International Journal of

Molecular Sciences

Article
Polyphenols with Anti-Inflammatory Properties: Synthesis and
Biological Activity of Novel Curcumin Derivatives
Yisett González 1,2 , Randy Mojica-Flores 3 , Dilan Moreno-Labrador 1 , Luis Cubilla-Rios 2,4 ,
K. S. Jagannatha Rao 5,6 , Patricia L. Fernández 1,2 , Oleg V. Larionov 7, * and Johant Lakey-Beitia 2,3, *

1 Center for Molecular and Cellular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de
Alta Tecnología (INDICASAT AIP), Clayton, City of Knowledge, Panama City 0843-01103, Panama
2 Sistema Nacional de Investigación (SNI), SENACYT, Panama City 0816-02852, Panama
3 Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta
Tecnología (INDICASAT AIP), Clayton, City of Knowledge, Panama City 0843-01103, Panama
4 Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Sciences and Technology,
University of Panama, Panama City 0824-03366, Panama
5 Center for Neuroscience, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT
AIP), Clayton, City of Knowledge, Panama City 0843-01103, Panama
6 Department of Biotechnology, Koneru Lakshmaiah Education Foundation (KLEF) Deemed to be University,
Vaddeswaram 522 302, India
7 Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249, USA
* Correspondence: [email protected] (O.V.L.); [email protected] (J.L.-B.);
Tel.: +1-210-458-6050 (O.V.L.); +507-517-0700 (J.L.-B.)

Abstract: Herein, we describe the synthesis and evaluation of anti-inflammatory activities of new cur-
cumin derivatives. The thirteen curcumin derivatives were synthesized by Steglich esterification on
Citation: González, Y.; Mojica-Flores, one or both of the phenolic rings of curcumin with the aim of providing improved anti-inflammatory
R.; Moreno-Labrador, D.; activity. Monofunctionalized compounds showed better bioactivity than the difunctionalized deriva-
Cubilla-Rios, L.; Rao, K.S.J.; tives in terms of inhibiting IL-6 production, and known compound 2 presented the highest activity.
Fernández, P.L.; Larionov, O.V.; Additionally, this compound showed strong activity against PGE2 . Structure–activity relationship
Lakey-Beitia, J. Polyphenols with
studies were carried out for both IL-6 and PGE2 , and it was found that the activity of this series of
Anti-Inflammatory Properties:
compounds increases when a free hydroxyl group or aromatic ligands are present on the curcumin
Synthesis and Biological Activity of
ring and a linker moiety is absent. Compound 2 remained the highest activity in modulating IL-6
Novel Curcumin Derivatives. Int. J.
production and showed strong activity against PGE2 synthesis.
Mol. Sci. 2023, 24, 3691. https://
doi.org/10.3390/ijms24043691
Keywords: curcumin; curcumin derivatives; succinate; difunctionalized; monofunctionalized; cytokine;
Academic Editors: Marco Fiore, IL-6; PGE2 ; anti-inflammatory activity; structure–activity relationship
Cláudia Nunes dos Santos,
Natasa Loncarevic-Vasiljkovic,
Inês Figueira and María
Ángeles Ávila-Gálvez
1. Introduction
Received: 11 December 2022 Polyphenols are natural products with several benefits for human health [1–4]. Polyphe-
Revised: 2 February 2023 nols consist of a wide range of compounds that have multiple therapeutic properties [1–4].
Accepted: 3 February 2023
Curcumin (1) is a fascinating polyphenol with several biological effects, such as anti-
Published: 12 February 2023
inflammatory, neuroprotective, antioxidant, and anticancer activities [2,5–12]. This pleiotropic
molecule is found in the rhizome of Curcuma longa, and is a diarylheptanoid with two
O-methoxyphenols attached to a β-diketone moiety connected by two symmetrical olefinic
Copyright: © 2023 by the authors.
bonds [13]. However, curcumin has low bioavailability due to its low solubility in water and
Licensee MDPI, Basel, Switzerland.
instability at physiological pH, and transforms into ferulic acid, vanillin, dehydrozingerone,
This article is an open access article and curcumin glucuronide [1,14–17]. Curcumin displays anti-inflammatory effects by mod-
distributed under the terms and ulating several pathways involved in the inflammatory process. This polyphenol inhibits
conditions of the Creative Commons the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and
Attribution (CC BY) license (https:// interleukins (ILs) 1, 2, 6, 8, and 12 [18], and regulates the activity of cyclooxygenase 2
creativecommons.org/licenses/by/ (COX-2) [19]. Previous studies have shown that the anti-inflammatory effect induced by
4.0/). curcumin in vitro is achieved by regulating the activation of transcription factors such as

Int. J. Mol. Sci. 2023, 24, 3691. https://doi.org/10.3390/ijms24043691 https://www.mdpi.com/journal/ijms

 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 19

and curcumin glucuronide[1,14–17]. Curcumin displays anti-inflammatory effects by mod-

ulating several pathways involved in the inflammatory process. This polyphenol inhibits the
Int. J. Mol. Sci. 2023, 24, 3691 2 of 17
production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and inter-
leukins (ILs) 1, 2, 6, 8, and 12 [18], and regulates the activity of cyclooxygenase 2 (COX-2)
[19]. Previous studies have shown that the anti-inflammatory effect induced by curcumin in
activating protein-1
vitro is achieved by (AP1) and nuclear
regulating factor (NF)
the activation κB [18–20].factors
of transcription Curcumin suchinhibits NFκB pro-
as activating by
blocking
tein-1 (AP1)IκB protein
and nuclearphosphorylation
factor (NF) κB [18–20]. TheCurcumin
[18–20]. curcumin-mediated
inhibits NFκB downregulation
by blocking of IκB
these
protein intracellular
phosphorylation signals[18–20].
leads to Thea reduction in the expression
curcumin-mediated of cytokines
downregulation [21].intracel-
of these It has
been
lular suggested
signals leads that
to curcumin
a reduction could beexpression
in the used as a of nonsteroidal anti-inflammatory
cytokines [21]. drug
It has been suggested
(NSAID)
that curcumin[22]. However,
could be usedits low
as abioavailability due to its susceptibility
nonsteroidal anti-inflammatory to degradation
drug (NSAID) in
[22]. How-
biological
ever, its low systems and poordue
bioavailability solubility in water and to
to its susceptibility plasma has prevented
degradation the medical
in biological systemsuse and
of
poorcurcumin
solubility [23].
in water and plasma has prevented the medical use of curcumin [23].
Protecting the reactive
reactive sites
sites of
ofcurcumin
curcumin(the (thearomatic
aromaticrings
ringsandandthetheketo-enol
keto-enolregion)
region)
(Figure 1)
(Figure 1) through the formation of derivatives
derivatives could
couldbe beananalternative
alternativestrategy
strategytotoimprove
improveits
stability
its andand
stability taketake
advantage
advantageof theof benefits of this
the benefits polyphenol
of this polyphenol[1,16,17]. Previous
[1,16,17]. studies
Previous
have shown
studies havethat shownthe phenolic
that the rings and β-diketone
phenolic rings and moieties
β-diketonein the curcumin
moieties structure
in the suffer
curcumin
from degradation
structure suffer from bydegradation
oxidation and hydrolysisand
by oxidation [5,24]. Thus, [5,24].
hydrolysis protectionThus,ofprotection
the hydroxylof
groups
the mightgroups
hydroxyl increase the increase
might bioavailability of this compound
the bioavailability by suppressing
of this compound degradation
by suppressing
[22,25,26]. In [22,25,26].
degradation this sense, someIn thisresearch
sense, groups have incorporated
some research groups have amino acids [27], amino
incorporated glucose
acids [27], glucose [28], alkyl [7] and succinyl groups into the curcumin structure [5].etInal.
[28], alkyl [7] and succinyl groups into the curcumin structure [5]. In 2011, Wichitnithad
demonstrated
2011, Wichitnithad that succinylation of curcuminoids
et al. demonstrated protects theofcurcumin
that succinylation curcuminoidsfrom hydrolysis
protects the and
is an effective
curcumin fromstrategy
hydrolysisagainst
andcolon
is ancancer [5]. strategy
effective Hence, we addedcolon
against the succinyl
cancer group to cur-
[5]. Hence,
cumin
we addedstructure to protect
the succinyl the to
group new molecules
curcumin from degradation
structure to protect theand new
to improve theirfrom
molecules bioa-
vailability. and to improve their bioavailability.
degradation

Figure 1. Curcumin
Figure Curcumin and
and its
its reactive
reactivesites.
sites.

In
In this
thisresearch,
research,new
newcurcumin
curcumin derivatives werewere
derivatives synthesized to establish
synthesized a structure–activity
to establish a structure–
relationship (SAR) between
activity relationship curcumin
(SAR) between and itsand
curcumin derivatives [19]. Specifically,
its derivatives we focus
[19]. Specifically, on
we focus
protecting
on protectingthe hydroxyl groups
the hydroxyl of the
groups of aromatic ring ring
the aromatic of curcumin through
of curcumin the incorporation
through the incorpora-
tion
of theofsuccinyl
the succinyl group.
group. We evaluated
We evaluated howanti-inflammatory
how the the anti-inflammatory activity
activity of theofderivatives
the deriva-
tivesaltered
was was altered as a result
as a result of theofstructural
the structural changes
changes compared
compared withwith curcumin.
curcumin.

2.
2. Results
2.1.
2.1. Synthesis of Novel
Synthesis of Novel Curcumin
CurcuminDerivatives
Derivatives
The
The synthesis
synthesis ofof the
the curcumin
curcuminderivatives
derivativeswas wascarried
carriedout
outinintwo
two stages.
stages. In the
In the first
first stage, the alkyl succinate derivative was synthetized through the interaction
stage, the alkyl succinate derivative was synthetized through the interaction of an alcohol of anof
alcohol
interest of interest
with with theanhydride
the succinic succinic anhydride (Supplementary
(Supplementary Figure S1).Figure
In theS1). In the
second second
stage, each
stage, each alkylderivative
alkyl succinate succinate(S1–S9)
derivative
was(S1–S9)
coupledwaswithcoupled
curcuminwithtocurcumin
produce theto produce
curcuminthe de-
curcumin derivative (Supplementary Figure S2). Thirteen novel curcumin derivatives
rivative (Supplementary Figure S2). Thirteen novel curcumin derivatives (3–15) were syn- (3–15)
were synthesized
thesized to determine
to determine their biological
their biological activity andactivity andaestablish
establish a structure–activity
structure–activity relationship
relationship
(SAR). (SAR).
Compound 1 was obtained commercially from Alfa Aesar with a 95% total curcumi-
noid content from turmeric rhizome. In this study, compound 1 was used as a reference
to compare its activity to that of the novel curcumin derivatives. It is thought that the
modification of curcumin, in addition to protecting its reactive sites (Figure 1), provides
improvement in activity. We previously described a curcumin derivative, compound 2
(Figure 2), with a prominent inhibitory effect on IL-6 production [16]. Compound 2 has
a different structural modification than the novel curcumin derivatives presented herein.
Thus, this compound was also used as a reference to evaluate which modifications are more
relevant to the anti-inflammatory activity of curcumin derivatives.
 pare its activity to that of the novel curcumin derivatives. It is thought that the modification
of curcumin, in addition to protecting its reactive sites (Figure 1), provides improvement in
activity. We previously described a curcumin derivative, compound 2 (Figure 2), with a
prominent inhibitory effect on IL-6 production [16]. Compound 2 has a different structural
modification than the novel curcumin derivatives presented herein. Thus, this compound
Int. J. Mol. Sci. 2023, 24, 3691 3 of 17
was also used as a reference to evaluate which modifications are more relevant to the an-
ti-inflammatory activity of curcumin derivatives.

