Synthesis and characterization of 18F-labeled hydrazinocurcumin derivatives for tumor imaging

Article abstract of DOI:10.1039/c5ra15380h

Fluorine-substituted hydrazinocurcumin derivative 1 and its dimethyl-substituted form at the C2 and C6 positions (2) were synthesized and their radiolabeled forms, [¹⁸F]1 and [¹⁸F]2, were evaluated for tumor imaging. In vitro and in vivo metabolism studies showed that the two radioligands were resistant to reductive metabolism, probably due to the presence of a pyrazole ring. In cellular uptake studies, [¹⁸F]1 and [¹⁸F]2 exhibited comparable uptake by human umbilical vascular endothelial cells and rat C6 glioma cells. Inhibition of radioligand uptake to a similar extent by HC and curcumin suggests that these radioligands may share the same binding sites as those for HC and curcumin. Positron emission tomography imaging of C6 glioma xenografted mice acquired 30 and 60 min after radioligand injection showed that [¹⁸F]2 had markedly higher tumor uptake than [¹⁸F]1, which was consistent with biodistribution data (3.20 ± 0.35% ID per g vs. 0.98 ± 0.31% ID per g, respectively). However, the two radioligands showed similar levels of tumor-to-background uptake ratio, except for the significantly higher uptake of [¹⁸F]1 by the small intestine, indicating its more rapid clearance. The results of this study will guide further structural modifications of these radioligands to enhance tumor-to-background uptake ratios.

Full text of DOI:10.1039/c5ra15380h

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18
Synthesis and characterization of F-labeled hydrazinocurcumin derivatives for tumor
imaging
ab
a
a
*ab
a
Sarah Shin, HyunꢀJung Koo, Iljung Lee, Yearn Seong Choe, Joon Young Choi, Kyungꢀ
ab
a
Han Lee and ByungꢀTae Kim
†
Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University
School of Medicine, 81 Ilwonꢀro, Kangnamꢀku, Seoul 06351, Korea
‡
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul
06351, Korea
Submitted to: RSC Advances
*Corresponding author: Tel: +82ꢀ2ꢀ3410ꢀ2623; Fax: +82ꢀ2ꢀ3410ꢀ2667; Email:
ysnm.choe@samsung.com
1

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Abstract
Fluorineꢀsubstituted hydrazinocurcumin derivative 1 and its dimethylꢀsubstituted form at C2
18
18
and C6 positions (2) were synthesized and their radiolabeled forms, [ F]1 and [ F]2, were
evaluated for tumor imaging. In vitro and in vivo metabolism studies showed that the two
radioligands were resistant to reductive metabolism, probably due to the presence of a
18
18
pyrazole ring. In the cellular uptake studies, [ F]1 and [ F]2 exhibited comparable uptake by
human umbilical vascular endothelial cells and rat C6 glioma cells. Inhibition of radioligand
uptake to a similar extent by HC and curcumin suggests that these radioligands may share the
same binding sites as those for HC and curcumin. Positron emission tomography imaging of
C6 glioma xenografted mice acquired 30 and 60 min after radioligand injection showed that
18
18
[
F]2 had markedly higher tumor uptake than [ F]1, which was consistent with
biodistribution data (3.20 ± 0.35% ID/g vs. 0.98 ± 0.31% ID/g, respectively). However, the
two radioligands showed similar levels of tumorꢀtoꢀbackground uptake ratios, except for the
1
8
significantly higher uptake of [ F]1 by the small intestines, indicating its more rapid
clearance. The results of this study will guide further structural modifications of these
radioligands to enhance tumorꢀtoꢀbackground uptake ratios.
2

