Chemical conjugation of 2-hexadecynoic acid to C5-curcumin enhances its antibacterial activity against multi-drug resistant bacteria

Article abstract of DOI:10.1016/j.bmcl.2015.10.022

The first total synthesis of a C5-curcumin-2-hexadecynoic acid (C5-Curc-2-HDA, 6) conjugate was successfully performed. Through a three-step synthetic route, conjugate 6 was obtained in 13% overall yield and tested for antibacterial activity against methi

Full text of DOI:10.1016/j.bmcl.2015.10.022

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Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical conjugation of 2-hexadecynoic acid to C5-curcumin
enhances its antibacterial activity against multi-drug resistant
bacteria
a
a
a
David J. Sanabria-Ríos a, , Yaritza Rivera-Torres , Joshua Rosario , Ricardo Gutierrez ,
Yeireliz Torres-García a, Nashbly Montano b, Gabriela Ortíz-Soto c, Eddy Ríos-Olivares c,
José W. Rodríguez c, Néstor M. Carballeira b
⇑
a Faculty of Science and Technology, Inter American University of Puerto Rico, Metropolitan Campus, PO Box 191293, San Juan 00919, Puerto Rico
b Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan 00931-3346, Puerto Rico
c Department of Microbiology and Immunology, Universidad Central del Caribe School of Medicine, PO Box 60327, Bayamón 00960, Puerto Rico
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of a C5-curcumin–2-hexadecynoic acid (C5-Curc–2-HDA, 6) conjugate was suc-
cessfully performed. Through a three-step synthetic route, conjugate 6 was obtained in 13% overall yield
and tested for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains.
Our results revealed that 6 was active against eight MRSA strains at MICs that range between 31.3 and
Received 17 August 2015
Revised 6 October 2015
Accepted 8 October 2015
Available online xxxx
62.5 lg/mL. It was found that the presence of 2-hexadecynoic acid (2-HDA, 4) in conjugate 6 increased
4–8-fold its antibacterial activity against MRSA strains supporting our hypothesis that the chemical con-
nection of 4 to C5-curcumin (2) increases the antibacterial activity of 2 against Gram-positive bacteria.
Combinational index (CIn) values that range between 1.6 and 2.3 were obtained when eight MRSA strains
were treated with an equimolar mixture of 2 and 4. These results demonstrated that an antagonistic
effect is taking place. Finally, it was investigated whether conjugate 6 can affect the replication process
of S. aureus, since this compound inhibited the supercoiling activity of the S. aureus DNA gyrase at min-
Keywords:
C5-curcuminoids synthesis
Antibacterial agents
MRSA
Antagonism
DNA gyrase
imum inhibitory concentrations (MIC) of 250 lg/mL (IC50 = 100.2 13.9 lg/mL). Moreover, it was
observed that the presence of 4 in conjugate 6 improves the anti-topoisomerase activity of 2 towards
S. aureus DNA gyrase, which is in agreement with results obtained from antibacterial susceptibility tests
involving MRSA strains.
Ó 2015 Elsevier Ltd. All rights reserved.