Figure 2.
Figure 2. Curcumin
Curcumin derivatives
derivativesto
todetermine
determinetheir
theiranti-inflammatory
anti-inflammatoryactivity.
activity.

Compounds S1–S9
Compounds S1–S9 were
weresynthesized
synthesizedviaviaa asuccinylation
succinylationreaction (Supplementary
reaction (Supplementary Fig-
Figure S1). The reaction started with the interaction between adamantan-2-ol and DIPEAin
ure S1). The reaction started with the interaction between adamantan-2-ol and DIPEA
pyridine
in pyridine(Py).
(Py).After, succinic
After, anhydride
succinic anhydride(SA) andand
(SA) 4-dimethylaminopyridine
4-dimethylaminopyridine (DMAP) were
(DMAP)
addedadded
were to giveto4-((adamantan-2-yl)oxy)-4-oxobutanoic
give 4-((adamantan-2-yl)oxy)-4-oxobutanoicacid (S1, 86%). The 86%).
acid (S1, same outcome was
The same
observed was
outcome whenobserved
2-(hydroxymethyl)anthracene-9,10-dione and DIPEA in dichloromethane
when 2-(hydroxymethyl)anthracene-9,10-dione and DIPEA in
(DCM)
dichloromethane reacted
(DCM) reacted with SA DMAPand
with SA and DMAP to
to give 4-((9,10-dioxo-9,10-dihydroan-give
4-((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)-4-oxobutanoic
thracen-2-yl)methoxy)-4-oxobutanoic acid (S2,occurred
acid (S2, 89%). The same reaction 89%). Thewith
same
reaction occurred with
diphenylmethanol diphenylmethanol
to generate to generate 4-(benzhydryloxy)-4-oxobutanoic
4-(benzhydryloxy)-4-oxobutanoic acid
acid (S3, 88%). Similarly,
(S3, 88%). Similarly, cyclohexyl(phenyl)methanol
cyclohexyl(phenyl)methanol produced 4-(cyclohexyl(phenyl)methoxy)-4-oxobutanoic acid produced
4-(cyclohexyl(phenyl)methoxy)-4-oxobutanoic
(S4, 98%). On the other hand, when 9H-fluoren-9-ol acid (S4,
with98%).
DIPEA Oninthe otheradded
Py was hand,towhen
SA
and DMAP, 4-((9H-fluoren-9-yl)oxy)-4-oxobutanoic acid (S5, 77%) was formed. When a
solution of 2,3-dihydro-1H-inden-2-ol and DIPEA in DCM was added to SA and DMAP,
the reaction produced 4-((2,3-dihydro-1H-inden-2-yl)oxy)-4-oxobutanoic acid (S6, 94%).
Similarly, when a solution of (1R,2S,5R)-(-)-menthol and DIPEA in DCM was added to SA
and DMAP, 4-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)-4-oxobutanoic acid (S7,
94%) was produced. In the same conditions, methanol generated 4-methoxy-4-oxobutanoic
acid (S8, 67%). Finally, the addition of a solution of 1,2,3,4-tetrahydronaphthalen-1-ol
and DIPEA in DCM to SA and DMAP afforded 4-oxo-4-((1,2,3,4-tetrahydronaphthalen-1-
yl)oxy)butanoic acid (S9, 84%).
Compounds 3–15 (Figure 2) were synthesized by Steglich esterification. The reac-
tion between curcumin and 4-(((1R,3 R,5 R,7R)-adamantan-2-yl)oxy)-4-oxobutanoic acid
(S1) in the presence of DMAP, N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hy-
drochloride (EDC), and DCM produced the difunctionalized (3, 37%), and monofunc-
tionalized (4, 62%) succinate analogs. However, the reaction between curcumin and 4-
((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)-4-oxobutanoic acid (S2) in the presence
Int. J. Mol. Sci. 2023, 24, 3691 4 of 17

of DMAP, EDC, and DCM produced a monofunctionalized (5, 13%) succinate analog
(Figure 2). The same outcome was observed when curcumin and 4-(benzhydryloxy)-
4-oxobutanoic acid (S3) were reacted with DMAP, EDC, and DCM, which generated a
monofunctionalized (6, 14%) succinate analog (Figure 2). The reaction between curcumin
and 4-(cyclohexyl(phenyl)methoxy)-4-oxobutanoic acid (S4) in the presence of DMAP, EDC,
and DCM produced difunctionalized (7, 16%), and monofunctionalized (8, 45%) succinate
analogs (Figure 2).
On the other hand, when curcumin and 4-((9H-fluoren-9-yl)oxy)-4-oxobutanoic acid
(S5) reacted in the presence of DMAP, EDC, and DCM, difunctionalized (9, 4%) and mono-
functionalized (10, 20%) succinate analogs formed (Figure 2). However, when curcumin
and 4-((2,3-dihydro-1H-inden-2-yl)oxy)-4-oxobutanoic acid (S6) reacted in the presence
of DMAP, EDC, and Py, only the monofunctionalized (11, 16%) succinate analog was
produced (Figure 2). The reaction between curcumin and 4-(((1R,2S,5R)-2-isopropyl-5-
methylcyclohexyl)oxy)-4-oxobutanoic acid (S7) in the presence of DMAP, EDC, and DCM
generated both difunctionalized (12, 9%), and monofunctionalized (13, 57%) succinate
analogs (Figure 2). However, when curcumin was treated with 4-methoxy-4-oxobutanoic
acid (S8) in the presence of DMAP, EDC, and DCM, only the monofunctionalized (14, 11%)
succinate analog was produced (Figure 2). Finally, when curcumin reacted with 4-oxo-4-
((1,2,3,4-tetrahydronaphthalen-1-yl)oxy)butanoic acid (S9) in the presence of DMAP, EDC,
and DCM, only the monofunctionalized (15, 4%) succinate analog formed (Figure 2). The
characterization of compounds S1-S9 and 2-15 is available in the Supplementary Material.

2.2. Anti-Inflammatory Activity of the Curcumin Derivatives In Vitro

To evaluate the anti-inflammatory activity of curcumin and its derivatives (2–15),
we measured the production of inflammatory mediators by murine macrophages stim-
ulated with LPS in the presence or absence of the compounds. We previously reported
a curcumin derivative 2, which preserves the effect of curcumin on IL-6 production [16].
That compound and curcumin (1) were used as references when determining activity in
this study. We first evaluated the effect of a single concentration of each compound on
the production of IL-6 and TNF-α. Monofunctionalized compounds 4, 6, 10, and 13–15
inhibited the production of IL-6 at 30 µM. Although the effect of compound 8 was not
statistically significant, we observed a conserved trend throughout the experiments. Di-
functionalized compounds 3, 7, 9, 11, and 12 did not show this activity (Figure 3B). In our
experimental conditions, none of the compounds influenced TNF-α production (Figure 3A).
Compounds 2–13 and 15 were not cytotoxic at this concentration (Figure 3C). However,
under our experimental conditions, 1 and 14 affected cell viability. All monofunctionalized
compounds were selected for further experiments.
We then evaluated the effects of the monofunctionalized compounds on IL-6 produc-
tion at different concentrations. All compounds, including compounds 1 and 2, inhibited
the production of IL-6 in a dose-dependent manner (Figure 4). Greater inhibitory effects
were observed at 10 and 30 µM, although compounds 1 and 2 still showed stronger ac-
tivity. In general, at lower concentrations (1 and 3 µM), the effects of the compounds
were similar to that of compound 1. Compound 2 exhibited a statistically significant in-
hibitory effect even at the lowest concentration (1 µM), which was better than that of the
rest of the compounds, including compound 1 (Figure 4). The effect of the compounds
was not due to cellular death, as none of the compounds were cytotoxic at any of the
evaluated concentrations.
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 19

compounds influenced TNF-α production (Figure 3A). Compounds 2–13 and 15 were not
cytotoxic at this concentration (Figure 3C). However, under our experimental conditions, 1
Int. J. Mol. Sci. 2023, 24, 3691 5 of 17
and 14 affected cell viability. All monofunctionalized compounds were selected for further
experiments.

Figure 3. Anti-inflammatory activity of the curcumin derivatives. Peritoneal macrophages from

Figure 3. Anti-inflammatory activity of the curcumin derivatives. Peritoneal macrophages from
C57BL/6mice
C57BL/6 mice were
were pretreated
pretreated with
with 30 μM of
30 µM of each
eachcompound
compound11hhbeforebeforestimulation
stimulationwith
with1010 ng/mL
ng/mL
LPS. After 6 h, the concentration of TNF-α (A) and IL-6 (B) in the supernatant of
LPS. After 6 h, the concentration of TNF-α (A) and IL-6 (B) in the supernatant of the cells was deter-the cells was
determined. (C) Cell viability was tested by MTT assays after supernatant collection.
mined. (C) Cell viability was tested by MTT assays after supernatant collection. All results are pre- All results
sented as the mean
are presented as the± S.E.M.
mean from two independent
± S.E.M. experimentsexperiments
from two independent performed inperformed
triplicate. *,inp <triplicate.
0.05; **, p
< 0.01; ***, p < 0.001; ****, p < 0.0001 relative to LPS stimulus alone (black bar). C, negative control.
*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 relative to LPS stimulus alone (black bar). C,
negative control.
We then evaluated the effects of the monofunctionalized compounds on IL-6 production
at different concentrations.
In addition All compounds,
to IL-6, we evaluated whetherincluding compounds
the compounds 1 and the
could reduce 2, inhibited
production the
production of IL-6 in a dose-dependent manner (Figure 4). Greater inhibitory
of prostaglandin E2 (PGE2 ). PGE2 is secreted by macrophages that have been exposed to effects were
observed at 10stimuli,
inflammatory and 30andμM,itsalthough
synthesiscompounds 1 and
is influenced 2 still
by the enzymeshowed
COX-2stronger
[29]. Itactivity.
has beenIn
general, at lower
shown that concentrations
curcumin can suppress(1 and 3 μM),
COX-2 the effects
expression andofPGE
the compounds were similar to
2 production in models of
inflammation [30]. We decided to examine whether these curcumin derivatives, including
compound 2, preserved the effect on PGE2 production. We stimulated macrophages with
LPS in the presence or absence of compounds and determined the levels of PGE2 six hours
after stimulus. All monofunctionalized compounds inhibited the production of PGE2 in
a dose-dependent manner, and the effect was statistically significant at concentrations of
10 and 30 µM (Figure 5). Among these, only compound 15 significantly reduced PGE2
production at a concentration of 3 µM. Compound 2 also showed an inhibitory effect on
PGE2 production at all concentrations tested. At a concentration of 1 µM, only compound 2
showed an effect (Figure 5).
 that of compound 1. Compound 2 exhibited a statistically significant inhibitory effect even at
the lowest concentration (1 μM), which was better than that of the rest of the compounds,
Int. J. Mol. Sci. 2023, 24, 3691 including compound 1 (Figure 4). The effect of the compounds was not due to cellular
6 of 17death,
as none of the compounds were cytotoxic at any of the evaluated concentrations.

