as the thermal and oxidative stabilities of enzymes, ligand binding abilities, and

intracellular delivery (Bhutani et al., 2021; Fang et al., 2019). They can also use their bulk to
have agonistic or antagonistic activities on bioactive targets (Liu et al., 2020; Zhou et al.,
2018b). Thus, ADME (adsorption, distribution, metabolism, and excretion) parameters of
compounds like drug binding affinity, membrane permeabilization, and lipophilicity can be
altered by halogen bonding (Fig. 2) (Hernandes et al., 2010). Interestingly, halogenated
antimicrobial agents contribute significantly to the natural defense systems of several plants,
marine algae, fungi, aquatic animals, and halobionts, which supports the notion that
halogenation plays and important role in antimicrobial activity (Fig. 2) (Mardirossian et al.,
2021).
Previous reviews have addressed diverse synthetic and naturally occurring
halogenated compounds, their modes of action, and therapeutic potentials. The roles and
8
importance of halogens, especially fluorine, and chlorine, in medicinal chemistry and
marketed FDA-approved drugs (Böhm et al., 2004; Fang et al., 2019; Gillis et al., 2015;
Mardirossian et al., 2021; Swallow, 2015), and the contribution of halogen bonding to the
functionalities of halogen-containing compounds (Cavallo et al., 2016; Wilcken et al., 2013)
have been previously reviewed. However, this is the first comprehensive review of the
antimicrobial and anti-virulence effects of halogenated antibiotics and pharmacological
scaffolds activity on resistant microbes (Fig. 2). The structural features, structure-activity
relationships (SARs), and the mechanism of actions (MOA) of halogenated pharmacological
scaffolds are provided. In addition, halogenated polymeric materials, proteins,
carbohydrates, and lipids exhibiting antimicrobial activity against resistant microbes and
factors affecting the bioactivities, pharmacokinetic and pharmacodynamic properties of
halogenated antimicrobials and their relationships with gut microbiota are reviewed. We also
provide an overview of the rejuvenation of impotent antibiotics by halogenation and of the
resistance evasion conferred by halogenated scaffolds and offer perspectives regarding the

clinical use of halogenated antimicrobials.
II. Halogenated Antibiotic Classes
Antibiotics are a cornerstone of chemotherapy against bacteria and fungi. The
discovery of penicillin by Alexander Fleming in the 20th century revolutionized general
medicine and led to the discovery of several other classes of antibiotics. However, antibiotic
abuse resulted in the development of microbe drug resistance, and today novel antibiotics are
in constant demand to combat increasing trends of microbial resistance (Pulingam et al.,
2022). Starting with the discovery of chloramphenicol in 1947, halogens have now become
prominent components of several classes of antibiotics and antibiotic scaffolds. Furthermore,
several other halogenated metabolites have been isolated from various sources like
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microorganisms, algae, and several plants and animal sources (Hutchings et al., 2019).
Antibiotic classes are usually classified by molecular structure, mode of action, or activity
spectrums. Here, we discuss and classify halogenated antibiotic classes, in which the halogen
is responsible for the antimicrobial activity or its enhancement against drug-resistant
microbes, based on their modes of action.
A. Cell Wall Synthesis Inhibitors (Penicillins, Cephalosporins, Glycopeptides, and
Lipopeptides)
Inhibitors of cell membranes and cell wall synthesis are among the most effective
antibiotics (Bhattacharjee, 2016). Inhibition of the cell envelope at various stages of cell
development provides one of the most efficient antibacterial strategies as these agents lodge
themselves in cell membranes, compromise membrane integrity, and cause cell swelling and
lysis (Sarkar et al., 2017). Major antibiotic classes like penicillins, lipopeptides,
cephalosporins, and glycopeptides inhibit bacteria in this manner.
Penicillin was the first class of antibiotics discovered in penicillium moulds and

possesses broad-spectrum anti-bactericidal activity. Halogenated isoxazolyl penicillin
derivatives like cloxacillin, dicloxacillin, and flucloxacillin are members of this class of
antibiotics (Fig. 3A-C) (Yasuda and Shimada, 1971). Chloro or dichloro substitutions of
phenyl-, naphthyl-, or quinolyl-penicillins are potent inhibitors of Gram-negative E. coli and
Klebsiella aerogenes (Cole et al., 1972).
Penicillins inhibit the cross-linking of peptidoglycans during cell wall biosynthesis by
inhibiting the activities of associated penicillin-binding proteins, thereby disrupting cell
structural integrity (Yocum et al., 1980). Penicillins contain the β-lactam ring, which binds to
the penicillin-binding protein (PBP) and disrupts peptidoglycan synthesis (Fig. 3D) (Hou and
Poole, 1971). However, this ring is susceptible to degradation by β-lactamases produced by
bacteria. Oxacillin derivatives like cloxacillin, dicloxacillin, and flucloxacillin are impervious
10
to β-lactamase activity because their large chains prevent enzyme binding. Chlorine
substitution in these antibiotics aid PBP binding and augments the antimicrobial effects of
these antibiotics (Fig. 3L) (Neu, 1986). Although a clear role of halogens in preventing β-
lactamase binding is not mentioned, halogen substitution being the only structural difference
in the above-mentioned antibiotics might play a larger role in the reduced binding affinity
with the enzyme.
Cephalosporins are also β-lactam antibiotics and inhibit the peptidoglycan synthesis
required for cell wall formation. Several halogenated cephalosporins like cefazaflur,
cefazedone, loracarbef, cefaclor, flomoxef, and cefiderocol (Fig. 3E-J) are present across
generations, although halogen components are not part of their core structures. The
cephalosporin cephem nucleus contains a β-lactam ring which is essential for antibacterial
activity. Other groups attached to the cephem nucleus, like the aminothiazole, carboxylic acid,
or dimethyl oxime groups, enhance binding to penicillin-binding proteins (Fig. 3K) (Aoki et
al., 2018). Halogens are present in different parts of the sub-structure of cephalosporins, such

as in the cephem nucleus in cefaclor or the chlorocatechol group in cefiderocol, which makes
the role of the halogen atom difficult to determine (Aoki et al., 2018). The locations of
halogen atoms in these structures may dictate the effects of these antibiotics, for example,
strengthening binding to penicillin-binding proteins in cefaclor or augmenting the chelation
of iron in cefiderocol (Fig. 3K, L) (Sato and Yamawaki, 2019). As cephalosporins have a β-
lactam ring in their substructure which is subject to enzymatic degradation, strategic
halogenation of the cephalosporins has the potential to attenuate resistance as observed in
penicillins.
Glycopeptide antibiotics include glycosylated monocyclic or polycyclic non-
ribosomal peptides with effects similar to those of β-lactam antibiotics. They inhibit cell wall
peptidoglycan synthesis by binding to the d-Ala-d-Ala termini of pentapeptide-ending

11
precursors localized at the outer surfaces of cytoplasmic membranes, which blocks the
reticulation of peptidoglycan by inhibiting associated transglycosylases and transpeptidases
(Kang and Park, 2015) (Van et al., 2017). Halogenated glycopeptides include first-generation
teicoplanin (Fig. 4A) and vancomycin (Fig. 4B), and the second-generation semisynthetics
dalbavancin and oritavancin (Binda et al., 2014) (Allen, 2010). Pentapeptide binding is
further enhanced by dimerization, and the presence of a halogen or a sugar moiety is believed
to be responsible for the stronger interaction conferred by homo-dimerization, as was
reported for vancomycin (Fig. 4B, E) and oritavancin (Kang and Park, 2015). The addition of
lipid groups to the side chains of the core structures of glycopeptides enhances binding to the
lipid bi-layer and thus increases permeability (Fig. 4B) (Kang and Park, 2015). Although
halogen atoms are secondary to target binding, they play significant roles by enhancing
bioactivity, as was demonstrated by the dehalogenation and halogenation of balhimycin,
which contains a chlorine atom. Interestingly, fluorobalhimycin, bromobalhimycin, and
balhimycin were reported to be up to 8 times as potent as dechlorobalhimycin at combating

microbial infections validating a crucial role in glycopeptides (Ashford and Bew, 2012).
Vancomycin-resistant Enterococci species were found to alter the d-Ala-d-Ala termini to d-
Ala-d-Ser or d-Ala-d-Lac thereby preventing vancomycin binding. But these resistant species
were sensitive to teicoplanin, which also has halogen substitutions on its sugar moiety but at
different positions in comparison to vancomycin (Yushchuk et al., 2020). Therefore,
halogenation of key binding sites of the sugar moiety can increase binding to the termini of
the resistant strains and restore efficacy of the glycopeptides.
Lipopeptides also include peptides in their core like glycopeptides, bound to lipid
entities giving them their name. The action mechanisms of lipopeptides differ from those of
glycopeptides. Lipopeptides insert themselves into cell membranes in a phosphatidylglycerol-
dependent manner, aggregate, and produce holes that leak ions (Reynolds et al., 2018),
12
causing rapid depolarization, membrane potential loss, and ultimately cell death. This
mechanism was observed in the daptomycin series of lipopeptide antibiotics. The taromycin
series, which was derived from the marine bacterium Saccharomonospora sp., is the only
halogenated antibiotic series in the lipopeptide antibiotic class. Taromycin A and B displayed
potent antimicrobial activity against methicillin-resistant Staphylococcus aureus and
vancomycin-resistant Enterococcus faecium (Fig. 4C-D) (Reynolds et al., 2018). Daptomycin
has tryptophan and kynurenine groups in its peptide structure, and these are essential for
membrane binding. The additional chlorine substitution in the taromycin lipopeptide series
takes place at key tryptophan and kynurenine groups (Fig. 4D), suggesting chlorine might be
responsible for their antimicrobial activity, although the impacts of these substitutions on
efficacy have yet to be determined (Reynolds et al., 2018). SAR studies of the halogenated
tryptophan and kynurenine groups would give better insights into the role of halogens, and
their incorporation into other lipopeptide-based antibiotics. Halogen substitution might have
aided in improved activity against the methicillin-resistant Staphylococcus aureus and

vancomycin-resistant Enterococcus faecium. Lipopeptide-resistant strains are not a
development yet and halogenation of the lipopeptides especially the tryptophan and
kynurenine groups could afford minimal resistance and enhanced binding to the bacterial
membrane.
B. Cell Proliferation Inhibitors (Sulfonamides, Trimethoprims, and Quinolones)
Cell division is an attractive antibacterial target, as the bacterial cell divisome is
essential and conserved in many infectious pathogens. Also, access to associated protein
targets of the cell divisome is made easier by their external locations (Lock and Harry, 2008).
Common strategies for suppressing bacterial cell proliferation include inhibiting
deoxyribonucleic acid (DNA) synthesis or proteins, such as those of the Fts family associated
13
with cell division, and targeting metabolic processes (Lock and Harry, 2008). Different
antibiotics like sulfonamides, trimethoprim, and quinolones inhibit cell proliferation by
inhibiting DNA synthesis by disrupting different stages of the DNA biosynthetic pathway.
Sulfonamides, more commonly known as sulfa drugs, are sulfonamide-derivatized
anilines. Sulfonamide antibiotics disrupt enzymatic reactions involving para-aminobenzoic
acid (PABA) by competitively inhibiting the enzyme dihydropteroate synthase (Kumar
Verma et al., 2020). Bacteria require PABA to produce folic acid, which is required for
purine and pyrimidine synthesis. The para-amino group attached to the aromatic benzene ring
and the sulfanilamide group are essential for the activities of these antibiotics as they mimic
the binding site of dihydropteroate synthase, which is responsible for the synthesis of PABA
(Fig. 5D) (Kumar Verma et al., 2020). The native scaffold of sulfonamides is devoid of
halogens, but a series of di-halogenated sulfonamides were found to be potent inhibitors of
the three β-class carbonic anhydrases in drug-resistant Mycobacterium tuberculosis (Fig. 5A-
C) (Maresca et al., 2013). Typically, sulfonamides are ineffective against mycobacteria, in

part, because of their low membrane permeabilities. Halogenation might aid permeability and
increase the antimicrobial activity of the sulfonamide pharmacophore. Furthermore,
substitution patterns on the aromatic ring affect activity; for example, halogen substitution at
ortho positions enhances antimicrobial activity (Fig. 5D, S). Also, these halogenated
derivatives have potential use against other multidrug resistant pathovars like Staphylococcus
aureus and Streptococcus pneumoniae (Aspatwar et al., 2019). Since halogenated
sulfonamides were active against several MDR pathogens, these derivatives might be
effective at bypassing MDR-associated efflux pumps and decreasing membrane permeability
commonly associated with MDR pathovars.
Trimethoprim is another antibiotic that inhibits DNA synthesis by inhibiting the
conversion of dihydrofolic acid to tetrahydrofolic acid, which acts as a precursor in the

14
thymidine synthesis pathway (an important requirement for bacterial DNA synthesis). The
amine group present in the fourth position of trimethoprim is essential for antimicrobial
activity as it enables the competitive inhibition of the dihydrofolate reductase enzyme by
mimicking the carbonyl oxygen of the 5,6-dihydrofolate substrate (Blaney et al., 1984;
Watson et al., 2007). Trimethoprim has no halogen atoms in its core structure, but
halogenated derivatives (Fig. 5E-G) were successfully synthesized (Nilchan et al., 2018).
Halogenated trimethoprim derivatives synthesized by substituting one of its methoxy groups
with a halogen resulted in 5 times more activity against the panel of microbes tested (Fig. 5H)
(Kompis and Wick, 1977). Furthermore, docking analysis revealed that this increased
potency was the result of bond formation between the Ser49 amino acid residue of
dihydrofolate reductase and the halogen atom, which supported the notion that the inclusion
of a halogen provides additional enzyme binding affinity and increases antimicrobial activity
(Fig. 5S) (Nilchan et al., 2018). This can be an effective way to minimize trimethoprim
resistance as halogenated trimethoprim derivatives can facilitate and enhance binding affinity

for modified dihydrofolate reductase in resistant strains.
Quinolones employ a different strategy to inhibit cell division. Fluoroquinolones
constitute one of the largest halogenated antimicrobial classes and are commonly used
clinically (Fig. 5I-Q). Fluoroquinolones inhibit the ligase activity of type II topoisomerase,
DNA gyrase, and topoisomerase IV, which are responsible for negative supercoiling in the
replication fork and the decatenation of DNA after replication. The inhibition of these
enzymes leads to single or double-stranded breaks in the DNA and eventually to cell death
(Fàbrega et al., 2009). Binding to DNA gyrase is achieved by the two carbonyl groups
present at the third and fourth positions of the bicyclic core of fluoroquinolones (Fig. 5R).
The fluorine atom, which is generally located at the sixth position of the bicyclic core,
enhances antimicrobial activity by 10-fold for gyrase inhibition and increases antimicrobial
15
activity by 100-fold. In this case, the halogen plays a subsidiary role by enhancing
antimicrobial activity (Fig. 5R) (Domagala, 1994).
Despite the substantial potency of fluoroquinolones, microbes were able to develop
resistance by reducing membrane permeability, efflux of compounds, or modifying target
configurations, though this increased resistance can be counteracted using adjuvant efflux
pump inhibitors or strategies that improve permeability. Interestingly, halogenation of efflux
pump inhibitors also increased the efficacy of halogenated chalcone adjuvants of
fluoroquinolones against NorA and MepA multidrug efflux pumps in Staphylococcus aureus
(Freitas et al., 2021) and halogenated pyridylpiperazine-based allosteric inhibitors of RND-
type multidrug efflux pump in E. coli (Plé et al., 2022). In addition, a novel bactericidal 8-
chloroquinolone was synthesized that was 30 and 128 times more potent than trovafloxacin
against two clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) (Fig. 5L)
(Kuramoto et al., 2003). Mutation in DNA gyrase also imparts quinolone resistance by
substitution of Leu or Trp for Ser-83 and Asn or Tyr for Asp-87 in the GyrA protein, leading

to an electron-deficient helix microenvironment. But halogenation of the quinolone at the C8
position reduces the probability of the development of quinolone resistance (Lu et al., 1999)
and can aid in higher efficacy and activity of several quinolone antibiotics against resistant
strains.
C. Protein Synthesis Inhibitors (Aminoglycosides, Tetracyclines, Macrolides,
Lincosamides, and Oxazolidinones)
Protein synthesis inhibitors act at different translation stages to inhibit the synthesis of
proteins. These inhibitors are highly specific for prokaryotic 70S ribosomes. Translation in
prokaryotes involves the assembly of ribosomes, binding to mRNA, and tRNA binding to A,
P, and E sites to further translate to polypeptide sequences. Common antibiotics like

16
aminoglycosides, tetracyclines, macrolides, lincosamides, and oxazolidinones disrupt this
pathway at different translation stages.
Aminoglycosides are a Gram-negative combating class of antibiotics that contain an
amino-modified glycoside, which acts by penetrating the bacterial cell wall and disturbing
peptide elongation at the 30S subunit of the ribosome, which in turn, leads to improper
mRNA translation and truncated protein biosynthesis or amino acid alterations.
Aminoglycoside-to-ribosome binding is mediated by the aminoglycoside neamine core,
which binds specifically to the decoding site (site A) of ribosomes (Carter et al., 2000;
François et al., 2005). The OH and amine groups in ring 1 of aminoglycosides are required
for stacking interactions with ribosomes (Fig. 6B) (Hobbie et al., 2006; Hobbie et al., 2005).
Antibiotic resistance is due to aminoglycoside structural modifications by catalytic enzymes
like aminoglycoside phosphotransferases, nucleotidyltransferases, and acetyltransferases
(Azucena and Mobashery, 2001; François et al., 2005).
The aminoglycoside class of antibiotics does not contain any halogen, but fluorination

produced derivatives that successfully recovered the potencies of parent aminoglycosides
against resistant bacteria (Fig. 6A-B). Fluorinated derivatives of many mainstream
aminoglycosides like kanamycin A, tobramycin, amikacin, dibekacin, gentamycin,
paromomycin, and neomycin have significant antimicrobial activities against drug-resistant
and non-drug-resistant microbes (Fig. 6F) (Mingeot-Leclercq et al., 1999; Tsuchiya et al.,
1985). Fluorine substitution alters glycosidic bonds and reduces the binding affinities of
fluorinated aminoglycoside analogs with modifying enzymes rather than binding affinity with
the ribosome site (Fig. 6B) (Shitara et al., 1992). The fluorinated derivatives maintained their
similar intrinsic activity against both sensitive and resistant strains (Mingeot-Leclercq et al.,
1999).
17
Tetracyclines also target the 30S subunit of ribosomes and inhibit the formation of the
mRNA-ribosome complex by blocking the aminoacyl tRNA binding site (Chukwudi, 2016).
Tetracycline possesses four hydrocarbon rings with functional substitutions of alkyl, halogen,
hydroxyl, amine, and others on the upper and lower periphery of the tetracycline rings (Fig.
6C-E). Modifications on the lower periphery of the rings diminished bioactivity while
modifications of the upper periphery enhanced activity, as exemplified by substitutions of
highly electronegative groups, including halogens on the C7 and C9 positions of the D ring
(Fig. 6E) (Chopra and Roberts, 2001).
Tetracyclines do not have halogen atoms in their native structures, but several
important tetracyclines like chlortetracycline, demeclocycline, meclocycline, and
eravacycline and their associated semi-synthetic derivatives contain halogen atoms (Fig. 6C-
E) (Lamberth and Dinges, 2016). A bromine-substituted tetracycline 7-bromotetracycline
was synthesized, though the rate of halogenation was significantly less than that of its
chlorine analog (Doerschuk et al., 1959). Halogen substitution on the D ring improved

antibacterial activity but at the cost of stability. Furthermore, halogen substitution is not
essential for bioactivity and might only enhance binding to the ribosomal unit (Fig. 6F)
(Dong et al., 2012). Common tetracycline resistance mechanisms involve efflux pumps and
the ribosomal protection protein, which inhibits tetracycline binding (Hobson et al., 2021).
Although halogenation has not been used to counter these mechanisms, we hypothesize
strategic halogenation on the upper periphery of the tetracycline pharmacophore might thwart
the binding of resistance enzymes and the efficacy of efflux pumps and prevent recalcitrance.
In contrast to tetracyclines and aminoglycosides, macrolides target the 50s subunit of
ribosomes. Macrolides block the approach to the exit tunnel of elongated peptides, which
leads to the premature release of the peptidyl-tRNA complex (Gaynor and Mankin, 2003).
Macrolides are a class of natural polyketide antibiotics that typically contain a large
18
macrocyclic 15-16 membered lactone ring with one or more attached deoxy sugars. The C5
desosamine sugar moiety and the three hydroxy groups present on the lactone ring are
essential for antimicrobial activity as they form polar contacts and hydrogen bonds with the
residues of the 50S ribosomal subunit (Mazzei et al., 1993).
Macrolides have no halogen atom in their core structure, but several halogenated
ketolide derivatives have been synthesized, like flurithromycin and solithromycin, which are
the fluorinated members of this class of antibiotics (Fig. 7A-B) (Nguyen and Chung, 2005).
Macrolides also include brominated phorboxazoles isolated from the marine sponge Phorbas
sp. Furthermore, halogenated hydrophosphoryl derivatives of pimaricin and mycophetin were
reported to be much more potent against Candida .sp than their non-halogenated counterparts
(Belakhov et al., 2008; Belakhov and Shenin, 2007). The addition of fluorine at position 2 or
8 of the macrolide scaffold enhances binding to the ribosomal subunit and improves
pharmacokinetic properties (Fig. 7B, L). In this case, halogen substitution enhanced
macrolide antimicrobial activity (Chellat et al., 2016). To a large extent, macrolide resistance

