Synthesis and activity of novel homodimers of Morita–Baylis–Hillman adducts against Leishmania donovani: A twin drug approach

Article abstract of DOI:10.1016/j.bmcl.2016.07.022

It is reported here the synthesis of novel Homodimers 12–19 of Morita–Baylis–Hillman adducts (MBHA) from one-pot Morita–Baylis–Hillman Reaction (MBHR) between aromatic aldehydes as eletrophiles and ethylene glycol diacrylate as Michael acceptor (35–94% yields) using cheap and green conditions. The bioactivities were evaluated against promastigote form of Leishmania donovani. All homodimers showed to be more potent than corresponding monomers. It is worth highlighting that the halogenated homodimers 17 and 18 (0.50?μM) is almost 400 times more active than the corresponding monomer 10 and 1.24 times more potent than the second-line drug amphotericin B (0.62?μM). Moreover, the selectivity index to 18 is very high (SI_rb?>?400) far better than amphotericin B (SI_rb?=?18.73). This is the first report of twin drugs strategy applied on Morita–Baylis–Hillman adducts.

Full text of DOI:10.1016/j.bmcl.2016.07.022

Accepted Manuscript
Synthesis and activity of novel homodimers of Morita-Baylis-Hillman adducts
against Leishmania donovani: a twin drug approach
Wagner A.V. da Silva, Daniele.C. Rodrigues, Ramon G. de Oliveira, Rhuan
K.S. Mendes, Tayná R. Olegário, Juliana C. Rocha, Tatjana S.L. Keesen,
Claudio G. Lima-Junior, Mário L.A.A. Vasconcellos
PII:
S0960-894X(16)30724-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.022
BMCL 24066
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 June 2016
6 July 2016
7 July 2016
Please cite this article as: da Silva, W.A.V., Rodrigues, Daniele.C., de Oliveira, R.G., Mendes, R.K.S., Olegário,
T.R., Rocha, J.C., Keesen, T.S.L., Lima-Junior, C.G., Vasconcellos, M.L.A., Synthesis and activity of novel
homodimers of Morita-Baylis-Hillman adducts against Leishmania donovani: a twin drug approach, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.022
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and activity of novel homodimers of Morita-Baylis-Hillman adducts against
Leishmania donovani: a twin drug approach
Wagner A. V. da Silva a, Daniele. C. Rodrigues a, Ramon G. de Oliveira, a Rhuan K. S. Mendes a, Tayná R.
Olegário a,Juliana C. Rocha b, Tatjana S. L. Keesen b, Claudio G. Lima-Junior a,* and Mário L. A. A.
Vasconcellos a,*
a Departamento de Química, Universidade Federal da Paraíba, Campus I, 58059-900 João Pessoa, PB, Brazil.
b Departamento de Biotecnologia, Universidade Federal da Paraíba, Campus I, 58059-900 João Pessoa, PB, Brazil.
ARTICLE INFO
ABSTRACT
It is reported here the synthesis of novel Homodimers 12-19 of Morita-Baylis-Hillman adducts
(MBHA) from one-pot Morita-Baylis-Hillman Reaction (MBHR) between aromatic aldehydes
as eletrophiles and ethylene glycol diacrylate as Michael acceptor (35-94% yields) using cheap
and green conditions. The bioactivities were evaluated against promastigote form of
Leishmania donovani. All homodimers showed to be more potent than corresponding
monomers. It is worth highlighting that the halogenated homodimers 17 and 18 (0.50 µM ) is
almost 400 times more active than the corresponding monomer 10 and 1.24 times more potent
than the second-line drug amphotericin B (0.62µM). Moreover, the selectivity index to 18 is
very high (SIrb >400) far better than amphotericin B (SIrb=18.73). This is the first report of twin
drugs strategy applied on Morita-Baylis-Hillman adducts.
Article history:
Received
Revised
Accepted
Available online
Keywords:
Morita-Baylis-Hillman adducts
Homodimers
Twin drugs
Leishmania donovani
2009 Elsevier Ltd. All rights reserved
.
Leishmaniasis is an infectious disease caused by protozoan
Leishmania, and affects millions worldwide. As this parasite
infection leads to an immunological imbalance outcome. Deeper
studies on the new treatments and mechanisms involved in
intracellular pathogen control are essential. Infection with the
protozoa parasite Leishmania can cause several clinical forms of
disease such as tegumentary or visceral forms,1,2 this last one is
the severe form and the major cause of mortality This clinical
form is caused by Leishmania donovani 2 and the development of
new drugs and treatments is a necessity due to the resistance and
cytotoxicity of commercial drugs used in treatment of human
visceral leishmaniasis (HVL). In this context, the Morita-Baylis-
This approach is supported by an association of two identical
pharmacophoric entities generating an “identical twin drug”
which is equivalent to a homodimer derivative.9-11 Some authors
have been suggested that administration of twin drugs can be
Hillman reaction (MBHR)
is an important chemical
transformation which allows not only access to poly-
functionalized molecules with considerable synthetic potential as
also promising biological profile.4,5 Several Morita-Baylis-
Hillman adducts (MBHA) have been reported as bioactive
compounds against neglected diseases such as leishmaniasis,
schistosomiasis, malaria and Chagas disease.6
In connection with our continuum interest in synthesis of new
Morita-Baylis-Hillman adducts with potent antiparasitic activity
herein we report the synthesis of homodimers of Morita-
Baylis-Hillman adducts based on twin drug approach.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢁ
favorable when compared with two separated drugs presenting
own pharmacokinetic and pharmacodynamics proprieties.10-12
6-8
The antiparasitic Piperaquine (1), bis-aminoacridine (2) and
potent acetyl cholinesterase inhibitor bis-tacrine (3) are some
examples of twin drugs (homodimers) present in literature
(Figure. 1).12,13
ꢁ
#
Corresponding authors. E-mail addresses: claudio@quimica.ufpb.br.
(Lima-Junior, Claudio), mlaav@quimica.ufpb.br (Vasconcellos, Mário).