Figure
Figure 4. Monofunctionalized
4. Monofunctionalized curcuminderivatives
curcumin derivativesinhibit
inhibit the
the production
productionofofIL-6 IL-6ininmacrophages
macrophages in-
ducedinduced
by LPS. by LPS. Peritoneal
Peritoneal macrophages
macrophages fromC57BL/6
from C57BL/6micemicewere
were pretreated
pretreated with withdifferent
differentconcen-
concentra-
tionstrations
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW (1, 3, (1,
10, 3,
or 10,
30 or 30
μM) µM)
of of
thethe compounds
compounds 11 h
h before
before stimulation
stimulationwith 10
with ng/mL
10 ng/mL LPS. After
LPS. 6 h,
After the h, the
7 of6 19
concentrations of IL-6 in the supernatants of the cells were determined. All results
concentrations of IL-6 in the supernatants the cells were determined. All results are presented as the are presented as the
mean ± S.E.M.
± S.E.M.
mean fromfromthree independent
three independent experiments
experiments performed triplicate. *,*, pp << 0.05;
performedinintriplicate. 0.05; **, pp << 0.01;
0.01; ***, p
< 0.001;
***, p****, p < 0.0001
< 0.001; ****, p relative
< 0.0001 to LPS stimulus
relative alone (black
to LPS stimulus bar). C,
alone (black negative
bar). control.
C, negative control.

In addition to IL-6, we evaluated whether the compounds could reduce the production
of prostaglandin E2 (PGE2). PGE2 is secreted by macrophages that have been exposed to in-
flammatory stimuli, and its synthesis is influenced by the enzyme COX-2 [29]. It has been
shown that curcumin can suppress COX-2 expression and PGE2 production in models of in-
flammation [30]. We decided to examine whether these curcumin derivatives, including
compound 2, preserved the effect on PGE2 production. We stimulated macrophages with LPS
in the presence or absence of compounds and determined the levels of PGE2 six hours after
stimulus. All monofunctionalized compounds inhibited the production of PGE2 in a
dose-dependent manner, and the effect was statistically significant at concentrations of 10
and 30 μM (Figure 5). Among these, only compound 15 significantly reduced PGE2 produc-
tion at a concentration of 3 μM. Compound 2 also showed an inhibitory effect on PGE2 pro-
duction at all concentrations tested. At a concentration of 1 μM, only compound 2 showed an
effect (Figure 5).
Figure 5. Monofunctionalized curcumin derivatives inhibit the production of PGE2 . Peritoneal
Figure 5. Monofunctionalized
macrophages curcumin
from C57BL/6 mice derivatives
were pretreated inhibit
with the concentrations
different production of(1,PGE 2. Peritoneal macro-
3, 10, or 30 µM) of
phages
the compounds 1 h before stimulation with 10 ng/mL LPS. After 6 h, the concentrationsor
from C57BL/6 mice were pretreated with different concentrations (1, 3, 10, of 30
PGEμM) of the
2 in
compounds 1 h before stimulation with 10 ng/mL LPS. After 6 h, the concentrations of PGE2 in the su-
the supernatants of the cells were determined. All results are presented as the mean ± S.E.M. from
pernatants of the cells were determined. All results are presented as the mean ± S.E.M. from two inde-
two independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to
pendent experiments performed in duplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to LPS stim-
LPS stimulus alone (black bar). C, negative control.
ulus alone (black bar). C, negative control.
The IC50 values representing the effects of all of the compounds are listed in Table 1
Therange
and IC50 values representing
from 1.94 ± 0.66 µM tothe effects
10.6 ± 0.33ofµM
all for
of the
the compounds are listed
inhibition of IL-6 in Table
production and 1 and
range from
from 0.511.94 ± 0.66
± 0.08 µM μM to ±
to 5.93 10.6
2.29± µM
0.33for
μM thefor theon
effect inhibition of IL-6 production and from
PGE2 production.
0.51 ± 0.08 μM to 5.93 ± 2.29 μM for the effect on PGE2 production.

Table 1. Chemical structures and anti-inflammatory activity of the novel curcumin derivatives.
a IC50 ± S.D. (μM) a IC50 ± S.D. (μM)
Compound
IL-6 PGE2
1 6.85 ± 2.50 1.34 ± 0.067
2 3.59 ± 0.27 0.51 ± 0.08
4 10.5 ± 0.6 4.50 ± 3.18
5 7.11 ± 0.75 2.60 ± 1.75
6 4.21 ± 0.73 4.43 ± 0.85
8 1.94 ± 0.66 5.90 ± 2.38
Int. J. Mol. Sci. 2023, 24, 3691 7 of 17

Table 1. Chemical structures and anti-inflammatory activity of the novel curcumin derivatives.
a IC50 ± S.D. (µM) a IC50 ± S.D. (µM)
Compound
IL-6 PGE2
1 6.85 ± 2.50 1.34 ± 0.067
2 3.59 ± 0.27 0.51 ± 0.08
4 10.5 ± 0.6 4.50 ± 3.18
5 7.11 ± 0.75 2.60 ± 1.75
6 4.21 ± 0.73 4.43 ± 0.85
8 1.94 ± 0.66 5.90 ± 2.38
10 3.60 ± 0.21 5.93 ± 2.29
13 10.6 ± 0.33 5.42 ± 2.04
15 10.4 ± 0.75 3.07 ± 0.72
a Values represent the average IC50 from three independent experiments performed in duplicate ± S.D.

3. Discussion
Curcumin derivatives were prepared by first considering forming succinate and then
esterifying curcumin.
When macrophages are activated, they release a wide variety of mediators, including
proinflammatory cytokines such as TNF-α, IL-6, and prostaglandins (such as PGE2 ). These
mediators are usually used as markers of an inflammatory response. We determined
the anti-inflammatory effects of curcumin and its derivatives in macrophages stimulated
by LPS, an inducer of inflammation. An anti-inflammatory effect was observed after
treatment with the monofunctionalized curcumin derivatives with statistically significant
dose-dependent inhibition of IL-6 production.
A structure–activity relationship (SAR) evaluation indicated that the difunctionalized
compounds lost the anti-inflammatory activity of curcumin, while this property was main-
tained with the monofunctionalized compounds. Compounds 6, 8, and 10 showed the best
activity among the monofunctionalized, with IC50 values of 4.21 ± 0.73, 1.94 ± 0.66, and
3.60 ± 0.21, respectively. Curcumin has activity comparable to those previously mentioned.
Compound 2 exhibited the best activity with an IC50 of 3.59 ± 0.27. The compounds that
were less active were 4, 5, 13, and 15. The structural difference between compound 2 and
this new set of curcumin derivatives is the succinate linker between the curcumin structure
and the ligand (benzyl alcohol). The addition of this linker to the new derivatives decreased
the anti-inflammatory activity compared to compound 2. These results indicate that the
overall length of the derivative structure might influence activity [16].
The ligands of compounds 2, 6, 8, and 10 all contain aromatic rings in the benzylic
position. Among the compounds with two aromatic rings attaching to the benzylic position,
6 and 10 showed the greater effect on IL-6 production. However, when a single aromatic
ring was attached to this position (5, 15), the activity decreased [16].
A common feature of compounds 4, 13, and 15 is an aliphatic six-membered ring, and
these compounds showed a lower activity than curcumin, indicating that the six-membered
ring attached directly to the keto group of the ligand induces a loss in activity. However,
when a six-membered aromatic ring is attached at a benzylic position (8), the activity of
the compound increases. These results suggest that there are three key points to increasing
anti-inflammatory activity: (i) the phenolic ring of the unesterified curcumin derivatives
can form hydrogen bonds with another molecule, affecting the activity of these compounds;
(ii) a π-π interaction with the curcumin ligand is possible since compounds 2, 6, 8, and 10
showed high anti-inflammatory activity against IL-6, while compounds 4, 13, and 15 had
low activity. (iii) The length of the curcumin ligand can influence its activity, as observed
with compound 2 compared to compounds 6, 8, and 10, in which the succinate linker in the
latter compounds had an increased length and decreased activity compared to compound 2.
Several studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs)
inhibit LPS-stimulated PGE2 production [31]. Because curcumin is considered an NSAID,
we evaluated the effect of this polyphenol and its derivatives on PGE2 production. A
significant reduction in PGE2 secretion was observed, suggesting that these curcumin
Int. J. Mol. Sci. 2023, 24, 3691 8 of 17

derivatives have a broad effect on the production of proinflammatory mediators associated

with the LPS response.
Compound 2 was the most active compound, with a PGE2 IC50 value of 0.51 µM ± 0.08.
Among the monofunctionalized, compounds 5, 6, and 15 showed activity, while compounds
4, 8, 10, and 13 exhibited the lowest activity.
For PGE2 , structure–activity relationships similar to like those found with IL-6 were
observed, as a free hydroxyl group on the aromatic ring of curcumin enhanced activity.
Additionally, the length of the molecule remained an influential factor when the aromatic
rings in the benzylic position were present. The curcumin derivatives inhibited the produc-
tion of IL-6 and PGE2 without affecting TNF-α secretion. NSAIDs suppress the synthesis
of PGE2 by a mechanism that involves regulation of COX-2 activity [31]. Since curcumin
derivatives presented herein affect the production of PGE2 , further studies are necessary to
elucidate a mechanism engaged in this effect.
These results lead us to infer that the protection of both sides of the aromatic ring
results in the inactivity, seemingly because of degradation. However, protection of one
of the rings maintains the activity, and, depending on the type of the substituent, it can
enhance it to the point of exceeding the activity of the unprotected molecule, contradicting
the above conclusion. Thus, it remains unclear whether it suffers degradation in the
unprotected ring. Given the uncertainty, we plan evaluate the mechanism of action of the
monofunctionalized compounds in the near future and continue our search for protective
groups (succinyl or ether) that are more favorable for prevention of the degradation of
the curcumin derivative. At the moment, it can be seen that curcumin has in vitro activity
when it is protected on only one side, and that the ether group is the most active.