Although our knowledge about infectious diseases produced by
multidrug-resistant bacteria has advanced greatly, neither their
incidence of spread nor their rate of death has decreased in the last
10 years. The Center for Disease Control and Prevention (CDC) esti-
mated that in the United States, more than 1.2% out of 2 million
people die every year from infections with multidrug-resistant
bacteria (MDRB).1 Infectious diseases caused by MDRB add
substantial costs to the nation’s health care system since these
infections require prolonged and expensive treatments, extended
hospitalizations, additional physician visits, and healthcare use,
which results in increased rate of disability and death.1 Despite
the fact that several compounds are being evaluated as antibacte-
rial agents, it is urgent to develop new antibiotics. Among the
compounds that are being evaluated, curcumin (1, Fig. 1) has
demonstrated to have both anticancer and antibacterial proper-
ties.2–6
Although preclinical and clinical studies have shown that 1 is
not toxic against normal human cells, several pharmacokinetics
disadvantages, such as poor bioavailability, fast metabolism and
the requirement of repetitive oral doses has been reported, which
limits its pharmacological applications.7–9 However, 1 is still an
excellent lead compound for the drug design and development of
new drugs on the basis of its explicit bioactivities, non-toxicity
and easy synthesis.10–12 Several applications have been reported
in order to overcome the pharmacokinetics disadvantages men-
tioned above, which includes the synthesis of bioconjugates of 1
bearing fatty acids,13,14 preparation of liposome-encapsulated 115
,
and synthesis of mono-carbonyl analogs of
1 (C5-curcumi-
noids).16–18
In 2008, the synthesis of (1E,4E)-1,5-bis(4-hydroxy-3-methoxy-
phenyl)penta-1,4-dien-3-one (C5-curcumin, 2, Fig. 1) was achieved
⇑
Corresponding author. Tel.: +1 787 250 1912x2332; fax: +1 787 250 8736.
E-mail address: dsanabria@intermetro.edu (D.J. Sanabria-Ríos).
http://dx.doi.org/10.1016/j.bmcl.2015.10.022
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sanabria-Ríos, D. J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.022

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D. J. Sanabria-Ríos et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
compared to 2 alone. MRSA strains were selected for this study
because the reduction of infections caused by these bacteria in hos-
pital settings represents a priority for the CDC, since these threats
are responsible for the largest outbreak of hospital acquired infec-
tions.1,20 We are particularly interested in conjugating 4 to 2
because we have demonstrated that this aFA is cytotoxic against
several MRSA strains in vitro.19
Figure 1. Chemical structures of Curcumin (1) and C5-Curcumin (2).
The synthesis of 6 started with the carboxylation of 1-pentade-
cyne (3) affording 4 in a 73% yield (Scheme 1). Then, 2-HDA was
conjugated to vanillin, using Steglich esterification conditions as
with the goal of evaluating its antibacterial properties.16,17 It was
found that 2 was biologically active against Gram-positive and
Gram-negative bacteria, including Staphylococcus aureus,
Micrococcus luteus, Staphylococcus saprophyticus, Enterococcus sp.,
Enterobacter cloacae, and Escherichia coli.16 Aimed at improving
the antibacterial properties of 1, the synthesis of a curcumin–
palmitic acid conjugate (Curc–PA) was performed.14 In this study,
Singh et al. demonstrated that the Curc–PA conjugate was five-fold
more active against Streptococcus viridans, E. coli, Klebsiella
pneumoniae, and Proteus miraleilis than 1 itself. These authors pos-
tulated that the enhanced activity of the Curc–PA conjugate is due
to the presence of the PA moiety, which decreases the rate of
metabolism of curcumin through the hydrolysis of ester bonds
by carboesterases present in the cells, provides structural similar-
ity to the cell wall thus allowing a better uptake of 1, enhances its
effective concentration, and improves the biological activity of cur-
cumin after enzymatic hydrolysis of the ester group in the Curc–PA
conjugate.14 The results mentioned above prompted us to ask
whether the chemical conjugation of an antibacterial fatty acid to
described in the literature,21,22 affording
5 in a 40% yield.