Figure 1. Synthetic bioactive homodimers as twin drugs.
We began our experimental work preparing ethylene glycol
diacrylate 20 (Scheme 1). This compound was synthetized by
reaction between acrylic acid, ethylene glycol under p-toluene
sulfonic acid catalysis at 110 °C. The diacrylate 20 was obtained
in 86% after 6 hours of reflux.16 1H NMR and 13C NMR analysis
confirmed the structure 20 (see supplementary data).
The design of new homodimers of Morita-Baylis-Hillman
adducts was based on compounds 4-11 presented in Figure 2.
Except 9, all Morita-Baylis-Hillman adducts already presented
activity against promastigote s and amastigotes forms of
Leishmania amazonensis and Leishmania chagasi.7,15 The
structure of eight new homodimers synthesized in this work are
presented in Figure 3.
Scheme 1. (a) Acrylic acid (2 equiv.), TsOH (10%mol),
hydroquinone, cyclohexane, Dean-stark, 110 °C, 6h (86%); (b) 1
equiv. DABCO, Aldehydes (2 equiv.), optimized conditions in Table
3 (35-94%).
Due to higher reactivity of 2-nitrobenzaldehyde and
bioactivity against leishmaniasis of MBHA derivatives of this
starting material, we have decided to find an efficient
experimental protocol to optimize the synthesis of 13, and then
apply such protocol to synthesize other MBHA 12, 14–19.
Initially, to a mixture 2:1 of 2-nitrobenzaldehyde and 20 as
Michael acceptor in acetonitrile, DABCO as promoter was added
and stirred for 24 hour at room temperature. The reaction
afforded homodimeric MBHA 13 in 88%. After we have studied
the solvent effect. Thus, MBHR were performed between the 2-
nitrobenzaldehyde and acceptor 20 using some solvents, such as
Figure 2. leishmanicides MBHA previously described by us (4-8,
10, 11) and described here (9).
t
t
DMF, DMF: H2O (6:4), BuOH, BuOH:H2O (6:4) and DMSO.
The reactions time were fixed in 24 h. It was observed that 13
was obtained in better yield using DMF (94% yield). With the
solvent optimal conditions established, we investigated the
influence of temperature in this reaction. Thus, reactions were
performed at 80 °C under microwave irradiation and low
temperature (0 °C) 17,18 in the presence of DABCO and DMF. It
was observed that 13 was obtained in low yields in microwave
and temperature protocols (25% yields).
With the optimal conditions for the preparation of 13, we
examined the MBHR between aromatic aldehydes as electrophile
and ethylene glycol diacrylate 20 as Michael acceptor aiming to
obtain new homodimers of MBHA (12-19). All reactions were
carried out using DMF as solvent, DABCO (1 equiv.) as
promoter at room temperature. The results of 12-19 preparations
are presented in Table 1.
Figure 3. New Homodimers (12-19) of MBHA synthesized and biologically evaluated in this article.
Table 1. Synthesis of homodimers of Morita-Baylis-Hillman adducts (12 – 19).
Entry
Ar
Homodimer
Time
20 days
24 h
Yield %
60
1
2
3
4
5
C6H5
12
13
14
15
16
o-(NO2)C6H4
m-(NO2)C6H4
p-(NO2)C6H4
p-(F)C6H4
94
24 h
88
24 h
92
20 days
35
6
7
8
p-(Cl)C6H4
p-(Br)C6H4
2-C10H7
20 days
20 days
20 days
48
40
35
17
18
19
2