4. Materials and Methods

4.1. Synthesis
Chemical reagents were used as commercially available (Tedia, Applichem, Sigma
Aldrich, USA). All reactions were conducted in a borosilicate glass tube (20 mL or 16 mL)
fitted with screw-cap and magnetic stirring under the atmosphere of argon. The reaction
mixture was evaluated in an Agilent 1260 Infinity II HPLC system equipped with a quater-
nary pump, an Agilent diode array detector 1260 Series and normal phase silica gel column
(Phenomenex® Luna Silica (2), 250 mm × 10 mm, 5 µm) with gradient system n-hexane to
ethyl acetate in 20 min at 2 mL/min (Agilent Technologies, Santa Clara, CA, USA). The
reaction mixture was purified using a Buchi C-815 Flash HPLC system equipped with a
binary pump, a UV scan withan Evaporative Light Scattering (ELSD) detector, a closed
fraction collector bay, and normal phase silica gel cartridge (Buchi® FlashPure EcoFlex Silica
12 g, 40–63 µm) in an isocratic system n-hexane/ethyl acetate 7:3 (BÜCHI Labortechnik AG,
Meierseggstrasse 40 postfach, Switzerland). NMR characterization was performed on 1 H.
13 C NMR spectra were recorded at 500 (1 H), 125 MHz (13 C) on a Jeol JNM-ECZ500R/S1

500 MHz spectrometer in CDCl3 and DMSO-d6 (JEOL Ltd. 3-1-2 Musashino, Akishima,
Tokyo 196-8558, Japan). Chemical shifts (δ) are reported in parts per million (ppm) from the
residual solvent peak and coupling constant (J) in Hz. Proton multiplicity is reported in:
singlet (s), doublet (d), triplet (t), quartet (quart.), quintet (quint.), septet (sept.), multiplet
(m), broad (br). Infrared measurements were carried out on a Bruker Platinum ATR Alpha
instrument (Bruker, Billerica, MA, USA). The MS analyses were carried out on a Waters
Xevo TQD spectrometer with Electrospray Ionization (ESI) as an ion source (Waters Corpo-
ration, Milford, MA, USA). Melting point determinations were measured by triplicate on
an Automatic Melting Point apparatus Stuart SMP50 (Cole-Palmer, Staffordshire, UK). The
detailed synthesis procedure and spectral characterization are described below.

4.1.1. General Procedure 1 (GP1) for the Synthesis of Alkyl Succinate Monoesters (S1–S9)
An oven dried vial (20 mL) fitted with screw-cap with magnetic stirrer was flushed
with argon and charged with alcohol (854 mg, 14.7 mmol), dichloromethane (5 mL), and
N,N-diisopropylethylamine (1.3 mL, 7.35 mmol, 0.5 equiv.) at room temperature (rt). After
Int. J. Mol. Sci. 2023, 24, 3691 9 of 17

2 h, succinic anhydride (735 mg, 7.35 mmol, 0.5 equiv.), and 4-dimethylaminopyridine
(448 mg, 3.67 mmol, 0.25 equiv.) were added, and the reaction stirred at rt. After 48 h,
the reaction mixture was diluted with brine/1M HCl (3:1, 10 mL). The aqueous layer was
extracted with dichloromethane (3 × 20 mL), and the combined organic phases dried over
anhydrous sodium sulfate (Na2 SO4 ) and concentrated under reduced pressure.

Synthesis of 4-((Adamantan-2-yl)oxy)-4-oxobutanoic Acid (S1)

According to GP1, adamantan-2-ol (500.0 mg, 3.28 mmol), and N,N-diisopropylethylamine
(858 µL, 4.92 mmol, 1.5 equiv.) were stirred in pyridine (4 mL) at rt. After 2 h, succinic
anhydride (492.9 mg, 4.92 mmol, 1.5 equiv.) and 4-dimethylaminopyridine (601.8 mg,
4.92 mmol, 1.5 equiv.) were added, and the reaction mixture stirred for 48 h at rt to obtain
the monoester S1 (713.3 mg, 86%). 1 H NMR (500 MHz, DMSO-D6 ): δ 4.76 (t, J = 3.2 Hz,
1H), 2.49–2.44 (m, 5H), 1.95–1.84 (m, 4H), 1.79–1.63 (m, 8H), 1.47 (d, J = 11.5 Hz, 2H) ppm.
13 C NMR (125 MHz, DMSO-D6): 26.4, 26.6, 28.9, 29.3, 31.2, 35.7, 36.8, 76.2, 171.3, 173.5.

IR: 2901.31, 1702.61, 1699.69, 1322.74, 1162.63. mp: 87.8 ± 1.5 ◦ C. MS (m/z) calcd for
C14 H20 NaO4 : 275.30; found: 274.99 [M+Na+ ].

Synthesis of 4-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)methoxy)-4-oxobutanoic Acid (S2)

According to GP1, 2-(hydroxymethyl)anthracene-9,10-dione (200.9 mg, 0.84 mmol),
and N,N-diisopropylethylamine (500 µL, 2.87 mmol, 3.4 equiv.) were stirred in CH2 Cl2
(5 mL) at rt. After 2 h, succinic anhydride (251.9 mg, 2.52 mmol, 3.0 equiv.) and 4-
dimethylaminopyridine (307.6 mg, 2.52 mmol, 3.0 equiv.) were added, and the reac-
tion mixture stirred for 48 h at RT to yield monoester S2 (255.2 mg, 89%). 1 H NMR
(500 MHz, DMSO-D6 ): δ 8.14–8.05 (m, 4H), 7.87–7.82 (m, 2H), 7.79 (d, J = 9.7 Hz, 1H),
5.23 (s, 2H), 2.61–2.56 (m, 2H), 2.49–2.44 (m, 2H) ppm. 13 C NMR (125 MHz): 28.72, 28.74,
64.5, 125.4, 126.7, 126.8, 127.1, 132.5, 133.01, 133.04, 133.0, 133.1, 134.6, 134.7, 143.2, 172.1,
173.5, 182.2, 182.3 ppm. IR: 2932.30, 1684.14, 1671.36, 1652.92, 1591.01, 1175.53, 705.69. mp:
152.1 ± 1.1 ◦ C.

Synthesis of 4-(Benzhydryloxy)-4-oxobutanoic Acid (S3)

According to GP1, diphenylmethanol (1380.6 mg, 7.49 mmol), and N,N-diisopro-
pylethylamine (870 µL, 5.00 mmol, 0.67 equiv.) were stirred in CH2 Cl2 (4 mL) at rt. After
2 h, succinic anhydride (500 mg, 5.00 mmol, 0.67 equiv.) and 4-dimethylaminopyridine
(610.4 mg, 5.00 mmol, 0.67 equiv.) were added, and the reaction mixture stirred for 48 h at
rt to yield monoester S3 (1865.3 mg, 88%). 1 H NMR (500 MHz, DMSO-D6 ): δ 7.35–7.28 (m,
10H), 6.75 (s, 1H), 2.64–2.60 (m, 2H), 2.50–2.47 (m, 2H). 13 C NMR (125 MHz, DMSO-D6 ): δ
173.39, 171.19, 140.57, 128.48, 127.70, 126.48, 76.45, 29.02, 28.67 ppm. IR: 3059.77, 3028.62,
1733.35, 1240.98, 1154.35, 694.86. MS (m/z) calcd for C17 H16 NaO4 : 307.3; found: 306.9
[M+Na+ ].

Synthesis of 4-(Cyclohexyl(phenyl)methoxy)-4-oxobutanoic Acid (S4)

According to GP1, cyclohexyl(phenyl)methanol (50.0 mg, 0.26 mmol), and N,N-
diisopropylethylamine (100 µL, 0.57 mmol, 2.1 equiv.) were stirred in CH2 Cl2 (2 mL) at rt.
After 2 h, succinic anhydride (80.0 mg, 0.80 mmol, 3.0 equiv.) and 4-dimethylaminopyridine
(100 mg, 0.82 mmol, 3.0 equiv.) were added, and the reaction mixture stirred for 48 h at rt
to yield monoester S4 (74.8 mg, 98%). 1 H NMR (500 MHz, DMSO-D6 ): δ 7.35–7.24 (m, 5H),
5.45 (d, J = 7.1 Hz, 1H), 2.58–2.52 (m, 2H), 2.49–2.45 (m, 2H), 1.75–1.55 (m, 5H), 1.31–0.93
(m, 6H) ppm.13 C NMR (125 MHz, DMSO-D6 ): 25.3, 25.4, 25.8, 28.0, 28.6, 28.7, 28.9, 29.0,
42.5, 79.3, 126.6, 127.5, 128.1, 139.5, 171.3, 173.3, 173.6 ppm. IR: 2916.61, 2846.67, 1721.37,
1225.98, 1164.43, 695.52 cm−1 . mp: 156.1 ± 3.5 ◦ C. MS (m/z) calcd for C17 H22 NaO4 : 313.3;
found: 313.0 [M+Na+ ].
Int. J. Mol. Sci. 2023, 24, 3691 10 of 17

Synthesis of 4-((9H-Fluoren-9-yl)oxy)-4-oxobutanoic Acid (S5)

According to GP1, 9H-fluoren-9-ol (500 mg, 2.74 mmol), and N,N-diisopropylethylamine
(717 µL, 4.12 mmol, 1.5 equiv.) were stirred in pyridine (4 mL) at rt. After 2 h, succinic
anhydride (411.9 mg, 4.12 mmol, 1.5 equiv.) and 4-dimethylaminopyridine (502.8 mg,
4.12 mmol, 1.5 equiv.) were added, and the reaction mixture stirred for 48 h at rt to yield
monoester S5 (600 mg, 77%). 1 H NMR (500 MHz, DMSO-D6 ): δ 7.84 (d, J = 7.5 Hz, 2H),
7.52 (dd, J = 7.5, 1.0 Hz, 2H), 7.50–7.41 (m, 2H), 7.33 (td, J = 7.4, 1.1 Hz, 2H), 6.75 (s, 1H),
2.66–2.62 (m, 2H), 2.58–2.55 (m, 2H) ppm. 13 C NMR (125 MHz, DMSO-D6 ): δ 173.6, 173.4,
141.8, 140.4, 129.6, 127.9, 125.7, 120.4, 74.5, 28.9. mp: 130.5 ± 0.1 ◦ C. MS (m/z) calcd for
C17 H14 NaO4 : 305.2; found: 304.9 [M+Na+ ].

Synthesis of 4-((2,3-Dihydro-1H-inden-2-yl)oxy)-4-oxobutanoic Acid (S6)

According to GP1, 2,3-dihydro-1H-inden-2-ol (500 mg, 3.73 mmol), and N,N-diisopro-
pylethylamine (973 µL, 5.59 mmol, 1.5 equiv.) were stirred in CH2 Cl2 (4 mL) at rt. After
2 h, succinic anhydride (559.3 mg, 5.59 mmol, 1.5 equiv.) and 4-dimethylaminopyridine
(682.8 mg, 5.59 mmol, 1.5 equiv.) were added, and the reaction mixture stirred for 48 h at
RT to yield monoester S6 (817.8 mg, 94%). 1 H NMR (500 MHz, DMSO): δ 12.19 (s, 1H),
7.27–7.14 (m, 4H), 5.46–5.40 (m, 1H), 3.27 (dd, J = 17.1, 6.4 Hz, 2H), 2.89 (d, J = 17.0 Hz,
2H), 2.44 (s, 4H) ppm. 13 C NMR (125 MHz, DMSO): δ 173.4, 172.1, 140.4, 126.6, 124.5, 75.0,
28.8, 28.6 ppm. IR: 2943.05, 2910.11, 1716.85, 1652.88, 1398.03, 1165.52, 941.71, 748.10. mp:
112.8 ± 0.9 ◦ C. MS (m/z) calcd for C13 H14 NaO4 : 257.2; found: 256.9 [M+Na+ ].