involves efflux pumps, as is the case for quinolones (Schroeder and Stephens, 2016), and
adjuvant halogenated efflux pump inhibitors offer an excellent strategy for diminishing
macrolide resistance. However, no study has yet reported on the efficacies of halogenated
macrolide derivatives against multidrug-resistant strains. Halogenation of the macrolide
pharmacophore might be an effective strategy to subside AMR and increase antimicrobial
efficacy.
Lincosamides compete with macrolides as peptidyl transferase inhibitors and bind to
the peptidyl transferase center of the 23S portion of the 50S subunit of bacterial ribosomes.
Binding takes place via the mycarose sugar moiety, which has structures that overlap with
peptidyl transferase, and leads to the premature dissociation of peptidyl-tRNA containing two,
19
three, or four amino acid residues and ultimately inhibits peptidyl transferase activity by
steric hindrance, as is observed for lincomycin (Fig. 7C) (Schlünzen et al., 2001).
There are two lincosamide halogenated antibiotics, namely clindamycin and
pirlimycin (Fig. 7D-E) (Spížek and Řezanka, 2017). Chlorine atom substitution on the 7-
carbon importantly enhanced the activity of clindamycin (Fig. 7E), and E. coli was 20 times
more susceptible to clindamycin than lincomycin, elucidating halogen-promoted
enhancement (Douthwaite, 1992). Lincosmide and macrolides are competing antibiotics and
elicit similar patterns of antibiotic resistance involving efflux pumps and target site
modifications. A novel ketolide derivative was developed by linking macrolide and quinolone
units (macrolones) to combat resistance against macrolides and lincosamines. The
halogenated derivatives of these macrolones were the most potent derivatives of the
pharmacophore examined and potently reduced the viabilities of several multidrug resistant
strains of E. coli and S. pneumoniae (Fajdetić et al., 2010) (Paljetak et al., 2016). Also,
halogenated analogs of 4’’-O-(ω-quinolylamino-alkylamino) propionyl derivatives of

macrolides potently inhibited macrolide-resistant Streptococcus. Sp, Staphylococcus. Sp, and
Haemophilus influenzae (Fajdetić et al., 2010). This demonstrates the ability of halogen
derivatives in overcoming resistance mechanisms like efflux pumps and ribosomal
modifications associated with the tested resistant strains.
Like macrolides and lincosamides, oxazolidinones target the 50S subunit of
ribosomes, but they act by disrupting the formation of the initiation complex and the
translocation of peptidyl-tRNA from the A to the P site of the 50S ribosomal subunit to
ultimately inhibit protein synthesis (Bozdogan and Appelbaum, 2004). Most of the antibiotics
in this class have halogens, especially fluorine in their native structures. Linezolid, posizolid,
sutezolid, tedizolid, radezolid, and contezolid are examples of fluorinated oxazolidinones
(Fig. 7F-K) (Diekema and Jones, 2001). The n-aryl group in the 2-oxazolidone nucleus is
20
important for antimicrobial activity (Fig. 7H). Also, the fluorine atom of the fluorophenyl
core (a fluorinated phenyl ring) is essential for antimicrobial activity as the fluorine atom is
perfectly situated to form bonds with the 50s ribosomal subunit (Fig. 7H) (Kumar et al., 2015;
Phetsang et al., 2014). Mutations of the 23S rRNA of the 50S ribosomal diminish
oxazolidinone affinity and antibiotic resistance. Several synthesized halogenated
oxazolidinone derivatives, such as derivatives with a halogenated pyrrolidone moiety at α- or
β-position, have exhibited antimicrobial activity against drug-resistant MRSA, vancomycin-
resistant Staphylococcus aureus, and several other Gram-negative bacteria (Bhattarai et al.,
2012). No significant changes were observed in the MICs of these derivatives against drug-
sensitive and drug-resistant strains except S. aureus strains which had a two-fold increase in
MICs against resistant strains. Oxazolidinones containing a halostilbene pharmacophore
substitution demonstrated significant potency against Gram-positive bacteria, and increased
efficacy against drug-resistant pathogens (Sciotti et al., 2002), and fluorine-containing
benzoxazinyl-oxazolidinones have been used to treat multidrug-resistant tuberculosis (Zhao

et al., 2017). The use of halogenation to combat oxazolidinone resistance is largely
unexplored. However, strategic halogenation to facilitate binding to a modified resistant 50S
ribosome unit may be a judicious strategy to combat resistance.
III. Halogenated Antimicrobial Scaffolds
Natural antimicrobials isolated from different sources, including plants, animals, and
prokaryotic and eukaryotic organisms, and their derivatives account for a third of the drugs
approved by the FDA (Quave, 2016). Their applications are often limited by narrow
bioactivity spectrums, low toxicities and yields, enzymatic degradation, and microbial
resistance. Halogen incorporation represents an innovative means of enhancing or restoring
the pharmacological efficacies of simple and complex natural and synthetic products
21
(Hurtová et al., 2022; Molchanova et al., 2020). As a result, studies now focus on the control
of emerging and reemerging drug-resistant pathogens using halogenated products. Here, we
discuss the roles of these derivatives based on chemical groups and/or scaffold robustness.
This classification includes halogenated polyphenolics, essential oils, alkaloids,
benzopyrones, phenazines, azoles, repurposed drugs, and other bioactive compounds. Each
section provides details of efficacies against resistant pathogens, the roles of halogens, their
cytotoxicities, and their mechanisms.
A. Polyphenolic Compounds
Phenolics contain at least a benzene ring and a hydroxyl group attached directly to a
phenyl group. Natural phenolics are secondary metabolites produced via phenylpropanoid
pathways and are found in fruits, vegetables, and other food plants (Hamad, 2021; Laura et
al., 2019). These compounds include phenolic acids, tannins, lignans, flavonoids, stilbenes,
catechols, magnolols, hydroquinones, lignans, and chalcones and are used as food

supplements and intermediate or active ingredients in pharmaceutical products (Hamad,
2021). Furthermore, their halogenated derivatives (Fig. 8A-X) display a variety of
pharmacological properties, including antimicrobial activities.
1. Catechols
Catechol is a water-soluble compound found in plants and marine mussels and is used
in pesticides, bioadhesives, and as a starting material in the perfume and pharmaceutical
industries (Ahn, 2017; Razaviamri et al., 2021). Halogenated catechol, 6-chlorodopamine
methacrylamide loaded into hydrogel remarkably inhibited Gram-positive and -negative
drug-resistant bacteria. It showed >8-log colony-forming unit reduction against methicillin-
resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistant A.

22
baumannii, P. aeruginosa, and carbapenem-resistant K. pneumoniae as compared with its
non-halogenated analog (Liu et al., 2021). In addition, the biofilm-forming capacities of P.
aeruginosa and MRSA which aid in antibiotic resistance was inhibited by the chlorinated
catechol. The halogenated catechol being electrically neutral oxidizes to produce negatively
charged semiquinone capable of preventing biofilm formation. The incorporation of chlorine
in the catechol moiety, increased cell membrane rupture, and eventually cell death (Fig. 8Y).
This mechanism was independent of H2O2 production since the incorporation of the Cl-
group reduced rates of catechol oxidation (Liu et al., 2021). Notably, catechol itself enhanced
adherence for contact killing of bacteria while chlorine presence may have enhanced its
permeability and bioavailability.
In addition, chlorinated catechol inhibited bacterial fatty acid synthesis via the
formation of enoyl-acyl carrier enzyme complex (DMA-Cl/FabI/NAD+) to disrupt fatty acyl
carbon chain elongation and, importantly, it exhibited little toxicity against 3T3-E1
fibroblasts (Liu et al., 2021). Chlorocatechol (Fig. 8A) present at the C3 position was

identified as a core moiety in a drug (cefiderocol) recently approved for the treatment of
complicated urinary tract infections (Fig. 3E). This moiety is responsible for siderophore-iron
binding activities, improved potency, and stability against drug-degrading enzymes like the β-
lactamases (Nishimura et al., 2022; Sato and Yamawaki, 2019). Overall, chlorocatechol We
suggest chlorocatechol might also inhibit resistant bacteria by chelating iron, interfering with
fatty acid synthesis and membrane disruption.
2. Magnolols
Magnolol is a natural biphenolic with two para-allyl groups isolated from the bark of
Magnoliae officinalis. This scaffold has established pharmacological activities, including
antimicrobial, antioxidative, antianxiety, anti-inflammatory, and antiproliferative activities
(Khadke et al., 2019). Halogenation of magnolol enhanced its antimicrobial activity. More
23
specifically, dibromo-magnolol (Fig. 8B) had a low MIC (1-2 µg/mL), which was 8-64 times
more superior to magnolol, ciprofloxacin, and erythromycin (MIC: 8-64 µg/mL) against
MRSA and vancomycin-resistant Enterococcus (VRE). Similarly, dichloro- and diiodo
magnolols (Fig. 8C-D) at 4-8 µg/mL were 4-16 times more effective than magnolol (32
µg/mL) and erythromycin (32-64 µg/mL) against MRSA and VRE (Jada et al., 2012).
Dihalogenation, especially with bromine, on the aromatic rings of biaryl groups (Fig. 8F)
increased potency against resistant pathogens while the mono I- and Cl- substitution showed
lesser activity (Jada et al., 2012; Li et al., 2021b). Parent magnolol inhibits MRSA by binding
with FtsZ protein, which is involved in cell division (Liu et al., 2014). However, the
mechanisms of its derivatives against MRSA and VRE were not investigated. A similar
derivative 5,5'-diallyl-3,3'-dibromo-[1,1'-biphenyl]-2,2'-diol (Fig. 8E) inhibited the growth
and virulence of B. cinera by disrupting mitochondrial functions, membrane permeability,
and hyphal integrity (Li et al., 2021b). Bacterial structures present similar targets with the
exception of hyphal formation, which suggests that halogenated magnolol might target the

cell division process (FtsZ protein), cell membrane, and/or adenosine triphosphate (ATP)
generation in bacteria. We recommend a detailed investigation of mechanisms, toxicities, and
how halogens modulate uptake by the pathogen.
3. Resveratrols
Resveratrol is a phytoalexin found in red wine, grapes, soybeans, mulberries,
cranberries, and Japanese knotweed which possesses numerous activities against microbes
and other biological conditions (Kim et al., 2022; Zhang et al., 2021). Halogenated
resveratrols are considered active compounds that might overcome antimicrobial resistance
(Di Fermo et al., 2020; Li et al., 2012). For instance, 2-chloro-resveratrol (Fig. 8G) and 2-
bromo-resveratrol (Fig. 8H) at MIC (3.9 g/mL) inhibited C. albicans and were 30- and 3-
fold more potent than resveratrol (125 g/mL) and fluconazole (12.5 g/mL), respectively
24
(Li et al., 2012). Similarly, 4-[(E)-2-(4-chlorophenyl)ethenyl]phenol (Fig. 8I) inhibited
resistant strains of H. pylori 11F/11 and ATCC 43629 at 3.1 and 12.5 g/mL and were 64-
and 16- times more potent than the parent compound (200 g/mL) (Di Fermo et al., 2020).
Parent resveratrol was reported to show less activity against Gram-negative bacteria due to
the efflux pump system which reduced interaction with cytoplasmic or periplasmic targets in
the bacteria (Mattio et al., 2020; Vestergaard and Ingmer, 2019). Interestingly, halogenated
resveratrol (Fig. 8I) displayed the capacity to suppress the efflux system and restore
antimicrobial activity as evident by the synergism with levofloxacin to control two drug-
resistant H. pylori at sub-MIC concentrations (Di Fermo et al., 2020).
Virulence factors are promising targets for the control of resistant pathogens.
Halogenated resveratrol at sub-MICs suppressed H. pylori biofilm formation and motility,
which are essential for the colonization of gastric mucosa (Di Fermo et al., 2020).
Halogenated resveratrols exhibited diverse antimicrobial activities, which were attributed to
the introduction of Cl- or Br- atoms on the aromatic A ring. Furthermore, this introduction

improved lipophilicity, facilitated diffusion into the bacterial membrane, and subsequently
triggered the production of peroxyl radicals (LOO*) (Fig. 8Y). Similarly, the halogenated
resveratrols were reported to interfere with ATP generation and adhesion to confer
antivirulence effects (Di Fermo et al., 2020; Li et al., 2012). Chlorinated resveratrol (Fig. 8I)
caused cell elongation, indicating interference with cell division, and protected Galleria
mellonella against H. pylori infection (Di Fermo et al., 2020). In addition, chlorinated
resveratrol was non-toxic to Galleria mellonella larvae (Di Fermo et al., 2020), and was
reported to be non-hemolytic to human red blood cells which could be influenced by the size
or numbers of halogen present (Li et al., 2012).
Previously, -OH substituents located at the 4- and 4′- carbon positions of trans-
resveratrol were reported to be required for its antiproliferative activities (Savio et al., 2009).
25
Other defects associated with resveratrol are poor bioavailability, metabolite production, and
poor stability resulting from excessive metabolism in the intestine and liver (Francioso et al.,
2014; Walle, 2011). However, halogenated resveratrol was reported to show a
cardioprotective effect with a higher bioavailability and inhibited sirtuins associated with
neurological or metabolic disorders better than the native scaffold indicating halogen
tendency to alleviate pharmacological issues (Bourgault et al., 2011; Nguyen et al., 2013).
Hence, we believe that halogenation of resveratrol per se could favor the antibacterial and
antivirulence effects, potentiate activities as reported against H. pylori strains and alleviate
issues associated with their pharmacology. However, further study is required to elucidate
their mechanisms and to what extent halogenation could shield resveratrol from the problems
of excessive metabolism.
4. Flavonoids
Flavonoids are ubiquitously found in plants as phytoalexins, detoxifying agents, and
signal molecules (Rana and Gulliya, 2019). They play protective roles in plants and the same

capacity is being harnessed for the treatment of human infections (Biharee et al., 2020).
Halogenated flavonoid derivatives such as BrCl-flav (Fig. 8J) were targeted against resistant
ESKAPE pathogens. Gram-positive MRSA and S. pneumonia were highly susceptible to
BrCl-flav (Fig. 8J) at very low MICs (0.24-0.48 µg/mL) which were superior to gentamycin
(3.9-62.5 µg/mL) and chloramphenicol (3.9-31.2 µg/mL), respectively (Moldovan et al.,
2022). Conversely, it was less effective against Gram-negative pathogens such as
Enterobacter cloacae, K. pneumonia, S. enterica, and P. aeruginosa except for Enterococcus
faecium (7.8 µg/mL) (Moldovan et al., 2022). The presence of an outer membrane in Gram-
negative bacteria confers extra protection, and hence, the low activities observed suggest an
inability to traverse bacterial membranes. Interestingly, when used in combination with
26
fluconazole, BrCl-flav showed a synergetic effect against fluconazole-resistant C. albicans by
reducing fluconazole MIC by 128-fold (Babii et al., 2021).
Halogenated flavonoids such as BrCl-flav (Fig. 8J) and ClCl-flav (Fig. 8K) exhibited
antimicrobial activities against bacteria by causing the leakage of bacterial intracellular
materials, increasing cell permeability to biocidal agents, and damaging cell morphology (Fig.
8Y) (Babii et al., 2018; Moldovan et al., 2022). Also, compound 8J inhibited Candida spp.
by irreversibly damaging cellular morphology, and preventing hyphal transition (Babii et al.,
2021). The replacement of hydrogen with halogen at the C4 position of compound 8K in
addition to the 1,3-dithiolium ring increased bactericidal activities (Babii et al., 2018).
Additionally, upon co-incubation with the breakpoint concentrations of respective antibiotics,
flavonolignan 8-bromo-2,3-dehydrosilybin AB (Fig. 8L) reversed the colistin-resistant
phenotype in P. aeruginosa to a sensitive strain. A similar effect was reported for 6,8,21-
tribromosilybin A (Fig. 8M) and 6,8,21-tribromosilybin B (Fig. 8N) at 40 µM against the
gentamicin-resistant S. aureus phenotype (Hurtová et al., 2022). The bromination of

flavonolignan (Fig. 8L-N) was reported to significantly enhance the activity and reversed
resistant phenotypes probably by inhibition of the efflux pump and bacterial communication
as well as improving selectivity index (Hurtová et al., 2022).
Furthermore, interference with bacterial communication represents a viable strategy to
regulate bacterial virulence. Halogenated flavonolignan (Fig. 8L-N) inhibited the
autoinducer-1 (AI-1) and autoinducer-2 (AI-2) in Vibrio campbellii and controlled the
adhesion of S. aureus and P. aeruginosa to the surface, subsequently preventing biofilm
formation (Hurtová et al., 2022). Similarly, BrCl-flav caused loss of hyphal formation and
impeded the biofilm formation of C. albicans by 80% at 15.0 µg/mL. It also prevented and
disrupted biofilms of A. baumannii by 95 and 58% at 62.5 and 3.9 µg/mL respectively (Babii
et al., 2021; Moldovan et al., 2022). In addition, ClCl-flav reduced the biofilm formation of S.
27
aureus, E. coli and K. pneumonia by 75% at 0.97-7.81 μg/mL (Babii et al., 2018). The
forgoing also suggests the capacity of the halogenated flavonoids to combat resistance by
targeting the virulence factors.
Notably, halogenated flavonoids such as ClCl-flav (Fig. 8K) were non-toxic to Vero
or mammalian HeLa cells at low concentrations (2 μg/mL) but mildly toxic at higher
concentrations (4–32 μg/mL) (Babii et al., 2018). BrCl-flav (Fig. 8J) was slightly cytotoxic
with an IC50 value of ~80 µg/mL. Notably, it enhanced cell viability at doses ranging from 5
to 25 µg/mL, probably due to cellular metabolization. Taken together, halogenated flavonoids
could serve as adjuvants with known antibiotics or target the virulence factors to reverse or
control resistance in pathogens with low selective pressure.
B. Essential Oil Compounds
Essential oils (EOs) are highly concentrated hydrophobic liquids derived from a
variety of plants and classified based on their chemical and physical properties. The

pharmacological effects of EOs have been extensively examined and include antimicrobial,
antioxidant, anti-inflammatory, and immunomodulatory activities (Masyita et al., 2022).
Structurally, the components of EOs can be classified as; terpenes, terpenoids,
phenylpropanoids, and others (Pandey et al., 2017). Chemical halogenation is a prominent
strategy used to enhance the uptakes and activities of EOs and other natural products. Here,
we describe the bioactivities of halogenated essential oil compounds such as thymol and
cinnamaldehyde.
1. Thymols
Thymol is a bioactive compound found in thyme oil (Thymus vulgaris) and the
Origanum genus (Marchese et al., 2016). Thymol is an isomer of carvacrol which is used to
28
control zoonotic and food pathogens. Synthetic thymols have been reported to have antiseptic
effects in toothpaste and are used to flavor cough syrups as well as chewing gum (Kaur et al.,
2014; Kim et al., 2022). Chlorothymol (Fig. 8O) at 32 µg/mL had 16-fold inhibitory effects
against MRSA than carvacrol (512 µg/mL) while thymol was without activity (Kim et al.,
2022). However, chlorothymol inhibited the sensitive S. aureus strain at 12.5 µg/mL which is
3 times lower than what was required for MRSA control (Kaur et al., 2014). Chlorothymol
was more active against sensitive than resistant strains. Interestingly, the combination of
chlorothymol with antibiotic oxacillin further improved activity against MRSA (MIC: 8
μg/mL) suggesting its capacity to act optimally and overcome resistance via synergism (Kim
et al., 2022). In addition, C. albicans was better inhibited by chlorothymol than thymol at 100
μg/mL and in a manner comparable to amphotericin B (Kaur et al., 2014).
As part of strategies to control resistance, halogenated thymols (Fig. 8O) exhibited
antivirulence potential. The sub-MIC concentrations prevented MRSA biofilm formation,
disrupted mature MRSA biofilms, restricted motility, and decreased staphyloxanthin

production involved in bacterial evasions of oxidative stress and host immune system (Kim et
al., 2022; Xue et al., 2019). Furthermore, the SAR study revealed that the presence of
chlorine at the C4 position of thymol was crucial to antimicrobial activity, compared to the
bromo- and dibromo- derivatives which lost activity (Fig. 8P-Q), suggesting a small-sized
halogen is required for activity (Kaur et al., 2014; Kim et al., 2022). We surmise that
halogens like chlorine enhanced the lipophilicity and membrane localization of thymol to
improve membrane penetration. This is corroborated by reports that the hydrophobic nature
and enhanced affinity for the membrane lipid bilayer of chlorothymol were responsible for
MRSA inhibition via membrane integrity disruption (Kim et al., 2022; Kowalczyk et al.,
2020).
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2. Trans-cinnamaldehydes (CNMA)
CNMA largely accounts for the antimicrobial activities of Cinnamomum spp. and was
approved by FDA for application in the food industry as a flavoring agent (Friedman, 2017;
Shreaz et al., 2016). The halogenation of CNMA was reported to offer a potential means of
countering antibiotic-resistant pathogens. 2,4-dichloro- and fluoroCNMA (Fig. 8R-S)
inhibited penicillin-resistant Streptococcus pyogenes and S. aureus (16-64 µg/mL) and were
better than cinnamic acid and ciprofloxacin (˃128 µg/mL) against resistant S. pyogenes (Li et
al., 2015). Comparatively, the derivatives (Fig. 8R-S) as expected required lesser doses (4-16
µg/mL) to achieve inhibition against sensitive S. aureus. However, sensitive S. pyogenes was
more tolerant (≥128 µg/mL) and required 2 times doses for activity than the resistant strain
(Li et al., 2015). This may suggest a specific resistance mechanism that is not available in the
sensitive strain as the target of the derivatives. Also, p-brominated CNMA (Fig. 8T) with
MIC of 0.51 mM against S. aureus and E. coli, respectively, was ~4-5 fold more effective
than the parent CNMA (1.93-2.32 mM) (Doyle et al., 2019). Also, 4-bromophenyl-

