Synthesis, anticancer activity, and preliminary pharmacokinetic evaluation of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives

Article abstract of DOI:10.3390/molecules25030479

Compound 1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the α-position (C-4 position) of the central 1,3-diketone moiety of 1 with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure–activity relationship of compound 1 derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds 2–6 are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound 1, respectively. Compounds 2m–6m, the ester hydrolysis products of compounds 2–6, respectively, were synthesized and assessed for anticancer activity. Among all compound 1 derivatives, compound 2 emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of 2 (IC₅₀ = 3.10 ± 0.29 μM) and its ester hydrolysis product 2m (IC₅₀ = 2.17 ± 0.16 μM) against HCT-116. The preliminary pharmacokinetic evaluation of 2 implied that 2 and 2m are main contributors to the in vivo efficacy. Compound 2 was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure–activity relationship and pharmacokinetic outcomes of 2 is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

Full text of DOI:10.3390/molecules25030479

molecules
Article
Synthesis, Anticancer Activity, and Preliminary
Pharmacokinetic Evaluation of 4,4-Disubstituted
Curcuminoid
2,2-bis(Hydroxymethyl)Propionate Derivatives
Der-Yen Lee 1, Yu-Chi Hou 2, Jai-Sing Yang 3, Hui-Yi Lin 4, Tsu-Yuan Chang 2,
Kuo-Hsiung Lee 5,6 , Sheng-Chu Kuo 2,4,* and Min-Tsang Hsieh 2,4,6,
*
1
Graduate Institute of Integrated Medicine, China Medical University, No. 91, Hsueh-Shih Road,
Taichung 40402, Taiwan; deryen.lee@mail.cmu.edu.tw
2
3
4
5
6
School of Pharmacy, China Medical University, Taichung 40402, Taiwan; houyc@mail.cmu.edu.tw (Y.-C.H.);
u103003309@cmu.edu.tw (T.-Y.C.)
Department of Medical Research, China Medical University Hospital, China Medical University,
Taichung 40447, Taiwan; jaisingyang@gmail.com
Research Center for Chinese Herbal Medicine, China Medical University, Taichung 404, Taiwan;
pingababy@yahoo.com.tw
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599, USA; khlee@unc.edu
Chinese Medicinal Research and Development Center, China Medical University Hospital,
Taichung 40447, Taiwan
*
Correspondence: sckuo@mail.cmu.edu.tw (S.-C.K.); t21917@mail.cmu.edu.tw (M.-T.H.);
Tel.: +886-4-22030760 (S.-C.K.); +886-4-22053366-5605 (M.-T.H.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Ping-Chung Kuo
Received: 13 January 2020; Accepted: 22 January 2020; Published: 22 January 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Compound
in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency
than curcumin. Here, we modified the -position (C-4 position) of the central 1,3-diketone moiety
of with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid
2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear
structure–activity relationship of compound derivatives focusing on the functional groups at
the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231
and HCT-116 cell lines. Compounds are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated,
4,4-dipropargylated and 4,4-diallylated compound , respectively. Compounds 2m 6m, the ester
hydrolysis products of compounds , respectively, were synthesized and assessed for anticancer
activity. Among all compound derivatives, compound emerged as a potential chemotherapeutic
agent for colon cancer due to the promising in vivo anti-proliferative activities of (IC50 = 3.10
0.29 M) and its ester hydrolysis product 2m (IC50 = 2.17 0.16 M) against HCT-116. The
preliminary pharmacokinetic evaluation of implied that and 2m are main contributors to the
in vivo efficacy. Compound was further evaluated in an animal study using HCT-116 colon
1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant
α
1
1
2–6
1
–
2–6
1
2
2
±
µ
±
µ
2
2
2
tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to
tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The
established structure–activity relationship and pharmacokinetic outcomes of 2 is the guidance for
future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives
as anticancer drug candidates.
Keywords: curcuminoid derivatives; prodrug; colon cancer; breast cancer; active metabolites
Molecules 2020, 25, 479; doi:10.3390/molecules25030479
www.mdpi.com/journal/molecules

Molecules 2020, 25, 479
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1. Introduction
Curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione, the structure
of which is shown in Scheme 1], the naturally occurring phytochemical from the rhizome of Curcuma
longa L., is a polyphenol with a symmetrical structure composed of two ortho-methoxyphenol rings
connected to each other through a flexible conjugated hydrocarbon chain. It is a versatile therapeutic
agent against cancer [
antiviral [ ], analgesic [
gene expression [ ] and protein binding [
multi-target anticancer agent [ ] with the ability to interfere with several signaling pathways associated
with tumor progression, metastasis, apoptosis, and angiogenesis [10].
1
4
] and exhibits diverse pharmacological effects, including antidiabetic [
], nephroprotective [ ], and cardioprotective effects [ ], via regulating the
]. Similar to some phenolic natural products, curcumin is a
2],
3
5
6
7
8
9
O
OH
O
OH
MeO
O
OMe
O
OMe
OH
MeO
HO
PLE
HO
HO
O
O
OH
OH
1
curcumin
Scheme 1. PLE mediated hydrolysis of 1.
While curcumin shows promising therapeutic properties, its low solubility and poor chemical
and metabolic stability hinder further drug development [11]. The low chemical stability of curcumin
is demonstrated by its rapid decomposition upon exposure to light or high temperatures (>70 ◦C) [12].
The compound decomposes into several degradation products, including a bicyclopentadione
derivative, vanillin, feruloylmethane, and ferulic acid. The degradation of curcumin presumably
results from its autoxidation [13], wherein the starting radical species is formed through hydrogen
atom removal from the phenolic moiety. The phenolic group with an easily abstractable proton
and the keto-enol system display significant hydrogen-donating abilities [14] that could initiate the
autoxidative degradation of curcumin. The poor metabolic stability of curcumin can be ascribed to the
presence of two phenolic hydroxyl groups that are prone to the formation of glucuronide and sulfate
conjugates through the phase II metabolic process [15]. Moreover, the enone moiety of curcumin is
accessible to nicotinamide adenine dinucleotide phosphate (NADHP) cytochrome P450 reductase or
alcohol dehydrogenases, allowing curcumin to be readily reduced to the hydrogenated metabolites
dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, and hexahydrocurcuminol [16].
Medicinal chemists have performed structural optimizations of curcumin to improve its stability
and activity. Most of these attempts were focused on modifying the phenolic hydroxyl groups,
keto-enol system, and enone moiety of curcumin [17–20]. However, the first two abovementioned
structural features are required for radical scavenging activity, which is regarded as vital for curcumin
chemopreventive and anti-inflammatory activities [21]. In addition, the enone moiety of curcumin,
serving as the electrophilic Michael acceptor, can interact with cysteine or selenocysteine residues in
proteins to mediate biological activities [22]. Thus, finding a balance between structural stability and
biological potency can become challenging. According to the FDA [23], about 30% of the curcumin
clinical trials stalled at stage II, mostly due to discrepancies between in vitro activity and in vivo
response. Accordingly, to achieve success in clinical trials, obtaining a more definite picture of signaling
pathways and ADME profiling for curcumin is recommended.
We previously developed a series of curcuminoid prodrugs by modifying the phenolic hydroxyl
groups of curcumin into a 2,2-bis(hydroxymethyl)propionate group [24]. Among the derivatives,
the curcumin 2,2-bis(hydroxymethyl)propionate
of curcumin. Particularly, showed comparable anticancer activity against MDA-MB-231 cells in vitro
and in vivo and had 8 to 10 times more potency than curcumin. Subsequently, the -position in the
central 1,3-diketone moiety (C-4 position) of were alkylated with methyl, ethyl, benzyl, and propargyl
groups to give compounds , and , respectively. Among them, , and showed more potent
1 exhibited stability and solubility superior to those
1
α
1
2,
3,
4
5
2,
3
5