Compound 5 was subsequently reacted with acetone and lithium
hydroxide monohydrate to obtain 6 in a 45% yield. The synthetic
strategy displayed in Scheme 1 was also used to prepare the C5-
curcumin–palmitic acid (C5-Curc–PA) conjugate as described by
Sanabria-Ríos et al.22
The FT-IR analyses were performed in a Thermo Nicolet IS5 and
NMR analyses were performed in a Bruker Avance DPX-300 spec-
trometer. In FT-IR, two bands were observed at 1651 and
1580 cmꢀ1. These bands are generated by one carbonyl group from
the ester moiety and the other carbonyl group from the a,b-diun-
saturated ketone moiety in 6. In 1H NMR, two doublets at 7.3 and
6.4 ppm were also observed for 6. These signals have a coupling
constant (J) of 20 Hz, which are characteristic of compounds con-
taining a trans alkene moiety.23 Two signals at 188 and 168 ppm
were also observed in 13C NMR, which confirm the presence of
the
a
,b-unsaturated and ester carbonyl groups, respectively.23
The purity of 6 was determined to be >95% by 13C NMR.24
2
will improve its antibacterial activity against methicillin-
Once 6 was prepared, the conjugate was tested against E. coli
(ATCC 25922) and MRSA (ATCC 43300) strains, which were
obtained from the American Type Culture Collection (Manassas,
VA). In addition, 6 was tested against seven clinical isolates of
MRSAs, which were kindly donated from a community hospital
in San Juan, Puerto Rico (see Supporting information). It can be
observed from Table 1 that 2 displayed antibacterial activity
against eight MRSA strains at MICs that range between 250 and
resistant S. aureus (MRSA) strains.
Recently, Sanabria-Ríos et al. published the total synthesis of a
series of 2-acetylenic fatty acids (2-aFAs) and other synthetic ana-
logs such as 2-tetrahydropyranyl protected alkynols and 2-alky-
nols aimed at establishing a structure activity relationship (SAR)
with these compounds to find the acetylenic fatty acid (aFA) with
better antibacterial activity against both Gram-positive and
Gram-negative bacteria.19 Among all the compounds tested,
2-hexadecynoic acid (2-HDA, 4) displayed the best overall antibac-
terial activity against Gram-positive S. aureus (MIC = 15.6
Staphylococcus saprophyticus (MIC = 15.6 g/mL), and Bacillus
cereus (MIC = 31.3 g/mL), as well as against the Gram-negative
Klebsiella pneumoniae (7.8 g/mL) and Pseudomonas aeruginosa
(MIC = 125 g/mL). In addition, 4 displayed significant antibacte-
rial activity against methicillin-resistant S. aureus (MRSA) ATCC
43300 (MIC = 15.6 g/mL) and clinical isolates of MRSA
(MIC = 3.9 g/mL). Moreover, we recently determined that 4 is
more active than palmitic acid (MIC >1000 g/mL) against
500 lg/mL, which are in agreement with those results obtained
by Liang and collaborators.16 The data presented in Table 1 also
demonstrate that the presence of 4 in conjugate 6 increased 4–8-
fold the antibacterial activity against MRSA strains supporting
our hypothesis that the chemical connection of 4 to 2 increases
the antibacterial activity of 2 against Gram-positive bacteria.
According to Singh et al., fatty acids are natural components of cell
membranes, therefore by conjugating 4 to 2 it is expected that this
connection enhances the cellular uptake of 2, increases its
lipophilicity, improves its half-life, and reduces its rate of intracel-
lular metabolism by carboesterases, which ensures its low toxicity
and high bioavailability.14 It is important to mention, that the
antibacterial activity of both palmitic acid (PA) and the C5-Curc–
PA conjugate against eight MRSA strains was also tested (Table 1).
Results from this test revealed that this conjugate was not active
lg/mL),
l
l
l
l
l
l
l
S. aureus, which demonstrates that the presence of a triple bond
at C-2 is important for the antibacterial activity of 4. Taken
together, these findings highlight 4 as a novel agent that can be
used as a pharmacophore in synthetic applications.
