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Current Topics in Medicinal Chemistry, 2017, 17, 1044-1079

REVIEW ARTICLE
ISSN: 1568-0266
eISSN: 1873-5294

Hybrid Compounds as Direct Multitarget Ligands: A Review Impact

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Michelle de Oliveira Pedrosaa,c, Rayssa Marques Duarte da Cruza,c, Jéssika de Oliveira Vianaa,
Current Topics in Medicinal Chemistry

Ricardo Olímpio de Mouraa,e, Hamilton Mitsugu Ishikib, José Maria Barbosa Filhoc,
Margareth F. F. M. Dinizc, Marcus Tullius Scottic, Luciana Scottic,* and
Francisco Jaime Bezerra Mendonça Juniora,c,d,e,*

a
Laboratory of Synthesis and Drug Delivery, Department of Biological Science, State University of Paraiba, João Pes-
soa, PB, Brazil; bUniversity of Western São Paulo (Unoeste), Presidente Prudente, SP, Brazil; cFederal University of
Paraíba, Campus I, João Pessoa-PB, Brazil; dPos-Graduate Program in Therapeutic Innovation. Center of Biological
Sciences, Federal University of Pernambuco, Recife, PE, Brazil; ePos-Graduate Program in Chemistry, State University
of Paraíba, Campina Grande, PB, Brazil

ARTICLE HISTORY
Received: May 01, 2016
Revised: June 06, 2016
Accepted: June 19, 2016
DOI: 10.2174/1568026616666160927
160620
Keywords: Hybrid compounds, Multitarget, Anti-inflammatory, Antimicrobial, Anticancer, Antiprotozoal.
Abstract: Molecular Hybridization is an approach in rational drug design where new chemical enti-
ties are obtained by combining two or more pharmacophoric units from different bioactive com-
pounds into a single molecule. Through this approach, medicinal chemists hope that the new hybrid
derivative presents: better affinity and efficacy when compared to the parent drugs; a modified selec-
tivity profile with improvement over pharmacokinetic and pharmacodynamic restrictions; dual or
multiple modes of action; reduction of undesirable side effects; decreases in drug-drug interactions; re-
duced emergence or spread of drug resistance in microorganisms and protozoans; and lower cost. The
approach has been successfully used by many research groups around the world and has had very prom-
ising results with diseases having multifactorial profiles, like Alzheimer´s, Parkinson´s disease, cancer,
inflammation, and hypertension among others. The purpose of this paper is to conduct an updated re-
view of molecular hybridization and multitarget profiling (a rational drug design approach), and its ap-
plications to the design and discovery of novel hybrid compounds with anti-inflammatory, antimicrobial,
anticancer and antiprotozoal (leishmaniasis, malaria, and schistosomiasis) activities over the last six
years.

1. INTRODUCTION Directly Linked Hybrids

Molecular Hybridization (MH) is an approach to rational
drug design in which new chemical entities are synthesized
by combining two or more pharmacophoric units from dif-
ferent bioactive compounds, or in other words, two or more
known bioactive compounds into a single compound [1, 2].
By this approach, medicinal chemists hope that the new
chemical entity developed will maintain the pre-selected
characteristics of the original templates, and exert a dual or
even multiple drug action. Several reviews dealing with con-
cepts and applications, advantages and disadvantages, and
finally the promise, challenges and the perspectives of hy-
brid compounds are available in the literature [1, 3-9].
Based on the binding mode of the two subunits (pharma-
cophoric units, or bioactive compounds) hybrid compounds
can be classified into:
*Address correspondence to these authors at the Department of Chemistry,
State Universtiy of Paraiba, João Pessoa, Paraiba -58071-160, Brazil;
Tel: +55 83 99924 1423; E-mail: franciscojbmendonca@yahoo.com.br
The two subunits are directly connected to each other
(without a spacer) through hydrolysable bonds, most com-
monly ester, amide, carbamates and phosphates e.g., hybrid
dihydroartemisnin-quinine (A) (Fig. 1), as was obtained by
Walsh and co-workers [10]. The hybrid presented superior
antimalarial activity than the parent compounds against
Plasmodium falciparum 3D7.
Spacer Linked Hybrids
A space linker connects the subunits, providing two sub-
classes which may be cleavable or non-cleavable:
Cleavable Hybrids
The moieties are joined by a bond which in vivo provides
two parent drugs, which will individually act on their respec-
tive targets. The most common cleavable linkers are ester,
amides, carbamates and disulfide bonds e.g., hybrid cinnam-
ic ester (B) (Fig. 1) was obtained by combination of phenox-
yphenyl cinnamic acid and m-acetamidophenol (an aceta-
minophen analogue). The hybrid showed lipoxygenase

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Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1045

MeO

HO NC
O N
N
N O (CH2)2-7
O N N R
N N
N O Me H H
quinine spacer cyanoguanidine

O mepyramine
O MeO
A artemisnin C
éster
cleavable bond (ester)

H
O N
O

cinamic acid derivative O O

m-acetamidophenol
B

O O NH
triazole thiadiazole demethoxycurcumin
N S
N N
R H
N
N
HO
R1 melatonin OMe
D
E
Fig. (1). Examples of hybrid derivatives based on connecting two pharmacophoric units.

inhibition (IC50 = 0.34 μΜ) associated with analgesic activity
(98.1%) [11].
Non-cleavable Hybrids
An enzymatically stable and non-hydrolyzable spacer
linker connects the pharmacophores or parent compounds
e.g., a dual histamine H1 and H2 antagonist was synthesized
through combination (via a poly-methylene spacer) of N-
desmethylmepyramine (H1 antagonist) and a cyanoguani-
dine group (H2 antagonist), resulting in a series hybrid (C)
(Fig. 1) [12].
Fused Hybrids
Fused hybrids compounds do not present a linker be-
tween the two subunits and the two pharmacophores or bio-
active compounds literally are touching e.g., fused hybrid
from a triazole and a thiadiazole (D) (Fig. 1) inhibits TNF-
alpha, preventing oxidative and nitrosative stress in cell [13].
Merged or Overlapped Hybrid Drugs
Merged hybrid compounds are obtained by overlap-
ping/combining two drug pharmacophores generating a new,
smaller, and simpler chemical entity. Its chemical structure is
often substantially different from the original drugs or phar-
macophores but its pharmacological properties are main-
tained e.g., hybrid 5-(4-hydroxyphenyl)-3-oxo-pentanoic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide E (Fig. 1)
was synthesized by the association of demethoxycurcumin
and melatonin. The resulting hybrid presented neuroprotec-
tive and antioxidant effects and interactions with Aβ oligo-
mers within the mitochondria at nanomolar concentrations
[14].
These last two classes (Fused and Merged hybrid com-
pounds) are also know in medicinal chemistry as chimeric
drugs, however for the purposes of this article, they will be
treated as hybrid compounds.
Hybrid compounds can act through three different inter-
action profiles with their biological targets:
• Single Target – when the two subunits of the hybrid act
with the same target (also called double-edged-sword).
e.g., Trioxaquine derivatives obtained by the combination
of trioxane and quinoline moieties (Fig. 2) that are able to
stack with heme through the aminoquinoline moiety, and
can alkylate heme after its reductive activation [15].
• Independent and non-related targets – each of the subu-
nits that form the hybrid has an independent and non-
related biological target. e.g., Hybrid synthesized by a
combination of cloroquine and amino-acridine (DNA in-
tercalation, inhibition of mitochondrial bc1 complex, in-
hibition of topoisomerases) (Fig. 2) for malarial therapy,
increasing cloroquine activity [16].
• Related Targets – when both subunits simultaneously act
on two related targets or two biological domains present
on the same target. e.g., Hybrids which can interact with
DNA by intercalation and topoisomerase inhibition, as
observed in certain thiazacridine and imidazacridine de-
rivatives (Fig. 2) [17].
1046 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
O H
Me O O
trioxane
OMe
NH
HN
Cl N
cloroquine
amino-acridine
Cl
Fig. (2). Examples of hybrid derivatives based on their dual mode of action.
The era of multitarget drug research and development
began after publication in 2005 of the manuscript "Designed
multiple ligands. An emerging drug discovery paradigm"
published by Morphy and Rankovic [18]. They introduced
the concept of Multi Target Drug Discovery (MTDD) to the
Medicinal Chemistry community, a concept that has rapidly
evolved, becoming the object of study for various research
groups around the world. Three years later, the research
group coordinated by Cavalli and Bolognesi introduced the
concept of Multi Target Direct Ligands (MTDL) [19].
In sequence, two books “Polypharmacology in Drug
Discovery” [20] and “Designing Multi-Target Drugs” [21]
and several other reviews [22-29] were published and pro-
vided valuable information concerning: drugs in combination
versus multitarget drugs; polypharmacology; the paradigm of
mono- to multi-target-directed ligands; approach advantages
and disadvantages; applications and limitations. These
themes will not be presented in detail in this review.
The aim of this review is to describe the application of
molecular hybridization with multitarget profiles to drug
design and discovery, this of; anti-inflammatory, antimicro-
bial, anticancer and antiprotozoal (leishmaniasis, malaria and
schistosomiasis) agents in the last six years.
Multi-Target Hybrid Compounds for Cancer Therapy
Of the degenerative diseases, cancer is considered one of
the most devastating and frightening in the world. According
to WHO (2016) [30], cancer is a generic term for a large
group of diseases that is able to affect any part of the body.
One defining feature of cancer is the rapid creation of ab-
normal cells that grow beyond their usual boundaries, and
which can then invade adjoining parts of the body and spread
to other organs; a process known as metastasis that is the
major cause of death from cancer.
Pedrosa et al.
N
NH
N Cl
aminoquinoline
O
NH
thiazolidinadione
S
S
N
acridine
Covington and co-workers (2015) [31] stated that under-
standing the mutational mechanisms in cancer can yield fur-
ther insights into both the biology of cancer cells, and of
cellular processes regulating DNA damage and repair in
general. As the vast majority of mutations arising in any giv-
en tumor are neutral in function (so-called passenger muta-
tions), the study of mutations arising in tumor data is largely
unbiased by phenotypic selection.
Among the processes of mutation the inactivation of the
transcription factor p53, either through direct mutation or
aberrations in one of its many regulatory pathways, is a
hallmark of virtually every tumor. Screening for p53 activa-
tors and a better understanding of the molecular mechanisms
of oncogenic perturbations of p53 function have opened up a
host of novel avenues for therapeutic intervention in recent
years [32].
Circular RNAs (circRNAs) are a novel class of
non-coding RNA molecules ubiquitously present in the cy-
toplasm of eukaryotic cells, recent studies described by Peng
and co-workers (2015) [33] have demonstrated that a recent-
ly identified circRNA, ciRS-7, can regulate the activities of
miRNAs, mRNAs, and RBP towards specific biological ef-
fects. CiRS-7 also acts as a naturally competing endogenous
RNA, a.k.a. ‘super sponge’ of microRNA-7 (miR-7), that
sequesters and competitively inhibits the activity of miR-7.
This competition between ciRS-7 and miR-7 may have pro-
found effects on oncogenesis. The review summarized the
origin and functions of ciRS-7 and discusses the relation-
ships between ciRS-7, its target molecules, and cancer [33].
However, despite increased knowledge about cancer bi-
ology, there are countless types of cancers that do not re-
spond to our growing arsenal of chemotherapeutic agents; in
a phenomenon known as multidrug resistance (MDR).
Kathawala and co-workers (2015) [34] described one of the
Hybrid Compounds as Direct Multitarget Ligands
OH N O
O O
O N
O O
Camptothecin - Topoisomerase I inhibitor
R
1a: R=OCH3
1b: R=H
R
Fig. (3). Indenoisoquinoline-camptothecin hybrids (1a and 1b) result from the merging of two topoisomerase I inhibitors: Camptothecin and
5,11-diketoindenoisoquinoline (NSC 314622).
major causes of MDR and chemotherapeutic failure in can-
cer therapy. It is believed to be the ABC transporter-
mediated active efflux across the membrane of a multitude
of structurally and mechanistically distinct cytotoxic com-
pounds. It is postulated that ABC transporter inhibitors,
known as chemosensitizers, might be used in combination
with standard chemotherapeutic agents to enhance therapeu-
tic efficacy [34]. The need for new compounds to overcome
such resistance mechanisms encourages research in hybrids
compounds with multi-target action. In recent years many
researchers have broadened our understanding of the cellular
mechanisms of cancer, and its resistance, and have suggested
that one of the most viable options is to develop hybrid com-
pounds which can act in a dual way to fight the disease.
Chen, Cui and Ai (2012) [35], make it clear that the het-
erogeneous nature of cancer requires a comprehensive ap-
proach for attacking the multiple mechanisms underlying the
initiation and progression of cancers, and point out that ra-
tional design of multi-targeted inhibitors based on examina-
tion and manipulation of chemical structures [35], and de-
sign of hybrid compounds is a feasible alternative.
Bérube and Fortin (2013) [4] describe hybrid anticancer
drugs as of great therapeutic interest since they can potential-
ly overcome most of the pharmacokinetic drawbacks en-
countered when using conventional anticancer drugs. In fact,
the future of hybrid anticancer drugs, for the discovery of
potent and selective molecules that (in synergy) trigger two
or more cytocidal mechanisms of action to inhibit can-
cer/tumor growth, is very bright.
The same authors describe two main ways by which hy-
brid anticancer molecules can be designed and prepared. The
first approach merges and blends haptophoric moieties of
different drugs that are used to design new anticancer hy-
brids based on the ability of a combination of haptophoric
moieties on a new molecular structure to retain their affinity
and activity for their biological targets and ii) the second
approach combines two or more complete drugs, bound di-
rectly or connected through a linking arm which can be
achieved using cleavable or non-cleavable linkages. Some
hybrids use both design approaches and are therefore diffi-
cult to classify in one or the other category [4].
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1047
O
O
N
O
5,11-Diketoindenoisoquinoline (NSC 314622)
Topoisomerase I inhibitor
N
N
As an example of the first approach, Fox and co-workers
(2003) [36], synthesized anticancer hybrids aiming at an
identical biological target. This design aimed to improve the
activity, selectivity and biopharmaceutical properties of two
parental anticancer drugs. The indenoisoquinoline-
camptothecin hybrids (1a and 1b) respectively result from
the merging of two topoisomerase I inhibitors: camptothecin
and 5,11-diketoindenoisoquinoline (NSC 314622) (Fig. 3).
Compounds 1a and 1b exhibit antiproliferative activity in the
micromolar range and possess significant topoisomerase I
inhibitory activity but show less biological activity than their
parental compounds alone [36].
As another example: a series of philadelphia chromo-
some inhibitors, (2,4-disubstituted thiazole derivatives) syn-
thesized by Wang and co-workers (2011) [37], were de-
signed and synthesized as new Bcr/Abl inhibitors, hybrids
using structural moieties of the FDA approved drugs;
imatinib, nilotinib and dasatinib. The new inhibitors strongly
suppressed the activity of Bcr/Abl kinase and potently inhib-
ited proliferation of K562 and KU812 leukemia cancer cells.
The hybrid compound 2a exhibited antiproliferative activity
in the nanomolar range, which is in the same order of magni-
tude as nilotinib on the assessed cancer cell lines. Compound
2a also displays inhibitory potency against Bcr-Abl kinase
comparable to nilotinib, it arrests the cell cycle progression
in the G0/G1-phase, and induces apoptosis of K562 cancer
cells (Fig. 4) [37].
Multi-Target Hybrid Compounds for Cancer; Acting on
Different Therapeutic Targets
Multiple biological target hybrids act on differing targets;
hybrid anticancer molecules merge and integrate haptophoric
moieties from different drugs, separately addressing two or
even multiple biological targets [4].
As an example, Belluti and co-workers (2010) [38] de-
veloped stilbene-coumarin hybrids, synthesized using the
phenylethenyl moiety of resveratrol and the coumarin ring
system, the latter class being responsible for a variety of ac-
tion mechanisms in cells, and is recognized to exhibit potent
proapoptic effects. As representatives, compounds 3a-b
1048 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
Fig. (4). Chemical structures Bcr/Abl inhibitors and the designed hybrid 2a.
Fig. (5). Stilbene-coumarin hybrids as anticancer molecules with multiple biological targets.
(Fig. 5), all exhibit antiproliferative activity in the low mi-
cromolar range on the cell lines studied. They arrest cell cy-
cle progression in the G2-M-phase and induce apoptosis
[38].
Other coumarin hybrid compounds, were synthesized by
Sashidhara and co-workers (2010) [39], which fuse Chalcone
with coumarin ring systems, to generate new coumarin-
chalcone hybrids 4a-c (Fig. 6) that showed antiproliferative
activity in the micromolar range on KB, C33A, MCF-7,
A549 and NIH3T3 cell lines, with a superior activity on a
cervical carcinoma C33A cell line [39].
It is known that antitumor activity of doxorubicin and
daunorubicin are strongly dependent on the presence of the
daunosamine (aminocarbohydrate) moiety, so research was
proposed by Bednarek and co-workers in 2006 [41] to im-
prove the properties of indolo[2,3-b]quinolones (Fig. 7)
which are analogs of neocryptolepine DNA topoisomerase II
Pedrosa et al.
inhibitors. They linked indolo compounds to aminoglyco-
sides to produce new indolo[2,3-b]quinoline hybrids (exem-
plified in (a)) and described as a new family of potent anti-
cancer drugs, both compounds are topoisomerase II inhibi-
tors [40].
As an example of this approach, Matsuya and colleagues
(2009) [41] designed and synthesized Macrosphelide-
epothilone hybrids, results in a combination of 2-methyl-4-
(prop-1-en-1-yl) thiazole, (for stabilizing microtubules), and
Macrosphelide-A as (for inhibition of cell to cell adhesion)
(Fig. 8). Compound 6a displays antiproliferative activity on
human colon carcinoma (HCT116) and human gastric cancer
(AGS) cells, with no effects on normal human dermal fibro-
blasts. It also induces the formation of reactive oxygen spe-
cies, activation of Jun N-terminal kinase, and apoptosis in
human lymphoma (U937) [41, 42].
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1049

Fig. (6). Coumarin-chalcone hybrid as potent inhibitor of cervical carcinoma C33A cell line.

Fig. (7). Indolo[2,3-b]quinoline hybrid (a) as potent anticancer drugs, both compounds are topoisomerase II inhibitors.

Fig. (8). Macrosphelide-epothilone hybrids as candidate drugs acting with the same mechanism of action.

In “Hybrid Molecules Incorporating Natural Products:
Applications in Cancer Therapy, Neurodegenerative Disor-
ders”; Decker (2011) [43] suggests that the largest therapeu-
tic area (apart from neurodegenerative disorders) to which
natural product hybrid molecule design has been successful-
ly applied is the diverse field of antitumor agents where it
has shown a promising outlook [43].
Due to the eminent importance of natural substances in
cancer therapy, their applications in medicinal chemistry
have enjoyed much research. From a set of synthesized hy-
brids and investigated SARs, dehydrozingerone was coupled
through its phenolic OH group to the carboxy function of
Glycyrrhetinic acid (GA; obtained by hydrolysis of glycyr-
rhizic acid from the medicinal plant licorice, Glycyrrhiza
1050 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
Fig. (9). A hybrid synthesized from dehydrozingerone coupled to the carboxy group of Glycyrrhetinic acid.
Fig. (10). Dual-action compounds to target histone deacetylase (HDAC) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR).
glabra L), (Fig. 9). The hybrids were tested on 9 (nine) hu-
man tumor cell lines for their cytotoxicity as were their re-
spective parent compounds dehydrozingerone, and GA. The
dehydrozingerone-GA hybrids showed μM cytotoxicity
(ED50 0.9-2.5) in tumor cell lines and multi-drug resistant
cell lines [44].
Ai and co-workers (2012) [35] describe Histone deacety-
lase inhibitors (HDACi), a new class of emerging anticancer
agents, targeting biological processes including cell cycle,
apoptosis, and differentiation. Studies have revealed that
HDACi are synergistic with diverse classes of anticancer
therapies including targeted therapeutics and conventional
anticancer agents. Among the most advanced; HDACi, su-
beroylanilide hydroxamic acid (SAHA), and FK-228 (ro-
midepsin), have been approved by the Food and Drug Ad-
ministration (FDA), which validates HDACi as effective
anticancer agents [35].
Based on this and other information, a series of dual-
action compounds were designed to target histone deacety-
Pedrosa et al.
lase (HDAC), and 3-hydroxy-3-methylglutaryl coenzyme A
reductase (HMGR), using a hydroxamate group essential for
chelation with the zinc ion in the active HDAC site and key
structural elements of statin for binding with both proteins.
The compounds showed potent inhibitory activities against
HDACs and HMGR, with IC50 values in the nanomolar
range (Fig. 10). The compounds effectively reduced HMGR
activity as well as promoting histone and tubulin acetylation
in cancer cells, yet they were not toxic to normal cells. They
are considered hybrid compounds of great interest [45].
Hybrid compounds targeting histone deacetylase
(HDAC) was also synthesized by Zhang and colleagues
(2015) [46]. The class is represented as a new class of col-
chicine derivatives, which were designed and synthesized as
dual tubulin–HDAC inhibitors (Fig. 11). Biological evalua-
tions of these hybrids included HDAC inhibitory activity,
tubulin polymerization analysis, in vitro cell cycle analysis
in HCT-116 cells, and cytotoxicity against different cancer
cell lines. Hybrid 9b behaved as a potent HDAC–tubulin
dual inhibitor and showed cytotoxicity comparable to
Hybrid Compounds as Direct Multitarget Ligands
Fig. (11). Colchicine derivatives as tubulin–HDAC dual inhibitors.