Synthesis of 4-(((1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl)oxy)-4-oxobutanoic Acid (S7)

According to GP1, (1R,2S,5R)-(-)Menthol (500.0 mg, 3.20 mmol), and N,N-diisopro-
pylethylamine (836 µL, 4.80 mmol, 1.5 equiv.) were stirred in CH2 Cl2 (4 mL) at rt. After
2 h, succinic anhydride (480.2 mg, 4.80 mmol, 1.5 equiv.) and 4-dimethylaminopyridine
(586.3 mg, 4.80 mmol, 1.5 equiv.) were added, and the reaction mixture stirred for 48 h
at rt to yield monoester S7 (774.1 mg, 94%). 1 H NMR (500 MHz, DMSO): δ 4.63–4.54 (m,
1H), 2.47 (s, 4H), 1.90–1.79 (m, 2H), 1.67–1.58 (m, 2H), 1.50–1.28 (m, 2H), 1.08–0.90 (m, 2H),
0.90–0.83 (m, 7H), 0.71 (d, J = 6.9 Hz, 3H). 13 C NMR (125 MHz, DMSO): δ 73.2, 46.4, 40.5,
33.7, 30.8, 29.0, 28.7, 25.7, 23.0, 21.9, 20.5, 16.3. IR: 2949.05, 2868.19, 1706.67, 1699.11, 1652.74,
1387.20, 1169.71, 952.45. mp: not determined. MS (m/z) calcd for C14 H24 NaO4 : 279.33;
found: 279.03 [M+Na+ ].

Synthesis of 4-Methoxy-4-oxobutanoic Acid (S8)

According to GP1, methanol (616.8 µL, 15.6 mmol), and N,N-diisopropylethylamine
(2249 µL, 12.9 mmol, 0.8 equiv.) were stirred in CH2 Cl2 (4 mL) at rt. After 2 h, succinic
anhydride (1292.2 mg, 12.9 mmol, 0.8 equiv.) and 4-dimethylaminopyridine (1577.6 mg,
12.9 mmol, 0.8 equiv.) were added, and the reaction mixture stirred for 48 h at rt to yield
monoester S8 (1381.0 mg, 67%). 1 H NMR (500 MHz, DMSO-D6 ): δ 12.16 (s, 1H), 3.55 (s, 3H),
2.48–2.41 (m, 4H) ppm. 13 C NMR (125 MHz, DMSO-D6 ): δ 173.4, 172.6, 51.4, 28.6, 28.5 ppm.
IR: 2932.26, 1732.32, 1653.16, 1436.81, 1170.72, 942.87. mp: 56.8 ± 0.6 ◦ C. MS (m/z) calcd
for C5 H8 NaO4 : 155.11; found: 154.93 [M+Na+ ].

Synthesis of 4-oxo-4-((1,2,3,4-Tetrahydronaphthalen-1-yl)oxy)butanoic Acid (S9)

According to GP1, 1,2,3,4-tetrahydronaphthalen-1-ol (500 mg, 3.37 mmol), and N,N-
diisopropylethylamine (881 µL, 5.06 mmol, 1.50 equiv.) were stirred in CH2 Cl2 (4 mL) at rt.
After 2 h, succinic anhydride (506.4 mg, 5.06 mmol, 1.5 equiv.) and 4-dimethylaminopyridine
(618.3 mg, 5.06 mmol, 1.50 equiv.) were added, and the reaction mixture stirred for 48 h
at rt to yield monoester S11 (700.6 mg, 84%). 1 H NMR (500 MHz, DMSO): δ12.23 (s, 1H),
7.26–7.09 (m, 4H), 5.90–5.84 (m, 1H), 2.84–2.61 (m, 2H), 2.55–2.43 (m, 5H), 1.97–1.71 (m,
4H).13 C NMR (125 MHz, DMSO): δ 173.4, 171.8, 137.6, 134.4, 129.0, 128.8, 127.9, 125.9, 69.4,
29.1, 28.8, 28.6, 28.3, 18.5. mp: 91.8 ± 0.2 ◦ C. MS (m/z) calcd for C14 H16 NaO4 : 271.27;
found: 270.99 [M+Na+ ].
Int. J. Mol. Sci. 2023, 24, 3691 11 of 17

4.1.2. General Procedure (GP2) for the Synthesis of Dialkylcurcumin and

Monoalkylcurcumin (3–15)
A borosilicate glass tube (16 mL) fitted with screw-caps equipped with magnetic stirrer
was flushed with argon and charged with alkyl succinate (41.4 mg, 0.12 mmol, 0.9 equiv.),
4-dimethylaminopyridine (15.1 mg, 0.12 mmol, 1.0 equiv.), curcumin (50.1 mg, 0.13 mmol),
and dichloromethane (4 mL), which were combined and stirred at 0 ◦ C for 10 min. After,
N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (24.9 mg, 0.13 mmol,
1.0 equiv.) was added and stirred at rt for 24 h. The reaction was extracted with with EtOAc
and water (3 × 20 mL). The organic phases were concentrated under reduced pressure, and
the remaining material was purified by HPLC to obtain monoester (11.3 mg, 13%).

Synthesis of Di((1r,3r,5r,7r)-adamantan-2-yl) O,O0 -(((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-

triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)) Disuccinate (3) and
(1r,3r,5r,7r)-Adamantan-2-yl (4-((1E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-
oxohepta-1,4,6-trien-1-yl)-2-methoxyphenyl) Succinate (4)
According to GP2, succinate S1 (31.3 mg, 0.12 mmol, 1.0 equiv.), 4-dimethylamino-
pyridine (16.1 mg, 0.13 mmol, 1.0 equiv.), curcumin (50.5 mg, 0.13 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
N0 -ethylcarbodiimide hydrochloride (26.1 mg, 0.14 mmol, 1.0 equiv.) was added and stirred
at rt for 24 h. The crude product was purified by HPLC to obtain diester 3 (21.0 mg, 37%)
and monoester 4 (25.5 mg, 62%). Compound 3: 1 H NMR (500 MHz, CDCl3 ): δ 7.62 (d,
J = 15.9 Hz, 2H), 7.17–7.06 (m, 6H), 6.57 (d, J = 15.8 Hz, 2H), 5.89–5.83 (m, 1H), 4.97 (s,
2H), 3.87 (s, 6H), 2.96 (t, J = 7.4 Hz, 4H), 2.79 (t, J = 6.9 Hz, 4H), 2.01 (s, 8H), 1.90–1.66 (m,
20H). 13 C NMR (125 MHz, CDCl3 ): δ 183.2, 171.5, 170.5, 151.5, 141.3, 140.1, 134.1, 124.4,
123.4, 121.2, 111.5, 102.0, 77.7, 56.1, 37.5, 36.4, 31.9, 31.9, 29.8, 29.2, 27.3, 27.1.ppm. IR:
2909.0, 2854.6, 1765.5, 1729.4, 1630.3, 1508.2, 1416.5, 1256.1, 1129.7 cm−1 . MS (m/z) calcd for
C49 H56 NaO12 : 859.37; found: 859.6 [M+Na+ ]. Compound 4:1 H NMR (500 MHz, CDCl3 ): δ
7.60 (dd, J = 15.8, 5.7 Hz, 2H), 7.19–7.00 (m, 6H), 6.94 (d, J = 8.2 Hz, 1H), 6.52 (dd, J = 27.3,
15.8 Hz, 2H), 5.83 (s, 1H), 5.01–4.93 (m, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 2.96 (t, J = 7.0 Hz, 2H),
2.79 (t, J = 7.0 Hz, 2H), 2.00 (s, 4H), 1.87–1.71 (m, 10H). 13 C NMR (125 MHz, CDCl3 ): δ 184.6,
181.9, 171.5, 170.6, 151.4, 148.1, 146.9, 141.3, 141.2, 139.6, 134.2, 127.7, 124.3, 123.4, 123.2,
121.9, 121.1, 115.0, 111.5, 109.7, 101.7, 77.7, 77.4, 56.1, 56.0, 37.5, 36.4, 31.9, 31.9, 29.8, 29.2,
27.3, 27.1 ppm. IR: 3403.0, 2908.8, 2854.7, 1726.1, 1626.5, 1587.5, 1509.6, 1267.0, 1129.0 cm−1 .
MS (m/z) calcd for C35 H38 NaO9 : 625.24; found: 625.4 [M+Na+ ].

Synthesis of (9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl (4-((1E,4Z,6E)-5-hydroxy-7-(4-

hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-trien-1-yl)-2-methoxyphenyl) Succinate (5)
According to GP2, succinate S2 (41.4 mg, 0.12 mmol, 0.9 equiv), 4-dimethylaminopyr-
idine (15.1 mg, 0.12 mmol, 1.0 equiv.), curcumin (50.1 mg, 0.13 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-N0 -
ethylcarbodiimide hydrochloride (24.9 mg, 0.13 mmol, 1.0 equiv.) was added and stirred at
rt for 24 h. The crude product was purified by HPLC to obtain monoester 5 (11.3 mg, 13%).
Compound 5: 1 H NMR (500 MHz, CDCl3 ): δ 8.31–8.26 (m, 4H), 7.84–7.76 (m, 4H), 7.56 (dd,
J = 50.9, 15.8 Hz, 2H), 7.14–7.04 (m, 4H), 6.96 (dd, J = 23.4, 8.2 Hz, 2H), 6.49 (dd, J = 15.8, 5.2
Hz, 2H), 5.81 (s, 1H), 5.32 (s, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.00–2.96 (m, 2H), 2.89–2.85 (m,
2H) ppm.—13 C NMR (125 MHz, CDCl3 ): 29.1, 29.3, 56.0, 56.1, 65.6, 101.7, 109.7, 111.5, 115.0,
126.4, 127.40, 127.43, 127.9, 133.3, 134.4, 139.4, 141.0, 141.3, 142.5, 146.9, 148.1, 151.3, 170.2,
171.9, 181.8, 182.9, 183.0, 184.7 ppm. IR: 3046.5, 2925.0, 2849.9, 1757.0, 1731.0, 1671.7, 1588.7,
1511.3, 1444.4, 1294.6, 1203.3, 1136.8 cm−1 . MS (m/z) calcd for C40 H32 O11 : 688.6; found:
689.4 [M+H+ ].
Int. J. Mol. Sci. 2023, 24, 3691 12 of 17

Synthesis of Benzhydryl (4-((1E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-

oxohepta-1,4,6-trien-1-yl)-2-methoxyphenyl) Succinate (6)

According to GP2, succinate S3 (135.88 mg, 0.48 mmol, 1.2 equiv), 4-dimethylamin-
opyridine (65.4 mg, 0.54 mmol, 1.3 equiv.), curcumin (152.4 mg, 0.41 mmol), and dichlorome-
thane (4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopro-
pyl)-N0 -ethylcarbodiimide hydrochloride (84.87 mg, 0.44 mmol, 1.1 equiv.) was added
and stirred at rt for 24 h. The crude product was purified by column chromatography on
silica gel to obtain monoester 6 (37.7 mg, 14%). Compound 6: 1 H NMR (500 MHz, CDCl3 ):
δ 7.59 (dd, J = 15.8, 9.1 Hz, 2H), 7.35–7.30 (m, 10H), 7.13–7.03 (m, 4H), 6.93 (dd, J = 8.2,
4.6 Hz, 2H), 6.91 (s, 1H), 6.51 (dd, J = 24.4, 15.8 Hz, 2H), 5.82 (s, 1H), 3.94 (s, 3H), 3.79 (s,
3H), 2.97–2.92 (m, 2H), 2.90–2.86 (m, 2H) ppm. 13 C NMR (125 MHz, CDCl3 ): δ 184.7, 181.9,
171.2, 170.3, 151.4, 148.1, 146.9, 141.3, 141.1, 140.1, 139.6, 134.2, 128.7, 128.1, 127.2, 124.3,
123.4, 123.2, 121.9, 121.1, 115.0, 111.4, 109.7, 101.7, 77.6, 77.4, 56.1, 56.0, 29.6, 29.1 ppm. IR:
3511.7, 2922.4, 2850.8, 1740.0, 1624.7, 1586.0, 1510.1, 1302.8, 1261.5, 1121.9 cm−1 . MS (m/z)
calcd for C38 H34 NaO9 : 657.21; found: 657.4 [M+Na+ ].