substituted (Fig. 8U) and 2,4-dichlorinated CNMA (Fig. 8R) were effective against MRSA
and A. baumannii (64 µg/mL), respectively (Chai et al., 2022), but ineffective against
extensively drug-resistant A. baumannii. However, they became effective when co-incubated
with an efflux pump inhibitor, indicating drug efflux contributed to the intrinsic resistance
displayed. Of note, the outer membrane of A. baumannii was reported to be about 100 times
more impervious than those of other Gram-negative bacteria, especially E. coli due to the
absence of non-specific porins (Chai et al., 2022; Zgurskaya et al., 2015). Therefore, further
halogen modulation of the CNMA scaffold is required to enhance efficacy against multidrug-
resistant A. baumannii. Mechanistically, halogenated CNMAs (Fig. 8R-S, 8U) similarly
inhibited the bioactivities of S. aureus and A. baumannii strains by suppressing cell
proliferation. Specifically, they caused cell elongation and inhibited FtsZ protein
30
polymerization as well as GTPase activity, which led to abnormal cell division and
subsequent cell death (Chai et al., 2022; Li et al., 2015).
Halogenated CNMAs also target bacterial virulence factors. For instance, 4-
bromoCNMA, 4-chloroCNMA, 4-fluoroCNMA, and dichloroCNMA (Fig. 8T and 8V-X)
decreased protease activities, biofilm formation, cell surface hydrophobicities, motilities,
fimbria production, quorum sensing (QS), and pigment production in C. albicans and Vibrio
spp., and these effects were corroborated by downregulations of virulence- and biofilm-
related genes (Brackman et al., 2011; Faleye et al., 2021; Khadke et al., 2022).
The pre-clinical safety evaluations showed that the halogenated derivatives 8R, 8U,
and 8W were devoid of hemolytic activity in human blood and non-toxic as determined by
their effects on the growth, metabolism of HepG2 mammalian liver cells and the survival of
Caenorhabditis elegans model (Chai et al., 2022). Depending on concentrations, 4-
bromoCNMA (Fig. 8T) had a none to slightly toxic effect on the survival of Galleria
mellonella larvae. Notably, the dichlorophenyl- and bromophenyl CNMA (Fig. 8R, 8U)

lacked an off-target effect on human tubulin polymerization (Chai et al., 2022). Given the
non-toxicity observed in preliminary in vivo and in vitro toxicity assays of halogenated
CNMAs, we recommend further toxicity tests in advanced models, especially animals for
possible future applications like the parent CNMA in food applications.
C. Alkaloids
Alkaloids play an essential role in the natural defense systems of organisms and
human medicine and constitute ~ 20% of known secondary metabolites in plants and
microbes. In plants, alkaloids protect against predators and regulate growth. Therapeutically,
alkaloids are used as anesthetics, cardioprotective and anti-inflammatory agents. Well-known
clinical alkaloids include morphine, strychnine, quinine, ephedrine, and nicotine (Heinrich et
31
al., 2021). Here, we discuss the bioactivities, toxicities, and pharmacokinetic properties of
alkaloids functionalized with halogens (Fig. 9A-Z).
1. Indoles
More than 85 species of Gram-negative and Gram-positive bacteria possess the
tryptophanase (tnaA) gene and produce indole as a signaling molecule directing certain
bacterial functions (Kumar et al., 2021; Lee and Lee, 2010). Indole-based compounds such as
vincristine, reserpine, and amedalin are used clinically as anticancer, antihypertensive and
antidepressant agents, respectively (Kumar et al., 2020a). Halogenated indole derivatives
have been used as antimicrobial controls against antibiotic-resistant pathogens (Qin et al.,
2020; Raorane et al., 2020). CZ74 (Fig. 9A) at 2-4 μg/mL was reported to exhibit highly
potent activity against ampicillin-resistant S. aureus MRSA, and vancomycin-resistant
Enterococcus (VRE). The activity was attributed to the presence of the trifluoromethyl group
(CF3) (Yuan et al., 2019). Furthermore, 5-iodoindole (Fig. 9B) inhibited the resistant strains
of A. baumanii at a MIC of 50 µg/mL which was superior to gentamicin and colistin but

comparable to ciprofloxacin at a dose of 200 µg/mL (Raorane et al., 2020). Insertion of
iodine at the C5 position of indole resulted in more effective antimicrobial activity against A.
baumannii than other positions by enhancing selective transportation via the membrane
(Raorane et al., 2020). In addition, potent brominated metabolites from the Red Sea sponge
Callyspongia siphonella, viz. 5‐bromotrisindoline (Fig. 9C) and 6‐bromotrisindoline (Fig.
9D) inhibited MRSA at concentrations of 8 and 4 μM, respectively (Sayed et al., 2020). Some
halogenated indoles (Fig. 9A, 9C-D) exhibited limited activities against Gram-negative
bacteria probably because being neutral, they were unable to establish electrostatic
interactions (Sayed et al., 2020; Yuan et al., 2019), which led to the inability to transverse the
outer bacterial membrane. Thus, the addition of positively charged groups, such as primary
amines, was proposed to expand their activity spectra (Huigens Iii et al., 2013).
32
Halogenated indoles can also target some virulence factors as reported for 5-
iodoindole, 4-fluoroindole, and 7-chloroindole (Fig. 9B, 9E-F), which eradicated MRSA and
E. coli persister cells by multiple folds as compared to the parent indole (Lee et al., 2016).
Interestingly, these halogenated indoles inhibited staphyloxanthin production by S. aureus
strains at sub-MIC concentrations as well as the motility and pellicle formation of A.
baumannii at 25 and 50 µg/mL, respectively (Lee et al., 2016; Raorane et al., 2020). In
addition, 4-chloroindole, 5-chloroindole, and 5-chloro 2-methyl indole (Fig. 9J-L) inhibited
biofilm formation, restricted motility, curli, fimbriae, protease production, and the cell surface
hydrophobicity of V. parahaemolyticus and uropathogenic E. coli (Boya et al., 2022;
Sathiyamoorthi et al., 2021).
Various mechanisms underlie the antimicrobial activities of halogenated indoles on
resistant pathogens. Specifically, CZ74 (Fig. 9A) was reported to interfere with FtsZ
polymerization and inhibited GTPase activity, resulting in the inhibition of bacterial cell
division (Yuan et al., 2019). Also, 5-iodoindole (Fig. 9B) increased reactive oxygen species

(ROS) generation which caused the leakage of cytoplasmic contents leading to oxidative
stress-mediated A. baumannii cell membrane disruption (Raorane et al., 2020). However, 9B
did not affect the cell shapes or membranes of E. coli or S. aureus (Lee et al., 2016). We
believe this might be related to the dosages used and the pathogen intrinsic factors. Other
halogenated indoles (Fig. 9G-H) effectively inhibited enzymes related to protein synthesis
and mycobacterial wall synthesis in E. coli and Mycobacterium, respectively (Rathod et al.,
2018; Sapnakumari et al., 2017). Also, 5‐bromotrisindoline (Fig. 9C) acted as a dual enzyme
inhibitor of DNA gyrase and pyruvate kinase, which are involved in DNA replication,
staphylococcal biofilm formation, resistance, pyruvate, and ATP generation (Sayed et al.,
2020; Thomsen and Liu, 2018; Vasu et al., 2015). Conversely, 6‐ bromotrisindoline (Fig. 9D)
was found to be a potent gyrase‐B inhibitor, indicating that the location of bromine
33
contributed to selectivity and potency (Sayed et al., 2020). Importantly, fluorinated indole
(Fig. 9I) potently inhibited S. aureus (SA-1199B), known to overexpress norA - a drug efflux
protein responsible for efflux activity and viability (Lepri et al., 2016).
Halogenated indoles demonstrated non to mild toxicity in various in-vivo, in-vitro and
in-silico studies. An ADME study of chloroindoles (Fig. 9J-L) revealed they are non-
carcinogenic to mice, have minimal acute fish toxicities, and do not violate Lipinski’s rule of
five (Boya et al., 2022). Halogenated trisindoles (Fig. 9C-D) exhibited excellent drug‐like
attributes, good oral absorption, reasonable toxicities, and high bioavailabilities (Sayed et al.,
2020). Similarly, 5-chloroindole (Fig. 9K) was non-toxic to the MN9D mouse dopaminergic
cell line (Kholodar et al., 2021). Fluorinated indoles (Fig. 9E, 9M-N) were reported to be
non-toxic to C. elegans at 100 mg/L. However, at high doses (ca. 100 × MIC), 7-fluoroindole
was mildly toxic (Raorane et al., 2022). It is worth noting that halogenation levels may alter
the pharmacokinetics of the indole scaffold (Fig. 9A5). For example, a fluorine atom at the
C2 position of a fluorinated indole (Fig. 9O) improved the pKa and directly affected

pharmacokinetics more so than its difluorinated indole congener (Fig. 9P) (Lepri et al., 2016;
Manallack, 2007). Contrary to the notion that halogens enhance metabolic stability, the
presence of fluorine in an indole derivative (Fig. 9O) resulted in rapid oxidation, molecular
instability, and the production of two metabolites including M-90 (N-dealkylation) and M-68
(N-dealkylation) (Lepri et al., 2016). Given the diverse, versatile activities displayed by
halogen-containing indoles against various resistant pathogens and their virulence factors, we
speculate that these compounds offer promising solutions to bacterial resistance and that, to a
greater extent, the antimicrobial and pharmacological properties of halogenated indoles are
dependent on the positions, numbers, types of halogens present and the bacterial cell type.
However, further in vivo assessments are required to identify suitable drug candidates.
2. Streptochlorins
34
Streptochlorin (4-chloro-5-(1H-indol-3-yl)oxazole) is a small alkaloid of indole origin
found in marine bacteria like Streptomyces sp. The streptochlorin scaffold is privileged and it
is found in natural compounds like labradorin, pimprinine, and almazole. The scaffold
exhibits a variety of biological activities but with narrow-spectrum antimicrobial activity
(Song et al., 2021; Zhou et al., 2018a). However, halogen modification of the scaffold was
used to increase potency (Fig. 9Q-R, A5). For instance, 4-bromo-5-(1H-indol-3-yl)oxazole
(Fig. 9Q) and ethyl 2-(3-4-bromooxazol-5-yl)-1H-indol-1-yl)-2-fluoroacetate (Fig. 9R)
potently inhibited some fungal pathogens (Jia et al., 2018). Notably, the replacement of Cl-
with Br- at the C4-position of the oxazole ring improved the activities of these derivatives
(Jia et al., 2018). In addition, streptochlorin could bind strongly with Thermus thermophiles
Leucyl-tRNA synthetase to inhibit protein synthesis like Tavaborole (a small-molecule
medication for fungal infections) (Gao et al., 2021). Modified streptochlorin (Fig. 9Q-R)
were not investigated for their effects on drug-likeness, however, parent streptochlorin was
shown to exhibit high hepatic clearance using mouse liver microsomes and liver S9 fractions.

It also showed low oral bioavailability, reduced plasma concentrations, short half-life, high
systemic plasma clearance, and metabolization by glucuronidation or monooxygenation of
the indole structure (Zhou et al., 2018a). Hence, further structural tuning is required to avert
metabolic stability problems and improve other pharmacokinetic profiles or to develop
compatible topical applications.
3. Quinone and Quinolines
Quinones are aromatic cores containing two oxygen atoms that form two carbonyl
bonds and are found in plants, fungi, and bacteria (Niaz et al., 2020). They are metabolites
formed by the oxidation of hydroquinone and have found applications in approved clinical
drugs and those in the developmental pipeline. Quinones have considerable pharmacological
potential, and their biological activities are due to their involvement in redox cycling
35
processes (Campos-Xolalpa et al., 2021; Sahoo et al., 2022). Quinones have been modified to
enhance their efficacies and alter their physiochemical features (Fig. 9S-Z). Fluorinated
carbazoloquinones (Fig. 9S-T) inhibited MRSA at MIC 25-50 µg/mL, and the effect of
compound (9S) was comparable to linezolid and vancomycin (28-30 µg/mL) (Chakraborty et
al., 2017). Interestingly, hydrophilic fluorine at C6 on one benzene ring and the incorporation
of hydrophobic methyl group at the C2 or C3 of the other benzene in carbozoloquinones were
crucial for anti-MRSA activity (Chakraborty et al., 2017).
In addition, 7-bromohydroxyquinoline and 5,7-dichlorohyroxyquinoline (Fig. 9U-V)
exhibited similar inhibitory activities against clinically important Gram-negative pathogens
but weaker activities against Gram-positive bacterial and fungal isolates (Cherdtrakulkiat et
al., 2016). The presence of Cl- or Br- at the C5 and/or C7 positions of these compounds
enhanced lipophilicity and improved their effectiveness against Gram-negative bacteria.
However, this was not the case for Gram-positive pathogens as unsubstituted and low
lipophilic 8-hydroquinoline adequately penetrated the bacterial membrane (Cherdtrakulkiat et

al., 2016). This observation challenges the status quo, and it suggests the possibility of
improved affinity conferred by halogenation against a specific target protein in Gram-
negative species. Also, bromoquinol (Fig. 9X) at 1 µM demonstrated 95% killing against the
invasive fungus Aspergillus fumigatus by rapidly inducing oxidative stress and apoptosis
(Ben Yaakov et al., 2017). Bromoquinol functions optimally at low metal concentrations, as is
observed in infected hosts, but becomes chelated and inactive in the presence of iron, copper,
or zinc. Consistently, the incorporation of bromine residues at C5 and C6 accounted for its
efficacy to parent 8-hydroxyquinoline (Ben Yaakov et al., 2017). In addition, to improving
lipophilicity, halogenation might enhance the iron-chelating effect of the 8-hydroxyquinoline
moiety to display antibacterial activity (Fig. 9A5). This is corroborated by the activity of
clioquinol, a hydroxyquinoline drug which contains chlorine and iodine and was reported to
36
exert various antimicrobial activities by chelating metals including Cu, Zn, and Fe (Das et al.,
2018).
Preclinical evaluations have shown that halogenated quinoline (Fig. 9W) is not toxic
to HeLa cells at 100 µg/mL (Desai et al., 2017). Bromoquinol (Fig. 9X) protected the larvae
of G. mellonella against aspergillosis as much as amphotericin B but was ineffective against
invasive pulmonary aspergillosis in a murine model at 8 mg/kg (Ben Yaakov et al., 2017).
The disparity observed between in vivo and in vitro efficacies may have been due to
physiological responses, drug properties, or drug-pathogen interactions. According to
transcriptional profiling, A. fumigatus in response to bromoquinol repressed energy-
dependent cellular activities such as protein synthesis and activated genes responsible for
efflux, detoxification, and reactive radical formation (Ben Yaakov et al., 2017). While we are
aware that fungi and bacteria are continuously evolving due to exposure to diverse
environmental cues or stressors, further investigation might provide better insight.
Furthermore, halogenated aminoquinones (Fig. 9Y-Z) were found to be non-carcinogenic and

non-mutagenic, like the antibiotics ciprofloxacin, vancomycin, and cefoxitin, and were
predicted to have high gastrointestinal absorption profiles. Additionally, no toxic/hemolytic
effect was reported on healthy human blood, and the presence of a sugar moiety on the
halogenated scaffold contributed to the non-toxic properties and non-CYP3A4 inhibition
(Dias et al., 2018). Based on the forgoing, these halogenated derivatives were less toxic than
the parent quinone, suggesting the possible role of halogenation in alleviating side effects.
Conversely, certain halobenzoquinones were reported to demonstrate genotoxic, carcinogenic
and cytotoxic effects but confirmation from in-vivo studies is scarce (Matos et al., 2015).
Despite the suggested aforementioned toxicological effects, quinones demonstrated
cytoprotective potentials via anti-inflammatory activities, detoxification enzyme induction,
and redox status modification (Bolton and Dunlap, 2017). Due to these differential toxicity
37
profiles of the various halogenated quinone analogues, more detailed elucidation of in-vivo
toxicity is recommended.
4. 4-Oxoquinolizines
4H-4-oxoquinolizine scaffold represents the bioisostere of quinolones and its
derivatives were synthesized as alternatives to combat the limitations of fluoroquinolones
which include low intrinsic activity towards Gram-positive bacteria and the increased
quinolone-resistant pathogens (Ma et al., 1999). The halogenated 4-oxoquinolizine displayed
better antibacterial activity than fluoroquinolones. For example, CC-195776, CC-195767 and
CC-195820 (Fig. 9A1-A3) exhibited excellent antibacterial activities against drug-resistant A.
baumannii at very low MICs of 0.09-0.19 µg/mL and were superior to ciprofloxacin,
ofloxacin and levofloxacin (8-64 µg/mL) (Na et al., 2017). Also, GC-072 (Fig. 9A4) which is
currently undergoing clinical trials exhibited better antibacterial activities against drug-
resistant Burkholderia pseudomallei strains (MIC: ≤ 0.004-0.25 µg/mL) than ciprofloxacin
(0.25-16 µg/mL) (Shearer et al., 2019). Another prodrug TNP-2092 containing 4H-4-

quinolizine pharmacophore and rifamycin inhibited resistant H. pylori strains (0.008-16
μg/mL) and was superior to clarithromycin and levofloxacin (0.016 - 128 μg/mL) (Wang et
al., 2018).
Previously, mutations that occur at the quinolone resistance determining region
(QRDR) of GYrA and ParC subunits were attributed to fluoroquinolone resistance (Madurga
et al., 2008). Halogenated 4-oxoquinolizines displayed the capacity to bind these mutated
enzymes to exert antimicrobial activities against quinolone-resistant bacteria. For instance,
CC-195776, CC-195767, and CC-195820 exhibited notable inhibitory effects on resistant A.
baumannii despite amino acid mutations in the QRDR of GyrA and ParC (Na et al., 2017).
Similarly, GC-072 overcame trimethoprim and ceftazidime resistance caused by folA and
PenA β-lactamase point mutations respectively with activity better than ciprofloxacin
38
(Shearer et al., 2019). They also overcame antibiotic resistance by inhibiting efflux activities
as evident in the resistance of GC-072 to efflux export mediated by AmrAB-OprA and
BpeAB-OprB in B. pseudomallei. However, it may be susceptible to BpEF-OprC pump
activity but with a significantly lower degree of efflux than ciprofloxacin and doxycycline.
Regarding the SAR, GC-072 containing two fluorine atoms and an amine group with
conformation different from the piperidine ring in ciprofloxacin potentially contributed to the
enhanced activities against quinolone-resistant bacterial strains (Shearer et al., 2019).
Previously, the substituent in the C-8 position of the oxoquinolizine scaffold was reported to
influence target affinity, drug access to enzymes, DNA binding and overall activity (Peterson,
2001). Addition of free halogen at this location or in combination with methyl group
enhanced activity against certain anaerobes and fluoroquinolone-resistant bacteria (Ledoussal
et al., 1999).
Furthermore, halogenated 4-oxoquinolizines like fluoroquinolones target bacterial
DNA gyrase and the topoisomerases IV but display better selectivity and also possess a

separate binding mode from the fluoroquinolones (Shearer et al., 2019). Halogenated
derivatives including CC-195776, CC-195767, and CC-195820 showed a high degree of non-
toxicity. They exhibited strong selective toxicity towards A. baumannii and were non-toxic to
human HeLa and U937 cells at lower doses (25 µg/mL) but became toxic at 50 µg/mL which
is a hundred times higher than their MICs (0.09, 0.19, and 0.19 µg/mL) respectively (Na et al.,
2017). Overall, halogenated 4H-4-oxoquinolizines have shown the capacity to outcompete
fluoroquinolones in overcoming drug resistance. It is our opinion that increased interest in
halogenation of the C8 position will further improve tolerability, specificity, and
pharmacokinetics and increase the activity spectrum.
D. Benzopyrone and Phenazines
39
Benzopyrones are common phytochemicals and have produced promising results in
clinical applications, especially when used with other therapies. Coumarins and some
bioflavonoids are common agents which have been used to treat asthma, lymphedema,
infections, and neoplasms (Hu and Piller, 2017; Matos et al., 2015).
1. Coumarins
The coumarins are bicyclic heterocyclic benzopyrans that contain an aromatic ring
fused to a six-membered lactone ring (Rehuman et al., 2021). Both natural and synthetic
coumarins are commonly used in the perfume and cosmetics industries (Khan et al., 2019).
The charge-transport properties and electron-rich structure of coumarins confer several
pharmacological and biological properties (Matos et al., 2015). Novel halogenated
coumarins, such as bromoacetyl derivatives (Fig. 10A-C), inhibited a panel of clinically
important bacteria including Bacillus coagulans, Bacillus cereus, Micrococcus luteus,
Streptococcus faecalis, E. coli and S. aureus in the MIC range of 0.75-1.5 mg/mL. The

addition of bromine to the acetyl group - 3-(2-bromoacetyl) and 3-(2, 2-dibromoacetyl) at the
C3 position of the coumarin nucleus favored antimicrobial activities (Kasumbwe et al.,
2014). Also, dihalogenation at C6 and C8 of the chromene ring (Fig. 10D-E) enhanced
potency against fungal (A. fumigatus, A. flavus, and Rhizopus spp.) and bacterial pathogens
(E. coli and B. cereus) as compared with mono substitution at C6 (Khan et al., 2019).
Notably, the para- bromination increased lipophilicity and stability of the coumarin structure
due to less electronegativity (Fig. 10A1). However, substituting fluorine at the -ortho position
led to destabilization (ZavrŠNik et al., 2011). It appears that the efficacy of halogenated
coumarin depends on halogen number, position, and electronegativity.
As regards their MOAs, halogenated coumarins exhibited a binding affinity for GlcN-
6-P (glucosamine-6-phosphate) synthase and lanosterol 14α-demethylase (CYP51), which are