Fig. (12). Hybrid topoisomerases and histone deacetylase (HDACs) inhibitors.
colchicine. Compound 9a exhibited powerful tubulin inhibi-
tory activity, moderate anti-HDAC activity, and the highest
cytotoxicity (IC50 = 2–105 nM) [46].
In a recent review, Seo (2015) [47] highlighted the im-
portance of topoisomerase and histone deacetylase (HDAC)
hybrid compounds. This because toposimerases are enzymes
involved in the cleavage and relegation of DNA, while
HADCs regulate dynamic epigenetic modification of lysine
amino acids on various proteins. Seo highlighted the hybrid
compounds synthesized by Yu and co-works in 2014, that
bound an HDAC inhibitor (SAHA), described in this work
(Fig. 9) to a topoisomerase inhibitor DACA, generating the
hybrid 10a (Fig. 12). This new hybrid displays better activity
than SAHA or DACA against human hormone-refractory
metastatic prostate cancer (HRMPCs) PC-3 cell lines (IC50
values of the 0.31 µM) and DU-145 (IC50 values of the 0.16
µM). The compound induced anti-HDAC and anti-
topoisomerase I activities, caused profound DNA damage,
and arrested the cell cycle at phases G1 and G2.
Seo [47] reported a class of triple-inhibitors
(HDAC/TopI/TopII). They synthesized a series of novel hy-
brids using a molecular hybridization strategy, attaching the
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1051


HDAC inhibitor SAHA to a topoisomerase I and II inhibitor
to arrive at compound 10b. This compound exhibited excel-
lent anti-proliferative activities against MDA-MB (IC50 = 2.3
µM), HCT116 (IC50 = 0.41µM), and HLF (IC50 = 1.3 µ M)
cell lines, and provided good anti-HDAC inhibitory activities
against HDAC1 (IC50 = 24 nM), HDAC6 (IC50 = 13 nM),
and HDAC8 (IC50 = 2.5 µM) [47].
Another class of compounds that has excelled in the fight
against cancer is derived from pyrazoline and isatin. This
because the pyrazoline derivatives are considered to be a
potent tyrosinase and P-glycoprotein-mediated multidrug
resistance inhibitors, and isatin is well-known as a cyclin-
dependent kinase and carbonic anhydrase isozyme inhibitor.
Many 3,5-diaryl-N-acetyl pyrazoline-isatin hybrids were
synthesized using a click chemistry approach, and were
evaluated against HeLa (cervix cancer), CAKI-I (Renal can-
cer), PC-3 (Prostate cancer), and Miapaca-2 (pancreatic can-
cer) cell lines. The hybrids were classified into right-handed
and left-handed conjugates on the basis of the isatin ring
placement. The length of the alkyl armed triazole linker was
varied from 2 to 6. The work, a preliminary cytotoxic assay
was performed on the series of 3,5-diaryl-N-acetyl-
pyrazolines. Only the potent 3,5-diaryl-N-acetyl-pyrazolines
1052 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
Fig. (13). 3,5-Diaryl N-acetyl pyrazoline-isatin hybrids with great cytotoxic activity.
Fig. (14). Design strategy of benzoselenazole-stilbene hybrids as multifunctional anti-cancer agents.
were selected for their inclusion in the hybrid scaffold.
Among the cell lines, the HeLa cell line was the most sensi-
tive to test compound exposure. Compounds 11a (Fig. 13)
showed significant cytotoxic potential and exhibited an IC50
of 1.3 μM against HeLa cell line [48].
Yan and co-workers (2015) [49] described the synthesis
of novel multi-target-directed drug candidates for the treat-
ment of cancer. Their work showed a series of benzose-
lenazole-stilbene hybrids, synthesized by combining the
pharmacophores of resveratrol and ebselen (Fig. 14). The
biological assay indicated that all of the hybrids exhibit anti-
proliferative activity against four human cancer cell lines,
and demonstrate good thioredoxin reductase (TrxR) inhibito-
ry activity. The biological function maintains the cellular
antioxidant system at normal physiological conditions, help-
ing cellular defense against apoptosis by reducing oxidized
thioredoxin (Trx). The mechanism of cell apoptosis was in-
vestigated in G2/M cell cycle arrest induced using compound
12a and apoptosis in the human liver carcinoma Bel-7402
cell line. A significant increase in intracellular ROS levels
confirmed that these compounds cause oxidative stress-
induced apoptosis [49].
Recent studies have described small molecules that can
restore biological function to p53 mutants found in human
cancers, and that have been highly sought to increase anti-
cancer efficacy.
Punganuru and co-workers [50] described the synthesis
of piperlongumine (PL) derivatives, with an aryl group in-
serted at the C-7 position. The insertion bestowed a com-
bretastatin A4 like structure (an established microtubule dis-
ruptor), while retaining the piperlongumine configuration.
The new compounds exhibited potent antiproliferative activi-
Pedrosa et al.
ties against eight cancer cell lines; in particular, they were
more cytotoxic against SKBR-3 breast cancer cells which
harbor a R175H mutation in the p53 suppressor. Among
them, 13a (Fig. 15) strongly disrupted in vitro tubulin
polymerization, destabilized the microtubules in cells, and
induced a potentG2/M cycle block. The compound also
showed the ability to reactivate the p53 mutation and restore
biological activity to the R175H mutant protein present in
SKBR3 cells. So, mechanistically, the redox-perturbation in
cancer cells by the hybrid drug appears to underlie the p53
reactivation process [50].
A class of hybrid derivatives was synthesized and evalu-
ated against PC-3, SGC-7901, A549 and MDA-MB-435s
cancer cells lines. The series of the dihydroartemisinin-
cinnamic acid ester derivatives highlighted a compound 14
(IC50 = 0.20 μM) (Fig. 16) which was the most potent anti-
proliferative agent against human lung carcinoma A549
cells, although it displayed low cytotoxicity for normal he-
patic L-02 cells (IC50 = 80.25 μM), Selectivity Index (SI) =
401.25 μM. The mechanism of action of compound 14 was
further investigated by analysis of cell apoptosis and intra-
cellular ROS generation. The results indicated that both ROS
and ferrous ion contributed to cell death as induced by com-
pound 14. Also, it was found that high intracellular ferrous
ion and endogenous oxidative stress in A549 cells made
them more susceptible to compound 14-induced apoptosis
[51].
Acridine derivatives have also been used as a basis for
design of new hybrid compounds with anticancer activity.
Jiang and co-works designed and synthesized a new series of
9-anilinoacridines (containing phenyl-urea moieties), as po-
tential, novel, dual Src and MEK inhibitors, and evaluated
their anti-proliferative action against K562 and HepG-2
Hybrid Compounds as Direct Multitarget Ligands
Fig. (15). C7-Aryl piperlongumine derivative with potent antimicrotubule and mutant p53-reactivating properties.


Fig. (16). Dihydroartemisinin-cinnamic acid ester derivatives.
tumor cells. The study showed that most of the derivatives
displayed good in vitro cytotoxicity. In particular, kinase
inhibition assays showed that compound 15 (Fig. 17) inhibit-
ed Src (59.67%) and MEK (43.23%) at 10 μM, and dis-
played moderate inhibitory activity against ERK and AKT.
Moreover, the compound induced K562 cells apoptosis [52].
Multi-target Hybrid for Inflammatory Disorders
Inflammation is a multifactorial process. It reflects the
response of the organism to various stimuli and is related to
many disorders. The arachidonic acid (AA) metabolic net-
work produces a large family of inflammatory mediators,
including leukotrienes (LTs) and prostaglandins (PGs),
which contribute to numerous inflammatory-related diseases
such as asthma, rheumatoid arthritis, atherosclerosis, Alz-
heimer’s disease, and cancer [53, 54]. Leukotriene B4
(LTB4) and prostaglandin E2 (PGE2), which are synthesized
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1053


via two major pathways involving 5-lipoxygenase (5-LOX)
and cyclooxygenase (COX), are major mediators and play
crucial roles in the inflammatory response. Hence, enzymes
involved in LT and PG biosynthesis, are key targets in anti-
inflammatory drug research [55].
The pharmacological effects of nonsteroidal anti-
inflammatory drugs (NSAIDs) are due to inhibition of a
membrane enzyme called cyclo-oxygenase (COX). There are
two isoforms, COX-1 and COX-2 [56], which have been
targeted for many years to treat acute and chronic inflamma-
tion. However traditional NSAIDs, such as aspirin, ibuprofen
and indomethacin cause side effects, including gastrointesti-
nal toxicity by inhibiting COX-1 [56]. The selective COX-2
inhibitors can reduce gastrointestinal toxicity; however car-
diovascular side effects are associated with them [56]. Cur-
rently, the only approved 5-LOX inhibitor is Zileuton [54].
1054 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Fig. (17). Acridine derivative hybrid as a dual (Src and MEK) inhibitor.

Due to the moderate capacity of current drugs, pain and potency of the products was near their 5-LO inhibition po-
inflammation remain areas of substantially unmet patient tency ranges [57].
need. Inflammation is a complex disease that cannot be ef-
fectively controlled by single-target drugs, thus strategies for HO NH2
designing new drugs that interfere at multiple inflammation N
O
targets have been developed. CH3
S
In 2010, Bosch and colleagues [57] proposed new dual- 1
action products, obtained by combining of zileuton (+)-[1-(1-
HO NH2
benzo[b]-thien-2-yl)ethyl]-1-hydroxyurea) (1) (Fig. 18), and N
an anti-asthmatic drug, 5-Lipooxygenase (5-LO) inhibitor O
with NO donor nitrooxy or furoxan moieties. Compounds 2, n(H2C) CH3
O S
3, 4, 5, 6 and 7 were obtained and evaluated for their proper- O2NO
2n=4
ties of 5-LO inhibition, myorelaxation, vasodilation and car-
3n=6
rageenan-induced paw edema inhibition. The ability of the
hybrids 2–7, and of the reference drug zileuton to inhibit 5- HO NH2
LO was assessed, and the results (Table 1) showed that all N
the products were able to inhibit LTB4 production in a con- O
(CH2)n
centration dependent manner. The inhibition potencies, ex- O2NO O S CH3
pressed as IC50, cover the concentration range of 2–18 µM. ONO2 4n=2
Compound 2 was the most potent, however the data indicate 5n=4
that hybridization of zileuton with appropriate NO donor
moieties affords compounds with good levels of 5-LO inhi- HO NH2
bition [57]. N
R
All of the products were able to relax the contracted tis- O
sue (myorelaxation effect) in a concentration dependent O
S CH3
manner. The results (Table 1) revealed a concentration range O N
of 15–37 µM. In the nitric acid ester series, mono-nitrooxy N 6 R = Me
O
derivatives 2 and 3 displayed the same activity and were half 7 R = CONH2
as potent as the equipotent di-nitrooxy analogues 4 and 5. Fig. (18). Anti-inflammatory hybrids synthesized by Bosch and
From the two furoxan derivatives, only the furoxancarbox- colleagues [57].
amide 7 showed an EC50 value in the micromolar range.
Zileuton had no effect on the contracted tissue, suggesting The vasodilator effect results showed that the nitro-oxy-
that the myorelaxing effects observed for all the active prod- substituted derivatives are very potent vasodilators, with
ucts were mediated by NO [57]. EC50 values in the sub-micromolar range. The furoxancar-
In order to confirm this hypothesis, the authors evaluated boxamide 7 was a very potent vasodilator, about 150-fold
the activity of the hybrids on pre-contracted tracheal rings in more potent than the methyl furoxan 6. According to the
the presence of 10 µM 1H-[1,2,4]oxadiazolo[4,3- authors, the data indicate that in vitro NO-mediated vasodila-
a]quinoxalin-1-one (ODQ), a well-known inhibitor of solu- tor effects of the tested compounds dominate their 5-LO in-
ble guanylate cyclase (sGC). According to the authors, the hibitor capacity, with the exception of methylfuroxan 6.
suppression of sGC activity, (products tested up to a concen- All compounds, including zileuton, were tested through
tration of 30 µm), is in keeping with the NO messenger’s carrageenan-induced paw edema. Administered at an equi-
myorelaxation involvement. The NO-dependent myorelaxing molar dose to zileuton (45.12 mg/kg-1 - i.g.), compound 2
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1055

Table 1. Results of vasodilation, myorelaxation assays, and 5-LO inhibition properties of zileuton (1) and hybrids 2-7 [57].

Vasodilation [a] Myorelaxation [b] 5-LO Inhibition [c]

Compounds EC50 ±SE [µM] EC50±SE [µM] IC50 [µM]
(+ODQ 1 µM) (+ODQ 10 mM) (95% CL)

1 –[d] –[d] 1.6 (1.3–2.0)

2 0.039 ±0,007 (>30) 37±5 (–[d]) 2.0 (1.6–2.5)

3 0.023 ±0.003 (>30) 36±8 (–[d]) 5.8 (4.3–7.7)

[d]
4 0.015 ±0.004 (23 ±3) 16±3 (– ) 2.8 (2.0–4.1)
[d]
5 0.027 ±0.004 (14 ±4) 15±1 (– ) 3.5 (2.8–4.3)

6 28±2 (–[d]) –[d] 6.3 (4.9–8.3)

[d]
7 0.18 ±0.04 (6.4 ±0.7) 24±2 (– ) 17.9 (11.5–28.0)

[a] Determined on pre-contracted rat thoracic aorta (1 mm phenylephrine). [b] Determined on rat tracheal rings pre-contracted with 1 µm carbachol. [c] Measured as the ability of the
compound to inhibit biosynthesis of LTB4 in human whole blood challenged with the calcium ionophore A23187. [d] Inactive at the maximum concentration tested (30 µM).

induced significant paw edema inhibition, displaying the pendent (12.5–100 µM) inhibitory effect on LPS-induced
same activity as the reference drug. Compound 7 (49.94 NO2-production. Though compounds 12, 13, 14 were well
mg/kg-1 - i.g.) significantly decreased (~20%) edema at the considered, for compounds 10, and 11 the inhibition was
4th hour of testing alone. All of the other analogues proved to remarkably more powerful, than that of resveratrol [59]. The
be ineffective. authors explained that the six-membered nitroxides 10, 12,
The authors believe that higher doses of the zileuton hy- 13 and isoindoline nitroxide 14 with a lower redox potential
brid analogues should be tested since the solubility of the were more potent RNS scavengers than the five-membered
products in the vehicle may have been a limiting factor. The nitroxide 15.
compounds at the doses tested differed in their anti-
inflammatory activity, however they displayed similar in R
R
vitro 5-LO inhibition potency, suggesting differing pharma- R
O N
cokinetic profiles between them [57].
8 R = OH
New hybrids based on resveratrol, (paramagnetic ana- 9 R = Me R
10 R = Me
12 R = OH
logues), containing coupled five- and six-membered nitrox- R
ides and isoindoline nitroxides were synthesized by Heck
Me
and Suzuki in a study by Kálai et al., (2011) (Fig. 19) [58].
H N O N
Resveratrol has been reported as exerting a variety of biolog-
N O
ical activities, including antioxidant and anti-inflammatory
effects [58]. Nitroxides are stable free radicals that rapidly 11 13
Me
cross cell-membranes, and preempt free-radical formation by
oxidizing redox-active metal ions, and function both as intra- OH OH
and extracellular superoxide dismutase mimics [59].
OH
HO
To furnish nitroxide influence along with the antioxidant
activity of resveratrol, the authors synthesized paramagnetic
analogues of resveratrol. They incorporated a nitroxide N
O
moiety into ‘resveratrol-like’ molecules using the following OH
14 N
strategies: replacement of the resveratrol phenol ring with a
O 15
nitroxide ring, inserting a pyrroline ring between the phenol
and resorcine ring, and replacement of both phenol and Fig. (19). Hybrids of resveratrol with nitroxides and isoindoline
resorcine rings with nitroxides [58]. The anti-inflammatory nitroxides.
and antioxidant activity of the new compounds (10-15) and
resveratrol (8, 9) were then evaluated. The antioxidant activity of the new resveratrol deriva-
tives was compared to Trolox (6-hydroxy-2,5,7,8-
To investigate the anti-inflammatory effect of the para- tetramethylchroman-2-carboxylic acid) as the antioxidant
magnetic analogues as compared to resveratrol, RAW264.7 standard, and the results were given in Trolox Equivalent
cells were treated with LPS (lipopolysaccharide) (100 Antioxidant Capacity (TEAC) units. The results are present-
ng/mL) alone or together with resveratrol or its analogues ed in the Table 2. According to the authors, compounds with
(12.5–100 µM). After 24 h of incubation, nitrite production two phenolic hydroxyl groups in the presence of nitroxide
was measured with Griess-reagent [58]. All of the paramag- exhibited the same antioxidant activity as resveratrol itself in
netic analogues were found to possess a concentration de- EtOH [58].
1056 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Table 2. TEAC activity of resveratrol and its paramagnetic analogues [58].

Compound TEACa in EtOH TEACa in PBS

Resveratrol (8) 1.0 ± 0.05 1.3 ±0.20

9 0.04 ± 0.02 ND

10 0.2± 0.07 0.28 ± 0.05b

11 0.32 ± 0.05 0.02 ± 0.01b

12 0.92 ± 0.08 0.62 ± 0.10b

13 0.22 ± 0.01 0.37 ± 0.08b

14 0.98 ± 0.02 0.67 ± 0.12b

15 1.02 ± 0.03 ND

ND, not determined; a, n = 3; b, N-hydroxylamine HCl salt.

TNFα levels were also measured at 1.5 h after the LPS Within this group of compounds, the N-methoxyl ethanesul-
challenge, and a 10 to 12 fold increase in concentration was fonamide ester of ibuprofen (20) showed a higher COX-2
observed in the treated cells media. All of the paramagnetic selectivity index than the parent NSAID ibuprofen where the
analogues (50 µM) and resveratrol (50 µM) decreased the SI’s are respectively 23.3 and 2.9. The authors note that the
amount of TNFα, and compounds 11, 13, and 14 exhibited ethanesulfohydroxamic acid ester of ibuprofen (19) showed
the strongest effects [58]. a much lower COX-2 SI (0.07) compared to the N-methoxyl
According to the authors, the structure–activity relation- analogue 20 (23.3). According to the authors, this indicates
that the N-methoxyl group enhances binding to the COX-2
ship studies suggest that in a resveratrol-like compound, sub-
enzyme.
stitution of an aromatic ring with a tetrahydropyridine or an
isoindoline nitroxide is more advantageous than incorporat- O O H
ing a pyrroline nitroxide ring. N R2
To design new drugs with anti-inflammatory activity that S O
R1 O
are devoid of adverse ulcerogenic and/or cardiovascular side O
effects, Huang et al., (2011) [60] synthesized new hybrid
compounds. To furnish this group of hybrid ester prodrugs
(16-20) that release nitric oxide (NO) and nitroxyl (HNO) Me
(Fig. 20), the carboxylic acid group of the anti-inflammatory 2 1 Cl
(AI) drugs indomethacin, (S)-naproxen and ibuprofen was 16 R = H, R = MeO N
covalently linked via a two-carbon ethyl spacer to a sulfohy-
droxamic acid moiety (CH2CH2SO2NHOH). All of the new O
compounds were evaluated for NO and HNO release in vitro,
inhibition in vitro of COX-1/COX-2, and also evaluated as
anti-inflammatory agents [60].
17 R2 = H, R1 =
The % NO released from the N-hydroxy (methoxy)
MeO
ethanesulfonamides 16-20 and ethanesulfohydroxamic acid
(EA) (21) under incubation in phosphate-buffered saline
(PBS at pH 7.4), or PBS containing rat serum were measured 18 R2 = H, R1 = benzyl
by quantitation of nitrite using the Griess reaction (See re-
sults in Table 3). According to the authors, when compared
to previous studies, the % NO released from the ethanesul-
fohydroxamic acid esters 16-19 in PBS at pH 7.4 varied over 19 R2 = H, R1 =
a 44.5-54.3% range, this is indicative of relatively high NO 20 R2 = Me, R1 =
release. The % NO release from 16-19 and EA (21) was
suppressed (1.3-4.8% range) in PBS containing rat serum
[60]. The assay of Nitroxyl (HNO) release was held indirect- O H
ly by quantification of N2O (See results in Table 3). N
21 S OH
The in vitro results for COX-1/COX-2 isozyme inhibition
studies showed that the ethanesulfohydroxamic acids (16- O
17), and the N-methoxyl ethanesulfonamide (20) ester pro- Fig. (20). Hybrids of indomethacin, (S)-naproxen and ibuprofen
drugs were more potent inhibitors of COX-2 (IC50 = 0.4-15.8 with sulfohydroxamic acid.
μM range) than COX-1 (IC50 = 1.1-77.7 μM range) [60].
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1057

Table 3. Percent (%) Nitric Oxide and Nitrous Oxide Release from 16-20 and the Reference Compound Ethanesulfohydroxamic
Acid (EA)(21) [60].