Synthesis of Bis(cyclohexyl(phenyl)methyl) O,O0 -(((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-

triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)) Disuccinate (7) and
Cyclohexyl(phenyl)methyl (4-((1E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-
oxohepta-1,4,6-trien-1-yl)-2-methoxyphenyl) Succinate (8)

According to GP2, succinate S4 (114.8 mg, 0.40 mmol, 1.0 equiv), 4-dimethylamino-
pyridine (89.7 mg, 0.73 mmol, 1.8 equiv.), curcumin (150.3 mg, 0.40 mmol), and dichlorome-
thane (4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopro-
pyl)-N0 -ethylcarbodiimide hydrochloride (92.8 mg, 0.48 mmol, 1.2 equiv.) was added and
stirred at rt for 24 h. The crude product was purified by column chromatography on
silica gel to obtain diester 7 (29.6 mg, 16%) and monoester 8 (59.2 mg, 45%). Compound
7: 1 H NMR (500 MHz, CDCl3 ): δ 7.61 (d, J = 15.8 Hz, 2H), 7.33–7.27 (m, 10H), 7.17–7.09
(m, 4H), 6.99 (d, J = 8.1 Hz, 2H), 6.56 (d, J = 15.8 Hz, 2H), 5.52 (d, J = 8.0 Hz, 2H), 3.83
(s, 6H), 3.00–2.66 (m, 10H), 1.88–1.70 (m, 8H), 1.24–0.90 (m, 12H) ppm. 13 C NMR (125
MHz, CDCl3 ): δ 183.2, 171.4, 170.3, 151.4, 141.3, 140.1, 139.6, 134.1, 128.3, 128.3, 127.9, 127.2,
127.2, 124.4, 123.4, 121.2, 111.5, 102.0, 81.0, 56.0, 43.1, 29.5, 29.1, 29.1, 26.3, 26.0, 25.9 ppm.
IR: 2927.4, 2852.0, 1763.4, 1732.1, 1629.0, 1506.8, 1415.7, 1253.7, 1122.0 cm−1 . MS (m/z)
calcd for C55 H60 NaO12 : 935.40; found: 935.8 [M+Na+ ]. Compound 8: 1 H NMR (500 MHz,
CDCl3 ): δ 7.59 (dd, J = 15.8, 12.5 Hz, 2H), 7.35–7.26 (m, 5H), 7.14–7.02 (m, 4H), 6.95 (dd,
J = 26.7, 8.1 Hz, 2H), 6.51 (dd, J = 22.6, 15.8 Hz, 2H), 5.82 (s, 1H), 5.52 (d, J = 8.0 Hz, 1H),
3.92 (d, J = 0.9 Hz, 3H), 3.82 (s, 3H), 3.01–2.63 (m, 5H), 1.93–1.66 (m, 4H), 1.22–0.86 (m, 6H)
ppm. 13 C NMR (125 MHz, CDCl3 ): δ 184.6, 181.9, 171.4, 170.4, 151.4, 148.1, 146.9, 141.3,
141.1, 139.6, 139.5, 134.2, 128.3, 127.9, 127.2, 115.0, 111.4, 109.7, 101.7, 81.0, 56.0, 56.0, 43.0,
29.5, 29.1, 29.1, 29.0, 26.3, 25.9, 25.9 ppm. IR: 3430.5, 2930.5, 2851.9, 1763.9, 1733.4, 1627.0,
1587.9, 1510.0, 1267.5, 1130.9 cm−1 . MS (m/z) calcd for C38 H40 NaO9 : 663.26; found: 663.5
[M+Na+ ].

Synthesis of Di(9H-fluoren-9-yl) O,O0 -(((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-triene-1,7-

diyl)bis(2-methoxy-4,1-phenylene)) Disuccinate (9) and 9H-Fluoren-9-yl
(4-((1E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-trien-1-yl)-2-
methoxyphenyl) Succinate (10)

According to GP2, succinate S5 (34.5 mg, 0.12 mmol, 0.9 equiv), 4-dimethylaminopy-
ridine (15.7 mg, 0.13 mmol, 1.0 equiv.), curcumin (50.0 mg, 0.13 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
N0 -ethylcarbodiimide hydrochloride (24.2 mg, 0.13 mmol, 1.0 equiv.) was added and stirred
at rt for 24 h. The crude product was purified by HPLC to obtain diester 9 (4.4 mg, 4%) and
monoester 10 (22.3 mg, 26%). Compound 9: 13 C NMR (125 MHz, CDCl3 ): δ 183.2, 172.9,
170.4, 151.4, 148.0, 146.9, 141.9, 141.1, 134.1, 129.7, 129.7, 128.0, 128.0, 126.1, 126.0, 120.2,
120.2, 114.9, 111.5, 109.7, 75.6, 56.1, 56.0, 52.1, 29.6, 29.5, 29.2, 29.1 ppm. IR: 2958.3, 2923.4,
Int. J. Mol. Sci. 2023, 24, 3691 13 of 17

2852.1, 1760.7, 1730.9, 1627.7, 1587.8, 1505.3, 1451.3, 1249.6, 1117.3 cm−1 . MS (m/z) calcd
for C55 H44 NaO12 : 919.27; found: 919.30 [M+Na+ ]. Compound 10: 13 C NMR (125 MHz,
CDCl3 ): δ 184.7, 181.9, 172.9, 170.4, 151.4, 148.1, 147.9, 146.9, 146.9, 141.9, 141.1, 129.7,
128.0, 126.1, 123.0, 121.9, 121.8, 120.2, 115.0, 114.9, 109.7, 101.8, 75.6, 56.1, 56.0, 52.1, 29.6,
29.2 ppm. IR: 3426.6, 2959.8, 2926.3, 2850.6, 1760.2, 1730.6, 1625.1, 1585.5, 1507.1, 1450.4,
1250.8, 1117.8 cm−1 . MS (m/z) calcd for C38 H32 NaO9 : 655.19; found: 655.05 [M+Na+ ].

Synthesis of Bis(2,3-dihydro-1H-inden-2-yl) O,O0 -(((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-

triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)) Disuccinate (11)

According to GP2, succinate S6 (100.2 mg, 0.43 mmol, 3.1 equiv), 4-dimethylamino-
pyridine (23.9 mg, 0.20 mmol, 1.2 equiv.), curcumin (50.1 mg, 0.14 mmol), and pyridine
(2.5 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
N0 -ethylcarbodiimide hydrochloride (152.1 mg, 0.79 mmol, 5.8 equiv.) was added and
stirred at rt for 24 h. The crude product was purified by HPLC to obtain diester 11 (17.6 mg,
16%). Compound 11: 1 H NMR (500 MHz, CDCl3 ): δ 7.62 (d, J = 15.8 Hz, 2H), 7.24–7.19
(m, 8H), 7.16–7.10 (m, 4H), 7.01 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 15.8 Hz, 2H), 5.87 (s, 1H),
5.61–5.57 (m, 2H), 3.85 (s, 6H), 3.33 (dd, J = 16.9, 6.4 Hz, 4H), 3.03 (dd, J = 17.0, 2.9 Hz, 4H),
2.92 (t, J = 6.8 Hz, 4H), 2.71 (t, J = 6.8 Hz, 4H). 13 C NMR (125 MHz, CDCl3 ): δ183.2, 172.1,
170.5, 151.4, 141.2, 140.5, 140.1, 134.1, 126.9, 124.8, 124.8, 124.8, 124.3, 123.4, 121.2, 111.5,
102.0, 75.9, 56.0, 39.7, 29.5, 29.0 ppm. IR: 2922.7, 2851.5, 1760.9, 1727.1, 1505.8, 1415.1, 1252.5,
1117.6 cm−1 . MS (m/z) calcd for C47 H44 NaO12 : 823.27; found: 823.5 [M+Na+ ].

Synthesis of O,O0 -(((1E,3Z,6E)-3-Hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2-methoxy-

4,1-phenylene)) bis((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) Disuccinate (12) and
4-((1E,4Z,6E)-5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-trien-1-yl)-2-
methoxyphenyl((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) Succinate (13)