Molecules 2020, 25, 479
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in vitro anti-proliferative activity than
1
against HCT-116 cells; therefore, were recommended them
as potential therapeutic agents for colon cancer [24]. In our recent in vitro studies on the enzymatic
hydrolysis of , we demonstrated that treating with porcine liver esterase (PLE) cleaves its two ester
groups gradually to form curcumin (see supporting information). Compounds are ester-based
derivatives and structurally correlated to . Therefore, we predicted that a high concentration of
esterase, as particularly found in the intestinal tract and mucosa [25], would hydrolyze , and
into their metabolites 2m, 3m, 4m, and 5m, respectively (Scheme 2). However, the anticancer activity
of 2m 5m should be evaluated prior to the further in vivo investigations. Compounds were
substituted with two nonpolar alkyl chains at the C-4 position with the loss of keto-enol tautomerism.
The anticancer activity of compound derivatives, which were substituted with two hydrophilic units
or two polar side chains at the C-4 position, still remains unclear.
1
1
2-5
1
2,
3
,
4
5
–
2–5
1
O
O
O
O
In vivo
MeO
O
OMe
O
OMe
OH
MeO
HO
metabolism
R
R
R
R
HO
HO
O
O
OH
OH
2
3
4
5
, R = methyl
, R = ethyl
, R = benzyl
, R = propargyl
2m
3m
4m
5m
, R = methyl
, R = ethyl
R = benzyl
,
, R = propargyl
Scheme 2. In vivo enzymatic hydrolysis of 2–5.
Herein, we report the successful replacement of the acidic
of compound with polar or nonpolar functional groups to produce compounds
and 14. Compounds 2m 6m were prepared through the chemical hydrolysis of corresponding parent
compounds . The anticancer activities of 12 14 and 2m 6m were evaluated in vitro. A selected
α
-hydrogens at the C-4 position
1
6,
9
,
10 11 12,
,
,
–
2–
6
2–
6,
9–
,
–
compound was submitted for preliminary pharmacokinetic study and antitumor study. The detailed
descriptions of the synthetic design, in vitro screening, and in vivo study of the abovementioned
compounds are presented as follows.
2. Results and Discussion
2.1. Chemistry
The detailed synthesis of compound 1 derivatives (substituted at the C-4 position with allyl (6),
hydroxymethyl (10), acetoxymethyl (11), methoxymethyl (12)), novel 2,6-dimethyl curcumin (DMC)
derivatives (13 and 15), and the ester hydrolysis products of derivatives showing potential anticancer
activity (2m
Scheme 3, compound
Compound , prepared by the esterification of curcumin with 2,2,5-trimethyl-1,3-dioxane-5-carboxylic
acid, was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and formaldehyde in THF to produce
intermediary compound . The latter was used without column chromatographic purification in
,
3m
,
4m
,
5m, 6m, 16, and 17) are depicted in Schemes 3 and 4. As shown in
6
was synthesized according to a procedure established for the synthesis of
2–5.
7
8
acid-promoted hydrolysis at room temperature to produce
synthetic steps.
Compound
and presenting an NMR spectrum comprising a messy jungle of peaks after being dissolved in
DMSO-d6 for 1 week at room temperature. The low stability of may have been due to self- or
9 with an overall yield of 6% for two
9
, however, proved unstable, slowly decomposing even when kept at 4 ◦C
9
cross-hydrolysis, such that the two hydroxyl groups at the C-4 alkyl chains cleaved the ester linkage
of the 2,2-bis(hydroxymethyl)propionate group. Previously, we had found that upon exposure to
alcohol for several hours, the ester linkages of
to the conversion of to curcumin and several unknown products. Therefore, we modified the
hydroxymethyl group of and were able to produce compounds 10 and 11. To prepare 10, compound
was acetylated with acetyl chloride in the presence of Et3N, and the resulting di-acetylation intermediate
1 were broken under neutral or basic conditions, leading
1
9
9

Molecules 2020, 25, 479
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was hydrolyzed by hydrochloric acid. Compound 11 was obtained with an overall yield of 5% following
a similar three-step reaction modified for methylation. The stability of these two products at room
temperature may have been due to the conversion of the labile hydroxyl groups of the C-4 alkyl chains of
10 and 11 into non-nucleophilic methyl and acetyl groups, respectively, which prevented degradation.
O
O
O
OH
Ref. 21
MeO
O
OMe
O
MeO
HO
OMe
OH
HO
HO
O
O
OH
OH
curcumin
6
O
OH
O
O
MeO
O
OMe
O
MeO
O
OMe
O
a
O
O
O
O
O
O
O
7
O
O
OH OH
O
O
8
O
b
d
O
O
c
MeO
O
OMe
O
O
O
MeO
OMe
O
O
HO
O
O
OH
OH
OH OH
HO
O
O
OH
OH
OMeOMe
11
9
HO
HO
O
O
MeO
O
OMe
O
HO
HO
O
O
OH
OAcOAc
10
OH
O
O
O
OH
MeO
MeO
OMe
OMe
MeO
OMe
e
f, g
MeO
OMe
OH OH
dimethylcurcumin
12
O
O
O
O
MeO
MeO
OMe
OMe
O
Cl
O
O
O
Na
O
14
13
OH OH OH OH
Scheme 3. Reagents and Conditions: (a) Formaldehyde in THF, DBU, 0 ◦C to rt, 2 h; (b) HCl, MeOH, rt,
1 h, 6% for two steps; ( ) Et3N, acetyl chloride, DCM, rt, 12 h; then HCl, MeOH, rt, 1 h, 3% for three
steps; ( ) K2CO3, CH3I, DMF, rt, 6 h; then HCl, MeOH, rt, 1 h, 4% for three steps; ( ) Formaldehyde in
) HCl, MeOH, 1 h, 7% for
c
d
e
THF, DBU, 0 ◦C to rt, 2 h, 42%; (
two steps.
f) Compound 13, Et3N, DCM, rt, 12 h; and (g
Because DMC is a dimethylated derivative of curcumin [26] that exhibits better anticancer
activity and stability than curcumin, we synthesized a novel DMC derivative (compound 12) in
which C-4 was substituted with two bulky 2,2-bis(hydroxymethyl)propionate groups. To produce
this derivative, commercially available DMC was reacted with DBU and formaldehyde in THF.
The reaction of 12 with 2,2,5-trimethyl-1,3-dioxane-5-carbonyl chloride 13
provided a diol intermediate, which was then hydrolyzed by hydrochloric acid in MeOH to yield
compound 14. In addition, as illustrated in Scheme 4, compounds 2m 6m were prepared through the
NaOMe-mediated hydrolysis of their corresponding parent compounds 2–6, with high yield.
[27] in the presence of Et3N
–