% N2O release a
% NO releaseb
Compound MeOH/TBS c MeOH/base d MeOH/base/GSH e

PBS f PBS + serum g 2h 24h 2h 24h 2h 24h

16 44.5 3.2 0 0 20 1 1 1

17 52.9 1.3 0 0 24 23 2 4

18 54.3 4.8 0 0 23 21 0 7

19 46.3 2.1 0 0 25 24 0 7

20 4.3 0.9 0 0 0 0 0 0

EA 54.5 1.4 0 0 24 7 0 0
a
Percent N2O released, based on condensation of 2 mol of HNO→1 mol N2O +H2O. The result is the mean value of three measurements (n = 3). The HNO donor test compound (16-
20, EA) concentration is 50mM in each experiment unless otherwise noted. b Percent NO released based on a theoretical maximum release of 1 mol of NO/mol of sulfohydroxamic
acid test compounds (16-20), and the reference agent ethanesulfohydroxamic acid (EA). The result is the mean value of three measurements (n = 3) where variation from the mean %
value was ≤0.2%. c MeOH/TBS solvent is comprised of 0.6 mL MeOH and 0.2 mL of 700 mM Tris buffer solution (TBS) at pH 7.00. d MeOH/base solvent is comprised of 0.6 mL
MeOH and 0.2 mL of 1M NaOH. e MeOH/base/GSH experiments are 100mM in glutathione (GSH) and 250mM in NaOH except for compounds 16 and EA which were 25mM in
NaOH. f A solution of the test compound (2.4 mL of a 5.0x10-2 mM) in phosphate buffer at pH 7.4 was incubated at 37 ºC for 1.5 h. g A solution of the test compound (2.4 mL of a 5.0
x 10-2 mM) in phosphate buffer at pH 7.4, to which 90 μL rat serum had been added, was incubated at 37 ºC for 1.5 h.

At the highest test compound concentration tested (100 Altogether 12 compounds were synthesized in each
μM), the phenylacetic acid analogue 18, as expected, did not group, the first group included dihydropyrimidinyl-
inhibit the COX-1 or COX-2 isozymes. Similarly, EA did quinazolinone derivatives, the second group included pyrim-
not inhibit COX-1 or COX-2 at the highest tested concentra- idinyl-quinazolinone derivatives [61] (Fig. 21). Anti-
tion (100 μM) [60]. inflammatory activity evaluations of the synthesized com-
pounds were performed using the carrageenan-induced rat
The ID50 values, for anti-inflammatory activity (AI), ac-
paw edema model with sodium diclofenac as the reference
quired for compounds 16-20 were determined using a carragee-
nan induced rat foot paw edema model [60]. The results showed drug. From among the synthesized compounds, five showed
potencies greater than 90% of that of diclofenac: 22, 23, 24,
that the ester prodrugs of NSAIDs 16-17 and 19-20 showed
25 and 26. The IC50 (µmol/kg) of these five compounds are
potent AI activities. The (S)-naproxen analogue 17 showed a
in Table 4. The potencies of 4-anilinopyrimidine derivatives
more potent AI activity (ID50 = 11.8 μmol/kg po) than (S)-
24 and 25 were equal to diclofenac [61].
naproxen (ID50 = 29.7 μmol/kg), and compound 16 was a less
O
potent AI agent (ID50 = 19.1 μmol/kg) relative to indomethacin
1
(ID50 = 11.7 μmol/kg). Compounds 19 and 20 exhibited more R CN
O N
potent, yet similar, AI activities (79.5 and 78.9% inhibition of H
N
inflammation for a 327 μmol/kg oral dose) relative to the parent N S N
NSAID ibuprofen (AI ID50 = 327 μmol/kg). The phenylacetic
O
acid ester 18 exhibited weak AI activity (12.4% inhibition of N Cl R
inflammation for a 100 mg/kg) [60], as expected. O
22 R = R1 = H
The authors also showed that the ethanesulfohydroxamic 23 R = Me, R1 = OH
acid ester of indomethacin 16, in view of its COX-2 selectiv- Cl
ity and ability to release NO, is a beneficial mediator of GI
mucosal protection, unlike indomethacin (UI = 64 for a 80 R1

μmol/kg), since 16 showed no visible gastric lesions (UI = 0

for a 80 μmol/kg) [60]. NH

In an attempt to design and develop new anti- R 1

CN
O N
inflammatory agents, Abbas et al., (2012) [61] synthesized H
two groups of hybrid compounds: quinazolinone- N
N
S N
dihydropyrimidines and quinazolinone-pyrimidines. In their
O
review, the authors report the importance of quinazolines N Cl R
which have drawn great attention due to their wide range of O
therapeutic activities including anti-inflammatory, analgesic 24 R = Cl, R1 = OH
and COX-2 inhibitory activity. Data from their literature 25 R = OH, R1 = H
review revealed that many effective anti-inflammatory Cl 26 R = R1 = OH
agents have been derived from pyrimidine, dihydropyrimi- Fig. (21). Hybrid dihydropyrimidinyl-quinazolinone and pyrimidi-
dine, and tetrahydropyrimidine nuclei [61]. nyl-quinazolinone derivatives.
1058 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Table 4. IC50 values of the anti-inflammatory activity for compounds 22-26, and sodium diclofenac [61].

Edema inhibition
Compound Dose (mg/kg) (IC50 µmol/kg)
% after 3 h

20 42.25

Diclofenac-sodium 50 86.26 141.03

100 91.23

50 68.42

22 100 85.96 143.26

200 91.23

50 70.42

23 100 85.96 130.44

200 97.75

50 61.40

24 100 91.23 114.03

200 96.47

50 77.19

25 100 91.23 116.73

200 96.49

50 56.14

26 100 86.84 123.88

200 91.23

According to the authors, in terms of IC50, all of the
compounds except 6a were more active than sodium diclo-
fenac. They observed that the 4-anilino derivatives (24, 25,
and 26) were more active than either 23 or 22, and the most
active compound was 24, with an IC50 =114.03 µ mol/kg
[61].
The ulcerogenicity of the compounds was compared to
the classical NSAID, sodium diclofenac and to a non-
ulcerogenic drug, celecoxib. The results revealed that com-
pounds 22, 23, 24, 25, and 26 were less ulcerogenic than
sodium diclofenac, and compounds 23 and 25 were less ul-
cerogenic than celecoxib [61].
Compounds 22-26 were evaluated for their selectivity in
COX-2 and COX-1 isoenzyme inhibition. The results
showed that compounds 23, 24 and 25 are selective COX-2
inhibitors with respective ratios of COX-2 IC50 to COX-1
IC50 of 0.62, 0.40 and 0.44 [61]. According to the results
presented by Abbas et al., 2012 [62], it was possible to ob-
tain new compounds with anti-inflammatory activity and
fewer ulcerogenic effects.
Isolated from Rhubarb, Rheum palmatum L., Rhein is a
principle constituent and a well characterized anti-
inflammatory with bone affinity and recognized effective-
ness in a range of inflammatory diseases such as osteoarthri-
tis and diabetic nephropathy. However, Rhein displays side
effects such as gastrointestinal irritation [62, 63]. Rhein is an
active metabolite of Diacerein which is a clinically used pro-
drug medicine. Its main mechanism of action is to inhibit
interleukin-1b (IL-1b). NSAIDs are widely used in the
treatment of pain, fever and inflammatory diseases including
arthritis, and exert their therapeutic effects by inhibiting cy-
clooxygenase (COX) [63].
In a study by Cai et al., 2012 [63], Rhein was chosen as a
bone-targeting carrier and potentially anti-inflammatory lead
compound. The carboxyl group of Rhein or diacerein was
sterified with ethylene glycol (diethylene glycol or triethy-
lene glycol), and was then chemically linked through glycol
ester to certain NSAIDs such as Aspirin, Diclofenac,
Naproxen, Indomethacin, and Ibuprofen to form potential
bone-targeting anti-inflammatory prodrugs [63].
The authors synthesized 13 new Rhein-NSAID hybrid
compounds (Fig. 22) which were evaluated for their bone
binding capabilities. Hydrolytic activation of these prodrugs
under simulated in vitro physiological conditions; mice auri-
cle tumefaction experiments; and in vivo ulcerogenic activi-
ty, and pharmacokinetics [63] were tested. According to the
authors, most of the new compounds showed obvious bind-
ing capability to HAP (Hydroxyapatite), and could success-
fully carry the connected NSAIDs to absorption [63].
In simulated physiological conditions, HPLC analysis of
the reactions showed that all of the prodrug hybrids were
spontaneously hydrolyzed and released the active drug. The
Hybrid Compounds as Direct Multitarget Ligands
hybrids, were however more stable under acidic conditions
than under neutral conditions, especially the compounds de-
rivative of acetylated Rhein. Both Rhein and the NSAIDs
were released slowly within 24 h [63].
OAc O OAc
O R
O O
n O
O O
32 n= 1, R= ibuprofen
33 n= 2, R= ibuprofen
34 n= 3, R= ibuprofen
35 n= 1, R= aspirin
36 n= 1, R= naproxen
37 n= 1, R= diclofenac
38 n= 1, R= indometacin
39 n= 1, R= naproxen
40 n= 2, R= naproxen
41 n= 3, R= naproxen
42 n= 1, R= aspirin
43 n= 1, R= naproxen
44 n= 1, R= diclofenac
Fig. (22). Hybrid Rhein-NSAID derivatives.
In Table 5 are presented the in vivo acute anti-
inflammatory activities of the tested compounds at a dose of
0.2 mmol/kg; in the xylene-induced mice auricle tumefaction
model. Nearly all of the Rhein-NSAID ester hybrid prodrugs
32-38 and 39-44 demonstrated good anti-inflammatory activ-
ity except for 35 (19.92%) and 42 (19.64%). The anti-
inflammatory activity achieved for 37 (43.98%) and 44
(43.89%) was high, with inhibition percentages higher than
that of either diclofenac (41.92%) or diacerein (29.22%)
[63].
Compounds 39-41 and 43 were evaluated for acute ul-
cerogenic activity, in comparison to ibuprofen. The results
indicate that these four compounds 39-41 and 43 (with ulcer
index ranges from 5.8 to 6.0) caused less gastric ulceration
and disruption of gastric epithelial cells than ibuprofen (ulcer
index 8.3). Compound 43 was chosen for pharmacokinetic
studies [63].
According to the authors, this same prodrug 43 is a po-
tential candidate for a slower and sustained release form of
Rhein-NSAID, as demonstrated in the pharmacokinetic stud-
ies. They believe that Rhein-NSAID ester prodrugs may
provide a promising alternative to the use of NSAIDs as an
anti-osteoarthritis agent with more AI activity and fewer side
effects [63].
Hernandez et al., (2012) [64] reported the synthesis of fu-
roxanyl N-acylhydrazones (furoxanyl-NAH), applying a mo-
lecular hybridization approach, and performed biological
evaluation of the analgesic and anti-inflammatory activities
of the new hybrids.
The compounds were designed as hybrid entities combin-
ing differing aromatic residues, among others, the furoxan
system, connected by the N-acylhydrazone moiety (Fig. 23).
For the 34 compounds evaluated, the data showed a range of
results, of which the furoxan hybrids 45, 46, and 47 showed
the best results, and they were not mutagenic under Ames
testing [64].
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1059
Ph
H
O N
N
N
O
O N O
O
45
Me O
N
N
N H
N
46 R = N(CH3)2 R
47 R = COOH
Fig. (23). Hybrid furoxan-N-acylhydrazone derivatives.
Taking into consideration the variety of pharmacological
effects mentioned in the literature for hydantoin and indole
derivatives, Guerra et al., (2011) [65] evaluated the potential
anti-inflammatory and antinociceptive activities of hybrid
compounds containing imidazole and indole nuclei [65].
The synthesized hybrids, 5-(1H-Indol-3-ylmethylene)-2-
thioxo-imidazolidin-4-one (48) and 3-(4-bromo-benzyl)-5-
(1H-indol-3-yl-methylene)-2-thioxo-imidazolidin-4-one (49)
(Fig. 23), were evaluated using air pouch and carrageenan-
induced peritonitis models, as well as an acetic acid-induced
vascular permeability model followed by IL-1β and TNF-α
quantification. The antinociceptive activities of the com-
pounds were also tested [66]. Both 48 and 49 exhibited sig-
nificant anti-inflammatory activity (Table 6). The results
obtained in the carrageenan-induced peritonitis test (Table 6)
revealed that at the doses tested, both 48 and 49 caused a
significant reduction in the migration of polymorphonuclear
leukocytes (PMNL) in comparison to the control group [65].
Br
S H
N
O
HN N S
N
H
48 O
HN N
H
49
Fig. (24). Hydantoin-indole hybrid compounds.
To assess the activity of the compounds under study in
cells of the innate immune system, concentrations of TNF-α
and IL-1ß in inflammatory exudates were determined, ob-
tained through air pouch and peritonitis tests. In the IL-1β
and TNF-α quantification tests, in air pouch levels testing,
reductions were observed in the levels of TNF-α in the ani-
mals treated with 49 (to 488.32 pg/mL) or 48 (to 218.70
pg/mL), as compared to the control group (1096.47 pg/mL).
For comparison, indomethacin reduced the concentration of
TNFα to 575.43 pg/mL; thus, 48 was more effective in re-
ducing TNF-α than indomethacin. Both compounds also
reduced the production of IL-1β relative to untreated control
values [66]. In the quantification tests of IL-1β, and TNF-α
levels in peritonitis, both 48 and 49 promoted significant
1060 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Table 5. `In vivo inhibitory activity of the target compounds in xylene-induced mice auricle tumefaction assay (adapted from Cai et
al., 2012) [63].

Degree of Inhibition
Compounds
tumefaction (mg) percentage (%)

Blank 10.64 ± 1.90 -

Diacerein 7.22 ± 1.59 1.15

Ibuprofen 7.23± 1.28 3.75

Diclofenac 6.18± 1.35 4.12

32 6.89 ± 1.61 5.18

33 7.13 ± 1.77 5.03

34 7.52 ± 1.73 4.87

35 8.52 ± 1.27 2.61

36 8.04 ± 1.03 4.4

37 5.96 ± 1.18 5.55

38 8.15 ± 1.45 5.12

39 6.26 ± 0.83 5.23

40 7.16 ± 1.62 5.08

41 7.03 ± 1.77 4.92

42 8.55 ± 1.41 2.66

43 8.10 ± 0.64 4.45

44 5.97 ± 1.00 5.6

Table 6. Carrageenan-induced air pouch inflammation and Carrageenan-induced peritonitis tests (adapted from Guerra et al.,
(2011) [65]).

Effect of 49 and 48 on cell migration into peritoneal

Effects of 49 and 48 on carrageenan-induced air pouch.
cavity on carrageenan-induced peritonitis in mice.

Nº of PMNL/mL
Compound Dose (mg/kg) Inhibition % No. of PMNL/mL (x106) Inhibition %
(×106)

49 3 6.88±1.3* 56.1 - -

10 4.36±0.7* 72.1 4.80±0.7 * 56.1

30 6.24±0.5* 60.1 -

48 3 3.0±0.7* 80.8 4.96±0.9* 54.7

10 5.36±0.6* 65.7 - -

30 6.70±1.3* 57.2 - -

Indomethacin 10 2.08±0.4* 86.7 4.62±0.3* 57.7

Control - 15.64±1.9 - 10.93±1.6 -

Data are the mean ± SD of at least six animals. *P<0.05. Significance was determined with ANOVA one way followed by Bonferroni's post hoc test when compared with control
group.

decreases in TNF-α concentration as compared to the control The evaluated indole–imidazolidine derivatives signifi-
group. Both compounds also inhibited the production of IL- cantly inhibited vascular permeability in comparison to the
1β [65]. control group. No significant difference was observed be-
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1061

tween the derivatives, though they both exhibited lower lev-
els of inhibition than indomethacin [65].
The results further indicated that the compounds tested
have antinociceptive activity as was shown in the formalin
and writhing tests.
According to the results presented by the study of Guerra
et al., (2011) [65] the evaluated hybrid compounds exhibited
promising anti-inflammatory and antinociceptive activities
that likely involve modulation of the immune system.
Nitrogen containing heterocyclic compounds (Pyrazoles
and 1,2-diazoles) have drawn the attention of the researchers
due to their wide range of biological activities including anti-
inflammatory, antipyretic and analgesic activity [66]. In
modern medicinal chemistry the thiadiazole ring system is a
privileged structural fragment considering its broad biologi-
cal spectrum and affinity for various bio targets. Thiazoli-
dinones were recently reported as potential anti-
inflammatories [67, 68], as well as for being selective COX-
2 and LOX inhibitors [69, 70].
Alegaon et al., (2014) [66] described the synthesis and
SAR study of a series of novel pyrazole derivatives, thio-
semicarbazone, and 4-thiazolidinone; and 1,3,4-thiadiazole
derivatives as potential selective COX-2 inhibitors.
The in vitro COX-1/COX-2 inhibition studies revealed
that all of the evaluated compounds are more potent COX-2
inhibitors (IC50 = 1.33–17.5 µM range) than COX-1 (IC50 =
42.33–80.33 µM range). Compound 50 (Fig. 25), a pyra-
zole–thiadiazole hybrid was identified as the most potent and
selective COX-2 inhibitor (COX-1 IC50 = 80.33 µM, COX-2
IC50 = 1.33 µM), with a significant COX-2 selectivity index
(SI > 60). Molecular docking studies revealed that pyrazole
analogues exhibit good interactivity with the active site of
the COX-2 receptor. The results of the study suggest that
pyrazole–thiadiazole hybrids could be an interesting ap-
proach for the design of new selective COX-2 inhibitory
agents. The results relative to compound 50 are summarized
in Table 7 [66].
O
MeO HN
S N
N
O
N
N
50
Ph
Fig. (25). Pyrazole–thiadiazole hybrid.
Peperidou et al., (2014) [11], studied potential newly
synthesized multitarget hybrid agents, incorporating cinnam-
ic acids and paracetamol, 4-/7-hydroxycoumarin, benzo-
caine, p-aminophenol and m-aminophenol (Fig. 26).
The study results revealed that hybrid 51 (phenyloxy-
phenyl acid with paracetamol) has high analgesic activity

Table 7. Results of tests carried out for compound 50 (adapted from Alegaon et al., 2014 [66]).

Data from the in vitro COX-1/COX-2 enzyme inhibition assay of synthesized compounds

Compound COX-1a IC50 (µM) COX-2a IC50 (µM) (SI)b

50 80.33 1.33 60.39

Binding energy of compounds 5c after docking in to COX-1 and COX-2 active sites

COX-2
COX-1

Hydrogen bonding resi-

Compound Binding Energy Binding Energy Hydrogen bonding residues
dues

ARG 197, GLN 350, GLY ARG 120, ARG 513, TYR 355, GLU
50 -4.93 -10.08
354 524

Celecoxib -6.93 GLN 350, TYR 355 -10.37 ARG 120, ARG 513, HIS 90
c
Cytotoxicity of compounds against the HDF (Human dermal fibroblast) cell line

Compound % Cell viability ± SD at 100 µM

50 56.67 ± 2.6

Molecular parameters

Lipinski viola-
Compound TPSAd n-rotbe nONf nOHNHg miLogph MWi
tion

50 88.31 5 8 1 2.07 435.50 0

a
IC50 value = concentration required to produce 50% inhibition of COX-1/COX-2 by means of two determinations; deviation from the mean is <10% of mean values. bSelectivity
index (COX-1 IC50/COX-2 IC50). c Each experiment was independently performed three times, and data are shown as mean SD. d TPSA, topological polar surface area. e n-rotb,
number of rotatable bonds. f nON, number of hydrogen bond acceptors. g nOHNH, number of hydrogen bond donors. h miLogP, logarithm of compound partition coefficient between
n-octanol and water. i MW, molecular weight.
1062 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

O 51 X = O
O X
52 X = CH2
O
N
H

H
N O
O O
O O

53
O
O O
Br
O O
Me
54
Fig. (26). Hybrids containing cinnamic acids and paracetamol, 4-/7-hydroxycoumarin, benzocaine, p-aminophenol and m-aminophenol.

Table 8. % inhibition of lipid peroxidation (AAPH %); in vitro soybean lipoxygenase inhibition (IC50μΜ or % LOX Inh.); in vitro
trypsin induced proteolysis inhibition (IC50 or % Trypsin Inh.); in vivo analgesic-antinociceptive activity (% ANA); in vivo
carrageenin-induced rat paw edema inhibition (ICPE %). (Adapted from Peperidou et al., (2014) [11]).

IC50μΜ/% b % ANA (0.01 mmol/0.1 % ICPE (0.01

Compounds AAPH % a,b IC50μΜb LOX Inh.
Trypsin Inh. kg) c mmol/1 kg) d

51 90 100 μΜ 10 μΜ 91 ** 36.5 **

nt
52 60 0.34 μΜ na 98.1 **

nt nt
54 60 72 μΜ 43.6 μΜ

53 90 50 μΜ 5 μΜ nt Nt

Troxolon 63

Coumarin 13%
e
NDGA - - 51.5 μM - -

4-hydroxycoumarin 100 62*

7-hydroxycoumarin 92 100 μΜ - -

Hymechromone 93 60% - -

Salicylic acid - - 53.6% - -

Paracetamol 91 82 μΜ Na 66* 21*

p-aminophenol 99 nt 87% nt nt

m-aminophenol 95 nt 90% nt nt

Benzocaine 25 nt 28% nt nt

Indomethacin nt nt nt nt 47*
a b
na: no activity under the reported experimental conditions; nt: not tested; % inhibition at 100 μM; Values are means (±SD < 10%) of three or four different determinations. Means
within each column differ significantly (p < 0.05); c value represents the mean obtained from 5 to 7 animals (±SD < 10%); * p < 0.1 ** p < 0.01 (Student’s t-test) in comparison to
controls; d value represents the mean obtained from 6 to 15 animals in two independent experiments. In all cases, significant difference from control: * p < 0.1 ** p < 0.01 (Student’s
t-test). eNDGA (nordihydroguaiaretic acid).