In the present study, the Steglich esterification approach was
used to synthesize the C5-curcumin–2-HDA (C5-Curc–2-HDA, 6)
conjugate in order to determine its antibacterial activity against
Gram-positive and Gram-negative bacteria, including clinical iso-
lates of MRSA (CIMRSA). We hypothesize that by connecting 4 to
2 in 6 its antibacterial activity against MRSA will improve when
against MRSA strains (MIC >1000 lg/mL), which implies that the
triple bond at C-2 in 4 has an important role in the antibacterial
activity of conjugate 6 against MRSA strains. It can be noted from
Table 1 that conjugate 6 is 32–64-fold less active than 4. These
results suggest that the addition of a curcuminoid moiety to 4 does
not help in increasing its antibacterial activity against
Scheme 1. Total synthesis of C5-Curc–2-HDA conjugate (6). (i) CO2 (xs.), n-BuLi, THF, ꢀ78 °C, 24 h; (ii) 1 M HCl, (iii) vanillin, DIC, dry THF–CH2Cl2 (1:1 v/v), DMAP, rt; (iv)
acetone, LiOH–H2O, 24 h, rt.
Please cite this article in press as: Sanabria-Ríos, D. J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.022

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D. J. Sanabria-Ríos et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Antibacterial activity of 2, 4, and conjugate 6 against MRSA strainsa,b
Test organism
2
4
6
MIC/IC50 SEM,
Methc
lg/mL
PA
C5-Curc–PA
Ciprod
MRSA (ATCC 43300)
CIMRSA 1
CIMRSA 2
CIMRSA 3
CIMRSA 4
CIMRSA 5
CIMRSA 6
CIMRSA 7
E. coli (ATCC 25922)
250/111.3 10.8
250/141.0 12.4
250/133.4 15.8
250/123.8 26.2
500/184.9 8.2
500/202.4 24.5
500/194.7 29.4
500/303.1 33.6
>1000
7.8/5.1 0.1
7.8/4.2 0.1
7.8/5.0 0.5
15.6/6.3 0.2
15.6/6.5 0.1
7.8/5.1 0.2
7.8/5.7 0.8
7.8/4.7 0.3
>1000
62.5/39.5 1.9
62.5/30.3 2.1
62.5/34.9 1.6
62.5/34.2 1.0
31.3/24.9 1.5
31.3/25.4 1.0
62.5/38.9 1.6
62.5/34.2 0.4
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1.95/0.98 0.02
7.8/2.2 0.7
31.3/11.8 3.9/7.3
3.9/2.2 0.7
31.3/11.8 3.9
3.9/1.9 0.4
1.9/1.2 0.1
3.9/1.3 0.2
>1000
0.50/0.20 0.09
15.6/10.4 3.7
3.9/2.9 1.2
7.8/4.4 0.8
3.9/2.0 0.2
15.6/5.3 0.5
7.8/2.5 0.2
0.98/0.39 0.04
<0.008
a
b
c
Experiments were performed in triplicate (N = 3).
CIMRSA = Clinical isolate of methicillin-resistant S. aureus. CIMRSA stains were kindly donated by a community hospital in San Juan, Puerto Rico.
Meth = methicillin. Positive control.
Cipro = Ciprofloxacin. Positive control.
d
Table 2
Cytotoxic activity of 2 and 6 against both normal Vero cells and PBMC isolated from healthy volunteersa,b
Compound
Test MRSA strains
Vero cells cytotoxicity IC50 SEM (
lg/mL)
PBMC cytotoxicity IC50 SEM (lg/mL)
Therapeutic index (TI)c
ATCC 43300
CI 1
CI 2
CI 3
CI 4
CI 5
CI 6
CI 7
2
4
6
0.4
13.5
6.2
0.3
16.4
8.1
0.4
13.8
7.1
0.4
10.9
7.2
0.3
10.6
9.9
0.2
13.5
9.7
0.2
12.1
6.3
0.2
14.7
7.2
5.7 0.4
1.6 0.4
>100.0
46.9 0.8
68.9 0.7
246.8 12.8
a
b
c
Experiments were performed in triplicate (N = 3).
CI = Clinical isolate of methicillin-resistant S. aureus.
TI was determined by dividing PBMC cytotoxicity by the inhibitory concentration (IC50) of the drug.