Molecules 2020, 25, 479
5 of 18
O
O
O
R
O
MeO
O
OMe
O
MeO
OMe
a
R
R
R
HO
HO
O
O
OH
OH
HO
OH
2
4
6
3
ethyl
, R = methyl; , R =
2m
3m
4m,
R = benzyl
, R = methyl;
, R =
6m
, R =
ethyl;
allyl
5
, R = benzyl; , R =
propagyl
5m
, R =
propagyl;
allyl
, R =
O
O
OH
O
MeO
HO
OMe
MeO
O
OMe
b, c
O
R
R
OH
O
O
15
16
, R = n-propyl
, R = n-butyl
Curcumin bis-acetate 17
Scheme 4. Reagents and Conditions: (a) NaOMe, MeOH, rt, 2 h, 82–87%; (b) K2CO3, alkyl iodide, DMF,
rt, 20 h; and (c) NaOMe, MeOH, rt, 3 h, 28–33% for two steps.
For SAR analysis, compounds 15 and 16, which were substituted with two straight alkyl chains
with three or four carbons at the C4 position, were prepared from curcumin bisacetate 17 28] via
a two-step procedure comprising K2CO3-induced dialkylation and NaOMe-mediated hydrolysis.
For the synthesis of four possible phase I metabolites of compound , compound 2m was subjected to
[
2
hydrogenation reactions. As shown in Scheme 5, compound 18 is a partially hydrogenated product
that can be prepared in 19% yield by treating compound 2m with H2(g) (1.0 atm, balloon) and a
catalytic amount of 10% w/w Pd/C in ethyl acetate. When the solvent was changed from ethyl acetate
to methanol, the hydrogenation reaction yielded the fully hydrogenated diketone 19 in 52% yield
and
β-hydroxy ketone 20 in 23% yield. Finally, the reduction of 20 with NaBH4 in MeOH at room
temperature (rt) for 2 h provided the desired diol compound 21 in 31% yield.
O
O
O
O
MeO
HO
OMe
OH
MeO
HO
OMe
OH
a
2m
18
O
O
O
OH
b
MeO
HO
OMe
OH
MeO
HO
OMe
OH
+
(52%)
(23%)
19
20
OH OH
c
MeO
HO
OMe
20
OH
21
Scheme 5. Reagents and Conditions: (
a
) 10% w/w Pd/C, ethyl acetate, rt, 1 h, 19%; (
b
) 10% w/w Pd/C,
MeOH, rt, 2 h; and (c) NaBH4, MeOH, 0 ◦C to rt, 2 h, 31%.
2.2. Structure–Activity Relationship
As mentioned above, we synthesized 2–5 and evaluated their anti-proliferative activity against
the MDA-MB-231 and HCT-116 cell lines (72-h treatment). These compounds, along with all newly
synthesized compounds, were screened for anti-proliferative activity against the same TNBC and
colon cancer cell lines. The results of the in vitro assay are compiled in Table 1.

Molecules 2020, 25, 479
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Table 1. The anti-proliferative effects of curcumin derivatives against MDA-MB-231 and HCT-116
cell lines.
O
O
MeO
R1
R1
OMe
R1
R2
R2
a
IC50 (M)/ 48 h
R2
Compound
MDA-MB-231
HCT-116
Curcumin
1
OH
H
H
22.05 ± 2.97
26.33 ± 1.38
O
C
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
OH
CH3
OH
O
O
O
O
O
O
O
O
O
3.06 ± 0.18
2.25 ± 0.10
3.02 ± 0.15
2.45 ± 0.24
5.87 ± 0.10
3.13 ± 0.54
>100
6.25 ± 0.60
3.10 ± 0.29
1.93 ± 0.01
7.66 ± 0.61
5.81 ± 0.23
0.92 ± 0.06
>100
O
C
2
3
methyl
ethyl
O
C
O
C
4
benzyl
O
C
5
propargyl
allyl
O
C
6
O
C
9
CH2OH
CH2OAc
O
C
10
11
6.55 ± 0.04
84.92 ± 1.67
6.53 ± 0.01
69.08 ± 3.11
O
C
CH2OMe
CH2OH
12
14
OCH3
OCH3
53.50 ± 3.45
57.43 ± 2.06
58.00 ± 0.60
40.10 ± 1.61
O
C
OH
H2
C
O
OH
2m
3m
4m
5m
6m
15
OH
OH
OH
OH
OH
OH
OH
methyl
ethyl
benzyl
propargyl
allyl
propyl
butyl
4.17 ± 0.15
9.12 ± 0.40
36.52 ± 1.66
3.10 ± 0.15
6.88 ± 0.36
18.17 ± 0.53
>100
2.17 ± 0.16
8.18 ± 0.57
9.40 ± 0.02
1.38 ± 0.06
4.40 ± 0.53
10.73 ± 1.63
67.00 ± 3.18
16
a
All data presented are means from at least three experiments with standard deviations of the value quoted.
The anticancer effects of
, although the drug treatment duration was reduced to 48 h. The newly synthesized compound
had a higher anti-proliferative activity against HCT-116. Compound was less potent against the
cell lines than curcumin and , which was used as a reference compound in the current study. We
2–5 against both cell lines were still better or at least similar to that
of
1
6
9
1
hypothesize that the two polar and hydrophilic side chains at the C-4 position in
9
are the cause for the
weak anticancer effects observed for this compound. Another possibility is that
9 decomposed during
the screening due to its low stability. Compound 10, having two acetoxymethyl groups rather than
hydroxymethyl groups at the C-4 side chains, showed significant improvement in in vitro anticancer

Molecules 2020, 25, 479
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activities compared to
9
; however, it was still less potent than
1. Compound 11, substituted with two
methoxymethyl groups at the C-4 position, displayed poor anticancer activity with high IC50 values
for both cell lines used.
After listing the compounds in descending order based on their potency against HCT-116 (
10 >> 11 >> ), we concluded that higher polarity and hydrophilicity of the C-4 side chain
are detrimental to the anticancer activity of compound derivatives. Alternatively, the DMC derivative
6 > 3 > 2
>
5
>
1
>
>
4
9
1
4,4-dihydroxymethyl dimethoxycurcumin (compound 12) displayed low anti-proliferative activity.
When the two hydroxyl moieties of 12 both esterified with 2,2-bis(hydroxymethyl)propionic acid,
the resulting compound 14 exhibited activities comparable to those of 12, which correlates with our
previous findings regarding the unfavorable effect of having a hydrophilic moiety at the C-4 position.
Subsequently, the activities of the ester hydrolysis products 2m
5m displayed substantial improvement in anticancer activity by two- to four-fold relative to that of
parent . Compound 2m exhibited activity similar to that of , whereas 3m 4m, and 6m showed
inferior activity than their corresponding parent compounds , and . Further screening of 15 and 16
–6m were explored. Unexpectedly,
5
2
,
3
,
4
6
revealed an obvious SAR, such that the anticancer activity of these hydrolysis compounds decreased
with the increasing length of the C-4 alkyl side chains. In addition, lower structural volume was
seemingly preferred to sterically congested side chains, as demonstrated by the analysis of 2m
, 3m, 15,
4m, and 16.
Among all examined compounds,
anticancer activity against MDA-MB-231 and HCT-116; therefore,
Notably, the solubility of was more significant than that of curcumin as was evident from the vehicle
of for the following in vivo study. The maximum solubility of in the aqueous vehicle comprising
2
and its ester hydrolysis product 2m possessed significant
2
was chosen for further studies.
2
2
2
5% ethanol, 10% Tween 80, and 85% saline was approximately 200 mg/mL, while curcumin was almost
insoluble in the vehicle. Based on the logic for the metabolism of curcumin [29] and an ester-type
prodrug [30], we speculated that the in vivo metabolism of
the latter would be converted into glucuronide 22 and sulfate 23 through the phase II metabolism
conjugation of hydrogenated products 18 19 20, and 21 via the phase I metabolic pathway (Scheme 6).
To verify our assumptions, the preliminary pharmacokinetic evaluation of 2 was executed.
2 would readily produce 2m. Subsequently,
,
,
O
O
MeO
O
OMe
O
2
HO
HO
O
O
OH
OH
esterase hydrolysis
O
O
MeO
HO
OMe
OH
Phase II
2m
Phase I
metabolism
metabolism
O
O
O
O
MeO
HO
OMe
MeO
HO
OMe
OH
18
22
O
OH
OH
O
O
O
HOOC
MeO
HO
OMe
OH
OH
19
O
O
MeO
HO
OMe
OSO3H
O
OH
23
MeO
HO
OMe
OH
20
OH OH
MeO
HO
OMe
OH
21
Scheme 6. In vivo metabolism of 2.