Leishmania express many peptidases during their life, and
cysteine are the most abundant proteases.28
It is possible to highlight in Table 1 that homodimers 12-15
were obtained in moderate to excellent yields (Table 1, entries 1-
4). However, no efficient results were obtained preparing
homodimers 16-19, due to long reactions times, low yields were
obtained ranging from 35-48% (Table 3, entries 5-8). Up to date,
the Morita-Baylis-Hillman reaction with diacrylates like-20 was
reported only once in the literature.19 The synthesis described by
Shanmugam et al. performed in 35% yield using 1,6-hexanediol
acrylate and isomerics pyridinecarboxaldehydes.19 The
homodimers synthesized here were characterized by
spectroscopic analysis such as 1H, 13C NMR and HRMS (for
detailed information see the supplementary data). The adducts 4 -
11 also were prepared using procedures recently described by
us.20 Homodimers 12-19 and monomers MBHA 4-11 were
evaluated in vitro against the promastigote forms of Leishmania
donovani 21-24 .The IC50 values are shown in Table 2.
However, considering only the presence of two identical
pharmacophoric groups, this fact does not explain the enormous
difference in activity of 10 monomer and homodimer 18 (396.1
times more potent). We believe that the pharmacokinetic
properties (ADME) and especially the conformational change of
the dimeric 18 versus monomeric 10 can explain this difference
in bioactivity.
Table 2. Antileishmanial activity of compounds 4-19 reference drug
Amphotericin B.
c
MBHA
4
SIrb
miLogP d
1.57
2.64
1.48
2.46
1.50
2.51
1.53
2.56
2.25
2.97
2.25
4.00
2.38
4.26
2.75
5.01
-2.49
IC50a µM
953.13 47.66
HC50b µMꢀ
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Figure 4. Insight about possible mechanism of action of homodimers
of Morita-Baylis-Hillman adducts.
12
5
126.20
690.29 34.51
24.77 1.23
161.3 1 8.07
43.01 2.15
165.36 8.27
44.64 2.23
610.00 30.50
42.37 2.12
201.77 10.09
10.47 0.52
196.55 9.53
0.500 0.03
185.37 9.27
6.01
In Figure 5, the conformational minima of 10 and 18 are shown.29
We can note in this complex cage-Like conformation of 18
several intramolecular hydrogen bonds and interactions between
the aromatic rings in Like-T position.30 Moreover, unlike the 10
minimum, in conformational minimum of 18 there are a s-cis and
a s-trans geometries to the acrylates moieties. Noteworthy is the
direct attraction of the two bromine atoms which has great
importance in molecular geometry of the dimer 18. It is well
reported in the literature the importance of halogens in increasing
bioactivity due to chlorine and bromine connections in enzymatic
13
6
14
7
15
8
-
-
-
16
9
>200
>19.10
17
10
18
11
19
-
>200
-
-
31
environment However, we do not know the pharmacophoric
>400.00
conformations of 10 and 18. Studies on biological mechanisms of
action of 18 are now in progress.
-
-
38.98
0.62
1.95
0.03
-
Amphotericin
B
11.61
18.73
at The half maximal inhibitory concentration. b 50% hemolysis in red
blood cells . c SIrb (selectivity index on red blood cell) = HC50/IC50. d
miLogP
values
were
calculated
by
Molinspiration
Cheminformatics.25
We can observe in Table 2 that homodimers 12-19 are much
more potent than corresponding monomers MBHA 4μ11, proving
once again the successful strategy of twin drugs. 9-11 Surprisingly
homodimer 18 showed IC50 = 0.50 µM being almost 400 times
more active than respective monomer 10 and showing to be 1.24
times more active than reference drug amphotericin B (IC50
=
0.62 µM). Although amphotericin B is the second-line drug, it is
well known for its severe and potential lethal side effects, and too
expensive for the majority of the poor population. The
haemolytic assays (HC50 > 200µM) showed that the 17 and 18
did not exhibit toxicity towards human red blood cells, unlike
amphotericin B (HC50=11.61µM, Table 2).26 It is worth noting
here that this is the first report on the strategy of twin drugs
applied on Morita-Baylis-Hillman adducts.
Figure 5. Conformational minima of monomer 10 and homodimer
18. Since enantiomers present same energies, the calculations were
performed for (R)-10. However, homodimer 18 has two asymmetric
carbons and we show the conformational minimum of most stabled
diastereomer.
Up to now, there is no knowledge of the exact mechanism of
action MBHA in Leishmania targets. A proposal to rationalize
our results is that the two unsaturated α,β-carbonyl portion
present in homodimers does hereby as covalent irreversible
inhibitors of Leishmania proteases and more active than aromatic
Morita-Baylis-Hillman adducts 4 - 11 (Figure 4). This is based
on the fact, recently described, where similar MBHA were
classified as covalent irreversible thiol-trapping agents.27
In conclusion, we have synthesized eight new homodimers of
MBHA 12-19 from reaction between diacrylate 20 and aromatic
aldehydes as starting materials. All homodimers were designed
based on twin drug approach and 12–19 were much more active
against promastigote forms of Leishmania donovani than
respective monomers MBHA 4-11. The homodimer 18 (0.50
µM) is almost 400 times more active than the corresponding
monomer 10 and 1.24 times more potent than second-line drug
3