According to GP2, succinate S7 (33.1 mg, 0.13 mmol, 1.0 equiv), 4-dimethylaminopy-
ridine (17.5 mg, 0.14 mmol, 1.1 equiv.), curcumin (50.3 mg, 0.13 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
N0 -ethylcarbodiimide hydrochloride (23.7 mg, 0.12 mmol, 0.9 equiv.) was added and stirred
at rt for 24 h. The crude product was purified by HPLC to obtain diester 12 (5.3 mg, 9%)
and monoester 13 (23.6 mg, 57%). Compound 12: 1 H NMR (500 MHz, CDCl3 ): δ 7.62 (d,
J = 15.8 Hz, 2H), 7.17–7.06 (m, 6H), 6.57 (d, J = 15.8 Hz, 2H), 5.86 (s, 1H), 4.75–4.70 (m, 2H),
3.87 (s, 6H), 2.93 (t, J = 7.2 Hz, 4H), 2.75–2.72 (m, 4H), 1.99 (d, J = 11.5 Hz, 2H), 1.88–1.84 (m,
2H), 1.68 (dd, J = 8.9, 5.4 Hz, 4H), 1.46–1.35 (m, 4H), 1.03–0.96 (m, 4H), 0.89–0.87 (m, 12H),
0.74 (d, J = 6.9 Hz, 6H). 13 C NMR (125 MHz, CDCl3 ): δ 183.2, 171.7, 170.5, 151.5, 141.3, 140.1,
134.1, 124.4, 123.4, 121.2, 111.6, 102.0, 74.9, 56.1, 47.1, 41.0, 34.3, 31.5, 29.6, 29.2, 26.4, 23.5,
22.2, 20.9, 16.4 ppm. IR: 2954.7, 2929.4, 2869.3, 1765.2, 1727.9, 1630.7, 1508.4, 1462.4, 1416.6,
1129.9 cm−1 . MS (m/z) calcd for C49 H64 NaO12 : 867.43; found: 867.46 [M+Na+ ]. Compound
13: 1 H NMR (500 MHz, CDCl3 ): δ 7.60 (dd, J = 15.8, 7.0 Hz, 2H), 7.18–7.01 (m, 6H), 6.93
(d, J = 8.2 Hz, 1H), 6.51 (dd, J = 26.9, 15.7 Hz, 2H), 5.82 (s, 1H), 4.79–4.65 (m, 1H), 3.94 (s,
3H), 3.86 (s, 3H), 2.96–2.90 (m, 2H), 2.78–2.69 (m, 2H), 2.02–1.96 (m, 1H), 1.86 (qd, J = 7.0,
2.7 Hz, 1H), 1.70–1.64 (m, 2H), 1.49–1.34 (m, 2H), 1.29–1.21 (m, 1H), 1.08–0.95 (m, 2H), 0.88
(t, J = 6.9 Hz, 6H), 0.74 (d, J = 7.0 Hz, 3H) ppm. 13 C NMR (125 MHz, CDCl3 ): δ 184.7, 181.9,
171.7, 170.5, 151.4, 148.1, 146.9, 141.3, 141.2, 139.5, 134.2, 127.6, 124.3, 123.4, 123.2, 121.8,
121.1, 115.0, 111.5, 109.7, 101.7, 74.9, 56.1, 56.0, 47.1, 41.0, 34.3, 31.5, 29.6, 29.2, 26.3, 23.5,
22.1, 20.9, 16.4 ppm. IR: 2954.5, 2931.6, 2869.1, 1764.7, 1726.6, 1627.4, 1510.3, 1417.7, 1267.7,
1131.6 cm−1 . MS (m/z) calcd for C35 H42 NaO9 : 629.27; found: 629.17 [M+Na+ ].

Synthesis of 4-((1E,4Z,6E)-5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-
trien-1-yl)-2-methoxyphenyl Methyl Succinate (14)

According to GP2, succinate S8 (60.4 mg, 0.46 mmol, 1.1 equiv), 4-dimethylaminopy-
ridine (66.4 mg, 0.54 mmol, 1.3 equiv.), curcumin (151.6 mg, 0.41 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
Int. J. Mol. Sci. 2023, 24, 3691 14 of 17

N0 -ethylcarbodiimide hydrochloride (87.9 mg, 0.46 mmol, 1.1 equiv.) was added and stirred
at rt for 24 h. The crude product was purified by column chromatography on silica gel
to obtain monoester 14 (22.3 mg, 11%). Compound 14: 1 H NMR (500 MHz, CDCl3 ): δ
7.65–7.52 (m, 2H), 7.16–7.00 (m, 5H), 6.92 (dd, J = 8.1, 1.8 Hz, 1H), 6.56–6.42 (m, 2H), 5.82 (s,
1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.72 (s, 3H), 2.94 (t, J = 6.7 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H) ppm.
13 C NMR (125 MHz, CDCl ): δ 184.7, 181.8, 172.6, 170.5, 151.4, 148.3, 148.1, 147.0, 147.0,
3
141.3, 141.1, 140.7, 139.5, 134.2, 127.6, 127.5, 124.3, 123.4, 123.2, 123.0, 121.7, 121.7, 121.1,
115.0, 115.0, 111.5, 109.8, 109.7, 106.5, 101.7, 56.0, 52.1, 29.0 ppm. IR: 3405.4, 2950.9, 2845.6,
1760.1, 1735.3, 1625.9, 1586.8, 1509.7, 1417.8, 1126.9 cm−1 . MS (m/z) calcd for C35 H34 NaO9 :
621.21; found: 621.4 [M+Na+ ].

Synthesis of 4-((1E,4Z,6E)-5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-
trien-1-yl)-2-methoxyphenyl (1,2,3,4-tetrahydronaphthalen-1-yl) Succinate (15)

According to GP2, succinate S9 (95.91 mg, 0.39 mmol, 1.0 equiv), 4-dimethylaminopy-
ridine (52.9 mg, 0.43 mmol, 1.1 equiv.), curcumin (150.47 mg, 0.41 mmol), and dichloromethane
(4 mL) were combined and stirred at 0 ◦ C for 10 min. After, N-(3-Dimethylaminopropyl)-
N0 -ethylcarbodiimide hydrochloride (76.9 mg, 0.40 mmol, 1.0 equiv.) was added and stirred
at rt for 24 h. The crude product was purified by column chromatography on silica gel to
obtain monoester 14 (10.6 mg, 4%). Compound 15: 1 H NMR (500 MHz, CDCl3 ): δ 15.96
(s, 1H), 7.60 (dd, J = 15.8, 6.7 Hz, 2H), 7.24–7.09 (m, 7H), 7.05 (d, J = 1.9 Hz, 1H), 6.99 (d,
J = 8.1 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.52 (dd, J = 26.8, 15.8 Hz, 2H), 6.05 (t, J = 4.5 Hz,
1H), 5.83 (s, 1H), 3.95 (s, 3H), 3.86 (s, 3H), 2.99–2.93 (m, 2H), 2.79–2.76 (m, 2H), 2.03–1.92 (m,
3H), 1.87–1.78 (m, 1H).13 C NMR (125 MHz, CDCl3 ): δ 184.6, 181.9, 171.8, 170.5, 151.4, 148.1,
146.9, 141.3, 141.2, 139.5, 138.1, 134.4, 134.2, 129.7, 129.2, 128.3, 127.7, 126.3, 124.3, 123.4,
123.2, 121.9, 121.1, 115.0, 111.5, 109.7, 101.7, 70.7, 56.1, 56.0, 29.8, 29.2, 29.1, 18.9 ppm. IR:
3421.9, 2938.6, 2868.4, 2840.0, 1727.9, 1627.0, 1587.8, 1510.0, 1131.3 cm−1 . MS (m/z) calcd
for C35 H34 NaO9 : 621.21; found: 621.4 [M+Na+ ].

4.2. Mice
Female and male C57BL/6 mice, 8 weeks of age, were obtained from INDICASAT’s
animal facility. Mice were maintained with a 12 h light/dark cycle, at a constant temperature
of 24 ◦ C with free access to food and water.

4.3. Ethics Statement

All experiments were performed in strict accordance with guidelines from the Institu-
tional Animal Care and Use Committee and the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. The Institutional Animal Care and Use
Committee of INDICASAT approved the protocol (CICUA-18-007).

4.4. Macrophage Culture

Peritoneal macrophages were obtained four days after i.p. instillation of 2 mL of thio-
glycollate 3%, by peritoneal washing with chilled RPMI. Cells were seeded in RPMI with
10% FCS at 2 × 105 /well in 96-well plates and cultured for 2 h at 37 ◦ C in an atmosphere of
5% CO2 . Non-adherent cells were removed by washing and adherent cells were stimulated
as indicated in figure legends. Cells were treated with compounds 1–15 (30 µM) 1 h before
the stimulus with 10 ng/mL of LPS. For dose response experiments, cells were pre-treated
with different concentrations (1, 3, 10, 30 µM) of compounds 1, 2, 4, 5, 6, 8, 10, 13, and
15 before the stimulation with 10 ng/mL of LPS. All the treatments and controls were
performed in the presence of 0.5% of DMSO, as compounds are solubilized in this solvent.
Supernatants were collected 6h after the stimulus with LPS.

4.5. IL-6 and PGE2 Measurements

Peritoneal macrophages were cultured as previously described. The concentrations
of IL-6 were determined by ELISA (DuoSet kit, R&D System) according to the manufac-
Int. J. Mol. Sci. 2023, 24, 3691 15 of 17

turer’s protocol. The concentration of prostaglandin E2 (PGE2 ) was determined using the
“Prostaglandin E2 ELISA Kit - Monoclonal” from Cayman CHEMICAL, according to the
manufacturer’s protocol.

4.6. Cytotoxicity Assay

After the removal of supernatants, 100 µL of MTT (0.5 mg/mL) dissolved in RPMI
were added to each well and cells were incubated overnight at 37 ◦ C. The supernatants
were removed and formazan crystals were dissolved in 100 µL of 0.04 M HCl in isopropanol.
The color was analyzed at 570 nm using an ELISA plate reader. The percent of viable cells
was calculated using the formula: % viability: [(OD sample) × 100%]/(OD control). The
non-stimulated cells, cultured in medium plus 10% FCS and 0.5% DMSO, represented
100% viability.

4.7. Statistical Analysis

Results were analyzed using the statistical software package GraphPad Prism 5. Data
are presented as means ± S.E.M. Statistical analysis was performed by Student’s t-test. A
significant difference between groups was considered when p < 0.05. The half maximal
inhibitory concentration (IC50 ) was calculated by adjusting a sigmoidal dose–response
curve following GraphPad Prism5 procedure.

5. Conclusions
In this study, we synthesized 13 new curcumin derivatives and evaluated their effect
on the production of inflammatory mediators such as TNF-α, IL-6 and PGE2 . A common
feature among these derivatives was a succinate linker that was coupled to curcumin via
an esterification reaction. The alkoxide group attached to the connector varied, including
aliphatic substituents to aromatic rings, where the former included acyclic structures and
cyclic rings, and the latter had an increase in the presence of aromatic rings. We added the
succinyl group to curcumin structure to protect the new derivatives from degradation and
determine how this group affect the anti-inflammatory response. Our results indicate that
a free hydroxyl group on the curcumin ring, the absence of a linker, and aromatic ligands
all increase the anti-inflammatory activity of this series of compounds. In this investigation,
we analyzed two different kinds of curcumin derivatives, the ether group (2) and succinyl
group (3–15), where compound 2 continues to have the best anti-inflammatory effects of the
series that we have studied. Further studies will be needed to describe the mechanism of
action of this compound and to evaluate the stability and bioavailability in in vivo systems.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/ijms24043691/s1, General procedure 1 (GP1) for the synthesis of Alkyl succinate monoesters,
General procedure (GP2) for the synthesis of dialkylcurcumin and monoalkylcurcumin (3–15). Refer-
ence [16] is cited in the Supplementary Material.
Author Contributions: Conceptualization, J.L.-B., Y.G. and O.V.L.; Data curation, P.L.F., R.M.-F.,
D.M.-L. and L.C.-R.; Formal analysis, J.L.-B. and Y.G.; Funding acquisition, J.L.-B., Y.G. and K.S.J.R.;
Investigation, J.L.-B., Y.G., R.M.-F. and D.M.-L.; Methodology, J.L.-B., Y.G. and O.V.L.; Project admin-
istration, J.L.-B. and K.S.J.R.; Resources, J.L.-B. and Y.G.; Supervision, J.L.-B. and O.V.L.; Validation,
J.L.-B., Y.G. and P.L.F.; Visualization, J.L.-B., Y.G. and O.V.L.; Writing—original draft preparation,
J.L.-B., Y.G. and O.V.L.; Writing—review and editing, J.L.-B., Y.G., O.V.L., P.L.F., L.C.-R. and K.S.J.R.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Secretaría Nacional de Ciencia, Tecnología e Innovación
[SENACYT grant number FID17-002], Secretaría Nacional de Ciencia, Tecnología e Innovación
[SENACYT grant number ITE18-R1-006], Instituto de Investigaciones Científicas y Servicios de
Alta Tecnología [INDICASAT AIP grant number JR04-2020], and the Welch Foundation grant
number AX-0047.
Data Availability Statement: Data is contained within the article and Supplementary Materials.
Int. J. Mol. Sci. 2023, 24, 3691 16 of 17