Molecules 2020, 25, 479
8 of 18
2.3. The Preliminary Pharmacokinetic Evaluation of 2
The preliminary in vivo pharmacokinetic evaluation of
2
was designed to identify the anticipated
metabolites and evaluate the plasma stability of
20, and 21 were synthesized and subjected to anti-proliferative screening against MDA-MB-231 and
HCT-116. Compound 18 exhibited moderate to weak anti-proliferative activity (MDA-MB-231, IC50
2 and 2m. The four possible phase I metabolites 18, 19,
=
36.48
(IC50
±
0.34
100
µ
M; HCT-116, IC50 = 9.64
±
0.21
and relevant metabolites was performed in male Sprague-Dawley
at a dose of 100 mg/kg. The LC signals of 2m, and 18 21 was
µM) and the others are inactive against both cell lines
≥
µM). The assessment of 2
rats after the oral administration of
2
2,
–
recognized unequivocally by comparison to the signals of standard compounds. The direct analysis of
22 and 23 was not easily attainable due to the low extraction efficiency and high probability of sample
loss in LC column. It has been well-documented that curcumin glucuronide and curcumin sulfate can
be transformed back into curcumin by the enzyme-mediated hydrolysis reaction [31]. Accordingly, the
treatment of 22 and 23 with enzyme that contains sulfatase and glucuronidase is supposed to produce
2m. In the current study, the amounts of 22 and 23 were not calculated individually but counted as a
summary value based on the disparities between the LC signal of 2m in enzyme-treated and -untreated
serum samples.
In practice, LC-MS analysis of one serum sample which was collected at 20 min post-dosing
demonstrated the existence of
Shown in Figure 1 are the LC signal count-time profiles for
and 23, following oral administration. The analysis of 20 and 21 is not illustrated due to their low LC
signal response and non-significant anticancer activity. The maximum blood content of was achieved
at 20 min, while the second and third highest appeared at 120 min and 360 min, respectively, following
2,
2m
,
18
,
19
,
20,
21,
22 and 23 (Figure S1 in the Supporting Information).
2,
2m 18 19, and the comparison of 2m 22
,
,
,
,
2
administration. The multiple peaks of
circulation cycle.
2
observed in the profile could be attributed to an enterohepatic
Figure 1. The amount of
oral administration of
2
,
2m
,
18
,
19
,
22, and 23 in serum at different time points following a single
2
at a dose of 100 mg/kg. The blood samples were collected at 5, 10, 20, 30, 60,
120, 240, 360, and 480 min after the administration of
by HPLC.
2. The samples were deproteinated and analyzed

Molecules 2020, 25, 479
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Such behavior, extensively studied in the phenolic drugs, was beneficial in maintaining the
circulating concentration of free-form curcumin [32] and may extend the pharmacological effect of
drug [33]. The hydrolysis metabolite 2m emerged from 10 min and reached its maximum blood content
at 120 min, indicating that a medium-to-high esterase-mediated biotransformation proceeded in vivo
to yield the efficient absorption of 2m. Subsequently, the phase II metabolic process of 2m progressed
quickly to produce 22 and 23. The ratio of 2m to its phase II metabolites (22 and 23) is about 1:4 based
on the area under the curves in part (e) of Figure 1.
The LC signal count-time profiles of 18 and 19 are analogous to that of 2m, implying that phase
I reduction reaction also occurred rapidly. Compared to non-formulated curcumin with a short
elimination period of 28 min in the rat model [34],
active metabolite 2m is equally potent as with regard to anticancer activity, and the maximum blood
content of 2m appeared after that of could be considered as an extension of drug efficacy of . Thus,
2 exhibited superior plasma stability. Otherwise, the
2
2
2
the pharmacokinetic outcome was to guide the following in vivo efficacy study of 2.
2.4. In Vivo Antitumor Efficacy of 2
The therapeutic effect of
The oral administration of at doses of 50, 100, or 150 mg/kg bw per day were performed after tumors
reached approximately 100 mm3 and continued for 30 consecutive days. 5-Fluorouracil treatment
(30 mg/kg bw, i.p., QOD) was used as a positive control. The group treated with compound (100
2 was evaluated on HCT-116 colon tumor xenograft bearing nude mice.
2
2
mg/kg) had a statistically significant reduction in tumor volumes, with a 45% tumor inhibition ratio
observed when compared with the cancerous control mice. Positive control group exhibited a capability
to cause 40% tumor-growth inhibition. There was a clear dose- and time-dependent inhibitory effect
on tumor size in the other two groups, in which at 50 and 150 mg/kg doses the volume of the HCT-116
colon tumor was reduced to 73% and 40% of the control, respectively (Figure 2). The body weight of
the mice was recorded prior to dosing every 3 d, and the average body weight of each group is depicted
in Figure 3. Otherwise, they were monitored for visible signs of toxicity and behavioral changes 1 h
after each administration. No obvious adverse effects were observed between the 2-administrated and
control groups.
Figure 2. The efficacy study of
2
.
In vivo antitumor activity of compound
2 was evaluated in HCT-116
tumor xenograft bearing nude mice. Compound
2
(50, 100, or 150 mg/kg, p.o.) was administered once
daily and 5-Fluorouracil was administrated (30 mg/kg, i.p.) every other day. The results are the mean
SEM of six mice in each group. *** P < 0.001 compared to untreated control.
±

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Figure 3. Mean body weight-time profile of 2- administrated and control groups.
3. Materials and Methods
3.1. General Information
The reactions were performed under an air atmosphere unless otherwise stated. All solvents and
reagents were employed as received. Analytical thin layer chromatography (TLC) was performed
on SiO2 60 F254 plates and flash column chromatography was carried out using SiO2 60 (particle size
0.040–0.055 mm, 230–400 mesh), both of which are available from E. Merck (Darmstadt, Germany).
Visualization was performed under UV irradiation at 254 nm followed by staining with aqueous
potassium permanganate [KMnO4 (3 g) and K2CO3 (20 g) in 300 mL of H2O containing 5 mL of an
aqueous solution of NaOH (5%, w/v)] and charring by heat gun. 1H- and 13C-NMR spectra were
recorded on a 500 FT NMR instrument (Bruker, Billerica, MA, USA). Chloroform-d and methanol-d
were used as solvents and TMS (δ = 0.00 ppm) as an internal standard. Chemical shifts are reported as
δ
values in ppm as referenced to TMS. Multiplicities are recorded as s (singlet), d (doublet), t (triplet),
q (quartet), quint (quintet), sext (sextet), sept (septet), dd (doublet of doublets), dt (doublet of triplets),
br (broad), m (multiplet). Coupling constants (J) are expressed in Hz. LRMS and HRMS were measured
by a JMS-HX110 spectrometer (JEOL, Tokyo, Japan) and spectroscopic data were recorded as m/z values.
Melting points were measured using an Electrothermal instrument (Anatec Yanaco Inc., Kyoto, Japan).
3.1.1. [(1E,6E)-4,4-Diallyl-3,5-dioxohepta-1,6-diene-1,7-diyl]bis(2-methoxy-4,1-phenylene)
bis[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] (6)
Mp = 195–197 ◦C; 1H-NMR (DMSO-d6)
1.5 Hz, 2H), 7.12–7.04 (m, 4H), 5.59–5.52 (m, 2H), 5.12–5.04 (m, 4H), 4.82–4.80 (m, 4H), 3.78 (s, 6H), 3.60
δ 7.59 (d, J = 15.5 Hz, 2H), 7.48 (s, 2H), 7.35 (dd, J = 8.2,
(s, 8H), 2.84 (d, J = 7.2 Hz, 4H), 1.17 (s, 6H); 13C NMR (DMSO-d6)
δ 196.8, 173.3, 151.7, 143.1, 142.1,
133.3, 133.2, 123,9, 122.9, 122.8, 119.5, 113.4, 67.4, 64.0, 56.7, 51.0, 35.0, 17.3; HRMS [ESI]+ calculated for
C37H45O12: 681.2911 [M + H]+; found: 681.2903.
3.1.2. [(1E,6E)-4,4-bis(Hydroxymethyl)-3,5-dioxohepta-1,6-diene-1,7-diyl]bis(2-methoxy-4,1-phenylene)
bis(2,2,5-trimethyl-1,3-dioxane-5-carboxylate) (8)
To a stirred solution of
7 (1.00 g, 1.47 mmol) in THF (7.3 mL, containing 0.6 M formaldehyde) was
added DBU (38.9 mg, 0.04 mmol) at 0 ◦C. The reaction mixture was stirred at the same temperature for
30 min and then warm to room temperature. After 2 h, the solution was concentrated under vacuum
to provide crude compound, which was identified by LRMS [ESI]+ calculated for C39H49O14: 741.31
[M + H]+; found: 741.33 and used for next step without purification.