(91%), and also appears to be a promising agent for treating ty (IC50 = 0.34 μΜ and 98.1%), whereas hybrid 53 (bromo-
peripheral nerve injuries. Hybrid 52 derived from phenoxy- benzyloxy cinnamic acid and hymechromone) exhibited
phenyl cinnamic acid and -acetamidophenol showed the simultaneously good LOX inhibitory activity (IC50 = 50 μΜ)
highest lipoxygenase (LOX) inhibition, and analgesic activi- and the highest anti-proteolytic activity (IC50= 5 μΜ). Hy-
Hybrid Compounds as Direct Multitarget Ligands
brid 54 having ester and amide bonds presented an interest-
ing combination of anti-LOX and anti-proteolytic activity
[72]. According to the authors, the study shows that the syn-
thesized cinnamic acid hybrids represent a promising class of
multitarget compounds, influencing several biological tar-
gets, e.g., lipoxygenase and trypsin [11]. The results of the
study for the hybrids 51-54 are summarized in Table 8.
Hybrids of dopamine and alpha-lipoic acid (ALA),
ALA–dopamine (55) and its acetylated derivative, ALA-
acetyl dopamine (56) (Fig. 27) were synthesized by Hwang
et al., (2015) [71]. In their study, the effects of the new com-
pounds on inflammatory responses of BV-2 microglial cells
and RAW264.7 macrophage cells activated by lipopolysac-
charide (LPS) were evaluated.
OR
O FAAH, COX-1, and COX-2, with median inhibitory concen-
N OR
H
S S
55 R = H
56 R = Ac
Fig. (27). Hybrids of dopamine and alpha-lipoic acid (ALA).
The results of the study showed that 55 and 56 effective-
ly inhibited LPS-induced NO generation in BV2 and
RAW264.7 cells and the RNA expressions of iNOS, COX-2,
IL-1β and IL-6 in RAW264.7 cells alone [71].
The hybrid compounds, almost completely inhibited NO
production at concentrations below 5 and 10 μM in BV2 and
RAW264.7cells, respectively. Both 55 and 56 may be effec-
tive anti-inflammatory compounds [71].
Compound 56 (less than 10% cell death at 200 μM) was
less toxic to RAW264.7 cells at 48h when compared to 55
(more than 50% cell death at 200 μM). Similar results were
observed in BV2 cells at 48 h: 55 induced more than 70%
cell death vs 56 which induced less than 20% cell death at 40
μM. The authors believe that acetylation of 55 may inhibit
the autoxidation reaction of the dopamine moiety, which
might reduce cytotoxicity and increase cellular uptake, this,
because 56 is less ionized than 55 (pKa= 8.9 versus 10.6)
respectively, under physiological conditions (pH 7.3) [71].
Compounds 55 and 56 suppressed LPS-induced NF-κB,
but not activation of MAPK (mitogen activated protein ki-
nases), suggesting that the inhibition of NF-κB activity is
independent of MAPKs [71].
The authors agree that since NF-κB (nuclear factor kap-
pa-light-chain enhancer of activated B cells) activation is the
most important signaling pathway in inflammation, by inhi-
bition of NF-κB associated gene products, HBU-199 (55)
and HBU-200 (56) represent excellent candidate agents for
various inflammatory diseases, including neuro-degenerative
diseases, arthritis, sepsis, cancer, obesity, and diabetes [71].
In his recent study, Sasso et al., (2015) [72] describes a
novel class of hybrids that simultaneously inhibits FAAH,
COX-1, and COX-2 with high potency, selectivity and oral
bioavailability. The prototype of this new class was called
ARN2508 (Fig. 28), and causes no gastric damage, indeed it
protects the gastrointestinal (GI) epithelium from damage
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1063
induced by non-steroidal anti-inflammatory drugs (NSAIDs)
through a mechanism that requires inhibition of FAAH.
Me
COOH
H
* N O
n O F
Fig. (28). Structure of the hybrid ARN2508.
The new inhibitor integrates the alkyl carbamic acid
group needed for FAAH inhibition and the propionic acid
group needed for COX-1/COX-2 inhibition around a com-
mon biphenyl core [72]. ARN2508 is potent at inhibiting
trations: FAAH, 0.031 ± 0.002 µM; COX-1, 0.012 ±0.002
µM; and COX-2, 0.43 ±0.025 µM.
When tested at a concentration that fully inhibits FAAH,
COX-1, and COX-2 (10 mM), ARN2508 had no effect on a
panel of 30 biologically relevant targets, including lipid me-
tabolizing enzymes that contribute to the inflammatory re-
sponse, such as 5-lipoxygenase, 12-lipoxygenase, 15- lipox-
ygenase, and secreted phospholipase A2 (type V). Further,
ARN2508 had no effect on the activity of monoacylglycerol
lipase, nor did it significantly affect other inflammation-
related targets, such as iNOS, histone deacetylase (types 3, 4,
6, and 11), sirtuin 1 and 2, and the phosphodiesterases 1B,
2A1, 3A, 4D2, and 5.
Even further, ARN2508 did not interact with 75 common
receptors, ion channels, or neurotransmitter transporters,
comprising cannabinoid CB1 and CB2; prostaglandin EP1,
EP3, EP4, and prostacyclin receptor; adenosine A1, A2A,
and A3; adrenergic a1, a2, b1, and b2; bradykinin B1 and
B2; endothelin A and B; histamine H1 and H2; leukotriene
BLT1 and cysteinyl leukotriene receptor 1, and protease-
activated receptor-2 [72].
The study results indicate that ARN2508 is orally availa-
ble and systemically active and does not cause gastric injury.
In anti-inflammatory effect testing using models of colon
inflammation, ARN2508 was highly potent and effective at
reducing TNBS-induced inflammation and mortality in mice.
ARN2508 was also more effective than 5-ASA, which is
used clinically to treat IBD (inflammatory bowel disease).
A series of ten new hybrid compounds were synthesized
combining terpenes from Chilean medicinal plants (whose
anti-inflammatory activity is known), with synthetic anti-
inflammatory drugs [73]. The terpenes ferruginol, imbrica-
tolic acid, and leanolic acid present an OH function which
can be used to prepare derivatives with commercial anti-
inflammatory drugs ibuprofen and naproxen, leading to new
hybrid compounds (Fig. 29).
The topical anti-inflammatory activity of the compounds
was assessed in mice by the arachidonic acid (AA) and 12-
O-tetradecanoyl phorbol 13-acetate (TPA) induced ear ede-
ma assays. The basal cytotoxicity of the compounds was
assessed against the following human cell lines: lung fibro-
blasts (MRC-5), gastric epithelial AGS cells and hepatocytes
HepG2 [73].
1064 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

The results showed that the new compounds had anti-
inflammatory effect, reducing inflammation respectively by
10.0%–56.8% when AA-induced, and by 26.8%–80% when
TPA-induced [73]. At 1.4 μmol/mouse, strong anti-
inflammatory effects in the TPA assay were observed for
oleanoyl ibuprofenate (61) (79.9%) and oleanoyl naproxe-
nate methyl ester (62) (80.0%). In the AA assay, the best
activity was observed for 61, at 3.2 μmol/mouse, with a
56.8% reduction in inflammation [73].
COOR
NSAID
O Recently, Hoxha et al. (2016) [74] synthesized four new
61 R = H, NSAID = ibuprofen
62 R = Me, NSAID = naproxen
Fig. (29). Hybridization of terpenes with ibuprofen (61) and
naproxen (62).
The compound oleanoyl ibuprofenate had the best inhibi-
tion percentage and was more active than the parent com-
pounds in both inflammation models (Table 9). The cytotox-
icity assay for the new hybrids was either higher or lower
than that of the starting terpenes, depending on the structural
features of the diterpene moiety. Compounds 61 and 62 had
little toxicity, except against HepG2 cells, and may be re-
garded as non-toxic (Table 9) [73].
According to the authors, the new products present prop-
erties that differ from their separate molecular constituents.
This suggests the potential of further structure-activity rela-
tionship studies, (on compounds which combine natural
products and bioactive synthetic moieties), to lead to new
chemical entities. The low cytotoxicity of the active anti-
inflammatory hybrids suggests a potential application in the
treatment of inflammatory responses including allergic skin
disorders and atopic dermatitis [73].
compounds (63-66) (Fig. 30) by modulating the structure of
the most potent coxib, (Lumiracoxib), to obtain novel multi-
target NSAIDs endowed with balanced coxib and tromboxa-
no A2 prostanoid (TP) receptor antagonist properties. An-
tagonist activity at the TP receptor (pA2) was evaluated for
all four compounds in human platelets and in a heterologous
expression system by measuring aggregation prevention and

Table 9. Results for topical anti-inflammatory effects and cytotoxicity of compounds 61 and 62 (adapted from Theoduloz et al.,
2015 [73]).

Topical anti-inflammatory effect of compounds in the mice ear edema model induced by arachidonic acid (TAAA) and 12-O-tetradecanoylphorbol
13-acetate (TATPA).
Results are expressed as percent reduction of the ear edema.

Compounds % TAAA ± SEM % TATPA ± SEM

Oleanolic acid 2.7 ± 7.5 70.2 ± 8.3 *,a

Ibuprofen 7.2 ± 5.5 56.2 ± 3.4 *,a

Naproxen 1.8 ± 7.5 a 29.9 ± 6.2 *,a

Oleanoyl ibuprofenate (61) 56.8 ± 7.4 * 79.9 ± 10.6 *

a
Oleanoyl naproxenate methyl ester (62) 2.7 ± 8.1 80.0 ± 11.0 *

Nimesulide ↑48.9 ±5.5 * -

Indomethacin - ↑92.9 ± 13.2 *

Cytotoxicity of compounds 61-62 against confluent cultures of three human cell lines: lung fibroblasts (MRC-5), gastric epithelial cells (AGS) and hepato-
cytes Hep G2.

(IC50 ± SD, μM) b

Compounds
MRC-5 AGS HepG2

Oleanolic acid 186 ± 9 234 ± 16 170 ± 5

Ibuprofen >1000 >1000 >1000

Naproxen >1000 >1000 >1000

Oleanoyl ibuprofenate (61) 616 ± 32 526 ± 19 >1000

Oleanoyl naproxenate methyl ester (62) 816 ± 42 593 ± 31 >1000

Etoposide 3.9 ± 0.1 0.36 ± 0.02 2.4 ± 0.1

* p ≤ 0.05 compared with control group; a p ≤0.05 compared with reference drug; n = 8; ↑Maximal effect; SEM standard error of the mean values. b Results are expressed as mean
values ± SD.
Hybrid Compounds as Direct Multitarget Ligands
Gq-dependent production of intracellular inositol phosphate
induced by the stable thromboxane A2 (TXA2) agonist
U46619. The COX-1 and COX-2 inhibitory activities were
respectively assessed in washed human platelets, and lym-
pho-monocyte suspension. COX selectivity was determined
from dose–response curves by calculating a ratio (COX-
2/COX-1) of the IC50 values [74].
H
Cl
N N
63 R =
N N
NH R 64 R = CH2-NHSO2CF3
65 R = COOH
F
COOH
NHSO2 Cl
66
Fig. (30). Hybrids from Coxibs.
The results of their study were summarized in the Table
10 and showed that the hybrids 63 and 64 were the most po-
tent in terms of TXA2 antagonism, with pA2 values compa-
rable to that of diclofenac [74].
All of the evaluated compounds were able to inhibit the
COX-2 enzyme in a concentration-dependent manner with
Lumiracoxib and diclofenac displaying the highest absolute
potency. Compound 64 was the most potent of the new hy-
brids, with potency similar to that of naproxen. With respect
to COX 1 inhibition, derivatives 63 and 65 behaved very
similarly, it was also clearly observed that compounds 63
and 65 were the most selective coxibs from among the multi-
target molecules synthesized. According to the authors, the
sulfonamide derivative (64), (despite being only about 40
times more selective for COX-2 than COX-1 with respect to
inhibition), presented a very steep slope on the concentra-
tion-response curve (Hill coefficient»1). This characteristic
has no direct impact on the Therapeutic Index of the mole-
cule, and, thus, on its safety, but rather suggests that it would
be possible to find a compound 64 dosage that is selective
for COX-2 [74].
The authors have synthesized a new generation of multi-
target compounds, among which, two of the lumiracoxib
derivatives. The tetrazole compound 63 and the trifluoro-
methansulfonamido-isoster compound 64 are the most active
with COX-2 inhibitor activity and TP receptor antagonism
being fairly balanced [74].
Hybrid Antimicrobial Agents
Bacteria develop resistance mechanisms to the antibiotics
agents most used in clinical medicine. The search for new
chemical entities with antimicrobial activity remains an ar-
duous task for scientists around the world. Studies with hy-
brid compounds combining heterocyclic pharmacophores
have brought excellent results; the antibiotic effect increases,
leading to greater efficacy in the treatment (Table 10).
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1065
Asadipourand co-workers (2013) [75] studied the anti-
Helicobacter pylori activity of 2-α-alkylthio-5-(nitroaryl)-
1,3,4-thiadiazole derivatives of the drugs tinidazole, furazol-
idone, and metronidazole (Fig. 31 - A). These compounds
present promising anti- H. pylori activity.
The compounds with strong anti- H. pylori activity dis-
played nitroaryl groups: nitroimidazole and nitrofuran, with
inhibition zone diameters IZD > 20mm (at a concentration of
25 μg/disk). The nitrothiophen derivatives showed moderate
to weak activity (IZD= 16-20 mm, and IZD= 11-15 mm) at
25 μg/disk [75].
Nitroimidazole compounds that had α-methylbenzylthio-
side chains (position R) showed increased activity, with in-
hibition zone diameters of > 50mm in all concentrations
[75].
R2
S
N
NH
N
S
Nitroaryl S
O
R
N N
A N R1
S
B
Fig. (31). General structure of the antimicrobial agent hybrids.
Ramprasad et al., (2015) [76] also studied 1,3,4-
thiadiazole derivatives and found derivatives with activity
against Mycobacterium tuberculosis. A series of phenothia-
zine and 1,3,4-thiadiazole compounds were designed by mo-
lecular hybridization. The most active compounds showed
anti-Mycobacterium tuberculosis activity with MIC = 0.8
µg/mL (R2= methyl or n-propyl; R1 = H or Cl) (Fig. 31 - B).
Structure-activity relationship studies showed that alkyl
groups (methyl or n-propyl) and substituted phenyl groups
(4-methyl, 4-Cl and 4-F) enhance activity [76].
1,3-thiazine linked to 1,3,5-triazine led to hybrid deriva-
tives with activity against Gram-negative and Gram-positive
microorganisms (Fig. 32 - A) [78]. The compound with R1 =
4-NO2 and R2 = 4-NO2 showed the best activity for all spe-
cies, with MIC= 6.25 µg/mL against Bacillus subtilis, and
MIC= 12.5 µg/mL against Bacillus cereus, Staphylococcus
aureus and Escherichia coli. The nitro group contributed to
the improved activity of these hybrid compounds [77].
Hybrid triazoles have been studied and described by sev-
eral authors. Dixit et al., (2016) [78] report hybrid triazoles
with potent dual anti-Mycobacterium tuberculosis actitivity
(growth inhibition and efflux pump inhibition), and synergis-
tic effect with anti-tubercular agents. The best results
showed MIC = 4 µg/mL and 8µg/ mL against Mycobacte-
rium tuberculosis, and MIC= 1 µg/ mL and 2 µg/ mL against
Mycobacterium smegmati [78].
Hybrids using the pharmacophores 1,2,3-triazolide and
benzimidazole (linked by a benzene connector) were synthe-
sized by microwave irradiation and your antibacterial activi-
ty was evaluated against: Staphylococcus aureus, Enterococ-
cus faecalis, Enterococcus faecium, Streptococcus
1066 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Table 10. Results for antagonist activity at TP receptor (pA2) and COX-1 and COX-2 inhibitory activities (adapted from Hoxhaet
al., 2016 [74]).

TP receptor antagonism for human washed platelet aggregation and total IP production in transfected HEK 293 cells. pA2 values were deter-
mined by measuring aggregation inhibition response to the stable agonist U46619.

pA2 ± %CV
Compounds
Human washed platelet aggregation Total IP production in HEK293 cells

Lumiracoxib 5.0 ± 2.5 4.6 ± 5.2

Diclofenac 5.4 ± 4.9 5.3 ± 4.7

Naproxen 5.3 ± 4.7 3.9 ± 16.3

Terutroban 9.4 ± 4.1 9.3 ± 2.8

63 5.6 ± 3.5 5.5a ± 2.2

64 5.9a ± 4.1 5.7a ± 2.3

65 5.0 ± 1.8 4.9 ± 5.1

66 4.9 ± 5.1 4.8 ± 2.5

COX-2 and COX-1 inhibitory activities determined by in vitro assay in lympho-monocytes and washed human platelets.

COX-2 inhibition COX-1 inhibition

Compounds COX-2/COX-1 selectivity
IC50 (µM) ± % CV IC50 (µM) ± % CV

Lumiracoxib 0.0035 ± 26 3.22 ± 22 910

Diclofenac 0.0011 ± 30 0.0083 ± 6.2 7.6

Naproxen 0.19 ± 66 0.11 ± 10 0.58

Terutroban inactive at 10 µM N.D. N.D.

63 0.014 ± 23 13.2 ± 22 942

64 0.42 ± 32 16.1 ± 6 38

65 0.025 ± 46 25.5 ± 10 1020

66 1.20 ± 45 inactive at 60 µM N.D.

a
95% CI vs lumiracoxib, see results N.D.: not determined.

pneumoniae, Haemophilus Influenzae, Escherichia coli and R= para-methyl 6-(1-benzyl-5-methyl-1H-1,2,3-triazol-4-

Pseudomonas aeruginosa. The best activity was against
Haemophilus Influenzae (MIC = 32 µg/mL) (Fig. 32-B) [79].
Some of the designed compounds presented weak antifungal
activity against Fusarium oxysporum, inhibiting sporulation
by up to 30.62 %. The benzimidazole analogue (containing
trifluoromethyl) inhibited linear growth of Verticillium dahl-
iae by up to 29.76 %. The authors attributed the increased
activity to lipophilicity, which facilitates metabolism [79].
Triazoles were also present in triazolyl dihydropyrimi-
dine-2-thione hybrid derivatives (Fig. 32 - C). They exhibit-
ed potential antibacterial activity against Pseudomonas ae-
ruginosa, Salmonella typhi, Staphylococcus aureus, and
Streptococcus pneumonia. The best compounds showed MIC
values of 8-20 µg/mL [80].
The non-substituted hybrid (non-substituted phenyl) 6-(1-
benzyl-5-methyl-1H-1,2,3-triazol-4-yl)-4-phenyl-3,4-
dihydropyrimidine-2(1H)-thione was able to inhibit the mi-
croorganisms with MIC = 10-19 µg/mL. Compounds with
yl)-4-p-tolyl-3,4-dihydropyrimidine-2(1H)-thione, and R =
para-chloro 6-(1-benzyl-5-methyl-1H-1,2,3-triazol-4-yl)-4-
(4-chlorophenyl)- 3,4-dihydropyrimidine-2(1H)-thione pre-
sented the best inhibition, with respective MIC values of 8-
16 µg/mL and 9-20 µg/mL [80].
Gaikwad and colleagues [81] investigated hybrid com-
pounds containing thiazole and benzotriazole and evaluated
their in vitro antibacterial activity against Bacillus subtilis,
Staphylococcus aureus, Escherichia coli, and Pseudomonas
aeruginosa, and antifungal activity against Candida albicans
and Aspergillus niger.
Compounds with electron-withdrawing groups (F, Cl,
Br, CF 3, and NO2) exhibited moderate to good activity
against most of the pathogens (Gram-positive and Gram-
negative). The minimum inhibitory concentration (MIC)
against bacterial and fungal strains was 16–128 µg/mL
(Fig. 32 - D) [81].
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1067

H H R
O2N N N N N
N N R
OR2 N
N N S
O N
N X R1
O2N NH H

R1 B
A

S
S
N HN
N N
NH N N N
N H
N R
Me R N
R1
C D

Fig. (32). Hybrid derivatives: 1,3-thiazine-1,3,5-triazine (A), 1,2,3-triazole-benzimidazole (B); triazolyl-dihydropyrimidine-2-thiones (C) and
thiazole-benzotriazole (D).