40
responsible for viability in bacteria and ergosterol biosynthesis and viability in fungi
respectively. Furthermore, this affinity was greater for bacterial GlcN-6-P than fungal
lanosterol, indicating better efficacy against bacteria (Khan et al., 2019). The halogenated
coumarin-chalcone CC2 (Fig. 10F) was able to cross the blood-brain barrier and was non-
toxic to Vero cells at a concentration 100 times higher than the effective concentration used
for biological evaluations. In addition, it halted H2O2-induced cellular damage by scavenging
ROS (Rehuman et al., 2021). Typically, coumarin and some coumarin derivatives were
reported to undergo phase I metabolism and subsequently become hepatotoxic (Hu and Piller,
2017). However, the effects of coumarin halogenation on these contraindications have not
been determined, hence, further investigation is recommended.
2. Phenazines Phenazines are found in Streptomyces and Pseudomonads as secondary
metabolites and confer survival advantages (Huigens Iii et al., 2022). Two phenazines,
pyocyanin and phenazine-1-carboxylic acid, produced by P. aeruginosa serve as electron
acceptors for intracellular redox homeostasis (Schiessl et al., 2019). Phenazines have several

pharmacological properties, ranging from anti-inflammatory, neuroprotective, antimicrobial,
antimycobacterial, and antibiofilm to anticancer activities. The halogenated congeners were
reported to possess better antimicrobial activities against resistant pathogens (Huigens Iii et
al., 2022). Halogenated phenazines (Fig. 10G-L) demonstrated excellent inhibitory activities
against MRSA, methicillin-resistant Staphylococcus epidermis (MRSE), and VRE at MICs of
0.04 to 1.17 μM, and were ~3-23-fold superior to the parent phenazine (1.17-4.69 μM) and
antibiotics – vancomycin, linezolid, and daptomycin (0.39 - 100 μM) (Liu et al., 2022; Yang
et al., 2017). The presence of Cl- or -Br- at the C6 or C8 positions of chlorinated or tri-
brominated phenazines (Fig. 10G-H, 10M) enhanced membrane permeability and
metalloprotein targeting (Yang et al., 2017). Halogenated phenazines (Fig. 10M-O) also
exhibited 4-fold antitubercular potential (6.25 M) against slow-growing Mycobacterium

41
spp. compared to the parent (25 M) (Garrison et al., 2016; Yang et al., 2017). Two Cl-
atoms at the C7 and C8-positions of phenazine enhanced antimycobacterial activity, but their
replacement with two -Br atoms led to a loss of activity, suggesting halogen size critically
affected bioactivity (Fig. 10N-O) (Garrison et al., 2016). Conversely, these derivatives
displayed limited activity against Gram-negative A. baumannii and no activity against E. coli
(Liu et al., 2022). This is the opposite as observed for halogenated quinones, which showed
better activities against Gram-negative bacteria. We opine that the difference was due to
cellular uptake capacity and possibly the compound structural properties. For instance, the
importance of the pair of nitrogen atoms on the phenazine scaffold was evident by a multiple-
fold reduction in activity when one of the nitrogen atoms of 2,4-dibromo-7,8-
difluorophenazin-1-ol (Fig. 10P1) was replaced with C-H to obtain halogenated acridine -
1,3-dibromo-6,7-difluoroacridin-4-ol (Fig. 10P2) (Liu et al., 2022).
Regarding antivirulence potentials, halogenated phenazines (Fig. 10I-J, 10L)
eradicated MRSA, MRSE, and VRE biofilms (0.30-37.5 M) and their persister cells many

times more effectively than the membrane lysing quaternary ammonium cation-10 (2.3-93.8
M) (Garrison et al., 2016; Liu et al., 2022). Also, the control antibiotics (vancomycin,
daptomycin, and linezolid) failed to eradicate biofilms at a concentration of ˃2000 μM (Yang
et al., 2017). The replacement of bromine with iodide in 10L resulted in a significant increase
in biofilm-eradicating activity (Garrison et al., 2016). These reports demonstrate the
halogenation of phenazine is a promising source of antibiofilm agents capable of effectively
controlling chronic and persistent bacterial infections.
Halogenated phenazines employ several mechanisms against resistant pathogens and
their virulence factors. In addition to bacterial membrane crossing and metalloprotein
targeting, they enhance phenolic acidity, which may be essential for metal-chelation (Fig.
42
10A1). Furthermore, 2,4-dibromo-1-hydroxyphenazine inhibited RNA and protein
biosynthesis but not DNA synthesis in MRSA, obviously suggesting RNA as its target (Yang
et al., 2017). Also, the biofilm-forming ability of MRSA was inhibited by a non-membrane
disrupting mechanism, specifically, iron starvation, which was corroborated by the excellent
iron-binding properties of the fluorinated halogenated phenazines and multiple fold
upregulations of the iron uptake genes sbnC and isdB (Liu et al., 2022). Unlike the iron-
specific antibiofilm mechanism, other halogenated phenazines may also employ other metal
(II)-dependent mechanisms (Garrison et al., 2016; Liu et al., 2022). Of note, the presence of
the phenazine efflux pump gene hprS responsible for survival against P. aeruginosa in the
host environment was reported in S. aureus (Fu et al., 2021) suggesting the possibility of
evolutionary resistance to the basic phenazine scaffold. However, the resistance of S. aureus
to 2,4-dibromo-8-chlorophenazin-1-ol (10M) was difficult to develop, probably due to the
enhanced potency and reduced selective pressure conferred by the halogen (Fu et al., 2021)
which implies another merit of halogenation.

Different pre-clinical evaluations have revealed that halogenated phenazines are not
hemolytic to mammalian red blood cells (Liu et al., 2022; Yang et al., 2017), and reportedly,
they were mild to non-toxic against HeLa cells at IC50 > 100 μM with excellent selectivity
indices (>1000-2,000-fold) towards MRSA and MRSE cells (Garrison et al., 2016; Liu et al.,
2022; Yang et al., 2017). Generally, pathogens develop resistance to drugs with little or no
selectivity. Therefore, high selectivity indices, multitarget inhibition, improved activity
spectrum, and low tendency for resistance development exhibited by different halogenated
phenazines highlight their potential as drugs that target resistant pathogens (Fig. 10A1).
E. Azoles
43
The azoles are a class of heterocyclic compounds with a five-membered ring carrying
an atom of nitrogen, sulfur, or oxygen. Based on the heteroatoms present, azoles include
imidazole, oxadiazole, triazole, tetrazole, isoxazole, thiazole isoxazole, pyrazole, 1,2,3-
triazole, thiadiazole, and others (Emami et al., 2022). Importantly, azoles are often used in the
design of antifungal compounds including ketoconazole, fluconazole, and voriconazole. In
addition to their antifungal efficacies, azoles also possess anti-inflammatory, anticancer,
immunosuppressant, and antidiabetic effects (Ghani, 2020). They are known to target the
cytochrome P450-dependent enzymes required for sterol biosynthesis and the formation of
antifungal by-products (Vardanyan and Hruby, 2016). However, fungal pathogens have
developed countermeasures, such as the upregulation of lanosterol 14α-demethylase encoding
(CYP51) and efflux pump genes (Cowen et al., 2015). To address these developments and
minimize unfavorable pharmacokinetics, a series of azole derivatives, including halogenated
azoles, have been developed (Fig. 10Q-Z). Previously, the importance of fluorine in
benzoxazole compounds was reviewed (Al-Harthy et al., 2020) but we focused herein on the

influence of different halogens in the azole scaffold with respect to resistance inhibition,
mechanisms, and pharmacokinetics.
Halogenated benzothiazole-urea derivatives (Fig. 10Q-R) inhibited MRSA at 0.78
mol/L which was 8-128-fold better than the antibiotics cefoxitin and linezolid (6-100
mol/L) but comparable to ofloxacin (1.56 mol/L). Relative to the sensitive S. aureus (0.39
mol/L), 2MIC (0.78 mol/L) of 10Q-R was required to control MRSA (Zha et al., 2022).
Most importantly, the passage assay revealed that MRSA could not gain resistance against
10Q which supports its potential to combat drug resistance. Improved lipophilicity and
solubility probably mediated by the presence of para-CF3 and 3,4-difluoro on the phenylurea
moiety as shown in Fig. 10S, A1 were responsible for the activity (Zha et al., 2022).
44
Furthermore, halogen containing 1,2,3-trizole-derived naphthalimides inhibited E. coli at 0.5
μg/mL which was 1000-, 32- and 4-fold more potent than its precursor, chloromycin and
norfloxacin respectively. The introduction of -Cl on the C3 and C4 of the benzyl group of
compounds enhanced activity against E. coli (Lv et al., 2014). Similarly, halogenated
benzoxazoles (Fig. 10T-U) more strongly inhibited the clinical pathogens - E. coli, S. aureus
A. terrus, and Penicillium brocae than benzoxazole (Rodrigues et al., 2022). These improved
activities were attributed to the presence of -Br or -Cl and the aminopyrazole group
(Rodrigues et al., 2022).
Also, halogenated azoles are highly effective at controlling virulence factors.
Halogenated benzothiazole‒urea (Fig. 10Q-R) exhibited better biofilm eradicating effects
against MRSA at 4-5 M than antibiotics ofloxacin (7-8 M) and erythromycin (˃200 M)
(Zha et al., 2022). In addition, other derivatives (Fig. 10X-Y) inhibited biofilm formation by
S. epidermidis and had no hemolytic effect on human erythrocytes at 50 µM (Zhao et al.,
2014). Further modification of fluconazole with halogens resulted in broad-spectrum activity

against the in-vitro and systemic viabilities, and biofilm formation of fluconazole-resistant C.
albicans isolates at MIC: 0.25-2 µg/mL compared to fluconazole (≥ 64 µg/mL) (Shafiei et al.,
2021).
Regarding the MOA, halogenated azoles exhibited various antimicrobial activities by
targeting the bacterial membrane and DNA replication. For instance, compound 10R killed
MRSA by damaging its cell membrane and bound favorably to the active site of S. aureus
DNA gyrase to disrupt replication (Zha et al., 2022). In addition, 10T-U were predicted to
inhibit the active site of S. aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB),
which participates in peptidoglycan biosynthesis (Rodrigues et al., 2022). Similarly, the
halogenated azoles (Fig. 10V-W) targeted sterol 14-α demethylase to exhibit anticandidal
activity and DNA gyrase to inhibit E. coli (Kumar et al., 2022). Also, 10Z is effectively
45
intercalated into DNA to block replication in E. coli and could be transported by human
serum albumin in the blood plasma via electrostatic interactions to enhance the drug
distribution (Lv et al., 2014). Other halogenated derivatives (Fig. 10X-Y) interfered with the
enzymatic activity of YycG (histidine kinase), which is essential for the viability and cell
wall synthesis of Staphylococcus spp. (Zhao et al., 2014).
Pre-clinical examinations revealed that halogenated analogs (Fig. 10Q-R) exhibited
low cytotoxicity at 25 µM in HepG-2 cells with a ˃75% survival rate. Furthermore, 10R was
well tolerated in a mouse model and did not induce central hepatic hilum necrosis or notable
steatosis at a concentration of 200 mg/kg (Zha et al., 2022). Functionalized fluconazole was
non-toxic to mammalian cells - human dermal fibroblast (HDF), epidermoid (A-431), and
human pancreatic cancer (PANC-1) but could produce a mild effect at 200  MIC (100
µg/mL). It was predicted with no carcinogenic tendencies, medium risk hERG inhibition, and
bioavailability similar to antifungal drugs fluconazole and ravuconazole (Shafiei et al., 2021).
These diverse biological activities, coupled with reasonable toxicities, showed halogenated

azoles provide a promising means of combating resistance either alone or in combination
with natural or conventional antibiotics.
F. Repurposed Halogenated Drug Scaffolds.
The processes involved in drug discovery are expensive and time-consuming, and
these restraints have limited numbers of approved drugs amidst infection concerns. Drug
repurposing which entails the discovery of new medicinal applications or clinical indications
for existing clinically important drugs could play an essential role in reducing these
constraints and facilitating the discovery of drugs. Here, we review various halogen
modifications made to approved drugs. The scaffolds include but are not limited to, isoniazid,
aspirin, haloperidol, phenothiazines, and salicylanilides (Fig. 11A-T).

46
1. Isoniazids
Isoniazid (also known as isonicotinic acid hydrazide) is used to treat tuberculosis. Due
to the nature of the isoniazid structure, only the hydrazine moiety can be altered. Isoniazid
mainly targets the mycobacterial cell wall; however, since toxicity to hepatic cells and drug
resistance development have been reported, they are usually used in combination with other
anti-tuberculosis drugs (Gegia et al., 2017). To address these shortcomings, the aniline moiety
was halogenated (Pflégr et al., 2021). For instance, the 4-triflouromethoxy- and 4-iodophenyl
isoniazid (Fig. 11A-B) displayed inhibitory effects on multidrug-resistant M. tuberculosis
strains (8-64 M) while isoniazid was inactive. As expected, the sensitive M. tuberculosis
strain was more susceptible to the derivatives and isoniazid at lower MIC of 0.03-0.25 M
and 0.5 M respectively (Pflégr et al., 2021). Mutation inhA genes encoding enoyl-acyl
carrier protein reductase involved in mycolic acid synthesis was reported to cause isoniazid
resistance in M. tuberculosis (Palomino and Martin, 2014). Hence, the inhibition of MDR M.
tuberculosis by halogenated compounds (Fig. 11A-B) compared to the inactivity of isoniazid

suggests the capacity to counter mutated enoyl-acyl carrier protein enzyme (InhA) and
overcome resistance via improved permeability and drug uptake. Their application as
adjuvants with antibiotics is recommended to further potentiate inhibitory activity.
Interestingly, the enhancement of lipophilicity and mycobacterial cell wall penetration as
improved by the presence of trifluoromethoxy (-OCF3) and iodine (I) groups at the C4 of the
aniline ring of 11A-B supported the effect (Fig. 11U) (Pflégr et al., 2021).
Furthermore, unlike the hepatotoxic effect reported for isoniazid (Gegia et al., 2017),
halogenated isoniazids 11A-B showed no cytotoxic or cytostatic activity in HepG2 cells (a
human hepatocellular cell line) at >50 M in addition to high selectivity index towards
Mycobacterium spp. (Pflégr et al., 2021). Also, INH-c (brominated isonicotinoylhydrazone
47
derivative) had none to mild toxicity and good biocompatibility with fibroblasts at 1.75
mg/mL and its LD50 revealed it is 10 times less toxic than the parent isoniazid (Fig. 11U). It
also showed the lowest signs of hepatic parenchyma alteration and vascular congestion
compared to isoniazid (Dragostin et al., 2019). Thus, indicating halogenation as a promising
alternative for alleviating hepatic toxicity typical of antimycobacterial drugs.
2. Azo aspirins
This drug is otherwise known as acetylsalicylic acid and is a representative NSAID
(non-steroidal anti-inflammatory drug). Its precursor aspirin -salicylic acid is present in
willow tree bark. Aspirin is weakly acidic and has been used to treat fever, pain and as an
anti-inflammatory drug, but possesses weak antibacterial activity (Nordin et al., 2017).
Halogen-containing derivatives were synthesized to enhance their antibacterial activity and
interactions with targets. Halogenated azo aspirin (Fig. 11C) displayed considerable
antibacterial activity better than aspirin against E. coli and S. aureus (Ngaini and Mortadza,
2019). Iodine substitution in addition to hydroxyl and carboxylic groups increased

lipophilicity and subsequent easy cell membrane penetration to enhance the antibacterial
activity (Ngaini and Mortadza, 2019). Compound 11C was predicted to bind effectively with
phosphatidylinositol-specific phospholipase C of S. aureus and phospholipid-binding protein
MIaC of E. coli. A halogen bond interaction was observed with the active site of E. coli MIaC
through iodine binding with –C=O and–NH of Gln48 (Ngaini and Mortadza, 2019). While
other aspirin derivatives with weak antibacterial activities have been reported (Nordin et al.,
2017), this review shows that halogenation is suitable to potentiate the antimicrobial effects
of azo aspirin.
3. Haloperidols
Haloperidol is a butylbenzoic antipsychotic agent cleared by the liver via
glucuronidation and CYP3A4-mediated oxidation. Haloperidol is tightly bound in human

48
plasma and extensively metabolized by the liver (Osacka et al., 2022). Haloperidol showed
promising antifungal properties against fluconazole-resistant C. albicans. Specifically,
fluorinated derivative 11D at 32 μg/mL inhibited the invasive fungi better than fluconazole
(˃64 μg/mL). Fluorine substitution on the C4 position of the terminal benzene ring was
reported to be crucial for antifungal activity and may have improved the metabolic stability,
toxicity and good intrinsic clearance rate compared with haloperidol (Ji et al., 2019).
Haloperidol derivative 11D (4-fluorophenyl haloperidol) exhibited mechanisms targeting the
growth and virulence factors of pathogens. It also reduced the production of biofilms,
melanin, urease, and capsular polysaccharides, which are responsible for cell wall integrity,
host immunity resistance, host survival and morbidity, and the pathogenicity of C.
neoformans (Ji et al., 2019; O'Meara and Alspaugh, 2012; Revankar and Sutton, 2010).
Co-administration of compound 11D with fluconazole further reduced MIC to 4
μg/mL and minimized resistance development by inhibiting efflux pump (MDR1) gene
expression and interfering with ergosterol biosynthesis in fluconazole-resistant C. albicans (Ji

et al., 2019). Furthermore, bromoperidol derivatives in combination with posaconazole and
voriconazole synergistically inhibited azole-resistant C. albicans which otherwise would have
been resistant to either drug (Holbrook et al., 2017). These observations suggest that the
application of haloperidol and its halogenated derivatives as adjuvants may reverse antifungal
drug resistance by targeting a transporter/efflux pump (MDR1) as corroborated by other
studies (Iatta et al., 2017; Iwaki et al., 2006). Haloperidol is associated with severe
extrapyramidal side effects because of its strong affinity for the dopamine D2 receptor
(Sikazwe et al., 2003). However, 11D exhibited less affinity for this receptor which suggests a
low risk of haloperidol-related side effects (Ji et al., 2019).
4. Pyrrolopyrimidines
49
The pyrrolopyrimidine scaffold has proven to be versatile in the medicinal chemistry
field and has been utilized in many FDA-approved medications (Tian et al., 2019). The
pyrrolopyrimidines are structurally similar to purines and can bind to proteins and
antimetabolites during the metabolism of nucleic acids (Adel et al., 2018). Compounds
containing this scaffold have antineoplastic, antibiotic, and antiviral effects and are known to
bind to similar targets in bacteria but have narrow activity profiles (Olsen et al., 2022;
Zanello and Corsini, 2017). Different halogenated pyrimidines were synthesized to target
antimicrobial resistance. The halogenated pyrrolopyrimidines 11E and 11F inhibited S.
aureus at 8 μg/mL, which was 8-fold better than parent pyrrolopyrimidine (64 µg/L), but
lacked activity against Gram-negative E. coli (Olsen et al., 2022). This may have been due to
the outer membrane-mediated protection of Gram-negative E. coli. Interestingly, the
combinatory effects of 11E or 11F with the antimicrobial peptide betatide drastically lowered
MICs against S. aureus (1–2 mg/L) suggesting their potential use as adjuvants (Olsen et al.,
2022). The presence of bromine or iodine at the C4 of the benzylamine group and a -OH

group at the -meta or -para position on the 6-aryl unit (Fig. 11G) were essential for increased
potency towards S. aureus. The bromophenyl derivative 11E moderately inhibited
thymidylate monophosphate kinase, which is responsible for essential nucleoside synthesis
and a possible target in S. aureus. However, 11E showed very low activity against human
kinases, suggesting the possibility of low in vivo activity (Olsen et al., 2022). Since
pyrimidine is an essential component of nucleic acid, we envisage that halogenated
pyrrolopyrimidines would have better in vivo compatibilities, and thus be leveraged for
application in drug development.
5. Phenothiazines
Phenothiazines are tricyclic compounds and represent a large group of antipsychotic
medications. Some phenothiazine derivatives, including thioridazine, were reported to inhibit

50
extensively drug-resistant M. tuberculosis, increased MRSA susceptibility to beta-lactam
drugs, and controlled P. aeruginosa, K. pneumonia, and A. baumannii persister cells
(Mohiuddin et al., 2022; Thorsing et al., 2013). However, the difficulty of separating their
central effects and other pharmacological activities had limited their applications (Nizi et al.,
2020). The versatility of the phenothiazine scaffold makes it an important lead structure and
derivatives with specific biological targets were discovered after halogenation. Compounds
11H-I exhibited excellent antimycobacterial activities better than standard thioridazine or
chlorpromazine against M. tuberculosis. Importantly, 11H and 11I inhibited non-replicating
M. tuberculosis under hypoxic conditions, suggesting their potential use to prevent persistent
infection (Nizi et al., 2020). The poly halogenation (3,7-dibromo and 1,3,7,9-tetrachloro-) of
the phenothiazines enhanced biological activity. Additionally, phenothiazine chlorpromazine
inhibited MDR-resistant S. enterica serovar Typhimurium and synergized with norfloxacin
and ethidium bromide (Fig. 11U). It was reported to repress the expression of efflux pump
gene acrB in a manner that incapacitated the efflux pump to significantly export the drug,

hence, increasing the accumulation of chlorpromazine within the Salmonella cells (Bailey et
al., 2008).
Phenothiazine derivatives exhibited different mechanisms for their antimicrobial
actions. For example, 3,7-dibromo- (11H) and 1,3,7,9-tetrachloro- phenothiazines (11I)
disrupted oxygen consumption and nicotinamide adenine dinucleotide hydrogen (NADH)
oxidation, which are central to the survival of M. tuberculosis. It was inferred that poly-
halogenated derivatives targeted NDH-2 (type II NADH dehydrogenase) to exert their
antitubercular activities. Of note, NDH-2 transfers NADH-derived electrons into the
mycobacterial respiratory chain and thereby plays an essential role in ATP generation
through oxidative phosphorylation in mycobacteria (Nizi et al., 2020). These outcomes
suggest that halogenation may have conferred the scaffold with novel potential and
51
mechanisms other than blocking D2 dopaminergic receptors, muscarinic M1 receptors, and
histamine H1 receptors.
Compounds 11H and 11I were metabolically stable and showed synergism with
rifampin but may be hepatoxic only at high concentrations. Typically, phenothiazines are not
used as anti-infective agents because of their effects on the central nervous system
(Dougherty and Marraffa, 2014). However, halogenated phenothiazines showed lower
affinities for central nervous system receptors, and could probably alleviate side effects
(Fig.11U) (Nizi et al., 2020). Also, parent phenothiazine seems to produce photoallergic and
phototoxic effects which are usually triggered by exposure to UV radiation and resulting in
eczematous eruption at the exposed sites (Kowalska et al., 2021). However, reports regarding
the potential of halogen modulation to curb this anomaly are scarce, hence, further
investigation is recommended.
6. Salicylanilides
The salicylanilide scaffold contains the amide of salicylic acid and aniline which acts

by restricting the two-component regulatory systems in bacteria, limiting nutrient uptake and
structural damage to microbial cells (Paraskevopoulos et al., 2017). Although the phenolic
group on the scaffold seems to confer antimicrobial activity, it is sometimes responsible for
irritation and uncoupling activity. Hence, modification is required to temporarily block these
activities and improve membrane penetration, bioavailability, and bioactivity (Ienașcu et al.,
2022). Halogenated salicylanilide derivatives were reported as anthelmintic agents,
disinfectants, and antimicrobial agents against resistant pathogens. Niclosamide (Fig. 11J) as
an FDA-approved anthelmintic drug and oxyclozanide (Fig. 11K) effectively inhibited
daptomycin, linezolid, methicillin, and vancomycin-resistant S. aureus by disrupting the cell
envelope (Rajamuthiah et al., 2015). Other derivatives (11L-M) effectively controlled
multidrug and extensively drug-resistant Mycobacterium (0.5 μM) better than standard
52
isoniazid (16 μM). The monohalogenation at C4 of the salicylic ring (Fig. 11L-M) and the
introduction of -CF3 at C4 of the aniline ring improved antimycobacterial activities
(Paraskevopoulos et al., 2017).
Furthermore, a panel of MRSA and vancomycin-resistant S. aureus (VRSA) were
inhibited at very low MICs (0.03–0.50 μg/mL) by trifluoromethylphenyl and 4-chlorophenyl
analogs (Figs. 11N-P). Particularly, 4’-bromo-3’-trifluoromethylphenyl congener – 0.03-0.06
μg/mL (11N) was 32 to 1024 fold better than methicillin (32-64 μg/mL) and vancomycin (1-2
μg/mL) against MDR S. aureus and VRSA (Lal et al., 2021). Relative to the sensitive S.
aureus (0.25-0.50 μg/mL), 11N was more effective implying the capacity to break resistance
and exert strong antibacterial activity. As revealed by the SAR study (Fig. 11Q), the activity
of compound 11N-P against MDR strains was potentiated by the presence of 3’-
trifluoromethyl and 4’-bromo on the salicylanilide moiety (Lal et al., 2021). Also,
halogenated salicylanilides (11R-S) effectively controlled different strains of colistin-
resistant Klebsiella pneumonia and A. baumannii by 128-2048 fold at 5 μM (Nemeth et al.,