Table 3
Gram-positive bacteria. Taken together these results are in agree-
ment with the findings reported by Sanabria-Ríos et al., where they
demonstrated the importance of both the carboxylic acid moiety
and the triple bond at C-2 in the antibacterial activity of aFAs
against MDRB.19
Combination index (CIn) values for the equimolar mixture of 2 and 4 in MRSAa
Test organism
CIn values
MRSA (ATCC 43300)
CIMRSA 1
CIMRSA 2
CIMRSA 3
CIMRSA 4
CIMRSA 5
CIMRSA 6
CIMRSA 7
2.0
2.0
2.2
1.7
1.6
1.9
2.2
2.3
The toxicities of
2 and 6 were determined by the CTG
Luminescent Cell Viability Assay (see Supporting information). In
addition to Vero cells, the toxicity of 2 and 6 was also determined
in peripheral blood mononuclear cells (PBMCs) from healthy vol-
unteers as described by Sanabria-Ríos et al.19,22 The cytotoxicity
of 2 and 6 against both Vero cells and PBMCs are displayed in
Table 2. Therapeutic indexes (TIs) of the above-mentioned com-
pounds were calculated as reported by Sanabria-Ríos.19
a
CIn vales were determined by using the following equation
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
½2ꢁ
½4ꢁ
½2ꢁx ½4ꢁ
: CIn ¼
þ
þ
, where [2] and [4] are concentrations of 2 and 4 in
½2ꢁx
½4ꢁx
½2ꢁx ꢂ½4ꢁx
combination, inhibiting 50% of cell viability, while [2]x and [4]x are the doses of 2
Cytotoxicity results displayed in Table 2 reveal that 2 is toxic
against both Vero cells and PBMC at IC50’s of 5.7 and 46.9 lg/mL,
and 4 alone, respectively, inhibiting 50% of cell viability.
respectively. It can be appreciated that 2 is 8.2-fold more toxic
against Vero cells than PBMC suggesting that 2 is more toxic
towards monkey kidney cells. In terms of TIs, conjugate 6 is
14.7–48.0-fold more therapeutically viable than 2. Results in
Table 2 also reveal that 4 is more selective towards MRSA strains,
but more cytotoxic against both Vero cells and PBMC, which is in
agreement with results obtained by Sanabria-Ríos et al.19
Moreover, these results demonstrate that the chemical connection
between 4 and 2 enhance the antibacterial activity of 2 against
MRSA strains. The results presented in Table 2 are important
because these demonstrate that the conjugation of 2-alkynoic fatty
acids can improve the antibacterial properties of C5-curcuminoids
and decrease their cytotoxicity against eukaryotic normal cells,
which represents a step forward towards the development of a
new generation of antibacterial drugs against bacterial infections
caused by MDRB.
susceptibility tests were performed using clinical isolates of MRSA
(CIMRSA) that were exposed to an equimolar mixture of 2 and 4.
Synergism was assessed by constructing dose–response plots and
calculating a combinational index (CIn) as described by Chou.25
A
CIn <1 represents a case where synergism between 2 and 4 is
present, while CIn values of 1 and >1 represent additive and antag-
onistic effects, respectively. Based on the CIn values presented in
Table 3, we can appreciate an antagonistic effect when CIMRSA
strains were treated with an equimolar mixture of 2 and 4.
Because the equation for calculating CIn assumes that drugs act
through an entirely different mechanisms of action,25 we theorize
that the presence of 2, in an equimolar mixture, may block effec-
tively the active site of important proteins, such as DNA topoiso-
merases26 and enoyl-ACP reductase InhA27
, involved in the
antibacterial activity of 4, which could decrease the cytotoxicity
of 4 against MRSA strains. Therefore, all of this data strongly sup-
port the idea that different mechanisms of action are taking place
when CIMRSA are treated with an equimolar mixture of 2 and 4.