R4 O
N
S
N
N H Ar
N R5
N N N N
H N
Me
N
R6
N
B
A
R1 R3

R2 N CF3

CF3
Fig. (33). Hybrids of ravuconazole (A) and 1,2,3-triazole-quinoline (B);

Others compounds studied by Gaikwad et al., (2012) [84]
were hybrids of ravuconazole (containing thiazole and tria-
zole pharmacophores). They showed moderate to good anti-
microbial activities against Gram positive and Gram nega-
tive bacteria (Bacillus subtilis, Staphylococcus aureus, Esch-
erichia coli, and Pseudomonas aeruginosa), and the fungal
species (Candida albicans and Aspergillus niger) with MIC=
16 - >64 µ g/mL (Fig. 33 - A) [84]. In most of the com-
pounds, the halogen groups (F, Cl, Br) enhanced antimicro-
bial activity, and groups like NO2 and CF3 reduce such activ-
ity (moderated activity) [82].
Sumangala et al., (2010) [83] also studied triazoles with
antimicrobial activity. They synthesized and evaluated hy-
brid compounds containing 1,2,3-triazoles and quinoline
moieties against fungi and bacteria, and found MIC values in
the range of 3.125 - 25 µg/mL.
The lead compound (Fig. 33 - B, Ar = 3-methylthien-2-
yl) had an MIC = 3.125 µg/mL against S. aureus, and an
MIC = 6.25 µg/mL against E. coli, P. aeruginosa and K.
pneumoniae. The same compound had antifungal activity
with an MIC= 3.125 µg/mL against A. flavus and an MIC =
6.25 µg/mL against A. fumigatus, P. marneffei, and T. men-
tagrophytes. The authors suggest that a ligand on position 4
of the triazole ring is important to antimicrobial activity
[83].
Hybrid compounds between norfloxacin and various het-
erocyclic pharmacophores (1,2,4-triazole, 1,3-thiazole, 1,3-
thiazolidinone and 1,3-oksazole) synthesized by microwave
irradiation were evaluated as antimicrobial agents [86].
Compound A with an MIC = 3.4 µg/mL against Escherichia
coli and an MIC= 15.6 µg/mL against Saccharomyces cere-
visae; and B with an MIC= 15.6 µg/mL against Escherichia
coli were the most active derivatives (Fig. 34) [84].
Hybrid triazoles were present in the work of Thomas et
al., (2011) [85], that evaluated anti-tubercular activity of
three new series of quinoline-4-yl-1,2,3-triazoles with amide,
sulphonamide, and amidopiperazine moieties (Fig. 35).
Some compounds showed excellent MIC values (MIC =
0.625 µg/mL) equal to the standard drug isoniazid against
Mycobacterium tuberculosis. They have groups in their
structure such as: acetyl, fluoro, methoxy, and trifluorome-
thyl giving marked activity [85].
The heterocyclic quinoline was extensively studied in the
construction of hybrid antimicrobial agents [83, 85]. Thomas
et al., (2011) [86] describes a series of quinoline and oxazol-
idinone derivatives (based on docking studies) that were
evaluated for antibacterial and anti-mycobacterial activity
(Fig. 35). Most of the compounds were active against all
evaluated bacteria, with an MIC range of 0.1 - >12.5 µg/mL.
The best MICs by species were: S. aureus (MIC = 0.8
1068 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

O O S

F (A) R =
OH N N N O
H H

F N N
(B) R = N
N N
S
R
O
Fig. (34). Compounds: 1-ethyl-6-fluoro-7-{4-[2-fluoro-4-({[(3-morpholin-4-ylpropyl)amino]carbonothioyl}amino)phenyl]piperazin-1-yl}-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (A), and 7-[4-(4-{[3-benzyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}-2-fluorophenyl)piperazin-1-
yl]-1-ethyl-6-fluoro-4-oxo-1,4- dihydroquinoline-3-carboxylic acid (B).

N R
N
N O O O
H
MeO R= N S R3, N N ,
N R1
O R2 H

N Me
quinoline-triazole
O
O R NH
O
O
N O
HN O R
N
MeO
MeO

N Me
N Me
quinoline-oxazolidinones

OMe
R
N
N
O NH
N
N
S NH
N OMe
quinoline-thiourea
quinoline-oxadiazole
F3C CF3

Fig. (35). Hybrid Quinolines: quinoline--triazoles with amides, sulphonamides and amidopiperazines; quinolone-oxazolidinone; quinolone-
oxadiazole; and quinolone-thiourea.

µg/mL), S. pyogenes (MIC = 0.2 µg/mL), P. aeruginosa
(MIC = 0.2 µg/mL), K. pneumoniae (MIC = 0.1 µg/mL), and
E. coli (MIC = 0.1 µg/mL). The most active hybrid against
Mycobacterium tuberculosis had MIC= 0.625 µg/mL [86].
A quinoline ring with an attached thiourea was described
by Dolan and colleagues (2016) [87]. Quinolines or phenyl
thioureas alone show little or no activity, but as hybrids they
are potently antibacterial [89]. Certain molecule hybrids
were tested against Gram-positive and Gram-negative bacte-
ria. In this series, one molecule had excellent MICs for
Escherichia coli (MIC50 = 2.63–3.95 µM) and Staphylococos
aureus (MIC50 = 3.95–5.26 µM) (Fig. 35). The compound
also had anti-MRSA activity with an MIC50 = 11.44 µ M.
The new antibacterial agent is non-toxic to Galleria
mellonella larvae (concentrations of up to 1000 µg/mL). Ac-
cording to SAR studies, the presence of a methoxyl group
increases activity, and –CF3 is essential for activity. The
presence of a bulky (bi-cyclo) group promotes activity.
Jain et al., (2016) [88] investigated a novel series of nine-
teen quinoline–oxadiazole hybrid compounds (Fig. 35) for
anti-tuberculosis activity against Mycobacterium tuberculo-
sis. In this series of compounds, the molecules containing
aliphatic groups or alkyl attached to the aromatic ring groups
obtained better results (MIC= 0.4–1.05 µM). The compound
with the highest activity showed an MIC = 0.4 µM and a
selectivity (SI of >610). The compound has an R= methoxy-
methyl group, similar to the compound used as a model (that
has excellent anti-TB activity), which contributed to the in
vitro activity of this compound [88].
Hybrid Compounds as Direct Multitarget Ligands
S
S S
N
R O
COOH
O N
chalcone-rhodanine-3-acetic acid
Fig. (36). Hybrids of chalcone and rhodanine-3-acetic acid, and rhodanine-based 5-aryloxy pyrazoles.
Hybrids possessing chalcone and rhodanine-3-acetic acid
moieties were investigated by Chen and co-workers (2010)
[89]. These hybrids were evaluated against Gram-positive
and gram-negative microorganisms such as Streptococus
mutans, Escherichia coli and Staphylococcus aureus [89]
(Fig. 36).
They showed excellent results against S. aureus and S.
mutans strains, with MIC = 2-8 µg/mL. They were also
evaluated against multidrug-resistant strains of clinical iso-
lates (methicillin-resistant S. aureus– MRSA, and quinolone-
resistant S. aureus- QRSA). The most active compound for
these strains (MIC= 2 µg/mL) has two chloro groups (R =
2,4-(Cl)2), and it was more active than the standard drug
(norfloxacin). Derivatives with halogen groups were identi-
fied as more potent than compounds with donor electron
substituents. The position of these halogens on the phenyl
ring also contributed to the antibacterial activity, the para
position is the most active (para>meta>orto) [89].
In continuation of the work of Chen et al., (2010) [89],
Song et al., (2013) [90] synthesized a series of eight com-
pounds, connecting 5-aryloxy pyrazole and rhodanine. These
hybrids presented good inhibitory activity against resistant
bacterial strains (MRSA and QRSA) with MIC = 1-32
µg/mL. The compounds were also non-cytotoxic in the eval-
uated in vitro concentrations. One of the series, which had
two Cl groups replaced in an aromatic ring (2,4-Cl2), showed
the most potent anti-MRSA and anti-QRSA activity (MIC =1
- 2 µg/mL.) (Fig. 36) [90].
Sulfonamides are known as potent antibiotics. For exam-
ple, the organic–inorganic hybrid sulfanilamide sulphate
(Fig. 37) revealed great antibacterial activity (MIC = 8-16
µg/mL) against Listeria innocua, Escherishia coli, Pseudo-
monas aeruginosa, and Staphylococcus aureus. The com-
pound also presented antifungal activity against Candida
albicans with an MIC = 16 µg/mL [93]. The compound in-
hibits spore germination in Botrytis cinerea and Fusarium
oxysporum culmorum by 97.6% and 60%, respectively, de-
spite having a high MIC value (100µg/mL) against, [91].
Pyrimidine derivatives containing; urea thiourea, and
sulfonamide moieties with antifungal and antibacterial ac-
tivity were studied by Keche et al., (2012) (Fig. 37) [92].
Some of the compounds showed activity equal or greater to
the standard drugs Ciprofloxacin and Miconazole, with the
best MIC against bacteria being MIC=10 µg/mL, and for
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1069
O COOH
N
S
Me
O
N
R
rhodanine-5-aryloxy pyrazole
fungi MIC= 15 µg/mL. Few of the molecules presented co-
incident antifungal and antibacterial activity. The derivatives
with R= isopropyl, -CF3, -OCF3, -CN, and -OMe groups at
positions 2, 3, or 4 presented the best antibacterial and anti-
fungal profiles [92].
A heterocyclic pyrrole in the hybrid structure demon-
strated activity, as in the 1H-pyrrole-2-carbohydrazide deriv-
atives that showed antibacterial activity [93]. The series 4,5-
dichloro-N-arylidene-1H-pyrrole-2-carbohydrazide, with
MICs = 1.56 – 12.5 µg/mL, were the most active for E. coli
and S. aureus. They had were active against C. albicans
(MIC= 12.5 µg/mL). The series of chloropyrrole chalcone
analogues were able to inhibit E. coli and S. aureus (MIC=
1.56 – 6.25 µg/mL) (Fig. 37) [93].
Hybrid compounds that present macrolide and macrolac-
tone rings have potential antibacterial activity [94]. Desmond
and coworkers (2014) synthesized a macrolide-glycolipid
macrolactone hybrid (pyranose monosaccharide linked to the
macrocyclic ring) using the macrolide erythromycin and
sophorlipid macrolactone as a base. The compound showed
modest antibacterial activity against Gram positive organ-
isms, with an MIC = 115 μg/mL against Bacillus anthracis
[94].
Hybrid Antiprotozoal Agents
Antileishmanial
Benzimidazole derivatives, used clinically as anthelmin-
thic drugs have diverse biological activities because of their
privileged structures [95]. Pentamidine is used for primary
stage African trypanosomiasis, antimony-resistant leishman-
iasis, and AIDS associated Pneumocystis jirovecii pneumo-
nia [96-98].
Searching for basic information about structural require-
ments for antiprotozoal activity Torres-Gómez et al., (2008)
[99] synthesized a series of hybrids built with benzimidazole
and pentamidine. The compounds (Fig. 38) were tested, in
vitro, against intestinal protozoa (Giardia lamblia, Entamoe-
ba histolytica), (Trichomonas vaginalis) a urogenital tract
parasite, (Plasmodium berghei) a red blood cell parasite and
(Leishmania mexicana) an intracellular kinetoplastid parasite.
Comparisons were made between the synthesized compounds
and the antiprotozoal drug of choice: metronidazol. In order to
compare bioactivities, pentamidine was also tested.
1070 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Cl

NH3 H
N R
HN CF3 N
N
H
O
SO4 N
(A)
N
O S O R
NH2 R Cl
2 N
H
R = urea, thiourea, sulfonamide O
(B)
Fig. (37). Organic–inorganic hybrid di-(4-sulfamoyl-phenyl-ammonium) sulfate; hybrid pyrimidine containing urea, thiourea and sulfona-
mide; and pyrrole-2-carbohydrazide (A) and chloropyrrole-chalcone (B) derivatives.

R N N R

O O
N (CH2)5 N
H H
R1 R1
Fig. (38). General structure of unsubstituted hybrid compounds derived from the fusion of benzimidazole and pentamidine molecules.

H H
Against T. vaginalis compound 2 was two times more ac- N N
tive than metronidazole and 23-fold more potent than pen- HN

tamidine. Compounds 8 and 10 were 4- and 5-fold more po- O

tent than pentamidine, respectively. Against G. lamblia, R

compounds 2 and 8 were three and four times more active

than metronidazole, and respectively, 9 and 11 times more Cl N

active than pentamidine.
Against E. histolytica, compounds 1–3 and 7 were re-
spectively two, seven, five, and three times more active than
metronidazole. Compound 2 was 108-fold more potent than
pentamidine, whereas compounds 1, 3, and 7 were 27.77,
and 41 times more active. Against P. berghei only com-
pound 4 showed moderated activity.
These results are very promising considering that the
synthesized compounds showed activity comparable to the
current used antiprotozoal drugs metronidazole and pentami-
dine.
Nava-Zuazo et al., (2010) [100] synthesized a series of
novel chloroquine–ethambutol–isoxyl tripartite hybrids (Fig.
39) and tested, in vitro, the antiparasitic activity of these
compounds on intestinal unicellular parasites (Giardia intes-
tinalis and Entamoeba histolytica), (Trichomonas vaginalis)
a urogenital tract parasite, and kinetoplastid parasites Trypa-
nosoma cruzi and L. mexicana.
Chloroquine (CQ) is mainly used as an antimalarial drug
[100] and possesses moderate biological activity against M.
tuberculosis [101]. Ethambutol is a medication primarily
used to treat tuberculosis [102], and Isoxyl has shown con-
siderable anti-mycobacterial activity, however clinical use
was discontinued, apparently because of the poor bioavaila-
bility of the highly non-polar product [103, 104].
All the screened compounds showed high bioactivity
(IC50 < 1.8 μM) against G. intestinalis, being more potent
than metronidazole (IC50 = 5.36μM). Compound 6 was 670-
times more active than metronidazole, the antiprotozoal drug
of choice, and 50-fold more potent than chloroquine.
Fig. (39). General structure of unsubstituted hybrid compounds
derived from the combination of chloroquine, ethambutol and isox-
yl moieties.
None of the synthesized compounds was more active
than metronidazole against T. vaginalis. Compound 6 was
again the most active of the series against E. histolytica. It
was three-times more active than chloroquine but four fold
less potent than metronidazole. For L. mexicana, only com-
pound 6 was as active as pentamidine (the second-line an-
tileishmanial drug). These compounds were tested in vitro
for their anti-mycobacterial activity against M. tuberculosis
strain H37Rv. Compounds 3 and 6 were found to be the most
potent compounds with MIC’s of 4 and 2 μg/mL, respective-
ly. Compound 6 was 2-fold more potent than the reference
drugs ethambutol and isoxyl (MIC = 4μg/mL), whereas
compound 3 was as active as these two anti-mycobacterial
drugs.
Ibrar et al., (2015) [105] designed and synthesized a new
series of bis-coumarin–iminothiazole hybrids (Fig. 40) using
the molecular hybridization approach. The synthesized com-
pounds were prepared by the reaction of bromoacetyl cou-
marin and a range of thiosemicarbazide derivatives in meth-
anol and evaluated for their alkaline phosphatase inhibition,
and anticancer, and antileishmanial potential.
The synthesized hybrid compounds (5a–m) were assayed
for their alkaline phosphatase inhibition using potassium
dihydrogen phosphate as the standard drug (IC50 = 2.43 +
0.04 mM) [106]. The most active compound of the series
was 5j (R = 2,4-di-Me), with an IC50 value of 1.38 + 0.42
mM.
Hybrid Compounds as Direct Multitarget Ligands
O S
N
N using the MHOM/BR/76/LTB-012 strain [112]. The synthe-
H N
O
O O
R
O pounds were from five to fifty fold more selective than Am-
Fig. (40). General structure of the unsubstituted hybrid compounds
derived from the fusion of bis-coumarin–iminothiazole moieties.
The antileishmanial activity was measured by MTT
method [107, 108]. Compound 5i (R = 2,3-di-Me) showed
the highest inhibition of 70.4 + 2.2% at 100 mM, whereas
amphotericin B showed 79.8% inhibition.
The antiproliferative activity of the synthesized com-
pounds was measured in vitro at four different concentra-
tions (100, 10, 1, and 0.1 mM) in the cell growth inhibition
against kidney fibroblast (BHK-21), and lung carcinoma (H-
157) cell lines [109]. Vincristine was used as the standard
drug for comparison. Compound 5m (R = 3,5-di-Me)
showed the highest inhibition (66.8 + 1.1 and 64.2 + 2.3%)
at 100 and 10 mM, respectively, against BHK-21cells. For
the H-157 cell lines, of the tested thiazole hybrids, com-
pound 5l (R = 3,4-di-Me) was the most potent analogue with
inhibition values of 65.0 + 1.8 % at 100 mM, and 57.3 + 0.7
% at 10 mM.
Forty hybrid furoxanyl N-acylhydrazones, belonging to
seven different families of heterocyclics, were synthesized
by Hernández et al., (2013) [110]. The synthesized com-
pounds combined different heterocyclic (hetm) and furoxa-
nyl moieties (fxm) connected by an N-acylhydrazone (NAH)
linker. They were evaluated against M. tuberculosis, T. cruzi,
and Leishmania.
The anti-mycobacterial activities were observed against a
sensitive M. tuberculosis H37Rv strain and a multidrug re-
sistant (MDR) clinical isolate strain. Compound fx-NAH 9
was better as an anti-mycobacterial agent against the MDR
strain than isoniazid, the reference drug, with an MIC value
of 4.5 times lower. The results for compound fx-NAH 9
against the H37Rv strain and macrophages were comparable
to isoniazid. The absence of an N-oxide moiety revealed the
importance of this function to anti-mycobacterium activity
and macrophage toxicity.
Trypanosomicidal properties were verified using axenic
epimastigotes of the Tulahuen 2 strain, and amastigotes of
the Tulahuen C4 strain grown in VERO (normal African
green monkey epithelial) cells [111, 112]. The most relevant
fx-NAHs were the pyrimidin-4-yl derivatives 9 and 10 both
having activity and selectivity similar to that of nifurtimox,
the reference drug. Compound 9 has excellent selectivity
indexes, SI (>63) (SI= IC50, macrophages/IC50, amastigotes),
when murine macrophages are taken into account as a
mammal system, and a good SI (2.5) when VERO cells are
considered. The same occurred with compound 10 belonging
to the same family. The best anti-amastigote derivative was
fx-NAH 15, which was eleven-fold more active than nifur-
timox with an excellent SI. Once again it was observed that
the absence of an N-oxide moiety decreases the compound’s
activities.
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1071
Certain hybrid compounds were evaluated against L.
amazonensis axenic amastigotes in MTT colorimetric assays
sized compounds displayed less leishmanicidal activity than
Amphotericin B, the reference drug. Nevertheless, the cyto-
toxicity against murine macrophages revealed that the com-
photericin B.
Da Silva, et al., (2007) [113] synthesized certain quino-
linylarylsulfonamides using two of their copper and zinc
complexes (Fig. 41). The compounds were assayed against
Leishmania and Trypanossome.
Quinoline derivatives can be employed as antiprotozoal
agents [114], and sulfonamide derivatives have been report-
ed to possess anti-trypanosomal and antileishmanial activi-
ties [115]. The synthesized quinolinylarylsulfonamides were
evaluated, in vitro, against promastigotes of L. amazonensis,
L. chagasi, and epimastigotes of T. cruzi.
O
N HN S R
O
Fig. (41). General structure of the unsubstituted hybrid compound
derived from the fusion of quinolone –sulfonamide molecules.
The quinolinylarylsulfonamides 6, 10, 11 and 12 were
found to be the most active compounds against promastigote
forms of L. amazonensis and L. chagasi with IC50s ranging
from 2.12 to 2.85 μM and 0.45 to 2.99 μM, respectively. In
the L. chagasi bioassay, it was observed that the IC50s for
compounds 6, 10, and 12 were two times greater than the
IC50 of the control (Amphotericin B). A nitro group or
fluorine atom at 4-position, of the phenyl moiety, decreases
activity. The presence of dibromine, or methoxy and methyl
groups also decreases activity.
The copper complexed compound showed significant ac-
tivity against L. amazonensis (IC50 = 2.28 μM), L. chagasi
(IC50 = 1.10 μM) and T. cruzi (IC50 = 4.10 μM) whereas the
zinc complexed compound was slightly less active against L.
amazonensis (6.46 μM) and L. chagasi (3.00 μM). Com-
pounds 6 and 17 were more active than the control benznida-
zol which reduced the cell infection by 92.0%, at 25 μM.
The lipophilic compound 7 was the most active and pro-
moted a reduction of cell infection of 82.1% at the 25 μM
concentration. This observation is in accordance with a trend
observed for other classes of anti-parasitic compounds where
lipophilicity increases favor the compound’s distribution to
the intracellular target [116].
Otero et al., (2014) [117] synthesized twelve hybrids via
Williamson etherification of O-triclosan alkyl bromide plus
chalcone, and O-coumarin or O-chromone alkyl bromide
plus triclosan. Triclosan is an uncompetitive inhibitor of pu-
rified enoyl-acyl carrier protein reductase, which has a
demonstrated in vitro inhibitory activity against Plasmodium
falciparum [118, 119].
1072 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
Additionally, the antileishmanial activity of several chal-
cones has been reported in literature [120-122]. Coumarins
and chromones are important compound classes having ver-
satile biological activities [96,123-125].
The most active compounds were 25-27, with inhibitions
of 94.4%, 91.0%, and 75.5%, respectively, against intracellu-
lar amastigotes of L. (V) panamensis at 20 µg/mL. The same
compounds showed high toxicity, with LC50 < 26 µ g/mL.
The best SI´s were observed for compounds 7–9 and 17 with
respective values higher than of 21.3, 19.6, 14.8, and 7.3.
Compound 25 had better activity than meglumine antimoni-
ate, however its SI is affected by its high cytotoxicity.
Mowbray et al., (2015) [126] synthesized a series of
amino-pyrazole intermediates. Certain pyrazole-based com-
pounds with antileishmanial activity have been reported
[127-129].
Recently, certain aminothiazole derivatives with excel-
lent in vitro potencies against visceral leishmaniasis were
reported [130]. In the intracellular L. infantum amastigote
assay, compounds 7−10 were significantly more potent than
miltefosine, used as a reference drug (IC50 = 7.26 μM).
Compound 7 (IC50 = 2.02 μM) had a cyclopropyl group at 4-
position on the pyrazole ring, compound 8 (IC50 = 0.064 μM)
had a 2-methoxy benzamide group, compound 9 had a fluoro
at position 2 (IC50 = 0.965 μM), and compound 10 had a
dichloro at positions 2 and 6 (IC50 = 2.11μM).
However, the series showed poor metabolic stability in
hamsters, and was overcome with the sterically hindered
analogue 10. The analogues 18−26 possessed excellent lev-
els of antileishmanial activity and SIs, this combined with
good metabolic stability in human liver microsomes. Mow-
bray et al., (2015) [126] treated hamsters infected with L.
infantum with oral doses of compound 26 at 50 mg/kg b.i.d.
for 5 days. The treatment resulted in respective 92.7% and
95% reductions in parasite burden in the liver and spleen,
without obvious signs of toxicity. The treatment with
miltefosine at 40 mg/kg q.d. for 5 days, showed 97.8% (liv-
er) and 99.6% (spleen) reductions in parasite burden. The
results demonstrate high in vivo efficacy in a hamster model.
Antimalarial
Kumar et al., (2015) [131] synthesized twenty one 4-
aminoquinoline-pyrimidine hybrid derivatives (Fig. 42), and
evaluated their in vitro antimalarial activity against choro-
quine-sensitive (CQ-S) (D6 clone) and CQ-resistant (CQ-R)
(W2 clone) strains of P. falciparum using choroquine and
pyrimethamine as reference drugs. Aminoquinoline based
compounds stop hemozoin formation and pyrimethamine
inhibits the enzyme dihydrofolate reductase.
Generally, substitution with halogen groups on the phe-
nyl ring increased the antimalarial activity against both the
strains. Compounds 7c-9c and 7d-9d with either chloro or
bromo at the para position of the phenyl ring showed excel-
lent activity against both strains (D6 and W2). It was ob-
served that a methyl group at the para position of the phenyl
ring decreases antimalarial activity against both strains and a
methoxy substitution at position 4-, and position 3,5- of the
phenyl ring increase antimalarial activity.
Pedrosa et al.
HN
NH2 N
(CH2)n
N N
H
Cl N
Fig. (42). General structure of unsubstituted hybrid compound de-
rived from the fusion of aminoquinoline-pyrimidine molecules.
Compound 7g (with significant in vitro antimalarial ac-
tivity) was selected for further in vivo evaluation but no
significant in vivo antimalarial activity was observed. Only a
weak effect was noticed at the highest dose on day 5 (a
42.12% fall in parasitemia) but the effect disappeared on day
7 and the mean survival time was only 9 days compared to
7.6 days for vehicle treated animals, yet 26.2 days for chlo-
roquine treated animals.
Molecular docking studies were also performed on the
binding mode of these novel 4-aminoquinoline-pyrimidine
based molecular hybrids in the active site of wild type
PfDHFR-TS and quadruple mutant PfDHFR-TS protein
structures. The most active compounds in the study exhibited
significant binding affinities towards the wild and quadruple
mutant PfDHFR-TS structures with energy ranges similar to
the reference compounds: pyrimethamine, cycloguanil and
WR99210.
The number of carbon spacer linkers does not play an
important role in influencing antimalarial activity when Cl is
substituted with amino functionalities; yet the activity in-
creases when Cl is replaced with amino functionalities, as
such, since the comparison of antimalarial activity of two
groups of regio-isomers indicated that the potency against
both strains of P.falciparum is similar, another series was
synthesized by the same group [132].
A series of thirty-two 4-aminoquinoline–pyrimidine hy-
brids were prepared with the same amino functionalities, but
altering the carbon spacer from C2 to C3, and most of the
hybrids showed potent antimalarial activity against CQ-
sensitive (NF54), and CQ-resistant (Dd2) strains of P. falci-
parum.
From the total, nineteen hybrids (IC50 values ranging
from 0.003–0.198 mM), displayed activity superior to CQ
(IC50 = 0.222 mM), while four hybrids (IC50 values ranging
from 0.003–0.011 mM) exhibited activity superior to ar-
tesunate (IC50 = 0.013 mM) when tested against the CQ-
resistant (Dd2 clone) strain of P. falciparum. Six hybrids
(IC50 values ranging from 0.009–0.025 mM) also exhibited
greater activity than CQ (IC50 = 0.027 mM) when tested
against the CQ-sensitive (NF54 clone) strain of P. falcipa-
rum.
The cytotoxicity of the selected active hybrids was as-
sessed against CHO cells. The most toxic of the hybrids pre-
sented IC50 = 11.09 mM while the least toxic presented IC50
= 230.77 mM.
Hybrid Compounds as Direct Multitarget Ligands
Using Glide software, a molecular docking study of the
best active compounds was performed on the binding pocket
of both the wild type Pf-DHFR-TS (PDB ID: 3QGT), and
the quadruple mutant Pf-DHFR-TS (PDB ID: 3QG2) struc-
tures [133]. Compounds 19a and 20e were the most active
and exhibited significant binding affinities towards the wild
(energy range -51.64 kcal mol-1 to -40.52 kcal mol-1), and
quadruple mutants (energy range -54.09 kcal mol-1 to -42.81
kcal mol-1). The energy ranges are comparable to that of ref-
erence compounds and the native DHFR dihydrofolate sub-
strate.
Pandey et al., (2013) [134] have incorporated a tetrazole
moiety in the side chain of a 4-aminoquinoline pharmaco-
phore in order to develop new antimalarial agents active
against chloroquine resistant strains of P. falciparum. The
synthesized 4-aminoquinoline-tetrazole derivatives were
evaluated for their in vitro antimalarial efficacy against CQ-S
(3D7) and CQ-R (K1) strains of P. falciparum. All 22 tested
compounds exhibited moderate activity against the CQ-S
(3D7) strain, with IC50 values ranging from 10.66 to 216 nM.
From the total, 13 compounds were found to be more active
than CQ-S when screened against the CQ-R (K1) strain of P.
falciparum, showing IC50 values between 73.70 and 233.70
nM. The structure-activity relationship studies suggested that
the activity was greatly influenced by: the type of linker,
substitutions on the aromatic ring, as well as substitution on
the tetrazole ring. In general, most of the synthesized com-
pounds exhibited reasonably promising activity against K1
strain and less cytotoxic effects, since showing high selectiv-
ity indexes.
Tripathi et al., (2015) [135] synthesized a series of ami-
noquinoline-pyrimidine conjugates with substitutions on the
cyclic secondary amines attached to the pyrimidine nucleus
having various open chain aliphatic amino-alcohols. All 24
compounds showed potent antimalarial activity against both
CQ-sensitive (D6) and CQ-resistant (W2) strains of P. falci-
parum. Compound 8e exhibited activity (IC50 = 0.05 µ M)
comparable to CQ against the CQ-sensitive (D6) strain,
whereas hybrids 8b (0.47 µM), 8c (0.34 µM) and 8d (0.41
µM) exhibited comparable activity to the reference drug
against the CQ-resistant strain.
The resistance-index of the compounds with no pyrimi-
dine methyl group and of the compounds with a pyrimidine
methyl group at the 6th position indicates that the synthe-
sized hybrids were potent against both the sensitive and re-
sistant P. falciparum strains. All of the synthesized hybrids
were assessed for mammalian cytotoxicity by testing against
VERO cells and none of them exhibited cytotoxicity to the
highest tested concentration of 12 µM.
Molecular docking studies of the best active compounds
(8b-8f, 8u and 8v) were performed on the active sites of both
the wild type and quadruple mutant Pf-DHFR-TS structures
(PDB ID: 3QGT and 3QG2, respectively), using Glide v5.8
[133]. The best active compounds in the study displayed
excellent binding affinities towards both the quadruple mu-
tant (–62.49 kcal mol-1 to –42.01 kcal mol-1) and the wild
type (–55.27 kcal mol-1 to – 37.77 kcal mol-1) which were
comparable to those of the native substrate dihydrofolate and
standard P. falciparum inhibitors.
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1073
A series of novel chloroquinoline based hybrid molecules
(Fig. 43) bearing a core active 4-aminochloroquinoline moie-
ty, and a substituted acetamide linked together with a pipera-
zine were synthesized by Inam et al., (2015) [136] and
screened in vitro against the HM1 : IMSS strain of E. histo-
lytica, and the 3D7 strain of P. falciparum. The obtained
compounds: substituted anilines (A1–A15), substituted pi-
perazine (A16–A22), and aliphatic amines (A23–A27) were
evaluated against E. histolytica. The amoebicidal activity for
all of the compounds was found to be very promising. All of
the compounds were active against the 3D7 strain of P. fal-
ciparum (IC50 range: 0.30– 33.52 mM).
Among the 27 derivatives, 17 were more toxic than
methotrexate, the dihydrofolate reductase inhibitor, and all
showed more toxicity than chloroquine when tested against
human kidney epithelial cells.
Dambuza et al., (2015) [137] synthesized two hydroxy-
pyridone-chloroquine hybrids and tested against chloro-
quine-sensitive Plasmodium falciparum (D10 and 3D7) and
resistant strains (Dd2 and K1). Hydroxypyridones are iron-
chelating agents known to suppress malaria growth in vivo
and in vitro [138, 139]. The IC50 values calculated from the
dose response curves were in the range of 0.041–0.064 µM
against 3D7, and 0.047–0.122 µM against D10, while IC50
values for chloroquine were respectively 0.019 and 0.023
µM against 3D7 and D10. Compound 1 was less active than
chloroquine with respective IC50 values of 0.505 and 0.463
µM for K1 and Dd2, and with poor oral bioavailability, but it
proved to be an effective in vivo antimalarial compound
when intravenously dosed. Compound 2 was more active
than chloroquine with IC50 values of 0.089 and 0.076 µM for
K1 and Dd2, respectively.
Lödige and Hierrsch, (2015) [140] have synthesized nov-
el hybrid molecules consisting of the antimalarial drugs pri-
maquine and chloroquine. These drugs had been successfully
used in combination for the weekly chemoprophylaxis
against malaria during the Vietnam War [141, 142].
Two types of compounds were synthesized: i) molecules
with an authentic linkage in order to avoid additional struc-
tural moieties affecting the pharmacodynamic and pharma-
cokinetic properties of the hybrid compounds and ii) deriva-
tives consisting of the aromatic pharmacophore prepared by
a direct linkage of both motifs in a ratio of 1:1 and 1:2 of
primaquine to chloroquine moiety. We note that hybrid mol-
ecules often do not follow the structural activity relationship
rules of single target components. Against gametocytes of P.
falciparum, hybrid 30 was the most promising structure with
the highest activity against both the number and the diameter
of liver stages, and against blood stages of 3D7, Dd2, and K1
(lower than 0.1
M), and.
Hybrid compounds bearing the (2R,3S)-N-benzoyl-3-
phenylisoserine moiety coupled to an appropriately derived
artemisinin or quinoline scaffold via ester, amide, or triazole
linkages were synthesized by Njogu et al., (2013) [143], and
tested for in vitro anti-plasmodial activity against the eryth-
rocytic stages of K1 and W2 strains of Plasmodium falcipa-
rum. (2R,3S)-N-Benzoyl-3-phenylisoserine is a structural
component of the anti-microtubular drug paclitaxel (Taxol)
initially isolated from the bark of the Pacific yew Taxus
brevifolia Nutt [144].
1074 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 Pedrosa et al.