2020). The replacement of the 3,5-bistrifluoromethyl groups with dibromo substituents and
the addition of fluorine enhanced the activity of 11R and 11S respectively against the colistin
resistance (Nemeth et al., 2020).
However, niclosamide (11J) and oxyclozanide (11K) lacked activity against Gram-
negative ESKAPE pathogens like K. pneumoniae, A. baumannii, P. aeruginosa, and E.
aerogenes (Rajamuthiah et al., 2015). It seems that the presence of bulky halogens (Br, CF3)
on the salicylanilide scaffold (Fig. 11R-S) could facilitate membrane penetration and uptake
as compared to -Cl on niclosamide and oxyclozanide (Fig. 11J-K). Perhaps, the bromine and
trifluoromethyl groups were able to create better dipole moment and interact with polar
amino acids in the constriction zone of the outer membrane proteins of Gram-negative
bacteria. Typically, the biological activities of salicylanilides are influenced by their

53
hydrophobicities (Ienașcu et al., 2022), which suggests that different halogen additions might
have modulated this property.
Halogenated salicylanilides could also potentiate activity against microbial virulence
factors capable of increasing drug resistance (Fig. 11U). For instance, compound 11N
disrupted pre-formed S. aureus biofilms better than vancomycin and levofloxacin at the same
doses (Lal et al., 2021). Similarly, niclosamide (Fig. 11J) at 0.5-5 μM attenuated the
filamentation, hyphae, and biofilm formation of sensitive and azole-resistant C. albicans
(Garcia et al., 2018). Niclosamide downregulated the virulence genes related to the cell wall,
filamentation, ergosterol, sphingolipids, and phosphoglycerides in Candida spp., inactivated a
nucleotide release factor Mge1, and disrupted mitochondrial membrane potential, thereby
affecting mitochondrial functionality (Garcia et al., 2018). The excellent antivirulence
activity by 11J was attributed to the stability of the C-Cl bond which enabled effective
binding to virulence targets. Notably, niclosamide (Fig. 11J) is currently undergoing a phase
2 clinical trial for the treatment of S. aureus infections (Butler and Paterson, 2020).

Toxicity profiling of various halogenated salicylanilides demonstrated they are safe
for clinical applications (Fig. 11U). Niclosamide and oxyclozanide protected host cells
against fungal invasion, significantly reduced damage to HT-29 enterocytes, attenuated
damage to intestinal epithelial cells, and were non-toxic to sheep erythrocytes (Garcia et al.,
2018; Rajamuthiah et al., 2015). Also, niclosamide rescued C. elegans from MRSA infection
and prolonged worm survival but was toxic to cancer lines (HepG2 and HEK293). The
anticancer effect of niclosamide and its ability to interfere with signaling pathways could
explain this observation. (Rajamuthiah et al., 2015; Ye et al., 2014). Furthermore, analog 11R
had no measurable toxicity against epithelial 4T1 cells up to 200 μM while others (Figs. 11N-
O) showed better specificity towards bacterial cells than host cells and improved selectivity
indices (Lal et al., 2021; Nemeth et al., 2020). Typically, halogenated salicylanilides share
54
some pharmacological features, such as prolonged elimination half-life, high plasma binding,
and limited metabolism (Swan, 1999). Information as to whether halogens influence these
parameters is not available. However, insight was provided by the contribution of halogens to
toxicity minimization, as was reported for the trifluoromethylphenyl derivative (11T),
whereby the introduction of an additional halogen alleviated cytotoxicity (Paraskevopoulos et
al., 2017). It does suggest that halogenation might potentiate the activity of salicylanilides
against resistant pathogens, minimize toxicity, and provide a safe scaffold for in vivo
administration.
G. Other Halogenated Bioactive Compounds
1. β-Nitrostyrene and Nitrovinylfurans
β-Nitrostyrene is an aromatic compound and nitroalkene isolated from plants and
bacteria. 2-nitro-4-((E)-2-nitrovinyl) phenol was isolated from Sonneratia acids Linn. F and
an artic sea ice bacterium Salegentibacter sp. The bioactivities and ease of synthesis of β-

nitrostyrene generated renewed interest in their derivatives (Shafi et al., 2016; Tsai et al.,
2017). Also, nitrovinylfuran has biological activities that have been harnessed in clinical and
industrial settings. Dermofural ointment is used to treat nail/human skin infections, and
Furvinol in veterinary medicine (Allas et al., 2016). Brominated nitrovinylfuran could exert
broad-spectrum antibacterial activity against sensitive and resistant bacteria (Fig. 12U). For
example, Furvina (12A) and its conversion product bromonitromethane (12B) exhibited the
same MICs of 4 and 2 µg/mL against both sensitive and resistant S. aureus respectively
which was comparable to fosfomycin (4 µg/mL) but superior to cnicin (˃32 µg/mL) (Scholz
et al., 2013). The broad potency observed was attributed to the presence of a
bromonitromethyl group in the scaffold (Scholz et al., 2013).
55
Similarly, halogenated nitrostyrene (Fig. 12C-E) exhibited antibacterial and
antifungal activities against E. coli, B. subtilis, S. aureus, E. faecalis, and C. albicans (Cornell
et al., 2014; Lo et al., 2012). Bromine addition to β-nitrostyrenes (Fig. 12C) at the C4
position of the aromatic ring and the β-position of the alkene side chain of nitropropenyl
arenes resulted in improved activities (Cornell et al., 2014). Inhibition of E. coli by the
fluorinated analogs (12D-E) was due to the high electronegativity and hydrophilic properties
induced by fluorine, which alters the ability to penetrate Gram-negative bacteria cell walls
(Lo et al., 2012). A comparison of the inactivity of the fluorinated derivative 12F to the
potency of a similar compound (12D) against E. coli demonstrated the importance of the
scaffold to which halogens are attached. Specifically, direct attachment of fluorine to β-
methyl-β-nitrostyrene favored efficacy more than methylenedioxy attachment (Cornell et al.,
2014; Lo et al., 2012). This corroborates a report that the ability of fluorine to alter
physicochemical properties like lipophilicity (logP) depends on the scaffold and proximal
functionality (Al-Harthy et al., 2020). Regarding antivirulence potentials, Furvina inhibited

the 3-oxo-C12-homoserine lactone-dependent quorum sensing system of P. aeruginosa,
reduced biofilm formation and the production of QS-dependent virulence factors (Borges et
al., 2017).
MOA studies revealed that the nitrovinylfuran derivatives (12A-B) irreversibly
inhibited MurA (an enzyme involved in peptidoglycan biosynthesis) and E. coli MetAP. They
also exerted pleiotropic effects on numerous proteins by interfering with cysteine residues
like the standard cysteine residue inhibitor Fosfomycin. Notably, like antibiotics, Furviva
targeted actively growing E. coli cells rather than non-growing cells (Allas et al., 2016).
Additionally, it disrupted translation initiation by interfering with the P-site of the 30S
subunit (Fabbretti et al., 2012; Oliveira et al., 2021).
56
Furvina (Fig. 12A), however, displayed in vitro instability as it transformed rapidly
into different intermediates attributing its antimicrobial effects to the combinatory effects of
its immediate reactivity and those of its reaction products (Allas et al., 2016). The
phenomenon was improved in acidic conditions but may impair target binding or cellular
uptake (Scholz et al., 2013). Furvina and its conversion products showed long-term toxic
effects against mammalian cell lines and were rather suggested for use in the design of
cysteine residues inhibitors in protein targets as indicated by the irreversible inhibition of
MurA cysteines and bacterial methionine aminopeptidase (Allas et al., 2016; Scholz et al.,
2013). In addition, 4-chloro-β-nitrostyrene (12G), was reported to exhibit vasorelaxant
effects courtesy of the electrophilic capacity of the substituted chloro-nitrostyrene (Sousa‐
Brito et al., 2021). Overall, the halogenation of β-nitrostyrene and nitrovinylfuran improved
the antimicrobial and antivirulence properties but further studies on their pharmacokinetics
are recommended.
2. 2-Aminothiopenes

2-Aminothiophenes are a group of heterocyclic compounds with five rings and
exhibit diverse biological functions, including antifungal, antiviral, anti-tubercular, anticancer
effects and allosteric agents. The aminothiophene scaffold can be found in approved drugs
such as Zyprexa and drug candidates (Bozorov et al., 2017). Despite the versatility of this
scaffold, low water solubility remains a challenge. As a result, new derivatives with lower
octanol-water partition coefficients, improved water solubilities, antimicrobial activities, and
pharmacokinetic profiles were synthesized (Luna et al., 2021). The halogenated 2-
aminothiophene 12H at 1 μg/mL inhibited all dermatophyte fungal strains, including T.
tonsurans and T. rubrum, and other congeners (Fig. 12I-J) inhibited them in a manner
comparable to fluconazole (Luna et al., 2021). The position and size of the halogen
substituent at the benzylidene moiety determined antifungal activity, and the presence of
57
fluorine, chlorine, or bromine at the C4 position increased derivative potencies (Fig. 12U).
Halogenation type had a differential influence on the antimicrobial activities of the 2-
aminothiopenes. The presence of bromine (Fig. 12J) caused sensitivity in tested fungal
strains, whereas the dichloro- substitution (Fig. 12I) at the -ortho position increased the
spectrum of activity (Luna et al., 2021). Furthermore, two halogenated 2-aminothiopene
derivatives (Fig. 12K-L) reversed ciprofloxacin and erythromycin resistance in S. aureus by
acting as NorA and MrsA efflux pump inhibitors independent of growth inhibition. The
presence of 2-aminotrifluoroacetyl group was responsible for the increased potentiation of
ciprofloxacin and erythromycin to overcome S. aureus resistance (Fig. 12U) (da Cruz et al.,
2020).
Preclinical toxicity evaluations of halogenated aminothiophenes (Fig. 12I-J) showed
they were not toxic to three non-tumor cell lines (VERO, MRC-05, and 3T3) at 1-100 μM or
to Tetrahymena pyriformis, a protozoan employed to assess in vivo toxicity. Similarly, the 2-
aminotrifluoroacetyl derivative (Fig. 12K) was non-toxic to the murine macrophage cells

while (Fig. 12L) showed potential toxic effects and the cost-benefit of its application should
be considered (da Cruz et al., 2020). In addition, ADME analysis revealed that halogenated
aminothiophenes (Fig. 12H-J) had excellent oral bioavailabilities and complied with
Lipinski's rule of five and Veber's parameters. In addition, they were negative for hERG
inhibition, had high gastrointestinal absorptions, non-hepatotoxic, and no skin sensitization
effects but had limited brain-blood barrier permeabilities and low skin permeabilities (Luna et
al., 2021). This could however impair distribution and topical application, respectively. We
consider halogenated aminothiophenes promising oral drug candidates which could act as
antibiotic adjuvants for therapy against drug-resistant infection However, further structural
modulation and study are required to improve pharmacokinetic properties and unveil the
antimicrobial MOAs.
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3. Isophthalonitrile
Isophthalonitrile derivatives remain one of the most essential organic cyanides and
halogenated isosteres which are usually referred to as virtual analog halogens. These
derivatives have antiviral, anti-inflammatory, and insecticidal activities, and their
polyhalogenated derivatives, especially polyfluoro-isophthalonitrile derivatives, also possess
anti-cancer, anti-inflammatory, and insecticidal activities and other applications of
pharmaceutical interest (Senapati et al., 2017). Halogenated isophthalonitrile derivatives
showed excellent antimicrobial activities against clinically important pathogens, including S.
aureus, Bacillus cereus, and C. albicans. Specifically, these derivatives (Fig. 12M) inhibited
the pathogens at 0.4-0.7 µg/mL, which compared favorably with norfloxacin and fluconazole
in the range of 0.5-3 µg/mL (Huang et al., 2013). It was reported that fluorine substitutions at
the C2 and C4 positions and benzyl amino substitution at the C6 position contributed to
antibacterial and antifungal activities. The group speculated that the cell wall/membrane was
probably targeted by halogenated isophthalonitrile for antimicrobial activity (Huang et al.,

2013). The potency shown by halogenated isophthalonitriles further confirms the
indispensable role of halogens in medicinal scaffolds. However, available data on fluorinated
and chlorinated isophthalonitriles is limited, and thus further studies are required to fill gaps
in addition to investigating the effects of other halogens like bromine and iodine.
4. β-Amino amides and Alboflavusins
β-Amino acids are essential monomers present in natural products, including Taxol
and peptidomimetics (Paulsen et al., 2016), and the synthesis of peptidomimetics like α,α -
disubstituted β-amino amides, which mimic the activities of larger antimicrobial peptides
against bacterial cell membranes, has received great attention. Structurally, β-amino acids
contain two cationic moieties and a pair of bulky lipophilic groups and are resistant to α-
chymotrypsin degradation and maintain their stability in aqueous solutions even at pH 7.4
59
(Paulsen et al., 2019). β-Amino acids were reported to possess excellent antibacterial and
antibiofilm activity against S. aureus through membrane disruption (Ausbacher et al., 2014).
However, despite these features, they are susceptible to phase I oxidation, and importantly,
their electron-rich aromatic sidechains are easily oxidized. Halogenation of β-amino amides
was employed as a tool to improve activity and control metabolic instability (Fig. 12U)
(Paulsen et al., 2019). Halogenated disubstituted β-amino amides exhibited antibacterial
activities against a panel of bacterial strains, including 30 multidrug-resistant (MDR) clinical
isolates (Paulsen et al., 2019). In particular, halogenated diamine and diguanidine derivatives
(12N-Q) with respective substituents (3,5-Br-Ph) and (3,5-CF3-Ph) inhibited multidrug-
resistant S. aureus, E. faecium, and K. pneumoniae at low concentrations (MIC: 4-8 µg/mL)
but not P. aeruginosa (MIC - 32 µg/mL) (Paulsen et al., 2019), which may be related to the
intrinsic factors of the pathogens. Also, halogenated di-guanidine derivatives (12P-Q)
exhibited similar activity against S. aureus, E. faecium, E. coli, P. aeruginosa, K.
pneumoniae, and colistin-resistant A. baumannii (MIC: 2-16 µg/mL). Of note, diguanylated

analog 3,5-Br-Ph (Fig. 12P) demonstrated effects comparable to oxytetracycline (the positive
control) against reference strains (Paulsen et al., 2019). Notably, SAR analysis as shown in
Fig. 12Q revealed that halogenated lipophilic groups (3,5-Br-Ph and 3,5-CF3-Ph) contributed
to the antibacterial effects and low toxicities of the di-guanidine derivatives (Paulsen et al.,
2019).
Alboflavusin is a naturally occurring, novel, cyclic hexapeptide antibiotic containing a
chlorine atom. It has been isolated from marine S. alboflavus and shown to possess promising
anti-tumor and antibacterial activities and apoptosis induction (Crowe et al., 2021; Li et al.,
2021a). Brominated alboflavusins (AFN) 1 and 2 (Fig. 12S-T) isolated from Streptomyces
alboflavus sp. 313 exhibited antibacterial against various MRSA strains. Particularly, AFN 2
(Fig. 12T) at MIC of 3.1-6.2 µM was 4-8 times superior to the parent alboflavusin (12.5-25.0
60
µM) against four MRSA strains (Li et al., 2021a). Compared to the sensitive strain (6.2 µM),
the halogenated congener (12T) showed a comparable-to-superior effect (3.1-6.2 µM)
indicating the potential to overcome resistance and exert equipotent activity. According to the
SAR studies, the presence of bromine improved their anti-MRSA activities (Li et al., 2021a).
Regarding toxicity, the halogenated β-amino amides and alboflavusins showed non-
to-tolerable toxicity and improved metabolic stability. β-amino amides derivative (Fig. 12P)
had no major in vitro toxicity on human lung fibroblasts (MRC-5) or human
hepatocyte carcinoma cells (HepG2) and they (Fig. 12P-Q) were non-hemolytic to red blood
cells at an EC50 of >200 µg/mL in addition to high selectivity indices. Compared to the parent
compound, the lipophilic sidechains 3,5-Br-Ph of the diguanylated analog (Fig. 12O) showed
resistance to phase I oxidation, suggesting high in vivo metabolic stability (Paulsen et al.,
2019). Taken together, the halogenation of β-amino amides and alboflavusins conferred
improved antimicrobial efficacy and protected against metabolic degradation. However,
target molecules and mechanisms for the various antibacterial effects displayed need further

investigation.
IV. Halogenated Polymeric Materials
Biopolymers such as chitosan are non-toxic, biocompatible, bioactive, and
biodegradable (Zhang et al., 2018b). Also, the antibacterial polymers especially the cationic
ones including poly(ethyleneimine), poly(l-lysine), and poly(2-dimethyl(aminoethyl)
methacrylate were reported as promising alternatives to commercial antibiotics (Kyzioł et al.,
2020; Qiu et al., 2020). Despite the aforementioned, the majority of biopolymers do not
possess inherent antimicrobial activity and therefore require chemical modification (Ganie et
al., 2021). This section reviews various halogen-functionalized polymers and the roles played
by halogens in their antimicrobial activities (Fig. 13).