Results in Table 1 provoked us to ask whether a synergistic
effect exists when treating MRSA strains with an equimolar mix-
ture of both 2 and 4. In order to determine synergy, microdilution
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Figure 2. Representative ethidium bromide stained gels showing the inhibitory activity of C5-Curc–PA (A), 2 (B), 4 (C), and conjugate 6 (D) against the S. aureus DNA gyrase.
Dose–response plots showing the inhibitory activity of 4 and 6 against S. aureus DNA gyrase (E). Experiments were performed in triplicate (N = 3).
Several studies have demonstrated that curcumin and other
curcuminoids display inhibitory activity against DNA topoiso-
merases I and II.28–30 DNA topoisomerases are enzymes involved
in generating the necessary topological and conformational
changes in DNA, which are essential for several cellular processes
such as replication, recombination, and transcription.31 In addition
to their normal cellular functions, DNA topoisomerases are known
to be important molecular targets for some anticancer32,33 and
antibacterial drugs.34–36 In order to get more insight regarding
the mechanism of action of conjugate 6, we performed several
DNA topoisomerase II inhibitory tests by using the S. aureus DNA
gyrase kit (TopoGEN, Inc., Port Orange, FL). In addition to 6, the
ability of 2 and 4 to inhibit the supercoiling activity of S. aureus
DNA gyrase was also tested (see Supporting information).
active site of the S. aureus DNA gyrase resulting in an inhibitory
effect of the supercoiling activity of this enzyme, which could
affect the DNA replication process in MRSA.
In summary, we successfully performed the total synthesis of
conjugate 6 in three synthetic steps and in an overall yield of
13%. Our results clearly demonstrated that the chemical connec-
tion of 4 to 2 in conjugate 6 improves both the antibacterial activ-
ity of 2 against MRSA strains and its inhibitory effect in inhibiting
the supercoiling activity of DNA gyrase, an important enzyme
involved in the DNA replication process in bacteria. We are in
the process of synthesizing other C5-Curc–aFA conjugates contain-
ing different carbon chain lengths, triple bond positions, and
degree of unsaturation in order to further explore their antibacte-
rial properties and mechanism of action.
The results in Figure 2 show the ability of C5-Curc–PA, 2, 4 and
6 in inhibiting the supercoiling activity of the S. aureus DNA gyrase.
Although these results demonstrate that 4 (Fig. 2C) is 3.7-fold more
active against DNA gyrase than 6 (Fig. 2D), the results also support
that the chemical connection of 4 to 2 in conjugate 6 enhances the
anti-topoisomerase activity of 2 (Fig 2A–D). In addition, it can be
appreciated that C5-Curc–PA was not inhibitory against the
S. aureus DNA gyrase, which suggests that the presence of a triple
bond at C-2 in the fatty acid moiety is important for the anti-
topoisomerase activity. Results displayed in Figure 2 are consistent
with the findings presented in Table 1, whereby the antibacterial
Acknowledgements
This project was supported by the National Center for Research
Resources and the National Institute of General Medical Sciences of
the National Institutes of Health through Grant Number 5 P20 GM
103475-13. J. Rosario acknowledges the support of the PR-INBRE
program for an undergraduate fellowship. This project was also
supported in part by the PRCTRC Grant through the Grant Numbers
U54 RR026139 and 8U54MD 007587-03, and by the National Insti-
tute on Minority Health and Health Disparities of the National
Institutes of Health through Grant Number 8G12MD007583-28.
N. Montano thanks the UPR-Rio Piedras RISE program for a gradu-
ate fellowship.
activity of
2 towards MRSAs is improved when connecting
compound 4 to 2 in a C5-Curc–2-HDA conjugate. The anti-
topoisomerase results in Figure 2, suggest that the presence of 4
in conjugate 6 promotes the interaction of compound 2 with the
Please cite this article in press as: Sanabria-Ríos, D. J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.022