Cl
Cl Cl

R
H X
N H
N N N Ar N N
N N N R N N N
O
O O
(A1-A15) (A16-A22) (A23-A27)
Fig. (43). General structure of unsubstituted hybrid compounds derived from the fusion of aminochloroquinoline - piperazine molecules.

The artemisinin-bearing hybrid molecule compounds 7a CONCLUSION

and 7b were 3−4 times more active than that of dihydroarte-
misinin against the K1 P. falciparum strain. However,
against W2 strain the same activity was not observed. For
the quinoline-based series, hybrids 10c and 10d were the
most actives (IC50 = 0.13 and 0.16 μM, respectively) against
the W2 strain. These values were lower than that of the con-
trol drug chloroquine (IC50 = 0.05 μM).
Schistosoma
Mott et al., (2012) [145] synthesized five hybrid com-
pounds derived from the antimalarial drug amodiaquine that
was covalently joined with a nitric oxide-releasing furoxan
to achieve multiple mechanisms of action. Furoxan, 3-cyano-
4-phenyl-1,2,5-oxadiazole-2-oxide, has shown activity
against malaria, schistosomiasis, and hookworm [146, 147].
In this review, we presented the applicability and current
status of molecular hybridization for designing new chemical
entities with multitarget profiles for inflammation, cancer,
microbial and parasitic infections.
The approach has been widely (and successfully) used by
various medicinal chemistry groups, and in many cases the
hybrid compounds (synthesized from the combination of two
or more pharmacophoric sub-units) resulted in derivatives
with greater potency and selectivity than the parent drugs. In
other cases, the hybrids obtained promoted modulation of
undesirable drug side effects, or allowed reinsertion of “old-
er antibiotics or antimicrobial agents” into the therapeutic, or
hindered multi-resistant microorganism strains, among oth-
ers advantages.

Notwithstanding the potential for toxic side effects, The effectiveness of this approach is unquestionable,
considering the existence of drugs such as dasatinib and
amodiaquine has been used for treatment of malarial infec-
sunitinib that are multiple tyrosine kinase inhibitors for can-
tions due to the 4-aminophenol structural feature [148]. The
cer therapy, or amitifadine, a triple multitarget antidepressant
amodiaquine–furoxan hybrid 15 showed significant potency
that inhibits serotonin, norepinephrine and dopamine uptake.
in both the standard assay and in the delayed death format
However, not more than a dozen hybrid compounds with
(40 nM and 630 nM IC50, respectively). Hybrid 15 was fur- multitarget profiles may be cited as already on the market or
ther screened against S. mansoni, using both a thioredoxin to become available in the coming years.
glutathione reductase biochemical assay as well as an ex vivo
parasite killing assay. Therefore, molecular hybridization has been shown to be
an important and viable tool for new drug design and discov-
Compound 15 showed activity comparable to the previ- ery. However, in order to provide new drugs, more efforts
ously evaluated phenyl furoxan. Hybrid 15 achieved potent need to be realized, especially against multifactorial diseases,
inhibition at 50 μM, achieving parasite killing in 24 hours. those against which hybrids seem to show more promise.
Additionally, treatment of adult hookworms with 15 at 100
μM resulted in a greater percentage of parasite deaths over a LIST OF ABBREVIATIONS
120 hour time course, when compared with the current
standard of care, Albendazole. MH = Molecular Hybridization
TNF-α = Tumor Necrosis Factor Alpha
Praziquantel, used for the treatment of schistosomiasis
[149], and NO-donor furoxan derivatives bearing at the 3- DNA = Deoxyribonucleic Acid
position CN, CONH2, COOMe, or SO2C6H5 moieties have MTDD = Target Drug Discovery
been synthesized by Guglielmo et al., (2014) [150]. In the
first group of hybrids, the furoxan substructures were substi- MTDL = Multi Target Direct Ligands
tuted for the cyclohexyl group of praziquantel. In the second WHO = World Health Organization
group of hybrids the furoxan moiety was linked to 10-
position of praziquantel through appropriate bridges. The RNAs = Ribonucleic Acids
inhibitory activities of all of the products described in this MDR = Multidrug Resistance
paper were evaluated against recombinant S. mansoni TGR, FDA = Food and Drug Administration
and all of the hybrid furoxan products acted as potent TGR
inhibitors. Furoxans 6 and 7 emerged as the most interesting GA = Glycyrrhetinic Acid
products, considering the balance between PZQ and NO- HDACi = Histone Deacetylase Inhibitors
dependent activities. Furoxans 18 and 24 had better worm
killing activity than furazan compounds, suggesting that NO HDAC = Histone Deacetylase
is involved in this action. HMGR = 3-hydroxy-3-methylglutaryl Coenzyme
A Reductase
Hybrid Compounds as Direct Multitarget Ligands Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1075