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The cellulose-grafted, hyperbranched, chlorine-containing polymer shown in Fig.
13A inhibited E. coli, Proteus microbilis, P. aeruginosa, P. vulgaris, E. aerogenes, and S.
typhimurium, and this was attributed to the hydrophobicity caused by chlorine substitution
(Demircan and Zhang, 2017). Similarly, the halogenated chitosan analogs (Fig. 13B-E)
displayed excellent antifungal activities against Fusarium oxysporum and Phomopsis
asparagi, which was attributed to the electron-withdrawing of halogen groups and resulting
increases in membrane permeability and hydrophobicity. Furthermore, observed increases in
antimicrobial activity were proportional to degrees of substitution and halogen
electronegativity (Zhang et al., 2018a). In addition, the halogenated Schiff bases of chitosan
(Figs. 13F-H) inhibited Botrytis cinerea by 96.7, 96.0, and 95.8 %, respectively, compared to
23.8% inhibition by chitosan. Regarding the mechanism, halogen atoms established strong
electrostatic interactions with negatively charged fungal cells, causing the leakage of
intracellular materials and death (Wei et al., 2021). Moreover, fabric antiadhesive polymer
modified with fluorine (Fig. 13I) reduced the presence of S. aureus and E. coli on fabric

surfaces due to increased penetration into the lipid domains of bacterial membranes (Lin et al.,
2018).
N-halamines constitute another category of halogen-containing polymers. They exist
largely as imides, amines, or amides and covalently interact with halogens to produce
bactericidal halogen ions in the form of oxidative halogens (Demir et al., 2017). The
synthesis, application, and antibacterial roles of N-halamines have been reviewed (Dong et al.,
2017; Wang et al., 2020). Since new N-halamines have been described, we provide an update
and describe the specific roles played by halogens. N-halamine conjugate based on
polydopamine was formulated, and the chlorinated polydopamine film produced reduced the
viability of E. coli by ~99.5% after 3 h of treatment and its surface adhesion by 45% (Nazi et
al., 2020). Similarly, a polyurethane functionalized with an N-halamine precursor (2-amino-

62
5-(2-hydroxyethyl)-6-methyl pyrimidine-4-one) (AHM) produced a conjugated PU-AHM-Cl
(Fig. 13J) film that showed marked antibacterial activity against S. aureus and E. coli with a
100% reduction in viability after 5 min of contact while the parent polyurethane had no effect
(Peng et al., 2021). Also, cotton fabric doped with N-halamine-based
dopamine/polyethyleneimine (PDA/PEI-Cotton-Cl) containing 0.182% of active chlorine
reduced the adhesiveness of S. aureus and E. coli to fabrics by 5.0 and 7.2 Log, respectively
(Wan et al., 2022). The MOAs of N-halamine-based polymers are similar and involve the
chlorination of N-H bonds to form N-Cl bonds, and the bonds undergo hydrolysis to produce
oxidative chloride ions, which disrupt bacterial membranes (Wan et al., 2022).
Antimicrobial polymers are less susceptible to bacterial resistance, and their
antibacterial activities are due to cell membrane disruption (Qiu et al., 2020). In the current
review, halogenation either restored or further enhanced the penetration of polymers into the
cell barriers to display notable antimicrobial activities. A deeper understanding of the effects
of halogenated polymers on resistant pathogens is required before they can be considered

viable alternatives to traditional antibiotics.
V. Halogenated Biomolecules
Halogen sources are not common in ecosystems, and halogen bonds were not
recognized in biological systems during the early part of the 20th century. However, due to
the discovery that halogens bond to peptides, lipids, carbohydrates, and other biomolecules
(Ho, 2014), halogens are now considered important elements in biological systems.
A. Peptides
Antimicrobial peptides (AMPs) are components of the innate immune defense
responses of many organisms. Their net positive charges enable them to bind negatively
charged bacterial components and disrupt their activities. The structural makeup of AMPs
63
like the amino acid substitutions critically determines their functionalities (Ageitos et al.,
2017). For example, proline-rich AMPs penetrate bacterial cytoplasm rather than destroy
membranes (Mattiuzzo et al., 2007), and glycine-rich AMPs induce phagocyte-mediated
bactericidal mechanisms that differ from the conventional MOAs of AMPs (Huan et al.,
2020). Notably, amino acid halogenation enhances antimicrobial potency; for example, the
replacement of phenylalanine with a halogenated analog profoundly increased the anti-
biofilm and antibacterial effect of jelleine-1 (Jia et al., 2019). Halogenation can also be
utilized to specifically target microbes, as demonstrated by o-fluorine substitution in
phenylalanine residues, which promoted the antimicrobial effect of temporin L on E. coli but
had no effect on S. aureus or P. aeruginosa (Setty et al., 2017).
Halogenation of amino acids alters the structural and physiochemical properties of
peptide scaffolds influencing peptide membrane permeabilization and catabolic stability
(Parisini et al., 2011). Fluorination impacts the proteolytic stabilities of peptides, and bulky
fluorinated residues, such as hexafluoroleucine or trifluoroisoleucine, and fluorinated amino

acids shield polypeptides from proteolytic enzymes (Berger et al., 2017; Huhmann and
Koksch, 2018). Halogenated amino acids may be natural or synthetic. Most natural
antimicrobial amino acids are bound by sp2 carbons as the sp2 C-X bond is less likely to
react with biological nucleophiles like water and amino and hydroxyl groups. For this reason,
most of the halogenated amino acids in nature are aromatic, like tyrosine, histidine, and
tryptophan (Table 1 A-N)(Peng et al., 2005). Halogenation of majority of the amino acids
occurs post-translationally, and thus effects are only observed in peptide form (Bittner et al.,
2007). Although aromaticity favors halogenation, non-essential and aliphatic amino acids
with antimicrobial activities have been isolated from natural sources (Table 1 O-X).
Halogenated amino acids are also well-represented among non-ribosomal peptides and are
usually synthesized by one or more specialized non-ribosomal peptide synthases (Moravej et

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al., 2018). A deeper understanding of many halogenated amino acids in non-ribosomal
peptides and their peptidomimetic characteristics were provided in a previous review
(Mardirossian et al., 2021).
Halogenated antimicrobial peptides can be classified into two categories, namely
ribosomally synthesized naturally halogenated AMPs and synthetic halogenated AMPs.
Halogenation of ribosomally synthesized peptides occurs post-translationally. In most cases,
bromine substitution in tryptophan occurs at position 6 of the tryptophan indole ring, as
observed in styelin D, hedistin, strongylocin, and centrocins, and in hagfish AMPs isolated
from marine sources (Cruz et al., 2015; Li et al., 2010; Li et al., 2008; Maffioli et al., 2014;
Tasiemski et al., 2007; Taylor et al., 2000; Uzzell et al., 2003). The role of bromine
substitution on AMP activity has not been determined, though bromine substitution was
reported to protect peptides from proteases (Uzzell et al., 2003).
Synthetic halogenated AMPs have been produced to improve antimicrobial potency
and modify pharmacokinetics. Fluorination of maganin and melittin enhanced antimicrobial

activity and proteolytic resistance. Moreover, halogenated analogs of jelleine-1 displayed an
eightfold increase in in vitro activity, and halogenation of tripticin and its amidated analog
tritrp1 also increased antimicrobial activities (Arias et al., 2020; Gottler et al., 2008; Jia et al.,
2019; Lawyer et al., 1996). Peptidomimetics are synthetic peptide analogs resistant to
proteolytic degradation. Several halogenated peptidomimetic libraries have been synthesized,
such as halogen battacin derivatives with fluorinated aromatic L-serine substitutions, which
increased broad-spectrum activity against several clinically relevant bacteria like P.
aeruginosa and Campylobacter jejuni, and a 36 peptoid oligomer library, in which
halogenated N-substituted glycine exhibited a multifold increase in antimicrobial activity
against S. aureus, E. coli and P. aeruginosa (Glossop et al., 2021; Krenk et al., 2015;
Molchanova et al., 2020).

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The antimicrobial activity of halogenated peptides depends on several factors, such as
the type and aromaticity of the amino acid substituted, the ability to form halogen bonds with
biological systems, and the ease of peptide self-assembly (Molchanova et al., 2020). Protein
and peptide halogenation have been used to synthesize defensive compounds in organisms of
various ecosystems (Pizzi et al., 2020). A similar approach to the synthesis of AMPs might be
fruitful, though the applicability of halogenation in synthetic biology remains largely
unexplored due to a lack of knowledge of the impacts of steric and electronic factors.
Nonetheless, leveraging halogenation in synthetic biology for the synthesis of peptides would
appear judicious as halogenation is a simple modification that induces marked differences in
peptide supramolecular behavior. The limited number of reports on the activities of
halogenated AMPs against drug-resistant pathogens warrant future studies to determine
halogenated AMPs as an alternative therapeutic strategy against drug-resistant microbes.
B. Carbohydrates

Halogenated carbohydrates were mentioned earlier in the section on major classes of
aminoglycoside, macrolide, and glycopeptide antibiotics (section II). Here we discuss other
halogenated carbohydrates and polysaccharides in detail. A carbohydrate-based cellulose
nanopaper impregnated with chitosan and halogenated demonstrated good antibacterial
properties against S. aureus and E. coli due to the conversion of chitosan amino groups to
bactericidal halamines (Fig. 14A) (Du et al., 2021). Furthermore, halogenated methyl β-D-
galactopyranoside derivatives were significantly more potent than their non-halogenated
counterparts against Bacillus subtilis and E. coli and showed potential against SARS-Cov-2
(Fig. 14B) (Amin et al., 2022). In another study, yeast and aerobic microbe invasion and
accumulation on bamboo shoots were prevented using a coating of chitosan and chlorine
dioxide, which significantly extended the postharvest life of fresh bamboo (Yang et al., 2015).
66
Halogenated monosaccharides derived by chlorobenzoylation of methyl α-D-
mannopyranoside were reported to be antimicrobial against B. subtlis, S. aureus, E. coli, and
A. niger (Yasmin et al., 2021). Others reported that chlorinated sugar-based acyclo C-
nucleosides of 1, 2, 4-triazolo[4, 3-a]quinoxaline derivatives were antimicrobial against S.
aureus, P. aeruginosa, E. coli and C. albicans (Fig. 14C) (Ayoup et al., 2016) and that O-
acrylamidomethyl-N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC)
derivatives were bactericidal against S. aureus and E. coli within 20 minutes of contact (Fig.
14D) (Lim and Hudson, 2004). Novel halogenated carbohydrate-based phosphoramidate
derivatives were reported to be antibiotic adjuvants. In particular, a fluorinated glucose
derivative demonstrated a remarkable 32-fold decrease in the MIC of ampicillin against
methicillin-resistant S. aureus (Fig. 14E-F) (Subratti et al., 2021). The MOAs of these
halogenated carbohydrates are substantially unknown, but we believe the appropriate
positioning of halogen groups enables these derivatives to bind to or penetrate cell
membranes and attack sub-cellular targets.

C. Lipids
Fatty acids (FAs) encompass a large part of the lipid class. Various natural
halogenated antimicrobial FAs have been discovered, and others have been synthesized.
Several natural C18 and C16-FAs isolated from Xestospongia sp. exhibited antimicrobial
activity against S. aureus (Hirsh et al., 1987) (Fig. 15A), and a novel series of natural
brominated FAs isolated from the sponge Petrosia volcano were antifungal against the
pathogenic fungus Mortierella ramannianus (Fig. 15B) (Dembitsky and Srebnik, 2002).
Furthermore, natural lactylates of chlorinated FAs (chlorosphaerolactylates A–D) isolated
from the cyanobacterium Sphaerospermopsis sp. were inhibitory against S. aureus and
Candida parapsiloris (Gutiérrez-del-Río et al., 2020) (Fig. 15C), and halogenated FAs
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isolated from Asparagopsis taxiformis were antimycobacterial against Mycobacterium pheli
(El-Baroty et al., 2007).
Halogenated FAs have also been synthesized to enhance antimicrobial activity. Chlorine or
bromine-substituted vinyl halogenated FAs were antimicrobial against methicillin-resistant S.
aureus (Fig. 15D) (Sanabria-Rios et al., 2022), and brominated derivatives of vinyl FAs were
antileishmanial and inhibited topoisomerase IB enzyme of Leishmania sp. due to halogen
bond formation (Carballeira et al., 2019). In addition, fluorinated polyhydroxyalkanoate-
derived FAs at high concentrations had bacteriostatic and mild bactericidal effects against a
panel of bacteria (Snoch et al., 2019).
Halogenated FAs incorporated into complex lipopeptide structures were antimicrobial
against pathogens. Several chlorinated FAs were isolated from lipopeptides present in marine
sponges, for example, (S)-4,4,4-trichloro-3-methylbutanoic acid from Lamellodysidea
herbacea and (E)-6,6,6-trichloro-3-methoxy-5-methylhex-2-enoic acid and herbacic acid
[(E)-6,6,6-trichloro-5-methylhex-2-enoic acid, 62)] from Dysidea (Dembitsky, 2017). (S)-

7,7-Dichloro-3-hydroxy-2,2-dimethyloctanoic acid is incorporated into many lipopeptides
predominantly in cyanobacteria like Lyngby sp. and Moorea sp. (Dembitsky, 2022).
Interestingly, Gram-positive bacteria are sensitive to FAs, whereas Gram-negative bacteria
are usually resistant (Knapp and Melly, 1986). We observed a similar pattern for halogenated
FAs, that is, they were predominantly active against Gram-positive bacteria. We suppose
halogens enhance FA activity by forming halogen bonds in binding sites, which contrasts
with the usual practice in medicinal chemistry wherein halogenation is used to accentuate
permeability and warrants extensive studies on the effects of halogen and parent scaffold in
permeability enhancement.
The type of halogen-substituted may also influence microbial membrane permeability.
Fluorine substitution is primarily used to enhance permeability, whereas most halogenated

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FAs of marine origin identified are brominated. Also, the effect of FA length on antimicrobial
potency or cell permeability, the extent of halogen substitution, and the effects of halogen-
containing FAs on drug-resistant microbes remain subjects for study. Recently, several
studies reported that FAs exhibit antibiofilm and antivirulence activities at low concentrations
(Kim et al., 2021; Kumar et al., 2020b; Lee et al., 2021; Lee et al., 2017), which suggests
halogenated FAs could be utilized to target drug-resistant biofilms and their pathogenesis.
VI. Effects of Halogenation and Factors Affecting the Antimicrobial Activities
A. Halogen Bonding, Pharmacokinetics and Pharmacodynamics
Halogens are capable of halogen bonding which makes them useful in medicinal
chemistry and drug design (Cavallo et al., 2016). Halogen bonds are generally formed
between halogen ligands and Lewis bases present in binding sites (Fig. 16). Halogens are
capable of covalent interactions both as electrophiles and nucleophiles (Turunen and Erdélyi,
2020). These interactions were observed based on the electron density of the σ-hole region, in

which the electrons of heavy elements like halogens have anisotropic distributions and three
lone pairs of electrons form a cylindrical cloud of higher electron density around the atom
that generates a positive electrostatic potential creating the σ-hole region (Fig. 16) (Zhu et al.,
2019). This phenomenon is used to increase drug affinity in biological environments.
Halogenation can enhance enzyme activity by destabilizing staggered conformations
and stabilizing gauche conformations, thereby reducing activation energy barriers and
improving catalysis (Szell et al., 2019). This behavior is useful in enzyme and peptide
engineering as the substitution of hydrogen bonds in peptides with a halogen bond increases
binding (Parisini et al., 2011). Halogen substitution also increases thermal stability and
enzymatic activity at higher temperatures, as observed after chlorotyrosine substitution in T4
lysozyme (Carlsson et al., 2018). Conversely, halogenation can be used to design ligands that
69
inhibit enzymes, as was reported for E .coli gyrase, which was inhibited by compounds with a
halogen at the p-position, and for 5-hydroxytryptamine 2B receptor, which was inhibited by a
halogenated doxepin ligand (Kolarič et al., 2021; Zhou et al., 2018b).
Halogen containing ligand – protein interactions are highly dependent on the ligand
scaffold (Fig. 17). A study was conducted in which different substitutions of iodine, bromine,
chlorine, and trifluoromethyl groups on the chlorobenzene scaffold were evaluated. 3-Iodo-1-
pyrrole had greater activity than iodobenzene, signifying the importance of the scaffold itself
over substituted atoms for antimicrobial activity (El Kerdawy et al., 2012; Wilcken et al.,
2013).
Target site conformations also play critical roles in halogen interactions (Fig. 17), as
observed by the inhibition of plasminogen activator by 4-bromobenzylamine, inactive 4-
iodobenzylamine, and CF3-containing bisphenol AF, which acted as agonist and antagonist
against ERα and Erβ estrogen receptors, respectively (Jiang et al., 2018; Liu et al., 2020).
Proteins also contain halogen bonding "hot spots" which preferentially bind to ligand halogen

atoms and increase binding affinity as demonstrated by the 5-hydroxytryptamine receptor,
which possesses four halogen binding spots, that is, two in extracellular and transmembrane
regions (Kurczab et al., 2018).
Other stereochemical and bond formation properties of halogen atoms, such as
distance between the halogen and the target Lewis base, σ-hole-angle, and spatial orientation
of halogen atoms with respect to the Lewis base, also critically determine the strengths of
halogen bond interactions (Fig. 17) (Wilcken et al., 2013).
Understanding pharmacokinetic and pharmacodynamic properties is important when
manipulating scaffold behavior in medicinal chemistry. Inserting halogen atoms is a common
practice in many hits to lead or lead to drug conversions. The incorporation of halogen atoms
improves key pharmacokinetic properties like membrane penetrance, blood-brain barrier, and
70
central nervous system permeability (Nunes et al., 2021). This increase in permeability is
believed to be due to the presence of halogen bond donor sites in membrane lipid bilayers and
the resulting enhancement of anion transportation by the 'ion hopping' pathway. The use of
specific transporters that act as halogen-bond acceptors is another strategy for delivering
halogen-containing molecules to cells (Govindaraj et al., 2019). The influence of halogen
bonding on the permeabilities of biological membrane systems was confirmed by interactions
between halobenzene derivatives and phosphate or oxygen acceptors in the phospholipid
bilayer (Nunes et al., 2021). Further exploration of halogen bonding in membrane systems is
required to make further inroads into the pharmacokinetic manipulation of lead compounds or
drug scaffolds. Data on parameters such as drug clearance and toxicity are also necessary
when deciding on the fates of potential drug candidates.
The toxicity profiles of scaffolds largely depend on the scaffolds themselves, and thus
singling out atoms on scaffolds for toxic effects is probably ill-advised. For example, several
brominated metabolites from natural sources are non-toxic, whereas brominated haloesters

are toxic (Khan and Roy, 2021). However, high-throughput in silico scaffold screening
enables the safety implications of certain functional groups to be assessed. A thorough point-
by-point survey of potential drug candidates during lead-to-drug conversion was performed
on halogenated structures and only two candidates were rejected based on toxicity. This
statistic cannot be applied to other functional groups like nitro and sulfur groups, which are
metabolized to harmful byproducts (Hernandes et al., 2010).
While the carcinogenic and mutagenic profiles of halogenated antibiotics have been
extensively tested, those of newer halogenated pharmacological scaffolds remain largely
unexplored. Several organochlorines and halogenated hydrocarbons pose serious
carcinogenic threats (Fiore et al., 2019; Muslem et al., 2020). Halocarbons, especially
aliphatic haloalkanes, are capable of interacting with DNA, accumulating, and inducing
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mutagenicity (Della-Flora et al., 2023). However, over 3800 halogenated compounds of non-
anthropogenic origin found in various ecosystems have no harmful effects (Gul et al., 2020).
Thus, we suggest widespread testing be conducted to determine the carcinogenicities,
toxicities, and mutagenicities of halogenated antimicrobial compounds using modern in silico
toxicity predicting tools.
Xenobiotic metabolism is another important pharmacologic parameter. The strength
of carbon-halogen bonds raises questions regarding the clearance of organohalogens by the
cytochrome p450 system (Hernandes et al., 2010; Wang et al., 2019). Biotransformation of
halogenated drugs can produce harmful byproducts in some cases, such as the hepato- and
nephrotoxicity induced by the glutathione-dependent bioactivation of a small subset of
compounds (Anders, 2008). It has been reported that halocarbons can accumulate in rat
adipose tissue making it difficult for excretion (Kania-Korwel and Lehmler, 2016). However,
halogenation of the substrates with aromatic derivatives of fluoro, chloro and trifluoromethyl
groups increased microsomal clearing (Sun et al., 2011). Also, strategic halogenation on the

scaffold can increase binding affinity with the CYP enzymes, as observed in coumarin
derivatives substituted with heavy halogens like chlorine, bromine and iodine had enhanced
binding affinity with CYP2B enzymes (Liu et al., 2016). Therefore, the role of halogen in
drug clearance is ambiguous and an elimination study with diverse halogenated compounds is
recommended to ascertain the role of halogen atoms in drug clearance.
The topics discussed in this section, viz. halogen bonding, scaffold dependence, target
conformation, halogen bond distance, and angle, importantly determine the pharmacological
characteristics of halogenated compounds. Although the upsides of halogen bond formation
are far-reaching, careful consideration should be given to the toxicological profiles of
halogenated compounds to weed out potential offenders. In-depth analysis at the molecular
72
level is required to further probe the effects of halogenated compounds, especially in contexts
of toxicity and pharmacodynamics.
B. Factors Affecting the Antimicrobial Activities
An understanding of the properties likely to modulate the efficacies of halogenated
agents is critical during drug development. Some studies have attributed the limited number
of bromine or iodine-containing drugs approved by the FDA to size among other factors
(Wilcken et al., 2013). Adequate awareness of these factors is essential for improving
antimicrobial performance or combating antimicrobial resistance, as this would provide
researchers with proper rationales or guidance regarding the design, synthesis, and
applications of halogenated drugs. Based on the reviewed literature, several critical factors
such as halogen size, substitution patterns, pathogen natures, hydrophobicity, and other
physicochemical characteristics determined antimicrobial activities and are herein elaborated.
1. Halogen Size and Substitution Pattern

Halogen sizes increase from fluorine to iodine as do carbon-halogen (C-X) bond
lengths (Fig. 1A) and halogen size and substitution patterns of halogens in the modified
haloalkanes and haloarenes may determine antimicrobial potencies (Fig. 17). For instance,
the introduction of chlorine or fluorine specifically at the C5 positions of 8-hydroxyquinoline
and indole scaffolds strictly favored activities against certain Gram-negative and fungal
pathogens (Cherdtrakulkiat et al., 2016; Raorane et al., 2022). Also, the antifungal activity
spectrum of fluconazole was increased markedly by further fluorine substitution, whereas
chlorine or bromine substitution reduced activity. The group noted that the small size and
electronegativity were crucial to the activity (Shafiei et al., 2021). Of note, small halogens
such as fluorine and chlorine may also enhance synergism between halogenated compounds
and antibiotics or other antimicrobial agents. This is supported by a report that combinations
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of fluorinated bromperidol with posaconazole, an antifungal agent showed the highest
synergy against fungal pathogens. The small-sized fluorine atom was deemed better for
optimal interaction with the fungal cells (Holbrook et al., 2017). Typically, size is essential
for permeability and absorption by the cells as smaller molecules diffuse more rapidly and
easily across membranes than bulky analogs due to factors related to surface area and
volume. Interestingly, this may partially explain the reason why fluorinated and chlorinated
drugs have been prominently approved by regulatory bodies. On the other hand, heavy
halogens such as bromine and iodine have also been reported to enhance the activities of
tricyclic flavonoid derivatives (Bahrin et al., 2016). Similarly, short peptoids (6-8mers)
containing bromine or iodine exhibited better activity against resistant S. aureus and S.
epidermidis (Molchanova et al., 2020). This relationship between high molecular weight
halogen atoms and high bioactivity may be explained by their propensity for halogen bond
formation, which promotes ligand binding affinities and confers stability (Xu et al., 2014).
Furthermore, the substitution pattern of halogens on the parent scaffold is also crucial

to bioactivity. For example, fluorination at the -ortho position on the aminobenzene ring was
solely responsible for the potency of a quinone derivative against C. albicans as modification
other than that decreased activity (Wellington et al., 2019). Similarly, the presence of iodine
atoms specifically at the C5-position on the antibacterial activity of halogenated indole
against A. baumannii decreased from C5, C6, and C7 to the C4 position (Raorane et al.,
2020). It is envisaged that the interaction between the halogens and the parent scaffold at
these specific locations might have produced sufficient effect to enhance the target binding
affinity of the compounds. Therefore, the understanding of these variations could contribute
immensely to the drug development and improvement process.
2. Hydrophobicity
74
Hydrophobic interactions and the conformation of the ligand to the protein determine
antimicrobial activities (Yin et al., 2022). These interactions are made possible by halogen
bonding, which enhances inhibitory activity by modulating the potentials of halogenated
agents to bind to cells, cross membrane barriers, and even transported them within cells and
organs (Priimagi et al., 2013). Fluorine grafting into chitosan increased hydrophobicity,
lowered surface energy, and prevented bacterial surface adherence (Lin et al., 2018).
Hydrophobicity may also be influenced by the size and electronegativity of halogens. The
presence of bromine or iodine increased the hydrophobicity of peptoids more so than fluorine
and notably corresponded to antimicrobial activities (Molchanova et al., 2020). However,
hydrophobicity has a threshold beyond which no antimicrobial effect is observed. Iodinated
peptoids lost their antimicrobial activities against Gram-positive bacteria when optimal
hydrophobicity was attained by an increase in the number of halogen and residue lengths
(Molchanova et al., 2020). This loss of activity may have resulted from poor solubility and
low bioavailability typical of highly hydrophobic drugs. Prodrugs, emulsion preparation

methods, drug complexation, polymeric micelles, drug nanocrystals, and other techniques
have been reported to control these anomalies (Vimalson, 2016).
Furthermore, the distribution of halogens on the parent moiety could also affect the
hydrophobicity of halogenated agents. For example, two bromine atoms on the phenyl rings
of halogenated peptide compared to its presence on all the rings resulted in a hydrophobicity
sufficient to exert a 32-fold antibacterial effect against resistant P. aeruginosa (Molchanova
et al., 2020). Also, Cl and Br substituents were required for the hydrophobic binding and
inhibitory activity of halofuginone on eukaryotic prolyl-tRNA synthetase (ProRS), whereas
the presence of highly electronegative trifluoromethyl group exhibited poor enzyme
inhibitory activity (Guo et al., 2020). It thus appears that the space of the hydrophobic pocket
75
at target sites determines which halogen elicits the suitable hydrophobic interaction for
antimicrobial activity.
3. Physicochemical/Biophysical Properties
Biophysical/physiochemical properties, such as steric effects, electrophilicity, charge
distribution, electronic (σ), and lipophilic attributes are necessary for binding to target
biomolecules and can also influence the target site affinities (El Hage et al., 2011; Fang et al.,
2019). Firstly, steric effects may give rise to electronic conditions that facilitate interactions
or impair target protein functionality or biological activity (Fang et al., 2019). For example, -
para-substituted bromocinnamaldehyde was 10-fold more active against E. coli and S. aureus
than the ortho derivative. The ortho location of Br- and its steric bulk were believed to hinder
nucleophilic interaction at the electrophilic β-carbon as steric interference increased (Doyle et
al., 2019). Similarly, the inactivity of halogenated pyrimidines against S. aureus growth and
biofilm formation was attributed to steric hindrance and low electron-withdrawing potentials
that impaired electron distribution on the linked ring system (Provenzani et al., 2021).