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3.1.3. [(1E,6E)-4,4-bis(Hydroxymethyl)-3,5-dioxohepta-1,6-diene-1,7-diyl]bis(2-methoxy-4,1-phenylene)
bis[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] (9)
A solution of crude
8 (0.34 g) and 6 N HCl (0.15 mL) in MeOH (1.83 mL) was stirred at room
temperature for 1 h. The reaction mixture was concentrated under vacuum to give the crude product,
which was purified by flash chromatography on silica gel with MeOH/CH2Cl2 (1:20) to afford compound
1
9
(40 mg) as an orange oil. Yield 6% for two steps. H-NMR (DMSO-d6)
δ
7.52 (d, J = 15.6 Hz, 2H), 7.44
(s, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.08–7.04 (m, 4H), 5.16 (br s, 6H), 4.19 (s, 4H), 3.78 (s, 6H), 3.61 (s, 8H),
1.17 (s, 6H); LRMS [ESI]+ calculated for C33H40O14: 661.25 [M + H]+; found: 661.4.
3.1.4. [(1E,6E)-4,4-bis(Acetoxymethyl)-3,5-dioxohepta-1,6-diene-1,7-diyl]bis(2-methoxy-4,1-phenylene)
bis[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] (10)
To a stirred solution of crude 8 (0.450 g) in DCM (10 mL) were added NEt3 (1.0 mL) and acetyl
chloride (0.140 g, 1.81 mmol) at 0 ◦C. The reaction mixture was warmed to room temperature and
stirred for 12 h. Then H2O (10 mL) and diluted HCl(aq) (1 N, 3 mL) were added to quench the reaction.
The aqueous layer was separated and extracted with CH2Cl2 (3
×
8 mL). The combined organic extracts
were washed with brine, dried over MgSO4, filtered and concentrated to give the crude product,
which was then treated with HCl(aq) (6 N, 0.2 mL) in MeOH (2 mL) at room temperature. The reaction
mixture was stirred at room temperature for 1h and then concentrated under vacuum to give the crude
product, which was purified on silica gel with MeOH/CH2Cl2 (1:20) to afford 10 (33 mg) as a yellow
solid (mp = 194–196 ◦C). Yield 3% for three steps; 1H-NMR (DMSO-d6)
(s, 2 H), 7.37 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 15.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 4.85 (t, J = 5.0 Hz, 8H),
δ 7.68 (d, J = 15.5 Hz, 2H), 7.51
3.80 (s, 6 H), 3.65-3.59 (m, 8H), 1.97 (s, 6H), 1.19 (s, 6H); 13C-NMR (DMSO-d6)
δ 193.1, 173.3, 170.5, 151.7,
144.6, 142.5, 132.9, 123.9, 122,7, 121.7, 113.6, 64.0, 61.9, 56.7, 51.0, 20.9, 17.3; HRMS [ESI]+ calculated for
C37H44NaO16: 767.2527 [M + Na]+; found: 767.2521.
3.1.5. [(1E,6E)-4,4-bis(Methoxymethyl)-3,5-dioxohepta-1,6-diene-1,7-diyl]bis(2-methoxy-4,1-phenylene)
bis[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] (11)
To a stirred solution of crude
8 (0.453 g) in DMF (1.0 mL) were added K2CO3 (0.240 g, 1.73 mmol)
and MeI (0.247 g, 1.73 mmol) at 0 oC. The reaction mixture was warmed to room temperature and
stirred for 6 h. Then H2O (10 mL) and diluted HCl(aq) (1 N, 3 mL) were added to quench the reaction.
The aqueous layer was separated and extracted with EtOAc (3
×
8 mL). The combined organic extracts
were washed with brine, dried over MgSO4, filtered and concentrated to give the crude product, which
was then treated with HCl(aq) (6 N, 0.25 mL) in MeOH (2 mL) at room temperature. The reaction
mixture was stirred at room temperature for 1h and then concentrated under vacuum to give the crude
product, which was purified on silica gel with MeOH/CH2Cl2 (1:20) to afford 11 (73 mg) as a yellow
solid (mp = 187–188 ◦C). Yield 5% for three steps; 1H-NMR (DMSO-d6)
(s, 2 H), 7.31 (d, J = 10.0 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 6.69 (d, J = 16.0 Hz, 2H), 4.85 (t, J = 5.5 Hz,
δ 7.65 (d, J = 16.0 Hz, 2H), 7.49
4 H), 3.80 (s, 6H), 3.74 (s, 6 H), 3.66-3.60 (m, 8H), 1.20 (s, 6H); 13C-NMR (DMSO-d6)
δ 193.1, 173.4, 167.1,
151.6, 144.4, 141.8, 133.2, 123.8, 121,9, 118.4, 112.6, 64.0, 56.3, 51.9, 51.0, 17.3; HRMS [ESI]+ calculated for
C35H44NaO14: 711.2629 [M + Na]+; found: 711.2633.
3.1.6. (1E,6E)-1,7-bis(3,4-Dimethoxyphenyl)-4,4-bis(hydroxymethyl)hepta-1,6-diene-3,5-dione (12)
To a stirred solution of DMC (1.270 g, 3.20 mmol) in THF (20 mL, containing 0.6 M formaldehyde)
was added DBU (15 mg, 0.10 mmol). The reaction mixture was stirred at 0 ◦C for 30 min. The reaction
mixture was concentrated under vacuum and then purified by flash chromatography on silica gel with
MeOH/CH2Cl2 (1:32) to afford 12 (558 mg, 42% yield) as a yellow solid; mp = 174–176 ◦C; 1H-NMR
(DMSO-d6) δ 7.49 (d, J = 15.5 Hz, 2H), 7.48 (d, J = 2.0 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 6.97–6.91 (m, 4H),
4.78 (t, J = 4.5 Hz, 2H), 4.19 (d, J = 4.5 Hz, 4H), 3.79 (s, 12H); 13C-NMR (DMSO-d6)
δ 196.2, 151.7, 149.1,
142.8, 127.4, 123.8, 120,9, 112.1, 111.3, 71.6, 60.1, 56.1, 56.0; HRMS [ESI]+ calculated for C25H28NaO8:
479.1682 [M + Na]+; found: 479.1677.