Acknowledgments: K.S.J.R. and J.L.-B. would like to acknowledge SENACYT Project FID17-002 and
Y.G. would like to acknowledge SENA-CYT Project ITE18-R1-006. J.L.-B., Y.G. and P.L.F. would like
to acknowledge the National System of Investigators (SNI) for supporting their research. K.S.J.R. and
J.L.-B. would like to acknowledge Melo Brain Grant (Panama) and INDICASAT Internal Grant JR04-
2020. O.V.L would like to acknowledge Welch Foundation grant number AX-0047. Mass spectroscopic
analysis was supported by the University of Panama. K.S.J.R. and J.L.-B. would like to acknowledge
Ricardo Santamaría for NMR’s sample recording and Daniel Torres for the MS data recording.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Lakey-Beitia, J.; Kumar, D.J.; Hegde, M.L.; Rao, K.S. Carotenoids as Novel Therapeutic Molecules Against Neurodegenerative
Disorders: Chemistry and Molecular Docking Analysis. Int. J. Mol. Sci. 2019, 20, 5553. [CrossRef]
2. Lakey-Beitia, J.; Berrocal, R.; Rao, K.S.; Durant, A.A. Polyphenols as Therapeutic Molecules in Alzheimer’s Disease Through
Modulating Amyloid Pathways. Mol. Neurobiol. 2015, 51, 466–479. [CrossRef] [PubMed]
3. Murillo, E.; Agócs, A.; Nagy, V.; Király, S.B.; Kurtán, T.; Toribio, E.M.; Lakey-Beitia, J.; Deli, J. Isolation and identification of
sapotexanthin 5,6-epoxide and 5,8-epoxide from red mamey (Pouteria sapota). Chirality 2020, 32, 579–587. [CrossRef] [PubMed]
4. Lakey-Beitia, J.; Vasquez, V.; Mojica-Flores, R.; Fuentes, C.A.L.; Murillo, E.; Hedge, M.L.; Rao, K.S. Pouteria sapota (Red Mamey
Fruit): Chemistry and Biological Activity of Carotenoids. Comb. Chem. High Throughput Screen. 2021, 25, 1134–1147.
5. Wichitnithad, W.; Nimmannit, U.; Wacharasindhu, S.; Rojsitthisak, P. Synthesis, characterization and biological evaluation of
succinate prodrugs of curcuminoids for colon cancer treatment. Molecules 2011, 16, 1888–1900. [CrossRef]
6. He, Y.; Yue, Y.; Zheng, X.; Zhang, K.; Chen, S.; Du, Z. Curcumin, inflammation, and chronic diseases: How are they linked?
Molecules 2015, 20, 9183–9213. [CrossRef]
7. Shi, W.; Dolai, S.; Rizk, S.; Hussain, A.; Tariq, H.; Averick, S.; L’Amoreaux, W.; El Idrissi, A.; Banerjee, P.; Raja, K. Synthesis of
monofunctional curcumin derivatives, clicked curcumin dimer, and a PAMAM dendrimer curcumin conjugate for therapeutic
applications. Org. Lett. 2007, 9, 5461–5464. [CrossRef]
8. Parvathy, K.S.; Negi, P.S.; Srinivas, P. Curcumin-amino acid conjugates: Synthesis, antioxidant and antimutagenic attributes. Food
Chem. 2010, 120, 523–530. [CrossRef]
9. de C. F. Gomes, D.; Vilela Alegrio, L.; Edilson Freire de Lima, M.; Leon, L.L.; Araújo, C.A.C. Synthetic Derivatives of Curcumin
and their Activity against Leishmania amazonensis. Arzneim.-Forsch./Drug Res. 2002, 52, 120–124.
10. Jovanovic, S.V.; Boone, C.W.; Steenken, S.; Trinoga, M.; Kaskey, R.B. How Curcumin Works Preferentially with Water Soluble
Antioxidants. J. Am. Chem. Soc. 2001, 123, 3064–3068. [CrossRef]
11. Feng, L.; Li, Y.; Song, Z.-F.; Huai, Q.-Y.; Li, H.-J. Synthesis and biological evaluation of curcuminoid derivatives. Chem. Pharm.
Bull. 2015, 63, 873–881. [CrossRef]
12. Banuppriya, G.; Sribalan, R.; Padmini, V.; Shanmugaiah, V. Biological evaluation and molecular docking studies of new
curcuminoid derivatives: Synthesis and characterization. Bioorg. Med. Chem. Lett. 2016, 26, 1655–1659. [CrossRef] [PubMed]
13. Lozada-García, M.C.; Enríquez, R.G.; Ramírez-Apán, T.O.; Nieto-Camacho, A.; Palacios-Espinosa, J.F.; Custodio-Galván, Z.;
Soria-Arteche, O.; Pérez-Villanueva, J. Synthesis of curcuminoids and evaluation of their cytotoxic and antioxidant properties.
Molecules 2017, 22, 633. [CrossRef]
14. Marchiani, A.; Mammi, S.; Siligardi, G.; Hussain, R.; Tessari, I.; Bubacco, L.; Delogu, G.; Fabbri, D.; Ruzza, P. Small molecules
interacting with alpha -synuclein: Antiaggregating and cytoprotective properties. Amino Acids 2013, 45, 327–338. [CrossRef]
[PubMed]
15. Rajeswari, A.; Sabesan, M. Inhibition of monoamine oxidase-B by the polyphenolic compound, curcumin and its metabolite
tetrahydrocurcumin, in a model of Parkinson’s disease induced by MPTP neurodegeneration in mice. Inflammopharmacology 2008,
16, 96–99. [CrossRef] [PubMed]
16. Lakey-Beitia, J.; Gonzalez, Y.; Doens, D.; Stephens, D.E.; Santamaria, R.; Murillo, E.; Rao, K.S.; Larionov, O.V.; Durant-Archibold, A.A.
Assessment of Novel Curcumin Derivatives for the Prevention of the Inflammatory Process in Alzheimer’s Disease. J. Alzheimers
Dis. 2017, 2017, S59–S68. [CrossRef]
17. Orjuela, A.; Lakey-Beitia, J.; Mojica-Flores, R.; Hegde, M.L.; Lans, I.; Alí-Torres, J.; Rao, K.S. Computational Evaluation of
Interaction Between Curcumin Derivatives and Amyloid-β Monomers and Fibrils: Relevance to Alzheimer’s Disease. J. Alzheimers
Dis. 2020, 82, S321–S333. [CrossRef]
18. Abe, Y.; Hashimoto, S.; Horie, T. Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes
and alveolar macrophages. Pharmacol. Res. 1999, 39, 41–47. [CrossRef]
19. Surh, Y.J.; Chun, K.S.; Cha, H.H.; Han, S.S.; Keum, Y.S.; Park, K.K.; Lee, S.S. Molecular mechanisms underlying chemopreventive
activities of anti-inflammatory phytochemicals: Down-regulation of COX-2 and iNOS through suppression of NF-κB activation.
Mutat. Res. 2001, 480–481, 243–268. [CrossRef]
20. Jobin, C.; Bradham, C.A.; Russo, M.P.; Juma, B.; Narula, A.S.; Brenner, D.A.; Sartor, R.B. Curcumin Blocks Cytokine-Mediated NF-
κ B Activation and Proinflammatory Gene Expression by Inhibiting Inhibitory Factor I- κ B Kinase Activity. J. Immunol. 1999, 163,
3474–3483. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 3691 17 of 17

21. Jurenka, J.S. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: A review of preclinical and clinical
research. Altern. Med. Rev. 2009, 14, 141–153. [PubMed]
22. Chainani-Wu, N. Safety and Anti-Inflammatory Activity of Curcumin: A Component of Tumeric (Curcuma longa). J. Altern.
Complement. Med. 2003, 9, 161–168. [CrossRef] [PubMed]
23. Wu, J.; Zhang, Y.; Cai, Y.; Wang, J.; Weng, B.; Tang, Q.; Chen, X.; Pan, Z.; Liang, G.; Yang, S. Discovery and evaluation of piperid-
4-one-containing mono-carbonyl analogs of curcumin as anti-inflammatory agents. Bioorg. Med. Chem. 2013, 21, 3058–3065.
[CrossRef]
24. Masuda, T.; Hidaka, K.; Shinohara, A.; Maekawa, T.; Takeda, Y.; Yamaguchi, H. Chemical studies on antioxidant mechanism of
curcuminoid: Analysis of radical reaction products from curcumin. J. Agric. Food Chem. 1999, 47, 71–77. [CrossRef] [PubMed]
25. Tang, B.L. Alzheimer’s disease: Channeling APP to non-amyloidogenic processing. Biochem. Biophys. Res. Commun. 2005, 331,
375–378. [CrossRef] [PubMed]
26. Ukil, A.; Maity, S.; Karmakar, S.; Datta, N.; Vedasiromoni, J.R.; Das, P.K. Curcumin, the major component of food flavour turmeric,
reduces mucosal injury in trinitrobenzene sulphonic acid-induced colitis. Br. J. Pharmacol. 2003, 139, 209–218. [CrossRef]
27. Ouberai, M.; Dumy, P.; Chierici, S.; Garcia, J. Synthesis and biological evaluation of clicked curcumin and clicked KLVFFA
conjugates as inhibitors of β-amyloid fibril formation. Bioconjug. Chem. 2009, 20, 2123–2132. [CrossRef]
28. Dolai, S.; Sun, C.; Averick, S.; Obeysekera, D.; Banerjee, P.; Raja, K.; Shi, W.; Corbo, C.; Farid, M.; Alonso, A. “Clicked”
sugar.curcumin conjugate: Modulator of amyloid-βand tau peptide aggregation at ultralow concentrations. ACS Chem. Neurosci.
2011, 2, 694–699. [CrossRef]
29. Reddy, S.T.; Herschman, H.R. Ligand-induced prostaglandin synthesis requires expression of the TIS10/PGS-2 prostaglandin
synthase gene in murine fibroblasts and macrophages. J. Biol. Chem. 1994, 269, 15473–15480. [CrossRef]
30. Zhang, F.; Altorki, N.K.; Mestre, J.R.; Subbaramaiah, K.; Dannenberg, A.J. Curcumin inhibits cyclooxygenase-2 transcription in
bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis 1999, 20, 445–451. [CrossRef]
31. Nakatsugi, S.; Sugimoto, N.; Furukawa, M. Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 produc-
tion by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with
lipopolysaccharide. Prostaglandins Leukot. Essent. Fat. Acids 1996, 55, 451–457. [CrossRef] [PubMed]

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