IC50 = Half Maximal Inhibitory Concentration MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide
ROS = Reactive Oxygen Species
NAH = N-acylhydrazone
SI = Selectivity Index
Fxm = Furoxanyl Moieties
AA = Arachidonic Acid
MDR = Multidrug Resistant
LTs = Leukotrienes
CQ-S = Choroquine-sensitive
PGs = Prostaglandins
CQ-R = CQ-resistant
LTB4 = Leukotriene B4
Pf-DHFR-TS = Dihydrofolate Reductase of P. falcipa-
PGE2 = Prostaglandin E2
rum
5-LOX = 5-Lipoxygenase
PDB = Protein Database Bank
COX = Cyclooxygenase
DHFR = Dihydrofolate Reductase
COX-1 = Cyclooxygenase Isoform I
TGR = Recombinant S. mansoni TGR
COX-2 = Cyclooxygenase Isoform II
NSAIDs = Nonsteroidal Anti-Inflammatory Drugs CONFLICT OF INTEREST
sGC = Soluble Guanylate Cyclase The authors confirm that this article content has no con-
flict of interest.
LPS = Lipopolysaccharide
TEAC = Trolox Equivalent Antioxidant Capaci- ACKNOWLEDGEMENTS
ty
The authors acknowledge the Conselho Nacional de
EA = Ethanesulfohydroxamic Acid Desenvolvimento Científico e Tecnológico (CNPq) (grant
PBS = Phosphate Buffered Saline number 486044/2013-0; and concession of scholarships to
J.V.O. and R.M.D.C)
AI = Anti-Inflammatory Activity
IL-1β = Interleukin-1 β REFERENCES
HAP = Hydroxyapatite [1] Viegas-Júnior, C.; Danuello, A.; da Silva Bolzani, V.; Barreiro,
E.J.; Fraga, C.A.M. Molecular hybridization: a useful tool in the
HPLC = High-performance Liquid Chromatog- design of new drug prototypes. Curr. Med. Chem., 2007, 14(17),
raphy 1829-1852.
[2] Teiten, M.H.; Dicato, M.; Diederich, M. Hybrid Curcumin Com-
furoxanyl-NAH = Furoxanyl N-acylhydrazones pounds: A New Strategy for Cancer Treatment. Molecules, 2014,
19, 20839-20863.
PMNL = Polymorphonuclear Leukocytes [3] Nepali, K.; Sharma, S.; Sharma, M.; Bedi, P.; Dhar, K. Rational
approaches, design strategies, structure activity relationship and
SAR = Structure-Activity Relationship mechanistic insights for anticancer hybrids. Eur. J. Med. Chem.,
ALA = Alpha-Lipoic Acid 2014, 77, 422-487.
[4] Fortin, S.; Bérubé, G. Advances in the development of hybrid anti-
iNOS = Inducible Nitric Oxide Synthase cancer drugs. Exp. Opin. Drug Discov., 2013, 8(8), 1029-1047.
[5] Garuti, L.; Roberti, M.; Bottegoni, G. Multi-Kinase Inhibitors.
IL-6 = Interleukin-6 Curr. Med. Chem., 2015, 22(6), 695-712.
[6] Musso, L.; Dallavalle, S.; Zunino, F. Perspectives in the develop-
NF-κB = Nuclear Factor Kappa-light-chain En- ment of hybrid bifunctional antitumour agents. Biochem. Pharm.,
hancer of Activated B Cells 2015, 96(4), 297-305.
[7] Fraga, C.A.M. Drug hybridization strategies: Before or after lead
MAPK = Mitogen Activated Protein Kinases identification? Expert Opin. Drug Discov., 2009, 4(6), 605-609.
[8] Morphy, R.; Kay, C.; Rankovic, Z. From magic bullets to designed
IBD = Inflammatory Bowel Disease multiple ligands. Drug Discov. Today., 2004, 9(15), 641-651.
TPA = 12-O-tetradecanoyl Phorbol 13-acetate [9] Morphy, R.; Rankovic, Z. Designing multiple ligands – Medicinal
chemistry strategies and challenges. Curr. Pharm. Des., 2009, 15,
TP = Thromboxane A2 A2 prostanoid 587-600.
[10] Walsh, J.J.; Coughlan, D.; Heneghan, N.; Gaynor, C.; Bell, A. A
TXA2 = Thromboxane A2 novel artemisnin-quinien hybrid with potente antimalarial activity.
Bioorg. Med. Chem., 2007, 17, 3599-3602.
IZD = Inhibition Zone Diameter [11] Peperidou, A.; Kapoukranidou, D.; Kontogiorgis, C.; Hadjipavlou-
MIC = Minimum Inhibitory Concentration Litina, D. Multitarget Molecular hybrids of cinnamic acids. Mole-
cules, 2014, 19, 20197-20226.
MRSA = Methicillin-resistant S. aureus [12] Sadek, B.; Alish, R.; Buschauer, A.; Elz, S. Synthesis and dual
histamine H1 and H2 receptor antagonist activity of cyanoguani-
QRSA = Quinolone-Resistant S. aureus dine derivatives. Molecules, 2013, 18, 14186-14202.
[13] Sharma, M.; Deekshith, V.; Semwal, A.; Sriram, D.; Yogeeswari,
AIDS = Acquired Immune Deficiency Syn- P. Discovery of fused triazolo-thiadiazoles as inhibitors of TNF-
drome alpha: pharmacophore hybridization for treatment of neuropathic
pain. Pain Ther., 2012, 1(3), 1-17.
1076 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
[14] Chojnacki, J.E.; Liu, K.; Yan, X.; Toldo, S.; Selden, T.; Estrada,
M.; Rodriguez-Franco, M.I.; Halquist, M.S.; Ye, D.; Zhang, S. Dis-
covery of 5-(4-hydroxyphenyl)-3-oxo-pentanoic acid [2-(5-
methoxy-1h-indol-3-yl)-ethyl]-amide as a neuroprotectant for alz-
heimer’s disease by hybridization of curcumin and melatonin. ACS
Chem. Neurosci., 2014, 5, 690–699.
[15] Meunier, B. Hybrid molecules with a dual mode of action: dream
or reality? Acc. Chem. Res., 2008, 41(1), 69-77.
[16] Oliveira, R.; Miranda, D.; Magalhães, J.; Capela, R.; Perry, M.J.;
O’Neill, P.M.; Moreira, R.; Lopes, F. From hybrid compounds to
targeted drug delivery in antimalarial therapy. Bioorg. Med. Chem.,
2015, 23, 5120–5130.
[17] Lafayette, E.A.; de Almeida, S.M.V.; Pitta, M.G.R.; Beltrão,
E.I.C.; da Silva, T.G.; de Moura, R.O.; Pitta, I.R.; Júnior, L.B.C.;
de Lima, M.C.A. Synthesis, DNA binding and topoisomerase I in-
hibition activity of thiazacridine and imidazacridine derivatives.
Molecules, 2013, 18, 15035-15050.
[18] Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging
drug discovery paradigm. J. Med. Chem., 2005, 48, 6523-6543.
[19] Cavalli, A.; Bolognesi, M.L.; Minarini, A.; Rosini, M.; Tumiatti,
V.; Recanatini, M.; Melchiorre, C. Multi-target-directed ligands to
combat neurodegenerative diseases. J. Med. Chem., 2008, 51, 347-
372.
[20] Peters, J.U. Polypharmacology in Drug Discovery. Wiley & Sons:
New Jersey, 2012.
[21] Morphy J.R.; Harris, C.J. Designing Multi-Target Drugs. The
Royal Society of Chemistry: Cambridge, 2012.
[22] Bolognesi, M.L. Multi-target-directed ligands as innovative tools to
combat trypanosomatid diseases. Curr. Top. Med. Chem., 2011,
11(22), 2824-2833.
[23] Walsh, J.J.; Bell, A. Hybrid drugs for malaria. Curr. Pharm. Des.,
2009, 15, 2970-2985.
[24] Muregi, F.W.; Ishih, A. Next generation antimalarial drugs: Hybrid
molecules as a new strategy in drug design. Drug Dev. Res., 2010,
71, 20-32.
[25] Bolognesi, M.L.; Polypharmacology in a single drug: multitarget
drugs. Curr. Med. Chem., 2013, 20, 1639-1645.
[26] Dias, K.S>t; Viegas Jr, C. Multi-Target Direct Drugs: Amodern
Approach for Design of New Drugs for the treatment of Alz-
heimer´s Disease. Curr. Neuropharmacol., 2014, 12, 239-255.
[27] Leon, R.; Garcia, A.G.; Marco-Contelles, J. Recent advances in the
multitarget-directed ligands approach for the treatment of Alz-
heimer’s disease. Med. Res. Rev., 2013, 33(1), 139-189.
[28] Njogu, P.M.; Chibale, K. Recent developments in rationally de-
signed multitarget antiprotozoan agents. Curr. Med. Chem., 2013,
20, 1715-1742.
[29] Bansal, Y.; Silakari, O. Multifunctional compounds: Smart mole-
cules for multifactorial diseases. Eur. J. Med. Chem., 2014, 76, 31-
42.
[30] WORLD HEALTH ORGANIZATION. http://www.who.int/en/
(Accessed April 26, 2016).
[31] Covington, K.R.; Shinbrot, E.; Wheeler, D.A. Mutation signatures
reveal biological processes in human cancer. BioRxiv. 2016, doi:
http://dx.doi.org/10.1101/036541.
[32] Joerger, A.C.; Fersht, A.R. The p53 Pathway: Origins, Inactivation
in Cancer, and emerging therapeutic approaches. Ann. Rev. Bio-
chem., 2016, 85.
[33] Peng, L.; Yuan, X.Q.; Li, G.C. The emerging landscape of circular
RNA ciRS-7 in cancer (Review). Oncol. Reports, 2015, 33(6),
2669-2674.
[34] Kathawala, R.J.;Gupta, P.; Ashby, C.R. Jr.; Chen, Z.S. The modu-
lation of ABC transportes-mediated multidrug resistance in cancer:
a review of the past decade. Drug Resist. Updat., 2015, 18, 1-17.
[35] Ai, T.; Cui, H.; Chen, L. Multi-Targeted Histone Deacetylase In-
hibitors in Cancer Therapy. Curr. Med. Chem., 2012, 19(4), 475-
487.
[36] Fox, B.M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B.L.; Stewart, L.; Cushman, M. Design, synthesis, and bio-
logical evaluation of cytotoxic 11-alkenylindenoisoquinoline topoi-
somerase I inhibitors and indenoisoquinoline-camptothecin hy-
brids. J. Med. Chem., 2003, 15(46), 3275-3282.
[37] Wang, D.; Zhang, Z.; Lu, X.; Feng, Y.; Luo, K.; Gan, J.; Yingxue,
L.; Wan, J.; Li, X.; Zhang, F.; Tu, Z.; Cai, Q.; Ren, X.; Ding, Ke.
Hybrid compounds as new Bcr/Abl inhibitors. Bioorg. Med. Chem.,
2011, 21(7), 1965–1968.
Pedrosa et al.
[38] Belluti, F.; Fontana, G.; Dal, Bo. L.; Carenini, N.; Zunino, F. De-
sign, synthesis and anticancer activities of stilbene-coumarin hybrid
compounds: identification of novel proapoptotic agents. Bioorg.
Med. Chem., 2010, 18(10), 3543-3550.
[39] Sashidhara, K.V.; Kumar, A.; Kumar, M.; Sarkar, J.; Sinha, S.
Synthesis and in vitro evaluation of novel coumarin-chalcone hy-
brids as potential anticancer agents. Bioorg. Med. Chem. Lett.,
2010, 20, 7205-7211.
[40] Bednarek, E.; Bocian, W.; Sitkowski, J.; Ulkowska, A.; Kaczma-
rek, L.; Badowska-Rolonek, K.; Ciesielska, A.; Bukowska, A.;
Kozerski, L. 1H and 13C NMR data for indolo[2,3-b]quinoline-
aminoglycoside hybrids, a novel potent anticancer drug family.
Magn. Reson. Chem., 2006, 44(4), 459-462.
[41] Matsuya, Y.; Kobayashi, Y.; Kawaguchi, T.; Hori, A.; Watanabe,
Y.; Ishihara, K.; Ahmed, K.; Wei, Z.L.; Yu, D.Y.; Kondo, T.; Ne-
moto, H. Design, synthesis, and biological evaluation of artificial
macrosphelides in the search for new apoptosis-inducing agents. J.
Eur. Chem., 2009, 15(23), 5799-5813.
[42] Ahmed, K.; Matsuya, Y.; Nemoto, H.; Zaidi, S.F.; Sugiyama, T.;
Yoshihisa, Y.; Shimizu, T.; Kondo, T. Mechanism of apoptosis in-
duced by a newly synthesized derivative of macrosphelides with a
thiazole side chain. Chem. Biol. Inter., 2009, 177(3), 218-226.
[43] Decker, M. Hybrid Molecules Incorporating Natural Products:
Applications in Cancer Therapy, Neurodegenerative Disorders and
Beyond. Curr. Med. Chem., 2011, 18(10), 1464-1475.
[44] Tatsuzaki, J.; Taniguchi, M.; Bastow, K. F.; Nakagawa-Goto, K.;
Morris- Natschke, S. L.; Itokawa, H.; Baba, K.; Lee, K. H. Anti-
tumor agents 255: Novel glycyrretinic acid-dehydrozingerone con-
jugates as cytotoxic agents. Bioorg. Med. Chem., 2007, 15(18),
6193-6199.
[45] Chen, J.B .;Chern, T.R.; Wei, T.T. Design and synthesis of dual-
action inhibitors targeting histone deacetylases and 3-Hydroxy-3-
methylglutaryl coenzyme A reductase for cancer treatment. J. Med.
Chem., 2013, 56(9), 3645–3655.
[46] Zhang, X.; Kong, Y.; Zhang J.; Su, M.; Zhou, Y.; Zang, Y.; Li, J.;
Chen, Y.; Fang, Y.; Lu, W. Design, synthesis and biological evalu-
ation of colchicine derivatives as novel tubulin and histone
deacetylase dual inhibitors. Eur. J. Med. Chem., 2015, 95, 127–
135.
[47] Seo, Y.H. Dual inhibitors against topoisomerases and histone
deacetylases. J. Cancer Prev., 2015, 20(2), 85-91.
[48] Sharma, M.; Sharma, S.; Buddhiraja, A.; Saxena, R.; Nepali, K.;
Bedi, P. Synthesis and cytotoxicity studies of 3,5-diaryl N-
acetyl pyrazoline-isatin hybrids. Med. Chem. Res., 2014, 23,
4337-4344.
[49] Yan, J.; Guo, Y.; Wang, Y.; Mao, F.; Huang, L.; Li, X. Design,
synthesis, and biological evaluation of benzoselenazole-stilbene
hybrids as multi-target-directed anti-cancer agents. Eur. J. Med.
Chem., 2015, 95, 220-229.
[50] Punganuru, S.R.; Madala, H.R.; Venugopal, S.N.; Samala, R.;
Mikelis, C.; Srivenugopal, K.S. Design and synthesis of a C7-aryl
piperlongumine derivative with potent antimicrotubule and mutant
p53-reactivating properties. Eur. J. Med. Chem., 2015, 107, 233-
244.
[51] Xu, C.C.; Deng, T.; Fan, M.L.; Lv, W.B.; Liu, J.H.; Yu, P.O.R.
Synthesis and in vitro antitumor evaluation of dihydroartemisinin-
cinnamic acid ester derivatives. Eur. J. Med. Chem., 2016, 107,
192–203.
[52] Cui, Z.; Li, X.; Li, L.; Zhang, B.; Gao, C.; Chen, Y.; Tan, C.; Liu,
H.; Xie, W.; Yang, T.; Jiang, Y. Design, synthesis and evaluation
of acridine derivatives as multi-target Src and MEK kinase inhibi-
tors for anti-tumor treatment. Bioorg. Med. Chem., 2016, 24(2),
261-269.
[53] Balakumar, C.; Lamba, P.; Pran, K.D.; Lakshmi, N.B.; Venkat,
R.K.; Rajwinder, K.; Raghuram, R.A.; Shireesha, B.; Narsaiah, B.
Synthesis, anti-inflammatory evaluation and docking studies of
some new fluorinated fused quinazolines. Eur. J. Med. Chem.,
2010, 45, 4904-4913.
[54] Meng, H.; Liu, Y.; Lai, L. Diverse ways of perturbing the human
arachidonic acid metabolic network to control inflammation. Acc.
Chem. Res., 2015, 48, 2242-2250.
[55] Khanapure, S.P.; Garvey, D.S.; Janero, D.R.; Letts, L.G. Eico-
sanoids in inflammation: Biosynthesis, pharmacology, and thera-
peutic frontiers. Curr. Top. Med. Chem., 2007, 7, 311-340.
[56] Blobaum, A.L.; Marnett, L.J. Structural and functional basis of
cyclooxygenase inhibition. J. Med. Chem., 2007, 50, 1425-1441.
Hybrid Compounds as Direct Multitarget Ligands
[57] Boschi, D.; Giorgis, M.; Cena, C.; Chandra, T.N.; Stilo, A.D.;
Morini, G.; Coruzzi, G.; Guaita, E.; Fruttero, R.; Gasco, A. Multi-
target drugs: synthesis and preliminary pharmacological characteri-
zation of Zileuton analogues endowed with dual 5-LO inhibitor and
NO-dependent activities. Chem. Med. Chem., 2010, 5, 1444-1449.
[58] Kálai, T.; Borza, E.; Antus, C.; Radnai, B.; Gulyás-Fekete, G.;
Fehér, A.; Sümegi, B.; Hideg, K. Synthesis and study of new para-
magnetic resveratrol analogues. Bioorg. Med. Chem., 2001, 19,
7311–7317.
[59] Soule, B. P.; Hyodo, F.; Matsumoto, K.I.; Simone, N.L.; Cook,
J.A.; Krishna, M.C.; Mitchell, J.B. Therapeutic and Clinical Appli-
cations of nitroxide compounds. Antioxid. Redox Signal., 2007, 9,
1731-1744.
[60] Huang, Z.; Velázquez, C.A.; Abdellatif, K.R.A.; Chowdhury, M.
A.; Reisz, J.A.; DuMond, J. F.; King, S. B.; Knaus, E.E. Ethanesul-
fohydroxamic acid ester prodrugs of nonsteroidal antiinflammatory
drugs (NSAIDs), synthesis, nitric oxide and nitroxyl release, cy-
clooxygenase inhibition, anti-inflammatory, and ulcerogenicity in-
dex studies. J. Med. Chem., 2011, 54(5),1356–1364.
[61] Abbas, S.E.; Awadallah, F.M.; Ibrahin, N.A.; Said, E.G.; Kamel,
G.M. New quinazolinoneepyrimidine hybrids: synthesis, anti-
inflammatory, and ulcerogenicity studies. Eur. J. Med. Chem.,
2012, 53, 141-149.
[62] Tamura, T.; Shirai, T.; Kosaka, N.; Ohmori, K.; Takafumi, N.
Pharmacological studies of diacerein in animal models of inflam-
mation, arthritis and bone resorption. Eur. J. Pharmacol., 2002,
448(1), 81-87.
[63] Cai, J.; Duan, Y.; Yu, J.; Chen, J.; Chao, M.; Ji, M. Bone-targeting
glycol and NSAIDS ester prodrugs of Rhein : Synthesis, hydroxy-
apatite affinity, stability, anti-inflammatory, ulcerogenicity index
and pharmacokinetics studies. Eur. J. Med. Chem., 2012, 55, 409-
419.
[64] Hernández, P.; Cabrera, M.; Lavaggi, M.L.; Celano, L.; Tiscornia,
I.; Da Costa, T.R.; Thomson, L.; Bollati-Fogolín, M.; Miranda,
A.L.P.; Lima, L.M.; Barreiro, E.J.; González, M.; Cerecetto, H.
Discovery of new orally effective analgesic and anti-inflammatory
hybrid furoxanyl N-acylhydrazone derivatives. Bioorg. Med.
Chem., 2012, 20, 2158-2171.
[65] Guerra, A.S.H.S.; Malta, D.J.N.; Laranjeira, L.P.M.; Maia, M.B.S.;
Colaço, N.C.; Lima, M.D.C.A.; Gonçalves-Silva, T.; Galdino, S.L.;
Pitta, I.R.; Gonçalves-Silva, T. Anti-inflammatory and antinocicep-
tive activities of indole–imidazolidine derivatives. Int. Im-
munopharmacol., 2011, 11, 1816-1822.
[66] Alegaon, S.G.; Hirpara, M.B.; Alagawadi, K.R.; Hullatti, K.K.;
Kashniyal, K. Synthesis of novel pyrazole–thiadiazole hybrid as
potential potent and selective cyclooxygenase-2 (COX-2) inhibi-
tors. Bioorg. Med. Chem. Lett., 2014, 24, 5324-5329.
[67] Apostolidis, J.; Liaras, K.; Geronikaki, A.; Hadjipavlou-Litina, D.;
Gavalas, A.; Sokovic, M.; Glamoclija, J.; Ciric, A. Synthesis and
biological evaluation of some 5-arylidene-2-(1,3-thiazol-2-
ylimino)-1,3-thiazolidin-4-ones as dual anti-
inflammatory/antimicrobial agents. Bioorg. Med. Chem., 2013, 21,
532-539.
[68] Hu, J.; Wang, Y.; Wei, X.; Wu, X.; Chen, G.; Cao, G.; Shen, X.;
Zhang, X.; Tang, Q.; Liang, G.; Li, X. Synthesis and biological
evaluation of novel thiazolidinone derivatives as potential anti-
inflammatory agents. Eur. J. Med. Chem., 2013, 64, 292-301.
[69] Eleftheriou, P.; Geronikaki, A.; Litina, D. H.; Vicini, P.; Filz, O.;
Filimonov, D.; Poroikov, V.; Chaudhaery, S. S.; Roy, K. P.; Sex-
ena, A. K. Fragment-based design, docking, synthesis, biological
evaluation and structure-activity relationships of 2-
benzo/benzisothiazolimino-5-aryliden-4-thiazolidinones as cyclox-
ygenase/lipoxygenase inhibitors. Eur. J. Med. Chem., 2012, 47,
111-124.
[70] Unsal-Tan, O.; Ozadali, K.; Paskin, K.; Balkan, A. Molecular mod-
eling, synthesis and screening of some new 4-thiazolidinone de-
rivatives with promising selective COX-2 inhibitory activity. Eur.
J. Med. Chem., 2012, 57, 59-64.
[71] Hwang, J.S.; Um, J.M.; Cho, H.; Lee, S.H.; Park, J.H; Han, I.O. A
dopamine-alpha-lipoic acid hybridization compound and its acety-
lated form inhibit LPS-mediated inflammation. Eur. J. Pharmacol.,
2015, 746, 41-49.
[72] Sasso, O.; Migliore, M.; Habrant, D.; Armirotti, A.; Albani, C.;
Summa, M.; Moreno-Sanz, G.; Scarpelli, R.; Piomelli, D. Multitar-
get fatty acid amide hydrolase/cyclooxygenase blockade suppresses
intestinal inflammation and protects against nonsteroidal anti-
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1077
inflammatory drug-dependent gastrointestinal damage. J. FASEB,
2015, 29(6), 2616-2627.
[73] Theoduloz, C.; Delporte, C.; Valenzuela-Barra, G.; Silva, X.; Cá-
diz, S.; Bustamante, F.; Pertino, M.W.; Schmeda-Hirschmann, G.
Topical Anti-inflammatory Activity of new hybrid molecules of
terpenes and synthetic drugs. Molecules, 2015, 20, 11219-11235.
[74] Hoxha, M.; Buccellati, C.; Capra, V.; Garella, D.; Cena, C.; Rolan-
do, B.; Fruttero, R.; Carnevali, S.; Sala, A.; Rovati, G.E.; Bertinar-
ia, M. In vitro pharmacological evaluation of multitarget agents
forthromboxane prostanoid receptor antagonism and COX-2 inhibi-
tion. Pharmacol. Res., 2016, 103, 132-143.
[75] Asadipour, A.; Edraki, N.; Nakhjiri, M.; Yahya-Meymandi, A.;
Alipour, E.; Saniee, P.; Siavoshi, F.; Shafiee, A.; Foroumadi, A.
Anti-Helicobacter pylori activity and structure-activity relationship
study of 2-Alkylthio-5-(nitroaryl)-1,3,4-thiadiazole Derivatives.
Iran. J. Pharm. Res., 2013, 12(3), 281-287.
[76] Ramprasad, J.; Nayak, N.; Dalimba, U. Design of new phenothia-
zine-thiadiazole hybrids via molecular hybridization approach for