Furthermore, electrophilicity, which is the affinity of a molecule for electrons in its
environment might also determine antimicrobial potency. It was reported that the more
electrophilic p-bromocinnamaldehyde was biologically more potent than its parent compound
against S. aureus and E. coli. Notably, the presence of Br- and Cl- substituents caused
moderate electrophilic activation which was independent of para-, ortho-, or meta- position
on the phenyl ring indicating that the type of halogen rather than the position caused the
activation and improved activity (Doyle et al., 2019). Halogens can also influence the
electrophilicity of neighboring atoms, as was reported for melphalan flufenamide an FDA-
approved drug in which the presence of chlorine increased the electrophilicity of directly
attached carbon atoms to make endogenous nucleophilic interactions with components of
nucleotide bases (Benedetto Tiz et al., 2022).

76
Charge distribution on the halogenated compounds is another important biophysical
property (Fig. 17). This is dependent on molecular electrostatic potentials that determine
relationships between binding activities and electron distributions, which can influence
antimicrobial properties (Shafiei et al., 2021). Paulsen et al. reported that an increase in net
positive charge of +2 on halogenated diamines (3,5-Br-Ph, 3,5-CF3-Ph, and 4-F-1-Nal)
enhanced their aqueous solubilities and antimicrobial activities against E. coli and P.
aeruginosa (Paulsen et al., 2019). Conversely, the charge distribution could also be
responsible for a loss of antimicrobial activity, as was observed for some halogenated
fluconazole derivatives. Specifically, the moderate electron densities in a negative potential
region having fluorine would be expected to bind more strongly with a positive target region
than those containing Cl or Br atoms. This led to reduced antifungal activity against yeast and
filamentous fungi compared to fluorine (Shafiei et al., 2021). The higher affinity of fluorine
towards the targets and membrane in comparison to other halogens might be explained by the
high electronegativity which could create a more positive sigma-region. Generally, a negative

charge on a halogen has little effect on ligand-to-receptor binding energy. However, an
increase in the electrostatic potential of a halogenated compound would enhance solvation
and electrostatic contributions and thus potentiate ligand-to-receptor binding (Ibrahim et al.,
2018). Therefore, the electron distribution charge on the halogenated moiety plays a key role
in bioactivity.
Lipophilicity is a measure of the ability of a compound to interact with or dissolve in
lipophilic surfaces or substances. Lipophilicity determines antimicrobial effectiveness by
influencing the permeability and subsequent bioavailability of halogenated agents in tissues
and lipid membranes (Fang et al., 2019). For example, modifications of isoniazid with 4-CF3
or 4-OCF3 at C4 on the aniline ring enhanced lipophilicity, and thus facilitated the passive
diffusion of compounds into a mycobacterial envelope inaccessible to isoniazid (Pflégr et al.,

77
2021). Furthermore, chlorinated resveratrol penetrated cell membranes and inhibited
Helicobacter pylori due to its increased lipophilicity (Di Fermo et al., 2020). Additionally,
higher electronic and lipophilic values of 3,4-dichloro-cinnamaldehyde increased antiquorum
sensing activities against Vibrio spp. (Brackman et al., 2011). Usually, lipophilicity increases
with the number of halogens on aromatic rings, and when in excess, it leads to improper
antioxidant localizations in membranes and reduces radical scavenging potentials, which may
be detrimental as evidenced by the loss of antivirulence activities by 2, 4, 6-tribromo-3, 5, 4′-
trihydroxystilbene (Li et al., 2012). Excessive lipophilicity could also be detrimental to drug
absorption and expose drugs more to degradation by liver enzymes (e.g., cytochrome P450),
reduce effective drug delivery at target sites due to reduced solubility, and increase toxic
effects (Böhm et al., 2004; Khan, 2016). Hence, the need to control lipophilicity to ensure
adequate metabolic stability and optimum efficacy.
4. Bacterial Cell Type
Although halogens are known to enhance the membrane permeabilities of halogenated

compounds, their activities may be subjected to cellular uptake (Fig. 17). During this review,
we observed that the majority of new and existing compounds functionalized with halogens
had a greater affinity for Gram-positive bacteria and fungi than Gram-negative bacteria
(Doyle et al., 2019; Sanabria-Rios et al., 2022). In addition, despite exhibiting excellent drug-
like properties, including blood-brain penetration and high GI absorption, Br- and Cl-
substituted cyclohexanol benzo[b]thiophene derivatives lacked activity against Gram-
negative bacteria at high doses (Masih et al., 2021). However, this was not observed for the
majority of halogenated cationic polymers, peptides, or quinones (Molchanova et al., 2020;
Nazi et al., 2020; Paulsen et al., 2019; Peng et al., 2021). The presence of peptidoglycan
fortified by the outer membrane of Gram-negative bacteria may have been a cause of this
differential behavior (Doyle et al., 2019). Furthermore, parent structure properties, testing
78
conditions, pathogen physiological state, medium pH, halogenation pattern, and enzymatic
and thermal stabilities may have also contributed, but unfortunately, these factors were not
fully addressed in the studies reviewed. Consequently, it is difficult to infer that halogenated
compounds are selective towards certain microbes, and thus we recommend that these
properties be given adequate consideration during the design and the antimicrobial studies of
halogenated drug candidates.
Also, the efficacies of halogenated agents depend on the state of the pathogen. When
pathogens form biofilms, drug permeability is reduced, and virulence and antibiotic
resistance are increased. Interestingly, this review revealed that halogenated agents potently
control biofilm formation and virulence of several resistant bacteria and fungi. We suppose
increased affinity, permeability, and bioavailability induced by halogenation facilitated the
penetration of biofilm matrices and subsequent control.
C. Halogenated agents and gut microbiota

Halogen-containing scaffolds have applications in the food, pharmaceutical,
agrochemical, and personal care product sectors as primary or adjuvant agents (Dikeocha et
al., 2022). The gut microbiome represents all microbial groups inhabiting the human
gastrointestinal tract and is often referred to as a metabolic organ with respect to maintaining
the host's overall health (Li et al., 2016). The gut microbiome plays an essential role in
metabolism and affects the pharmacology of drug candidates (Dhurjad et al., 2022). Thus,
understanding gut microbe-drug metabolism can improve drug development processes.
Herein, we discuss the possible impacts of gut microbiota on the administration of
halogenated antimicrobial agents.
Gut microbiota contains several drug-metabolizing enzymes, including lyases and
oxidoreductases (Koppel et al., 2017), and their interactions with halogenated antimicrobials
79
could produce toxic intermediates, inactivate drugs, and cause cross-resistance. For example,
gut microbiota converted 5-fluoro-2-deoxyuridine into 5-fluorouridine-5’-monophosphate,
which increased toxicity and became fatal to the host (Ke et al., 2020). Gemcitabine, a
fluorinated nucleoside, was also inactivated by cytidine deaminase of duodenal Gamma
proteobacteria and resulted in resistance and impaired efficacy (Li et al., 2021c). Also, genes
related to resistance to several antibiotics were reported to be abundant in gut microbes
exposed to chlorinated water (Nadimpalli et al., 2022). This suggests that the interactions
between halogenated and gut microbiota could trigger resistance or produce detrimental or
beneficial metabolites.
The interplay could also result in dysbiosis, impaired gut microbial diversity, and
metabolome changes. Chlorpyrifos favored the growth of Bacteroides sp. but reduced the
growths of Lactobacillus sp. and Bifidobacterium sp. Exposure of mice to 2,4-
dichlorophenoxyacetic acid was also reported to increase gut bacteria counts (Nadimpalli et
al., 2022; Ribado et al., 2017; Tu et al., 2019). These variations in the gut microbiome are

closely associated with the host's response to drug therapy (Li et al., 2016). In addition, gut
exposure to halogenated agents could result in translocation whereby gut commensals are
introduced to other sites and trigger unwarranted immune responses. Furthermore,
halogenated antimicrobials can directly modulate metabolic processes such as pyruvate
metabolism, the tricarboxylic acid cycle, protein export, and carbon fixation by gut
microbiota (Sha et al., 2022; Tian et al., 2020), which could impair the metabolic functions of
gut microbiota and consequently impact host health.
Gut bacteria can also modulate drug metabolism through host functions and are
capable of altering drug absorption rates by modulating gut environmental conditions
(Enright et al., 2016). In addition, adhesins expressed by most gut bacteria interact with and
bind drugs, which could reduce bacterial adhesion to host cells and alter drug absorption and
80
plasma levels (Dhurjad et al., 2022). Although gut microbes are not in direct contact with the
liver, they are capable of modulating the expressions of liver-related genes and thus
modulating drug metabolism (Dhurjad et al., 2022; Selwyn et al., 2016). Another crucial
effect is the competition for active sites whereby host, drug, and microbial metabolites
compete for active sites of the host metabolic enzymes and thus influence their efficacies
(Dhurjad et al., 2022). Generally, antibiotics are less effective in differentiating between
commensals and pathogenic microbes and the same is being envisaged as a limitation for
these halogenated compounds. Also, certain commensals which acquire antibiotic resistance
genes were reported to shield pathogens from the impacts of antibiotics indicating further
complication in the management of infection (Gjonbalaj et al., 2020).
There are also reports that attest to the beneficial or indifferent interaction between
gut microbiota and halogenated compounds. For instance, the gut microbiota of Bangladeshi
children was not perturbed after consumption of chlorinated water but rather increased the
beneficial bacteria which favored gut health (Nadimpalli et al., 2022). Similarly, the exposure

to household triclosan in toothpaste and soaps did not cause dysbiosis (Ribado et al., 2017),
and at physiologic concentrations, triclosan did not show notable effects on endocrine and
metabolic markers in human oral and gut microbiota (Poole et al., 2016). Taken together, the
interplay between gut microbes and halogenated antimicrobials generates several effects.
Thus, their understanding should be a guide to improve the design and production of
halogenated antimicrobials.
VII. Impacts of halogenation on antibiotic resistance mechanisms
As halogenation increases the abilities of antimicrobials to penetrate membranes and
enhances ADME capabilities, it could also rejuvenate antimicrobials that are ineffective
against drug-resistant antimicrobial strains to restore antibiotic susceptibility. Halogenated

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compounds are capable of overcoming several types of resistance mechanisms including
active drug efflux, drug target modification, limiting drug uptake, or drug deactivation as
elaborated below (Fig. 18).
Efflux pumps, which are mainly responsible for drug extrusion from bacterial cells
before reaching the intracellular targets, are the most significant cause of antibiotic resistance
and have become important therapeutic targets (Nishino et al., 2021). Halogenated agents
circumvented antibiotic resistance by inhibiting arrays of efflux pump transporters. For
instance, the overexpressing resistance nodulation cell division (RND) pumps namely
AcrAB-TolC, MexAB-OprM, and MexXY-OprM were inhibited by 3,4-dibromopyrrole-2,5-
dione to reverse the multidrug resistance of E. coli to ciprofloxacin, levofloxacin, kanamycin,
erythromycin, oxacillin, piperacillin, tetracycline and chloramphenicol (Whalen et al., 2015).
Similarly, ethyl 4-bromopyrrole-2-carboxylate used in combination with antibiotics reduced
the overexpressing RND transporters to restore susceptibility to MDR E. coli and
Pseudomonas aeruginosa (Tambat et al 2022).

Another efflux family controlled by halogenated agents is the proton-dependent pump.
Trifluoromethyl-ketone enhanced the activities of ampicillin, tetracycline, and erythromycin
antibiotics and improved sensitivity to resistant wild-type E. coli AG100 and E. coli K12
LE140 strain with a tetracycline-resistant plasmid by proton pump inhibition (Spengler et al.,
2003; Wolfart et al., 2006). 3-(2-benzoxazolyl)-1,1,1-trifluoro-2-propanone also inhibited the
E. coli proton pump to enhance promethazine activity (Wolfart et al., 2006). Notably, the
trifluoro group is a common feature among these compounds and may suggest an important
role in proton pump inhibition, but this requires further investigation. Furthermore,
halogenated 1,3,5-triphenyl-2-pyrazolines inhibited p-glycoprotein efflux pumps in
Mycobacterium tuberculosis and other bacterial and fungal strains and thus mitigate
resistance (Sivakumar et al., 2010).

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Halogenated agents are capable of suppressing efflux pump-related genes to limit
efflux activities. Chlorpromazine was reported to synergize with norfloxacin to control MDR
S. enterica by repressing the expression of the efflux pump gene (acrB), limiting export
potential, and enhancing chlorpromazine uptake within the bacterial cell. Also, haloperidol
derivative combined with fluconazole overcame fluconazole resistance in C. albicans by
downregulating the efflux pump gene (MDR1) (Ji et al., 2019). Herein, the synergistic effect
of halogenated agents with antibiotics is notable in efflux pump inhibition. Therefore, the use
of halogenated agents as adjuvants with antibiotics wherein halogenated agents remove
resistance barriers to allow antibiotics to exert their therapeutic effects could be an effective
strategy to treat resistant pathogens. Minimization of efflux pump activities by halogenated
compounds has the potential to rejuvenate antibiotic activity against MDR strains via
reducing drug expulsion and increasing the accumulation of drugs sufficient to reach
intracellular targets and could also act as additives in inhibiting the pathogens themselves.
However, halogenation processes could also impair the efflux pump inhibitory

potentials of halogenated agents. It was reported that orfA-mediated halogenation in
Streptomyces facilitated the binding affinity of halogenated albofungin towards
transglycosylase but limited the antimicrobial potencies against Klebsiella pneumoniae and P.
aeruginosa by intrinsically altering the biological and physicochemical properties. This
affected molecular recognition, hence enhancing the orfL-dependent expulsion of chloro- and
bromoalbofungin. OrfL is a proton-dependent oligopeptide transporter belonging to the major
facilitator superfamily (Wang et al., 2022). It does suggest that adequate modulation of
physicochemical properties is essential when designing potential halogenated efflux pump
inhibitors.
Membrane impermeability leading to decreased uptake is a relatively uncommon
resistance mechanism. Although research on this aspect is limited, 3,4-dibromopyrrole-2,5-

83
dione and ethyl 4-bromopyrrole-2-carboxylate boosted the uptake and accumulation of
Hoechst 33342 (fluorescent efflux pump substrate) in attempts to reverse the MDR in E. coli
and P. aeruginosa (Tambat et al., 2022; Whalen et al., 2015). Additionally, halogenated
novel bacterial topoisomerase inhibitors against several resistant strains of Staphylococcus sp.
and E. coli achieved satisfactory results by adjusting lipophilicity/hydrophobicity ratios
(Kolarič et al., 2021). Halogenated agents may act as membrane permeabilizers capable of
improving the permeability of the outer membrane and increasing intracellular drug levels to
overcome antibiotic resistance.
Halogenation could also potentiate the inhibition of drug-deactivating enzymes
responsible for the breakdown of antimicrobials like β-lactamase providing another viable
strategy to combat AMR. Enzyme-inhibition combinatory therapies that included halogenated
β-lactamase CMY-10 inhibitors were found to be effectively therapeutic against multidrug-
resistant Enterobacteriaceae (Parvaiz et al., 2021). Halogen-substituted
triazolethioacetamides displayed potential as metallo-β-lactamase inhibitors (Zhang et al.,

2019). Halogen ions also served as potent inhibitors of OXA-enzymes, which are extended-
spectrum β-lactamases and can increase the efficacies of antibiotics (Stojanoski et al., 2015).
Halogenation controlled drug resistance mediated by drug target modification or
mutation by improving potency and molecular recognition. Fluoroquinolone resistance
caused by the modification of gyrase-topoisomerase was circumvented using sparfloxacin (a
C-8 halogen-substituted fluoroquinolone) against the gyrase-resistant mutant of
Mycobacterium smegmatis and the gyrase-topoisomerase IV double mutant of S. aureus as an
alternative to C-8 hydrogen-substituted ciprofloxacin (Lu et al., 2001). Similarly, quinolone-
35 (Q-35) and sitafloxacin exhibited potent activity against quinolone-resistant staphylococci
and ciprofloxacin-resistant gyrase (gyrA CipR) mutants of P. aeruginosa, respectively, when
substituted with fluorine at the 8th position (Ito et al., 1995; Kitamura et al., 1995). A
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modification at the 2-position of halogenated quinolines exhibited antibacterial and biofilm
eradication activities against MDR strains of Staphylococcus sp. and Enterococcus sp. (Basak
et al., 2016).
Several halogenated compounds and antibiotics were active against resistant pathogens
as reviewed in sections II and III. But the novelty of the scaffold itself should be considered
when reviewing activity against resistant strains as resistance is subject to only specific
antibiotics resulting in similar MIC values between sensitive and resistant strains. Although
the practice of finding novel compounds for chemotherapy against resistant strains is a good
initiative, a deeper understanding of the resistance mechanisms would encourage the
rejuvenation of older antibiotics, which can be economical, and efficient, and also
differentiates potential approaches for sensitive and resistant strains. The potential of
rejuvenating older antibiotics is possible in the case of resistant strains employing efflux
pumps, membrane permeability, and enzyme degradation type of resistance mechanisms as
drug target modification is an irreversible modification. Taken together, halogenated

compounds could minimize various AMR mechanisms and restore antibiotic susceptibility
via the inhibition of efflux pumps, drug-deactivating enzyme inhibition, enhancement of drug
uptake, and resistance to target modifications in MDR strains. A continuous understanding of
resistance mechanisms and the effects of halogenated compounds on them would provide
better insight into how to best leverage halogen atoms against resistant microbes.
VIII. Current Practices and Prospects
Microorganisms have demonstrated resistance to many approved drugs and evolved
multidrug-resistant strains, in addition to several drug contraindications. Herein, we have
elaborated on the importance of halogenation for the discovery of drugs targeting
antimicrobial resistance. The halogenation of various antimicrobial scaffolds has conferred