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3.1.7. 2,2-bis[(E)-3-(3,4-Dimethoxyphenyl)acryloyl]propane-1,3-diylbis[3-hydroxy-2-(hydroxymethyl)-2-
methylpropanoate] (14)
To a solution of 12 (0.170 g, 0.37 mmol) in CH2Cl2 (4 mL) was added NEt3 (0.15 mL, 1.10 mmol),
DMAP (0.110 g, 0.92 mmol) and 2,2,5-trimethyl-1,3-dioxane-5-carbonyl chloride 13 (0.210 g, 1.10 mmol)
sequentially at room temperature. The reaction mixture was stirred at the same temperature for 12h
and then concentrated under vacuum to give the crude product, which without purification was treated
with HCl(aq) (6 N, 0.1 mL) in MeOH (0.5 mL). The resulting mixture was stirred at room temperature
for 1h and then concentrated under vacuum to give the crude product, which was purified by flash
chromatography on silica gel with MeOH/CH2Cl2 (1:19) to afford compound 14 (16 mg, 7% for two
steps) as a yellow solid; mp > 300 ◦C; 1H-NMR (DMSO-d6)
4H), 7.03 (d, J = 15.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 4.74 (s, 4H), 4.63 (t, J = 5.5 Hz, 4H), 3.79 (s, 12H),
δ 7.64 (d, J = 15.5 Hz, 2H), 7.34–7.30 (m,
3.40-3.37 (m, 8H), 0.95(s, 6H); 13C-NMR (DMSO-d6)
δ 193.3, 174.6, 152.1, 149.4, 145.0, 127.1, 124.7, 119,5,
112.0, 111.4, 66.9, 63.9, 62.3, 56.1, 56.1, 50.7, 17.0; HRMS [ESI]+ calculated for C35H44NaO14: 711.2629
[M + Na]+; found: 711.2620.
3.1.8. General Procedure for the Synthesis of Compounds 2m–6m
The general procedure is illustrated immediately below with compound 2m as a specific example.
(1E,6E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-dimethylhepta-1,6-diene-3,5-dione (2m)
To a stirred solution of compound 2 (0.450 g, 0.72 mmol) in CH3OH (6 mL) were added NaOMe
(0.116 g, 2.16 mmol) at room temperature. The reaction mixture was stirred at the same temperature
for 2 h and then H2O (5 mL) and diluted HCl(aq) (1 N, 1 mL) were added to quench the reaction.
The aqueous layer was separated and extracted with CH2Cl2 (3
were washed with brine, dried over MgSO4, filtered and concentrated to give the crude product, which
×
6 mL). The combined organic extracts
was then purified by flash chromatography on silica gel with EtOAc/n-hexane/CH2Cl2 (1:2:1) to afford
2m (0.245 g, 86% yield) as an orange solid; mp = 104
−
106 ◦C; 1H-NMR (CDCl3)
δ 7.69 (d, J = 15.5 Hz,
2H), 7.12 (dd, J = 8.0, 1.5 Hz, 2H), 7.01 (s, 2H), 6.91 (d, J = 8.5 Hz, 2H), 6.65 (d, J = 15.5 Hz, 2H), 5.97
(s, 2H), 3.94 (s, 6H), 1.48 (s, 6H); 13C-NMR (CDCl3)
δ 198.2, 148.6, 146.8, 144.5, 126.8, 124.1, 119.1, 114.8,
109.8, 60.8, 56.0, 14.1; HRMS [ESI]+ calculated for C23H24NaO6: 419.1471 [M + Na]+; found: 419.1480
(1E,6E)-4,4-Diethyl-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (3m)
Yield: 87%; mp = 114–115 ◦C; 1H-NMR (CDCl3)
Hz, 2H), 6.95 (d, J = 2.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 6.59 (d, J = 15.5 Hz, 2H), 5.88 (s, 2H), 3.88
δ 7.64 (d, J = 15.5 Hz, 2H), 7.05 (dd, J = 8.3, 1.5
(s, 6H), 2.06 (q, J = 7.5 Hz, 4H), 0.71 (t, J = 7.5 Hz, 6H); 13C-NMR (CDCl3)
δ 197.9, 148.5, 146.7, 144.0,
126.8, 124.2, 119.6, 114.7, 109.7, 69.0, 56.1, 21.6, 7.7; HRMS [ESI]+ calculated for C25H28NaO6: 447.1784
[M + Na]+; found: 447.1788.
(1E,6E)-4,4-Dibenzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (4m)
Yield: 82%; mp = 134–135 ◦C; 1H-NMR (CDCl3)
7.15–7.13 (m, 4H), 7.07–7.05 (m, 2H), 6.90–6.89 (m, 4H), 6.58 (dd, J = 15.5, 1.0 Hz, 2H), 5.31 (s, 2H),
δ 7.72 (d, J = 15.0 Hz, 2H), 7.26–7.21 (m, 6H),
3.89 (s, 6H), 3.42 (s, 4H); 13C-NMR (CDCl3)
δ 196.9, 148.7, 146.8, 144.0, 136.6, 130.4, 128.1, 126.8, 126.6,
124.3, 120.8, 114.8, 109.8, 70.1, 56.1, 37.7; HRMS [ESI]+ calculated for C25H32NaO6: 571.2097 [M + Na]+;
found: 571.2090.
(1E,6E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-di(prop-2-yn-1-yl)hepta-1,6-diene-3,5-dione (5m)
Yield: 83%; mp = 201–203 ◦C; 1H-NMR (CDCl3)
Hz, 2H), 7.03 (d, J = 2.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 15.0 Hz, 2H), 5.97 (s, 2H), 3.95
(s, 6H), 3.19 (s, 4H), 2.04 (s, 4H); 13C-NMR (CDCl3)
193.5, 148.9, 145.9, 126.5, 124.7, 117.8, 114.8, 109.8,
79.1, 71.9, 66.9, 56.1, 21.2; HRMS [ESI]+ calculated for C27H28O6: 471.1784 [M + Na]+; found: 471.1783.
δ 7.75 (d, J = 15.0 Hz, 2H), 7.13 (dd, J = 8.0, 1.5
δ

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(1E,6E)-4,4-Diallyl-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (6m)
Yield: 85%; mp = 160–162 ◦C; 1H-NMR (CDCl3)
Hz, 2H), 6.95 (d, J = 2.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 6.61 (d, J = 15.5 Hz, 2H), 5.87 (s, 2H), 5.54–5.49
δ 7.65 (d, J = 15.5 Hz, 2H), 7.06 (dd, J = 8.3, 1.5
(m, 2H), 5.07–5.04 (m 4H), 3.88 (s, 6H), 2.78 (d, J = 7.5 Hz, 4H); 13C-NMR (CDCl3)
δ 194.6, 148.7, 146.8,
144.6, 132.3, 126.7, 119.3, 119.1, 114.7, 109.7, 67.8, 56.1, 34.4; HRMS [ESI]+ calculated for C27H24NaO6:
467.1471 [M + Na]+; found: 467.1477.
3.1.9. General Procedure for the Synthesis of Compounds 15 and 16
The general procedure is illustrated below with compound 15 as a specific example. To a solution
of curcumin bisacetate 17 (0.76 g, 1.68 mmol) in DMF (8 mL) was added K2CO3 (0.58 g, 4.20 mmol) and
propyl bromide (0.435 g, 3.53 mmol) sequentially at 0 ◦C. The reaction mixture was stirred at room
temeperatre for 20 h and then H2O (10 mL) and diluted HCl(aq) (1 N, 5 mL) were added to quench
the reaction. The aqueous layer was separated and extracted with EtOAc (3
×
10 mL). The combined
organic layer was dried over MgSO4, filtered and concentrated to give the crude product, which
was subjected without any purification to the base-promoted hydrolysis reaction [NaOMe (0.190 g,
3.53 mmol), MeOH (5 mL)]. The reaction was followed by TLC until no starting material was present.
The resulting mixture was then concentrated to give the crude product, which was then purified by
flash chromatography on silica gel with EtOAc/n-hexane/CH2Cl2 (1:2:1) to afford compound 15 (0.213 g,
28% yield for two steps) as a yellow solid.
(1E,6E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-dipropylhepta-1,6-diene-3,5-dione (15)
mp = 137–139 ◦C; 1H-NMR (CDCl3)
(d, J = 1.5 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 15.5 Hz, 2H), 5.90 (s, 2H), 3.94 (s, 6H), 2.05–2.01
δ 7.69 (d, J = 15.0 Hz, 2H), 7.11 (dd, J = 8.3, 1.5 Hz, 2H), 7.01
(m, 4H), 1.15–1.08 (m, 4H), 0.95 (t, J = 7.5 Hz, 6H); 13C-NMR (CDCl3)
δ 197.9, 148.5, 146.7, 144.0, 126.8,
124.1, 119.5, 114.7, 109.7, 68.4, 56.1, 31.8, 16.8, 14.7; HRMS [ESI]+ calculated for C27H32NaO6: 475.2097
[M + Na]+; found: 475.2100.
(1E,6E)-4,4-Dibutyl-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (16)
mp = 144–146 ◦C; 1H-NMR (CDCl3)
7.01 (s, 2H), 6.91 (d, J = 8.2 Hz, 2H), 6.65 (d, J = 15.5 Hz, 2H), 6.01 (s, 2H), 3.93 (s, 6H), 2.07–2.03 (m,
δ 7.68 (d, J = 15.5 Hz, 2H), 7.11 (dd, J = 8.3, 1.5 Hz, 2H),
4H), 1.37–1.33 (m, 4H), 1.10–1.03 (m, 4H), 0.90 (t, J = 7.3 Hz, 6H); 13C-NMR (CDCl3)
δ 198.0, 148.5,
146.8, 144.0, 126.9, 124.1, 119.5, 114.7, 109.7, 68.3, 56.1, 29.1, 25.5, 23.2, 13.9; HRMS [ESI]+ calculated for
C29H36NaO6: 503.2410 [M + Na]+; found: 503.2400.
3.1.10. (E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-dimethylhept-1-ene-3,5-dione (18)
To a stirred solution of compound 2m (0.740 g, 1.87 mmol) in EtOAc (10 mL) was added Pd/C
(37 mg, 5% w/w) in one portion. The resulting mixture was hydrogenated under 1 atmosphere of H2 at
room temperature. After 1 h, the mixture was filtered by celite and concentrated to give the crude
product, which was purified by flash chromatography on silica gel with EtOAc/n-hexane/CH2Cl2 (1:2:1)
to afford compound 18 (140 mg, 19% yield) as a light-yellow oil and recover compound 2m (500 mg,
1
67% yield). H-NMR (CDCl3)
δ
7.61 (d, J = 15.5 Hz, 1H), 7.07 (dd, J = 8.5, 1.5 Hz, 1H), 6.95–6.93 (m, 2H)
,
6.75 (d, J = 8.0 Hz, 1H), 6.65–6.62 (m, 2H), 6.49 (d, J = 15.5 Hz, 1H), 6.07 (s, 1H), 5.45 (s, 1H), 3.94 (s, 3H),
3.79 (s, 3H), 2.83 (t, J = 7.5 Hz, 2H), 2.74 (t, J = 7.0 Hz, 2H), 1.38 (s, 6H); 13C-NMR (CDCl3)
δ
209.4, 197.7,
148.7, 146.8, 146.3, 144.7, 143.8, 132.6, 126.9, 123.9, 121.0, 118.2, 114.9, 114.2, 111.0, 110.0, 61.7, 56.0, 55.7,
40.7, 29.6, 21.0; HRMS [ESI]+ calculated for C23H26NaO6: 421.1627 [M + Na]+; found: 421.1233.