µg/mL of Morita-Baylis-Hillman adduct. The plates were
incubated for 72 h in a biological oxygen demand (B.O.D.)
incubator at 26º C. Cytotoxicity to promastigotes was evaluated by
the 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay . The absorbance was measured using
an ELISA plate reader (ELx800, BIOTEK) at 540 nm.
Anphotericin® was used as a positive control. The viability of
promastigotes incubated in the presence of different
concentrations of adducts was determined by comparing to culture
control (L. donovani cultivated in Schneider only). The
amphotericin B (0.62 µM). Besides this, homodimer 18 does not
exhibit toxicity on red blood cell studies (SIrb >400) which
represents a major advance in the VL treatment. These new
homodimers are very easy and cheap to prepare in green
conditions. This is the first report of twin drugs strategy applied
on Morita-Baylis-Hillman adducts which have showed promising
as novel leishmanicidal drug candidates.
Acknowledgments
concentration that caused a 50% reduction in cell viability (IC50
)
was calculated by Probit analysis (SPSS 13.0 for Windows). Each
experiment was performed in duplicate and repeated at least 3
times.
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financial support.
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(MHOM/ET/1967/HU3) was provided by Oswaldo Cruz Institute
Leishmania Collection-Fiocruz-RJ-Brazil. Promastigotes were
maintained in vitro at 26º C in Schneider's medium, pH 7 (20%
heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin
and 100 mg/mL streptomycin and 2% of male urine), as
previously described
Supplementary data associated with this article can be found, in
the online version, at…
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23. Antipromastigotes activities: promastigotes in the logarithmic
growth phase were cultured in 96-well cell culture plates at 1 X
106 parasites per well in 100 mL of Schneider's medium with
increasing concentrations of 3,12, 6,25, 12,5, 25, 50, 100, 200, 400
4

33. Graphical Abstract
Synthesis and activity of
Leave this area blank for abstract
novel homodimers of
Morita-Baylis-Hillman
adducts against Leishmania
donovani: a twin drug
approach
Wagner A. V. da Silva, Daniele C. Rodrigues, Ramon G. Oliveira, Rhuan K. S. Mendes,
Tayná R. Olegário, Juliana C. Rocha, Tatjana S. L. Keesen, Claudio G. Lima-Junior and
Mário L. A. A. Vasconcellos.
5