85
several advantages, including broadening the activity spectrum against resistant pathogens,
inducing reductions in virulence factors, such as biofilm formation, and toxin production, and
substantially increasing antimicrobial scaffold adjuvant activities. Halogenation also
improved metabolic stabilities by inhibiting phase I oxidation and alleviating the side
effects/toxicities of parent scaffolds. Pharmacokinetic properties such as absorption,
distribution, bioavailability, and clearance rate were also significantly improved by
halogenation (Fig. 19). Additionally, halogenated derivatives displayed potential for
expanding the clinical usages of existing drugs, blocking scaffold defects, serving as efflux
pump inhibitors to reverse drug resistance, and restoring the potency of erstwhile ineffective
drugs (Ienașcu et al., 2022; Nizi et al., 2020).
Halogenated antimicrobials utilize diverse mechanisms (Fig. 20). For instance,
halogenated derivatives of indole, magnolol, flavonoids, and CNMA seems to trigger more
mechanisms than their parent scaffolds, probably because of their different physicochemical
properties and improved target affinity mediated by halogenation. Also, halogens form salts

by directly reacting with metals, and this seems to have conferred metal chelation as
antimicrobial mechanism on halogenated catechols, phenazines, and hydroxyquinolines.
Taken together, halogenated derivatives display a wide range of antimicrobial mechanisms.
Furthermore, factors such as degree of halogenation, halogen size, pathogen type, halogen
locations, and the natures of substituents co-existing with halogens determined the bioactivity
and pharmacological properties of halogenated antimicrobials. Nonetheless, certain essential
aspects of halogenated antimicrobial drug research are underexplored.
Certain halogenated moieties have found application in the development of
antibiotics. Chlorocatechol (8A) is a component of cefiderocol, which was recently approved
by the FDA, where it conferred novel MOA such as siderophore-iron binding and stability
against enzymatic action (Sato and Yamawaki, 2019). Also, the halogenated
86
hydroxyquinoline derivative 9V has a structure similar to the antibiotic clioquinol, which was
approved for antimicrobial treatment (Das et al., 2018). Although these applications further
support the importance of halogenated antimicrobial groups, the subject has been
underexploited and could be further substantiated to potentiate, modulate, or replace existing
antibiotics.
Research groups have synthesized new prodrugs by linking or fusing halogenated
scaffolds to known antibiotics to overcome inactivity (Fig. 21). This molecular hybridization
approach involves combining molecules to produce more potent drugs that benefit from
synergism (Ivasiv et al., 2019; Zha et al., 2022). For instance, halogenated phenazine-
erythromycin and cephalosporin-halogenated phenazine prodrugs (Fig. 21A-B) were reported
to enhance the translational release of phenazines. This design conferred on the prodrug's
dual MOA as ribosome inhibitor and iron-starving agents (Xiao et al., 2020; Yang et al.,
2021). Similarly, fluconazole was hybridized with a halogenated phenyl urea group to
overcome resistant C. albicans, and in a similar study, halogenated thiazolo[4,5-d]pyrimidine

was employed (Fig. 21C-D) (Shafiei et al., 2021; Sharapova et al., 2022). The hybridization
concept confers affinities for multiple targets and is expected to rejuvenate potencies, and
stabilities and to minimize the side effects of existing medications. In addition, it reduces the
risk of drug resistance due to multitargeting ability and provides an avenue for developing
drugs at lower risks and high-cost effectiveness. However, the selection of suitable
combinations and linkers to ensure substrate processing and active compound release remains
a major challenge (Ivasiv et al., 2019; Zha et al., 2022).
Furthermore, it is essential that the effects of the interplay between halogenated drug
candidates and the host be determined with respect to toxicity and other pharmacological
effects. Available reviews have sparingly addressed in vitro and in vivo assessments of
toxicities. This is a fundamental requirement of preclinical drug trials and should be

87
adequately integrated into future studies to ensure smooth transitions from bench to bedside.
Similarly, information on administration routes, excretion pathways, metabolic soft spots,
physiological clearance, drug interference, metabolite formation, and liabilities should be
considered.
The use of heavy halogens such as bromine and iodine in drug repurposing or
modification may be promising for antivirulence activities. Microbes produce several
virulence factors to cope with stressful conditions and as a result, become more pathogenic
(Ji et al., 2019). Antimicrobial agents targeting them in ways that do not affect growth are
unlikely to result in resistance development and are viewed as attractive means of combating
drug-resistant pathogens. This review shows heavy halogens are present in many scaffolds
such as BrCl-flavonoid (8J), 5-iodoindole (9B), and halogenated phenazines (10L), and these
agents effectively inhibited biofilm formation and eliminated persister cells by multiple folds
(Babii et al., 2018; Garrison et al., 2016; Raorane et al., 2020). Interestingly, these
compounds contain only Br or I atoms with or without a smaller halogen like chlorine, which

suggests larger halogens should be included in studies undertaken to reduce drug resistance
and control virulence. The utilization of Br and I in drug production has been limited by their
reactivities, tendencies to produce metabolites, cost, and individual bias (Herrera-Rodriguez
et al., 2011; Jeschke, 2022; Wilcken et al., 2013). Tuning reaction conditions or late
introduction of Br or I might resolve reactivity issues (Tan et al., 2017; Wilcken et al., 2013),
and also, we envisage that halogen blending might alleviate the limitations assigned to Br and
I application. Of note, the phenomenon of halogen bonding and how it tunes antimicrobial
activity has gained attention (Edis et al., 2019). However, its relationship with virulence is
unknown and worth investigating.
Halogens have the potential to contribute significantly to alternative therapies
designed to combat drug resistance. Antiseptics play a critical role in fighting antimicrobial
88
resistance, especially in hospital settings (Maillard et al., 2021). Halogens are used to produce
many antiseptics and disinfectants like povidone-iodine (PVP-I), triclosan, chloroxylenol,
dequalium chloride, and cetylpyridium chloride (Slaga et al., 2022; Virji et al., 2022; Wade et
al., 2021), and interestingly, resistance to common iodophor antiseptics like PVP-I and
cadexomer iodine is minimal despite their widespread use. Their MOAs revolve around the
release of free halide ions, which induce lipid membrane peroxidation, and DNA damage,
and inhibit protein synthesis (Shah et al., 2021). However, the precise reason for their
prolonged efficacies has not been determined, though it is clear that free halide ion-induced
oxidation is something microbes find difficult to counteract.
Cationic polymers are associated with minimal AMR, as indicated in section IV,
which raises expectations regarding oxidative halogen species released by polymers like N-
halamines and antiseptics. Constant long-term release of antimicrobial ions is also an
attractive AMR strategy to ensure complete microbe killing and prevent recurrent infection.
Furthermore, reactive halogen species (RHS) play a vital role in immune-based defense, and

interestingly it was reported that ozone-depleting halogen cyclic reactions amplified RHS
pathogen-killing some 100,000 fold by phagocytosis (Lu, 2020).
Preventative vaccine therapies significantly aid the fight against microbial
recalcitrance (Micoli et al., 2021). Halogens can enhance vaccine efficacies, as was observed
for a fluorinated cocaine vaccine, a fluorinated carbohydrate cancer vaccine, and a
brominated human respiratory syncytial virus vaccine, which was 6.3 fold more efficient than
the non-brominated vaccine (Cai et al., 2013; Xue et al., 2023; Yang et al., 2011).
Halogenation of vaccines is incipient, especially for antimicrobial therapy, and may
constitute a vanguard approach for combating antimicrobial resistance.
In the same vein, halogen compounds can also act as immunomodulators to trigger
immune responses, as evidenced by the use of halogenated aryl thiosemicarbazones against

89
leishmaniasis, halogenated thymoquinone analogs against Plasmodium falciparum, and the
Th1-immunostimulatory activity of halogenated α-galactosylceramide analogs against
parasites (da Silva et al., 2017; Hossain et al., 2016; Johnson-Ajinwo et al., 2018). Although
the concept of immunomodulation using halogenated compounds to achieve antimicrobial
activity is a relatively unexplored topic compared with parasitism, we feel that its exploration
might provide a means of combating AMR.
Halogenated compounds can also augment viral phage therapy by promoting microbe
accumulation and lysis. Halobenzenes bind within the internal non-polar cavity of the
Leu99Ala bacteriophage T4 lysozyme mutant, and halogen bonding provides better
stabilization than hydrogen bonding (Erdélyi, 2017). Although the implications of
halogenated T4 lysozyme analog on the antimicrobial activity are unreported, they may have
significant impacts on future phage-based therapeutics, proteins, and engineering. As
discussed in section VI.A, replacing hydrogens with halogens in peptides reduces activation
energies and increases enzyme binding affinities, and these phenomena could be used to

synthesize specific endolysins or phages against a diverse range of resistant or novel
pathogens. Similarly, as discussed in section V.A., using halogenation as a tool in peptide
engineering has great prospects with improved antimicrobial activity and peptide’s
biocompatible nature in comparison to traditional antibiotics.
Many properties, conditions, and actions of halogens may be responsible for the
enhanced antimicrobial potencies of halogenated antimicrobial agents, but regardless of the
number of halogenated agents available, studies are limited. Interestingly, several machine
learning and artificial intelligence-based software packages, such as DeepChem, DeltaVina,
Chemputer, Open Drug Discovery Toolkit (ODDT), AMPlify SCScore, and DeepNeuralNet-
QSAR (Matsuzaka and Yashiro, 2022; Peña‐Guerrero et al., 2021; Staszak et al., 2022), are
now used for lead optimization and predicting these properties. Application of these
90
platforms reduces the time required for lead optimization and may further advance the
halogenated antimicrobial research field by providing more insights into the roles of halogens.
IX. Conclusions
Antimicrobial resistance has multidimensional impacts on global mortality, morbidity,
and national economies, and could lead to fatalities by simple infections (Ikhimiukor et al.,
2022). In addition, to judicious surveillance of antibiotic administration and compliance with
current health policies, new strategies based on novel therapeutics are required. In this review,
we evaluated the benefits of halogen atoms and their impacts as compared with parent
antimicrobial scaffolds. In addition, we explored the structure-activity relationships, modes of
action, toxicities, and applications of a wide range of halogenated scaffolds, including
antibiotics, antiseptics, plant products, polymers, and biomolecules, and the roles of halogen
atoms.
The halogenation of scaffolds provides various advantages; for example, it can

generate novel antimicrobial mechanisms and targets and has the potential to enhance activity,
minimize toxicity, and overcome drug resistance (Fig. 19). Furthermore, the concept of
scaffold or molecular hybridization has the potential to improve affinities for multiple targets.
However, an extensive survey of antimicrobial agents revealed that the majority of
halogenated antimicrobials synthesized were not tested for toxicity, which we consider an
essential part of drug discovery. In addition, drug repurposing is gaining attraction because of
the slow rate of novel drug approval (Zhan et al., 2022). In our opinion, halogenated
compounds are excellent drug-repurposing candidates, given their broad-spectrum
capabilities and affinity to various biological targets.
Rejuvenation of existing antimicrobials deemed ineffective against resistant
pathogens is an increasing trend (Zhan et al., 2022). Halogenation offers a practical means of
91
rejuvenating impotent antimicrobials, as discussed in section VII. A deeper understanding of
the effect of halogenation on retired antimicrobials against specific resistant strains is
required. We also identified a need for the standardized testing of compounds against a more
diverse panel of microorganisms and resistant strains. In our opinion, halogenation and the
incorporation of halogen atoms in pharmacological scaffolds are feasible strategies for
combating antimicrobial resistance because halogenation is simple, economically viable, and
readily accessible.
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XI. Acknowledgement
We would like to thank Dr. John Roberts for English proofreading and anonymous referees’
insightful comments.
Data Availability
The authors declare that all the data supporting the findings of this study are contained within
the paper and its Supplemental Data.
Authorship Contributions
Conception and Supervision: Lee, J.
Design and Draft: Faleye, O. S., Boya, B. R., Lee, J-H.
Wrote or contributed to the writing of the manuscript: Faleye, O. S., Boya, B. R., Lee, J-H.,
Choi, I., Lee, J.
Funding acquisition: Lee, J-H., Choi, I., Lee, J.

Footnotes
This study was supported by grants from the Basic Science Research Program of the National
Research Foundation of Korea (NRF) funded by the Ministry of Education
(2021R1I1A3A04037486, 2020R1A6A1A03044512) and the NRF funded by the Korean
government (MSIT) (2021R1A2C1008368).
The authors have no conflict of interest to declare.
132
Figure legends
Fig. 1 Properties of halogens (A), proportions of drugs containing halogens approved by the
Food and Drug Administration (FDA) between 1988 and 2006 (Hernandes et al., 2010) (B),
and numbers of small-molecule drugs containing fluorine, sulfur, phosphorus, or boron
approved by the FDA from 2015 to June 2020 (Bhutani et al., 2021) (C). e- =
electronegativity value, Ar = atomic radius.
Fig. 2 The sources, applications, and advantages of halogenated antimicrobial agents.
Fig. 3 Cloxacillin (A), dicloxacillin (B), flucloxacillin (C), SAR of cloxacillin (D),
cefiderocol (E), cefaclor (F), loracarbef (G), cefazaflur (H), cefazedone (I), flomoxef (J),
SARs of cefiderocol (K) and effects of halogenation on penicillins and cephalosporins (L)

Fig. 4 Teicoplanin (A), SAR of vancomycin (B), taromycin A (C), SAR of taromycin B (D),
and effects of halogenation on glycopeptides (E). The DXDG motif refers to the conserved
peptide motif of Asp-X-Asp-Gly in the lipopeptide backbone.
Fig. 5 4-Amino-3-bromo-5-chlorobenzenesulfonamide (A), 4-amino-3-chloro-5-
iodobenzenesulfonamide (B), 4-amino-3,5-dibromobenzenesulfonamide (C), SAR of
dihalogenated sulfonamide (D), chlorotrimethoprim (E), bromotrimethoprim (F),
iodotrimethoprim (G), SAR of halogenated trimethoprim derivatives (H), sitafloxacin (I),
rufloxacin (J), Levofloxacin (K), 8-chloroquinolone (L), ciprofloxacin (M), flumequine (N),
133
moxifloxacin (O), norfloxacin (P), sparfloxacin (Q), SAR of ciprofloxacin (R), and effects of
halogenation on sulfonamides, trimethoprim and quinolones (S).
Fig. 6 SAR of aminoglycosides as represented by 5-deoxy-5-fluoro-kanamycin B (A), 3’-
deoxy-3’-fluoro-kanamycin A (B), demeclocycline (C), eravacycline (D), and SAR of
chlortetracycline (E), and effects of halogenation on aminoglycosides and tetracyclines (F).
Fig. 7 Solithromycin (A), SAR of flurithromycin (B), lincomycin (C), pirlimycin (D), SAR
of clindamycin (E), linezolid (F), posizolid (G), SAR of sutezolid (H), tedizolid (I), radezolid
(J), and contezolid (K), and effects of halogenation on macrolides and lincosamides (L).
Fig 8. Structures of halogenated polyphenols and essential oils. Catechols (A), magnolols (B-
F), resveratrols (G-I), flavonoids (J-Q), cinnamaldehydes (R-X) and effects of halogenation
on catechols, resveratrols and flavonoids (Y). Full chemical names are listed in

Supplementary Fig. S1
Fig. 9 Structures of halogenated alkaloids. Indoles (A-P), streptochlorins (Q-R), quinones
and quinolines (S-Z), 4-oxoquinolizines (A1-A4) and effects of halogenation on indoles,
streptochlorins and quinones (A5). Full chemical names are listed in Supplementary Fig. S2.
Fig. 10 Structures of halogenated benzopyrones, phenazines, and azoles. Coumarins (A-F),
phenazines (G-P), azoles (Q-Z) and effects of halogenation on coumarins, phenazines and
azoles (A1). Full chemical names are provided in Supplementary Fig. S3
134
Fig. 11 Structures of repurposed scaffolds. Isoniazids (A-B), azo-aspirins (C-D),
pyrrolopyrimidines (E-G), phenothiazines (H-I), salicylanilides (J-T) and effects of
halogenation on isoniazides, phenothiazines and salicylanilides (U). Full chemical names are
listed in Supplementary Fig. S4.
Fig. 12 Structures of other halogenated bioactive compounds. β-Nitrostyrene and
nitrovinylfuran (A-G), aminothiopines (H-L), isophthalonitrile (M), β-amino amides and
alboflavusin (N-T) and (effects of halogenation on β-nitrostyrene, aminothiopenes, β-amino
amides and alboflavusins U). Full chemical names are listed in the Supplementary Fig. S5.
Fig. 13 Structures of halogenated polymeric materials. Halogenated cellulose grafted
hyperbranched polyethyleneimine (A), N,N,N-trimethyl chitosan trifluoroacetate (B), N,N,N-
trimethyl chitosan trichloroacetate (C), N,N,N-trimethyl chitosan dichloroacetate (D) and
N,N,N-trimethyl chitosan chloroacetate (E), chitosan bearing 2-fluoro-phenylimino)-

acetaldehyde (F), 2-chloro-phenylimino)-acetaldehyde (G), 2-bromo-phenylimino)-
acetaldehyde (H), fluorinated fabric polymer (I), and polyurethane-AHM-chlorine (J).
Fig. 14 A cellulose nano paper-chitosan derivative (A), a methyl β-D-galactopyranoside ester
derivatives (B), derivatives of the acyclo C-nucleosides of 1, 2, 4-triazolo [4, 3-a] quinoxaline
(C), O-acrylamidomethyl-HTCC derivative (D), and phosphoramidate adjuvants (E) and (F).
Fig. 15 Halogenated FAs isolated from Xestospongia sp (A), natural brominated FAs isolated
from the sponge Petrosia volkano (B), natural lactylates of chlorinated FAs-
chlorosphaerolactylates (C), and chlorine or bromine substituted vinyl halogenated FAs (D).
135
Fig. 16 Formation of a halogen bond between the less electron-dense σ-hole region and a
Lewis base facilitated by formation of R-X bond. Formation of the covalent bond between
the carbon and halogen leads to the depopulation of the valence pz orbital and creates an
electropositive crown while the pxy orbitals maintain their electrons to balance the negative
charge of the halogen (Mendez et al., 2017).
Fig. 17 Factors affecting the antimicrobial activities of halogenated scaffolds.
Fig. 18 Effects of halogenated compounds on various modes of antimicrobial resistance.
Fig. 19 Advantages of scaffold halogenation
Fig. 20 Mechanisms underlying the antimicrobial activities of halogenated antibiotics and
antimicrobial scaffolds.

Fig. 21 Structures of approved drugs conjugated with halogenated compounds developed to
resistant pathogen antimicrobials. Erythromycin–halogenated phenazine (A), cephalosporin-
halogenated phenazine prodrug (B), fluconazole-halogenated urea group (C), and
fluconazole- halogenated thiazolo[4,5-d] pyrimidines prodrugs (D).
136
Table 1. Halogenated aromatic, non-essential, and aliphatic amino acids and their sources.
Compound Halogenated amino acid Source Reference

A 3-chlorotyrosine
B 3-bromotyrosine
C 3,5-dichlorotyrosine,
D 3-iodotyrosine
Aplysina cavernicola (Ueberlein et al., 2014).
E 3-bromo-5-chlorotyrosine
(Ueberlein et al., 2017)
F 3-chloro-5-iodotyrosine
G 3,5-dibromotyrosine
H 3-bromo-5-iodotyrosine
I 3,5-diiodotyrosine
J 2-bromotryptophan
K 4-bromotryptophan
Various marine sponges and
L 5-bromotryptophan (Bittner et al., 2007)
invertebrates
M 6-bromotryptophan
N 7-bromotryptophan
O γ-chloronorvaline Streptomyces griseosporeus (Narayanan et al., 1980)
P 4-fluorothreonine Streptomyces cattleya (Sanada et al., 1986)
Q 4-amino-3-chloro-2- Streptomyces viridogenes; (Chaiet et al., 1984;
pentenedioic acid Streptomyces xanthocidicu Kuroda et al., 1980)
R 2-amino-5-chloro-hex-4Z-

Amanita solitaria
enoic acid
S (S)-2-amino-4-chloropent-4-
Amanita pseudoporphyria
enoic acid
(Chilton and Tsou, 1972)
T (2S,4R)-2-amino-5-chloro-4- Amanita onusta; Amanita
hydroxyhex-5-enoic acid miculifera
U (2S)-2-Amino-5-chloro-4-
Amanita gymnopus
hydroxy-5-hexenoic acid
V (S)-2-amino-5-chlorohex-5-
Amanita abrupta (Ohta et al., 1987)
enoic acid
W N-chlorotaurine (Marcinkiewicz et al.,
Neutrophils
X N-bomotaurine 2022)
137



Halogenated Antimicrobial Agents To Combat Drug

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Pharmrev Fast Forward. Published on 16 October 2023 as DOI 10.1124/pharmrev.123.

Halogenated Antimicrobial Agents to Combat Drug-Resistant Pathogens

University, 280 Daehak-Ro,

Running title: Halogenated Antimicrobial Agents

Jintae Lee, School of Chemical Engineering, Yeungnam University

Downloaded from pharmrev.aspetjournals.org at ASPET Journals on January 9, 2024

E-mail: [email protected]. Tel.: +82-53-810-2533. Fax: +82-53-810-4631

Number of text pages: 90

Number of references: 388

Number of words in the Abstract: 149

II. Halogenated Antibiotic Classes

A. Cell Wall Synthesis Inhibitors (Penicillins, Lipopeptides, Cephalosporins, and

B. Cell Proliferation Inhibitors (Sulfonamides, Trimethoprim, and Quinolones)

C. Protein Synthesis Inhibitors (Aminoglycosides, Tetracyclines, Macrolides,

Lincosamides, and Oxazolidinones)

III. Halogenated Antimicrobial Scaffolds

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B. Essential Oil Compounds

3. Quinones and Quinolones

D. Benzopyrones and Phenazines

G. Other Halogenated Bioactive Compounds

1. β-Nitrostyrene and Nitrovinylfurans

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IV. Halogenated polymerics

VI. Effects of Halogenation and Factors Affecting the Antimicrobial Activities

A. Halogen Bonding, Pharmacokinetics and Pharmacodynamics

B. Halogenated Agents and Gut Microbiota

C. Factors Affecting Antimicrobial Activities.

1. Sizes and Substitution Pattern

2. Hydrophobicity and Lipophilicity

3. Physicochemical and Biophysical Properties

4. Bacterial Cell Types

VII. Halogenation for Antibiotic Rejuvenation and Resistance Minimization

VIII. Current Practices and Prospects

ADME- Adsorption, distribution, metabolism, and elimination

AHM- 2-Amino-5-(2-hydroxyethyl)-6-methyl pyrimidine-4-one

AMP- Antimicrobial peptide

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ATP- Adenosine triphosphate

DNA- Deoxyribo nucleic acid

ESKAPE- Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

FA- Fatty acids

FDA- Food and Drug Administration

HIV- Human immunodeficiency virus

HTCC- N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride

MDR- Multidrug resistant

MIC- Minimum inhibitory concentration

MOA- Mode of action

MRSA- Methicillin-resistant Staphylococcus aureus

MRSE- Methicillin-resistant Staphylococcus epidermis

NADH- Nicotinamide adenine dinucleotide hydrogen

NDH-2- Type II NADH dehydrogenase

PABA- Para-aminobenzoic acid

PBP- Penicillin binding proteins

PVP-I- Povidone iodine

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RHS- Reactive halogen species

RNA- Ribonucleic acid