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3.1.11. 1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-dimethylheptane-3,5-dione (19) and 5-hydroxy-1,7-bis(4-
hydroxy-3-methoxyphenyl)-4,4-dimethylheptan-3-one (20)
To a stirred solution of compound 2m (0.885 g, 2.23 mmol) in MeOH (10 mL) was added Pd/C
(45 mg, 10% w/w) in one portion. The resulting mixture was hydrogenated under 1 atmosphere of
H2 at room temperature. After 2 h, the mixture was filtered by celite and concentrated to give the
crude product, which was purified by flash chromatography on silica gel with EtOAc/n-hexane/ (1:2)
to afford compound 19 (465 mg, 52% yield) as a colorless oil and compound 20 (206 mg, 23% yield) as
a colorless oil.
1
Compound 19: H-NMR (CDCl3)
δ 6.83 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 2.0 Hz, 2H), 6.63–6.61 (m,
2H), 5.51 (s, 2H), 3.87 (s, 6H), 2.76 (t, J = 7.0 Hz, 4H), 2.58 (t, J = 7.0 Hz, 4H), 1.29 (s, 6H); 13C-NMR
(CDCl3)
208.9, 146.4, 143.9, 132.7, 120.9, 114.3, 114.9, 111.7, 62.4, 55.9, 40.5, 29.4, 21.0; HRMS [ESI]+
calculated for C23H28NaO6: 423.1784 [M + Na]+; found: 423.1780.
Compound 20 6.86–6.83 (m, 2H), 6.72–6.66 (m, 4H), 5.62–5.61 (br s, 2H),
1H-NMR (CDCl3)
3.89 (s, 3H), 3.87 (s, 3H), 3.71–3.69 (m, 1H), 2.90–2.71 (m, 5H), 2.61–2.51 (m, 2H), 1.71–1.55 (m, 2H),
δ
:
δ
1.13 (s, 3H), 1.08 (s, 3H); 13C-NMR (CDCl3)
δ 216.5, 146.4, 143.9, 143.7, 133.9, 133.1, 120.9, 120.8, 114.4,
114.3, 111.2, 111.1, 55.9, 51.7, 40.3, 33.7, 32.6, 29.4, 21.7, 19.3; HRMS [ESI]+ calculated for C23H30NaO6:
425.4768 [M + Na]+; found: 425.4760.
3.1.12. 1,7-bis(4-Hydroxy-3-methoxyphenyl)-4,4-dimethylheptane-3,5-diol (21)
To a solution of compound 20 (110 mg, 0.27 mmol) in MeOH (3 mL) was added NaBH4 (30 mg,
0.81 mmol) at 0 ◦C. The resulting mixture was allowed to stir at room temperature for 2 h, and then
H2O (3 mL) was added. The aqueous layer was separated and extracted with CH2Cl2 (3
The combined organic extracts were washed brine, dried over MgSO4, filtered and concentrated to give
×
6 mL).
the crude residue, which was purified by flash chromatography on silica gel with EtOAc/n-hexane/
1
(1:1) to afford compound 21 (34 mg, 31% yield) as a colorless oil. H-NMR (CDCl3)
δ
6.86 (d, J = 8.0
Hz, 2H), 6.74–6.71 (m, 4H), 5.63 (s, 2H), 3.89 (s, 6H), 3.59–3.56 (m, 2H), 3.11 (s, 2H), 2.85–2.71 (m, 2H),
2.61–2.55 (m, 2H), 1.81–1.72 (m, 4H), 0.90 (s, 6H); 13C-NMR (CDCl3)
δ 146.5, 143.7, 134.1, 134.0, 120.9,
114.3, 111.1, 78.7, 55.9, 40.3, 33.9, 32.8, 21.0; HRMS [ESI]+ calculated for C23H32NaO6: 427.4928 [M +
Na]+; found: 427.4933.
3.2. Biological Assays
3.2.1. In Vitro MTT [3-(4,5-Dimethylthiazaol-2-yl)-2,4-diphenyltetrazolium bromide] Assay
Cell viability was evaluated by measuring the reduction in MTT to yield blue formazan.
Cells were cultured in 96-well plates, allowed to attach overnight, and then treated with compounds.
After treatment, MTT solution (1 mg/mL) was added to each well, and plates were incubated for
another 2 h. Medium was removed, blue formazan was dissolved in DMSO, and the absorbance was
read at 570 nm by Multiskan Go spectrophotometer (Thermo Scientific, Madison, WI, USA).
3.2.2. In Vivo Antitumor Activity Assay
Male nu/nu mice (5 weeks old) from National Laboratory Animal Center (Taipei, Taiwan) were
maintained under the procedures and guidelines provided by the Institutional Animal Care and
Use Committee of the National Health Research Institutes (Taipei, Taiwan). All experiments were
supervised under the Institutional Animal Care and Use Committee, China Medical University,
Taichung, Taiwan with a protocol number (CMUIACUC-2018-278). HCT-116 colon cancer cells (5 × 106
cells per mouse) were suspended in 0.1 mL of Matrigel solution (50% v/v Matrigel in PBS) and inoculated
into the mammary fat pads of the mice. When the tumor size reached 100 mm3, the tumor-bearing
mice were randomly divided into four groups for treating with vehicle (5% ethanol and 10% tween 80
in 85% saline, 0.1 mL) and
2 (p.o. 50, 100 and 150 mg/kg/day body weight) respectively. Compounds
was administered via oral route daily for 30 days. 5-Fluorouracil-treated group (i.p. 30 mg/kg) is the