the development of potent antitubercular agents. Eur. J. Med.
Chem., 2015, 106, 75-84.
[77] Singh, U.P.; Pathak, M.; Dubey, V.; Bhat, H.R.; Gahtori, P.; Singh,
R.K. Design, synthesis, antibacterial activity, and molecular dock-
ing studies of novel hybrid 1,3-thiazine-1,3,5-triazine derivatives as
potential bacterial translation inhibitor. Chem. Biol. Drug. Des.,
2012, 80, 572-583.
[78] Dixit, P.P.; Thore, S.N. Hybrid triazoles: design and synthesis as
potential dual inhibitor of growth and efflux inhibition in tubercu-
losis. Eur. J. Med. Chem., 2016, 107, 38-47.
[79] Ouahrouch, A.; Ighachane, H.; Taourirte, M.; Engels, J.W.; Sedra,
M.H.; Lazrek, H.B. Benzimidazole-1,2,3-triazole hybrid molecules:
synthesis and evaluation for antibacterial/antifungal activity. Arch.
Pharm. Chem. Life Sci., 2014, 347, 748-755.
[80] Sangaraiah, N.; Murugan, S.; Poovan, S.; Raja, R.; Alagusundaram,
P.; Ramakrishnan, V.; Vellasamy, S. Facile water promoted syn-
thesis of 1,2,3-triazolyl dihydropyrimidine-2-thione hybrids e high-
ly potent antibacterial agents. Eur. J. Med. Chem., 2012, 58, 464-
469.
[81] Gaikwad, N.D.; Patil, S.V.; Bobade, V.D. Synthesis and biological
evaluation of some novel thiazole substituted benzotriazole deriva-
tives. Bioorg. Med. Chem. Lett., 2012, 22, 3449-3454.
[82] Gaikwad, N.D.; Patil, S.V.; Bobade, V.D. Hybrids of ravuconazole:
synthesis and biological evaluation. Eur. J. Med. Chem., 2012, 54,
295-302.
[83] Sumangala, V.; Poojary, B.; Chidananda, N.; Fernandes, J.; Kuma-
ri, N.S. Synthesis and antimicrobial activity of 1,2,3-triazoles con-
taining quinoline moiety. Arch. Pharm. Res., 2010, 33(12), 1911-
1918.
[84] Mentese, M.Y.; Bayrak, H.; Uygun, Y.; Mermer, A.; Ulker, S.;
Karaoglu, S.A.; Demirbas, N. Microwave assisted synthesis of
some hybrid molecules derived from norfloxacin and investigation
of their biological activities. Eur. J. Med. Chem., 2013, 67, 230-
242.
[85] Thomas, K.D.; Adhikari, A.V.; Chowdhury, I.H.; Sumesh, E.; Pal,
N.K. New quinolin-4-yl-1,2,3-triazoles carrying amides, sulphon-
amides and amidopiperazines as potential antitubercular agents.
Eur. J. Med. Chem., 2011, 46, 2503-2512.
[86] Thomas, K.D.; Adhikari, A.V.; Chowdhury, I.H.; Sandeep, T.;
Mahmood, R.; Bhattacharya, B.; Sumesh, E. Design, synthesis and
docking studies of quinoline-oxazolidinone hybrid molecules and
their antitubercular properties. Eur. J. Med. Chem., 2011, 46, 4834-
4845.
[87] Dolan, N.; Gavin, D.P.; Ahmed Eshwika, A.; Kavanagh, K.;
McGinley, J.; Stephens, J.C. Synthesis, antibacterial and anti-
MRSA activity, in vivo toxicity and a structure–activity relation-
ship study of a quinoline thiourea. Bioorg, Med. Chem. Lett., 2016,
26, 630-635.
[88] Jain, P.P.; Degani, M.S.; Raju, A.; Anantram, A.; Seervi, M.;
Sathaye, S.; Ray, M.; Rajan, M.G.R. Identification of a novel class
of quinoline–oxadiazole hybrids as anti-tuberculosis agents.
Bioorg. Med. Chem. Lett., 2016, 26, 645-649.
[89] Chen, Z.; Zheng, C.; Sun, L.; Piao, H.; Synthesis of new chalcone
derivatives containing a rhodanine-3-acetic acid moiety with poten-
tial anti-bacterial activity. Eur. J. Med. Chem., 2010, 45, 5739-
5743.
[90] Song, M.; Zheng, C.; Deng, X.; Sun, L.; Wu, Y.; Hong, L.; Li, Y.;
Liu, Y.; Wei, Z.; Jin, M.; Piao, H. Synthesis and antibacterial eval-
1078 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9
uation of rhodanine-based 5-aryloxy pyrazoles against selected
methicillin resistant and quinolone resistant Staphylococcus aureus
(MRSA and QRSA). Eur. J. Med. Chem., 2013, 60, 376-385.
[91] Essghaier, B.; Naouar, A.; Abdelhak, J.; Zid, M.F.; Sadfi-Zouaoui,
N. Synthesis, crystal structure and potential antimicrobial activities
of di(4-sulfamoyl-phenyl-ammonium) sulphate. Microbiol. Res.,
2014, 169, 504-510.
[92] Keche, A.P.; Hatnapure, G.D.; Tale, R.H.; Rodge, A.H.; Birajdar,
S.S.; Kamble, V.M. A novel pyrimidine derivative with aryl urea,
thiourea and sulfonamide moieties: Synthesis, anti-inflammatory
and antimicrobial evaluation. Bioorg. Med. Chem. Lett., 2012, 22,
3445-3448.
[93] Rane, R.A.; Telvekar, V.N. Synthesis and evaluation of novel
chloropyrrole molecules designed by molecular hybridization of
common pharmacophores as potential antimicrobial agents. Bioorg.
Med. Chem. Lett., 2010, 20, 5681-5685.
[94] Desmond, R.T.; Magpusao, A.N.; Lorenc, C.; Alverson, J.B.;
Priestley, N.; Peczuh, M.W. De novo macrolide-glycolipid macro-
lactone hybrids: synthesis, structure and antibiotic activity of car-
bohydrate-fused macrocycles. Beilstein J. Org. Chem., 2014, 10,
2215-2221.
[95] Horton, D.A.; Bourne, G.T.; Smythe, M.L. The Combinatorial
Synthesis of Bicyclic Privileged Structures of Privileged Substruc-
tures. Chem. Rev., 2003, 103, 893-930.
[96] Sands, M.; Kron, M.A.; Brown, R.B. Pentamidine: a review. Rev.
Infect. Dis., 1985, 7, 625-634.
[97] Boykin, D.W. Antimicrobial activity of the DNA minor groove
binders furamidine and analogs. J. Braz. Chem. Soc., 2002, 13,
763-771.
[98] Balaña-Fouce, R.; Álvarez-Velilla, R.; Fernández-Prada, C.; Gar-
gía-Estrada, C.; Reguera, R.M. Trypanosomatids topoisomerase re-
visited. New structural findings and role in drug Discovery. Int. J.
Paras. Drugs Drug Resist., 2014, 4(3), 326-337.
[99] Torres-Gómez, H.; Hernández-Núñez, E.; León-Rivera, I.; Guerre-
ro-Alvarez, J.; Cedillo-Rivera, R.; Moo-Puc, R.; Argotte-Ramos,
R.; Rodríguez-Gutiérrez, M.C.; Chan-Bacab, M.J.; Navarrete-
Vázquez, G. Design, synthesis and in vitro antiprotozoal activity of
benzimidazole-pentamidine hybrids. Bioorg. Med. Chem. Lett.,
2008, 18(11), 3147-3151.
[100] Nava-Zuazo, C.; Estrada-Soto, S.; Guerrero-Alvarez, J.; León-
Rivera, I.; Molina-Salinas, G.M.; Said-Fernández, S.; Chan-Bacab,
M.J.; Cedillo-Rivera, R.; Moo-Puc, R.; Mirón-López, G.; Navar-
rete-Vazquez, G. Design, synthesis, and in vitro antiprotozoal, an-
timycobacterial activities of N-{2-[(7-chloroquinolin-4-
yl)amino]ethyl}ureas. Bioorg. Med. Chem., 2010, 18(17), 6398-
6403.
[101] de Souza, M.V.; Pais, K.C.; Kaiser, C.R.; Peralta, M.A.; Ferreira,
M.L.; Lourenço, M.C.S. Synthesis and in vitro antitubercular activ-
ity of a series of quinoline derivatives. Bioorg. Med. Chem., 2009,
17, 1474-1480.
[102] Upadhayaya, R.S.; Kulkarni, G.M.; Vasireddy, N.R.; Vandavasi,
J.K.; Dixit, S.S.; Sharma, V.; Chattopadhyaya, J. Design, synthesis
and biological evaluation of novel triazole, urea and thiourea deriv-
atives of quinoline against Mycobacterium tuberculosis. Bioorg.
Med. Chem., 2009, 17(13), 4681-4692.
[103] Yendapally, R.; Lee, R.E. Design, synthesis, and evaluation of
novel ethambutol analogues. Bioorg. Med. Chem., 2008, 18(5),
1607–1611.
[104] Bhowruth, V.; Brown, A.K.; Reynolds, R.C.; Coxon, G.D.; Mac-
kay, S.P.; Minnikin, D.E.; Besra, G.S. Symmetrical and unsymmet-
rical analogues of isoxyl; active agents against Mycobacterium tu-
berculosis. Bioorg. Med. Chem. Lett., 2006, 16(18), 4743-4747.
[105] Ibrar, A.; Zaib, S.; Khan, I.; Jabeen, F.; Iqbal, J.; Saeed, A. Facile
and expedient access to bis-coumarin– iminothiazole hybrids by
molecular hybridization approach: synthesis, molecular modelling
and assessment of alkaline phosphatase inhibition, anticancer and
antileishmanial potential. RSC Adv., 2015, 5, 89919-89931.
[106] Iqbal, J. An enzyme immobilized microassay in capillary electro-
phoresis for characterization and inhibition studies of alkaline
phosphatases. Anal. Biochem., 2011, 414(2), 226-231.
[107] Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods, 1983, 65, 55-63.
[108] Niks, M.; Otto, M. Towards an Optimized MTT Assay. J. Immu-
nol. Methods, 1990, 130(1), 149-151.
Pedrosa et al.
[109] Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R.
New colorimetric cytotoxicity assay for anticancer-drug screening.
J. Natl. Cancer Inst., 1990, 82(13), 1107-1112.
[110] Hernández, P.; Rojas, R.; Gilman, R.H.; Sauvain, M.; Lima, L.M.;
Barreiro, E.J.; González, M.; Cerecetto, H. Hybrid furoxanyl N-
acylhydrazone derivatives as hits for the development of neglected
diseases drug candidates. Eur. J. Med. Chem., 2013, 59, 64-74.
[111] Rodrigues, C.; Batista, A.A.; Ellena, J.; Castellano, E.E.; Benítez,
D.; Cerecetto, H.; González, M.; Teixeira, L.R.; Beraldo, H. Coor-
dination of nitro-thiosemicarbazones to ruthenium(II) as a strategy
for anti-trypanosomal activity improvement. Eur. J. Med. Chem.,
2010, 45, 2847-2853.
[112] Aponte, J.C.; Vaisberg, A.J.; Castillo, D.; Gonzalez, G.; Estevez,
Y.; Arevalo, J.; Quiliano, M.; Zimic, M.; Verástegui, M.; Málaga,
E.; Gilman, R.H.; Bustamante, J.M.; Tarleton, R.L.; Wang, Y.;
Franzblau, S.G.; Pauli, G.F.; Sauvain, M.; Hammond, G.B. Trypa-
noside, anti-tuberculosis, leishmanicidal, and cytotoxic activities of
tetrahydrobenzothienopyrimidines. Bioorg. Med. Chem., 2010,
18(8), 2880-2886.
[113] da Silva, L.E.; Joussef, A.C.; Pacheco, L.K.; da Silva, D.G.; Stein-
del, M.; Rebelo, R.A.; Schmidt, B. Synthesis and in vitro evalua-
tion of leishmanicidal and trypanocidal activities of N-quinolin-8-
yl-arylsulfonamides. Bioorg. Med. Chem., 2007, 15, 7553-7560.
[114] Carroll, F.I.; Berrang, B.D.; Linn, C.P. Synthesis of 2,4-
disubstituted 6-methoxy-8-aminoquinoline analogues as potential
antiparasitics. J. Med. Chem., 1980, 23, 581-584.
[115] Bhattacharya, G.; Herman, J.; Delfín, D.; Salem, M.M.; Barszcz,
T.; Mollet, M.; Riccio, G.; Brun, R.; Werbovetz, K.A. Synthesis
and antitubulin activity of N1- and N4-substituted 3,5-dinitro sul-
fanilamides against African trypanosomes and Leishmania. J. Med.
Chem., 2004, 47(7), 1823-1832.
[116] Guillon, J.; Forfar, I.; Mamani-Matsuda, M.; Desplat, V.; Saliège,
M.; Thiolat, D.; Massip, S.; Tabourier, A.; Léger, J.M.; Dufaure,
B.; Haumont, G.; Jarry, C.; Mossalayi, D. Synthesis, analytical be-
haviour and biological evaluation of new 4-substituted pyrrolo[1,2-
a]quinoxalines as antileishmanial agents. Bioorg. Med. Chem.,
2007, 15, 194-210.
[117] Otero, E.; Vergara, S.; Robledo, S.M.; Cardona, W.; Carda, M.;
Vélez, I.D.; Rojas, C.; Otálvaro, F. Synthesis, leishmanicidal and
cytotoxic activity of triclosan-chalcone, triclosan-chromone and
triclosan-coumarin hybrids. Molecules, 2014, 19(9), 13251-13266.
[118] Kapoor, M.; Reddy, C.C.; Krishnasastry, M.V.; Surolia, N.; Suro-
lia, A. Slow-tight-binding inhibition of enoyl-acyl carrier protein
reductase from Plasmodium falciparum by triclosan. Biochem. J.,
2004, 381, 719-724.
[119] Perozzo, R.; Kuo, M.; Sidhu, A.; Valiyaveettil, J.T.; Bittman, R.;
Jacobs, W.R. Jr.; Fidock, D.A.; Sacchettini, J.C. Structural elucida-
tion of the specificity of the antibacterial agent triclosan for malari-
al enoyl acyl carrier protein reductase. J. Biol. Chem., 2002,
277(15), 13106-13114.
[120] Kayser, O.; Kiderlen, A.F. In vitro leishmanicidal activity of natu-
rally occurring chalcones. Phytother. Res., 2001, 15(2), 148-152.
[121] Liu, M.; Wilairat, P.; Croft, S.L.; Tan, A.L.; Go, M.L. Structure-
activity relationships of antileishmanial and antimalarial chalcones.
Bioorg. Med. Chem., 2003, 11(13), 2729-2738.
[122] Boeck, P.; Bandeira Falcão, C.A.; Leal, P.C.; Yones, R.A.; Filho,
V.C.; Torres-Santos, E.C.; Rossi-Bergmann, B. Synthesis of chal-
cone analogues with increased antileishmanial activity. Bioorg.
Med. Chem., 2006, 14(5), 1538-1545.
[123] Montagner, C.; de Souza, S.M.; Groposoa, C.; Delle Monache, F.;
Smânia, E.F.; Smânia, A.Jr. Antifungal activity of coumarins. Z.
Naturforsch., 2008, 63, 21-28.
[124] Morabito, G.; Trombetta, D.; Singh Brajendra, K.; Prasad Ashok,
K.; Parmar Virinder, S.; Naccari, C.; Saija, A.; Cristani, M.; Firuzi,
O.; Saso, L. Antioxidant properties of 4-methylcoumarins in vitro
cell-free systems. Biochimie, 2010, 92(9), 1107-1107.
[125] Mallick, S.; Dutta, A.; Ghosh, J.; Maiti, S.; Mandal, A.K.;
Banerjee, R.; Bandyopadhyay, C.; Pal, C. Protective therapy with
novel chromone derivative against Leishmania donovani infection
induces Th1 response in vivo. Chemotherapy, 2011, 57(5),388-393.
[126] Mowbray, C.E.; Braillard, S.; Speed, W.; Glossop, P.A.; Whitlock,
G.A.: Gibson, K.R.; Mills, J.E.; Brown, A.D.; Gardner, J.M.; Cao,
Y.; Hua, W.; Morgans, G.L.; Feijens, P.B.; Matheeussen, A.; Maes,
L.J. Novel amino-pyrazole ureas with potent on vitro and in vivo
antileishmanial activity. J. Med. Chem., 2015, 58(24), 9615-9624.
Hybrid Compounds as Direct Multitarget Ligands
[127] Dos Santos Faiões, V.; Leon, L.L.; Canto-Cavalheiro, M.M.;
Torres-Santos, E.C.; Bernardino, A.M.; Vegi, P.F.; dos Santos,
M.S. Effectiveness of novel 5-(5-Amino-1-Aryl-1H-Pyrazol-4-yl)-
1Htetrazole derivatives against promastigotes and amastigotes of
Leishmania Amazonensis. Chem. Biol. Drug Des., 2014, 83(3),
272-277.
[128] Borges, J.C.; Carvalho, A.V.; Bernardino, A.M.R.; Oliveira, C.D.;
Pinheiro, L.C.S.; Marra, R.K.F.; Castro, H.C.; Wardell, S.M.S.V.;
Wardell, J.L.; Amaral, V.F.; Canto-Cavalheiro, M.M.; Leon, L.L.;
Genestra, M. Synthesis and in vitro evaluation of new nenzenesul-
fonamides as antileishmanial agents. J. Braz. Chem. Soc., 2014,
25(5), 980-986.
[129] Marra, R.K.; Bernardino, A.M.; Proux, T.A.; Charret, K.S.; Lira,
M.L.; Castro, H.C.; Souza, A.M.; Oliveira, C.D.; Borges, J.C.; Ro-
drigues, C.R.; Canto-Cavalheiro, M.M.; Leon, L.L.; Amaral, V.F.
4-(1H-Pyrazol-1-yl) Benzenesulfonamide derivatives: identifying
new active antileishmanial structures for use against a neglected
disease. Molecules, 2012, 17, 12961-12973.
[130] Bhuniya, D.; Mukkavilli, R.; Shivahare, R.; Launay, D.; Dere,
R.T.; Deshpande, A.; Verma, A.; Vishwakarma, P.; Moger, M.;
Pradhan, A.; Pati, H.; Gopinath, V.S.; Gupta, S.; Puri, S.K.; Martin,
D. Aminothiazoles: hit to lead development to identify antileish-
manial agents. Eur. J. Med. Chem., 2015, 102, 582-593.
[131] Kumar, D.; Khan, S.I.; Tekwani, B.L.; Ponnan, P.; Rawat, D.S. 4-
Aminoquinoline-pyrimidine hybrids: synthesis, antimalarial activi-
ty, heme binding and docking studies. Eur. J. Med. Chem., 2015,
89, 490-502.
[132] Manohar, S.; Pavan, V.S.; Taylor, D.; Kumar, D.; Ponnan, P.;
Wiesner, L.; Rawat, D.S. Highly active 4-aminoquinoline–
pyrimidine based molecular hybrids as potential next generation
antimalarial agents. RSC Adv., 2015, 5, 28171-28186.
[133] Yuvaniyama, J.; Chitnumsub, P.; Kamchonwongpaisan, S.;
Vanichtanankul, J.; Sirawaraporn, W.; Taylor, P.; Walkinshaw.
M.D.; Yuthavong, Y. Insights into antifolate resistance from malar-
ial DHFR-TS structures. Nat. Struct. Biol., 2003, 10(5), 357-365.
[134] Pandey, S.; Agarwal, P.; Srivastava, K.; Rajakumar, S.; Puri, S.K.;
Verma, P.; Saxena, J.K.; Sharma, A.; Lal, J.; Chauhan, P.M. Syn-
thesis and bioevaluation of novel4-aminoquinoline-tetrazole de-
rivatives as potent antimalarial agents. Eur. J. Med. Chem., 2013,
66, 69-81.
[135] Tripathi, M.;Khan, S.I.; Thakur, A.; Ponnan, P.; Rawat, D.S. 4-
Aminoquinoline-pyrimidine-aminoalkanols: Synthesis, in vitro an-
timalarial activity, docking studies and ADME predictions. New J.
Chem., 2015, 39, 3474-3483.
[136] Inam, A.; Van Zyl, R.L.; Van Vuuren, N.J.; Chen, C.T.; Avecilla,
F.; Agarwal, S.M.; Azam, A. Chloroquinoline-acetamide hybrids: a
promising series of potential antiprotozoal agents. RSC Adv., 2015,
5, 48368-48381.
[137] Dambuza, N.S.; Smith, P.; Evans, A.; Norman, J.; Taylor, D.; An-
dayi, A.; Egan, T.; Chibale, K.; Wiesner, L. Antiplasmodial activi-
ty, in vivo pharmacokinetics and anti-malarial efficacy evaluation
Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 9 1079
of hydroxypyridinone hybrids in a mouse model. Malar. J., 2015,
14, 505-512.
[138] Hershko, C.; Theanacho, E.N.; Spira, D.T.; Peter, H.H.; Dobbin, P.;
Hider, R.C. The effect of N-alkyl modification on the antimalarial
activity of 3-hydroxypyridin4-one oral iron chelators. Blood, 1991,
77(3), 637-643.
[139] Andayi, W.A.; Egan, T.J.; Gut, J.; Rosenthal, P.J.; Chibale, K.
Synthesis, antiplasmodial activity and β -hematin inhibition of hy-
droxypyridone-chloroquine hybrids. ACS Med. Chem. Lett., 2013,
4(7), 642-646.
[140] Lödige, M.; Hiersch, L. Design and synthesis of novel hybrid mol-
ecules against Malaria. Int. J. Med. Chem., 2015, 2015. 1-23.
[141] Baird, J.K.; Fryauff, D.J.; Hoffman, S.L. Primaquine for prevention
of malaria in travelers. Clin. Infect. Dis., 2003, 37(12), 1659-1667.
[142] Fisher, G.U.; Gordon, M.P.; Lobel, H.O.; Runcik, K. Malaria in
soldiers returning from Vietnam. epidemiologic, therapeutic, and
clinical studies. Am. J. Trop. Med. Hyg., 1970, 19(1), 27-39.
[143] Njogu, P.M.; Gut, J.; Rosenthal, P.J.; Chibale, K. Design, Synthesis
and antiplasmodial activity of hybrid compounds nased on
(2R,3S)-N-Benzoyl-3-phenylisoserine. ACS Med. Chem. Lett.,
2013, 4(7), 637-641.
[144] Kingston, D.G.I. The shape of things to come: structural and syn-
thetic studies of taxol and related compounds. Phytochemistry,
2007, 68(14), 1844-1854.
[145] Mott, B.T.; Cheng, K.C.; Guha, R.; Kommer, V.P.; Williams, D.L.;
Vermeire, J.J.; Cappello, M.; Maloney, D.J.; Rai, G.; Jadhav, A.;
Simeonov, A.; Inglese, J.; Posner, G.H.; Thomas, C.J. A furoxan–
amodiaquine hybrid as a potential therapeutic for three parasitic
diseases. Medchemcomm, 2012, 3(12), 1505-1511.
[146] Sayed, A.A.; Simeonov, A.; Thomas, C.J.; Inglese, J.; Austin, C.P.;
Williams, D.L. Identification of oxadiazoles as new drug leads for
the control of schistosomiasis. Nat Med., 2008; 14(4), 407-412.
[147] Rai, G.; Sayed, A.A.; Lea, W.A.; Luecke, H.F.; Chakrapani, H.;
Prast-Nielsen, S.; Jadhav, A.; Leister, W.; Shen, M.; Inglese, J.;
Austin, C.P.; Keefer, L.; Arnér, E.S.J.; Simeonov, A.; Maloney,
D.J.; Williams, D.L.; Thomas, C.J. Structure mechanism insights
and the role of nitric oxide donation guide the development of
oxadiazole-2-oxides as therapeutic agents against schistosomiasis.
J. Med. Chem., 2009; 52(20), 6474-6483.
[148] Bisby, R.H. Reactions of a free radical intermediate in the oxida-
tion of amodiaquine. Biochem. Pharmacol., 1990; 39(12), 2051-
2055.
[149] Gonnert, R.; Andrews, P. Praziquantel, a new broad-spectrum
antischistosomal agent. Z. Paras., 1977; 52, 129-150.
[150] Guglielmo, S.; Cortese, D.; Vottero, F.; Rolando, B.; Kommer,
V.P.; Williams, D.L.; Fruttero, R.; Gasco, A. New praziquantel de-
rivatives containing NO-donor furoxans and related furazans as ac-
tive agents against Schistosoma mansoni. Eur. J. Med. Chem.,
2014, 84, 135-145.