Molecules 2020, 25, 479
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positive control. Tumor size and mouse body weight were measured once every 3 days, and tumor
volume (mm3) was calculated using the equation: length
mice were sacrificed.
×
(width)2
×
0.5. At the end of experiments,
3.2.3. The Preliminary Pharmacokinetic Evaluation of 2 in Rat
Drug administration and blood collection
Male Sparague-Dawley rat (420 g) from National Laboratory Animal Center, Taipei, Taiwan were
maintained under the regulations of the Institutional Animal Care and Use Committee of the National
Health Research Institutes, Taiwan. The animal studies were supervised under the Institutional
Animal Care and Use Committee, China Medical University, Taichung, Taiwan with a protocol number
(CMUIACUC-2018-278). Rat was fasted for 16 h before dosing. Compound 2 was dissolved in aqueous
solution (5% ethanol and 10% Tween80 in 85% saline) and administrated via oral gavage at a dosage of
100 mg/kg. Blood samples were collected at selected time points (5, 10, 20, 30, 60, 120, 240, 360, 480 min)
post dose and centrifuged at 10,000 g for 15 min to provide serum samples, which were stored at
×
−80 ◦C until subsequent sample extraction.
3.2.4. Analysis of 2, 2m, 18, 19, 20, 21, 22 and 23 in Serum
Methods for the preparation of
L of acetate buffer (pH 5.0), 50
was added into 100 L of serum which was collected at different time points). The resulting mixture
was partitioned with 250 L of ethyl acetate (containing 5.0 g/mL of butyl paraben as the internal
standard). After centrifuging at 10,000 g for 15 min, the upper organic layer was separated and dried
2,
2m
,
18
,
19,
20 and 21 serum sample are delineated as follows.
50
µ
µL of ascorbic acid (200 mg/mL), and 50
µL of 0.1 N HCl solution
µ
µ
µ
×
under nitrogen gas to provide the crude sample, which was diluted with an appropriate volume of
acetonitrile for LC-MS analysis.
The amount of 22 and 23 were analyzed by the serum samples before and after treatment with
enzyme solution. The procedure for enzyme treatment is shown as follows. 100
treated with enzyme solution (50 L; containing 1000 units/mL of sulfatase and 39861 units/mL of
-glucuronidase in pH 5.0 acetate buffer), 50 L of 0.1 N HCl solution and 50 L of ascorbic acid
(200 mg/mL) in the light protected tubes at 37 ◦C. For optimal hydrolysis efficiency, the incubation time
was decided as 2 h based on a previous study [35]. The mixture was then partitioned with 250 L of
ethyl acetate (containing 5.0 g/mL of butyl paraben as the internal standard). After centrifuging at
10,000 g for 15 min, the upper organic layer was separated and dried under nitrogen gas to provide
µL of serum was
µ
β
µ
µ
µ
µ
×
the crude sample, which was diluted with an appropriate volume of acetonitrile for LC analysis.
3.2.5. LC-MS Analysis Method
All samples obtained from in vivo pharmacokinetic studies were assayed using LC-MS for the
analysis of
2 and the corresponding metabolites. The Waters ACQUITY UPLC I-Class system (Waters
Corp., Milford, MA, USA) was utilized. System control and all of the mass spectrometry data were
acquired and analyzed using UNIFI software (Waters Corp.). For sample separation, the LC was
equipped with an Inertsil ph-3 (50
Japan) in which the column temperature was held at 35 ◦C, injection volume was 7.5
rate of the mobile phase was 200 L/min. The elution started from 50% mobile phase A (ultrapure
×
2.1 mm, 2
µ
m particle size) RPLC column (GL Sciences Inc. Tokyo,
µL, and the flow
µ
water + 0.1% formic acid) and 50% mobile phase B (100% methanol + 0.1% formic acid), held at 50% B
for 0.5 min, raised to 95% B in 5.5 min, held at 95% B for 1 min, and then lowered to 50% B in 1 min.
Before sample injection, the column was equilibrated by pumping 50% B for 4 min. The analysis of
target compounds for all samples was performed by using a Waters Vion IMS QTof mass spectrometer.
Data were acquired in the electrospray ionization (ESI) positive ion MSE mode with range of m/z
100–1000 and 0.5 s scan time. Parameters were set as capillary voltage of 2.5 kV, source temperature of
100 ◦C, desolvation temperature at 250 ◦C, cone gas maintained at 10 L/h, desolvation gas maintained

Molecules 2020, 25, 479
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at 600 L/h. The acquired m/z and isotope pattern were processed by UNIFI software to illustrate
chromatogram, and the amounts of compound was calculated with the integrated peak area of signals.
4. Conclusions
Here, structural modifications at the C-4 position of compound
propionate curcumin prodrug, with polar and nonpolar functional groups were performed to form
a series of 4,4-disubstituted analogs of and DMC. The in vitro anti-proliferative assay of these
derivatives elucidated an obvious SAR in which the enhancement of polarity on C-4 resulted in the
erosion of anticancer activity. The ester prodrug characteristics of motivated us to synthesize their
hydrolysis compounds 2m 6m 15, and 16, whose anticancer activities were highly dependent on the
volume and length of the C-4 alkyl chains. The preliminary pharmacokinetic evaluations indicated that
the esterase-mediated hydrolysis of proceeded smoothly to produce 2m, which in turn was reduced
1, a 2,2-bis(hydroxymethyl)-
1
2–6
–
,
2
to 18 and 19 as major phase I metabolites. The phase II metabolic pathway was confirmed by the
detection of glucuronide 22 and sulfate 23. However, it is still possible that reduction precedes ester
hydrolysis to provide the hydrogenated counterparts of
with unknown biological activity.
2, which possess novel chemical skeletons
Our preliminary pharmacokinetic results also shed light on future directions for the structural
modification of the original compounds. Additional modifications have been scheduled to reduce
the high propensity towards ester hydrolysis by converting the methyl of 2,2-bis(hydroxymethyl)-
propionate group into more bulky alkyl groups. Pharmacokinetic studies have been designed to
identify, biologically evaluate, and quantify the possible metabolites. Apart from this, compound
and 2m may behave like which acts on multiple targets and signaling pathways. The differences
between the mechanism of action of 2m and are not definitively understood. Currently, we are
actively investigating the mechanism of action and pharmacokinetic parameters of and 2m, including
T1/2, AUC, and Cmax, and the results will be reported in due course. The animal study showed
that compound inhibits tumor growth by 45% at a dose of 100 mg/kg in HCT-116 xenograft nude
2
1
2,
1
2
2
mice. Thus, twice-a-day (BID) dosing formulations for achieving higher therapeutic efficacy at lower
doses should be considered in further development of title curcuminoid derivatives as anticancer
drug candidates.
Supplementary Materials: The following are available online. Scheme S1: PLE mediated hydrolysis of curcumin
2,2-bis(hydroxymethyl)propionate
1. Figure S1: The HPLC analysis of compound 18, 19, 20, 21, 22 and 23. Copies
of 1H and 13C NMR spectra of the new products.
Author Contributions: D.-Y.L. performed the preliminary pharmacokinetic analysis and wrote experimental
section and supplementary material file. Y.-C.H. designed the preliminary pharmacokinetic study and integrated
the experimental data. J.-S.Y. and H.-Y.L. performed the in vivo assay and in vivo animal study. K.-H.L. and T.-Y.C.
reviewed and revised the manuscript. S.-C.K. designed the target compounds and interpreted the experimental
data. M.-T.H. designed and synthesized targeted compounds. S.-C.K. and M.-T.H. are the responsible researchers,
who wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: We are grateful to Ministry of Science and Technology, Taiwan (MOST 107-2320-B-039-028) for financial
support. Partial support was also provided by the “Chinese Medicine Research Center, China Medical University”
from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project
by the Ministry of Education (MOE) in Taiwan (CMRC-CHM-5) awarded to S. C. Kuo.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).