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Introduction
It is known that 5ꢀhydroxyꢀ1,7ꢀbis(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ1,4,6ꢀheptatrienꢀ3ꢀone
(curcumin), a major ingredient of the curry spice turmeric, has anticancer, antioxidant, and
1
,2
antiinflammatory activities.
Despite its multiple activities, curcumin has poor
3
ꢀ5
bioavailability, which may result from its rapid metabolism in the liver and intestinal walls.
1
8
We previously synthesized a curcumin derivative substituted with [ F]fluoropropyl group at
6
one of the 4’ꢀaromatic OH groups for βꢀamyloid plaque imaging. Despite its suitable
lipophilicity and excellent in vitro binding affinity for βꢀamyloid aggregates,
18
[
F]fluoropropylcurcumin showed poor penetration through the bloodꢀbrain barrier (BBB),
probably due to rapid metabolism of curcumin resulting in formation of its reductive
metabolites, conjugation products, and reductive metabolites with conjugation.
In addition, curcumin has high reactivity at the central carbon and limited solubility,
therefore, a more stable and hydrophilic derivative, 3,5ꢀbis[βꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ
ethenyl]pyrazole (hydrazinocurcumin; HC) has been synthesized and evaluated for diverse
biological activities. Initial studies of HC demonstrated a more potent inhibitory activity than
curcumin for 5ꢀlipoxygenase (IC50 = 1.0 ꢁM vs. 8.0 ꢁM), which converts arachidonic acid to
7
a host of lipoxygenase products. HC was also shown to have antioxidant, cyclooxygenase
(COX) inhibitory, and antiinflammatory activities, with even higher activity and COXꢀ
3

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8
2
/COXꢀ1 selectivity than curcumin. Its activity has been expanded to the field of neurology;
HC showed more potent inhibitory activity than curcumin against calcium/calmodulinꢀ
dependent protein kinase II, which is known to be associated with various brain functions
9
including learning and memory, and also exhibited potent activity against multiple targets
1
0
associated with pathologic biomarkers of Alzheimer’s disease. Anticancer activity of HC
has been reported, with more potent cytotoxicity (ED50 = 1.0 ꢀ 3.9 ꢁg/mL) than curcumin
against various human cancer cell lines including MCFꢀ7, A549, KB, U87MG, CAKIꢀ1, 1A9,
11
HCTꢀ8, SKꢀMElꢀ2, PCꢀ3, HepG2, and LNCaP (clone FGC). Antiangiogenesis activity of
HC has also been reported, as shown by its inhibitory activity on endothelial cell proliferation
12
and in vivo neovascularization of chorioallantoic membrane. Another study showed that HC
was more potent than curcumin in inhibiting signal transducer and activator of transcription 3,
which in turn resulted in inhibition of cell proliferation, migration and invasion, and
13
induction of cell apoptosis. A recent study demonstrated that HC was more effective than
1
4
curcumin for prevention of diethylnitrosamineꢀinduced hepatocarcinogenesis in rats.
Recently, radiolabeled curcumin and HC derivatives were developed for βꢀamyloid plaque
1
8
imaging and cancer cellular uptake studies. Fꢀlabeled HC derivative, 2ꢀ[3,5ꢀbis(4ꢀhydroxyꢀ
18
3
ꢀmethoxystyryl)ꢀ1Hꢀpyrazolꢀ1ꢀyl]ꢀNꢀ{1ꢀ[2ꢀ(2ꢀ(2ꢀ[ F]fluoroethoxy)ethoxy)ethyl)ꢀ1Hꢀ1,2,3ꢀ
triazolꢀ4ꢀyl]methyl}acetamide had low BBB penetration in a WT and transgenic APP23 mice,
4

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even though it labeled βꢀamyloid plaques of APP23 mouse brain sections using in vitro
15
nat/68
autoradiography.
Gaꢀlabeled homodimers of curcumin, diacetylcurcumin, and
bis(dehydroxy)curcumin were tested for binding affinity to βꢀamyloid fibrils and uptake by
16
A549 lung cancer cells.
Unlike curcumin, the in vivo effects of HC and its derivatives have not been extensively
studied and there are few reports on their metabolism. We recently found that introduction of
methyl groups at both C2 and C6 positions of curcumin significantly diminishes reductive
1
7
metabolism by creating steric hindrance against reductive metabolizing enzymes. In
addition, HC as well as curcumin has never been applied to tumor imaging, despite its wellꢀ
known anticancer activity. In the present study, therefore, we synthesized 4ꢀ((E)ꢀ2ꢀ(5ꢀ(4ꢀ(2ꢀ
1
8
(
2ꢀ[ F]fluoroethoxy)ethoxy)ꢀ3ꢀmethoxystyryl)ꢀ1Hꢀpyrazolꢀ3ꢀyl)vinyl)ꢀ2ꢀmethoxyphenol
18
18
(
[ F]1) and its 2,6ꢀdimethyl derivative ([ F]2), elucidate their metabolism, and evaluated
them as tumor imaging agents in a C6 glioma xenograft model using micro positron emission
tomography (microPET).
Results
Chemistry
1
8
We designed and synthesized six fluorineꢀsubstituted HC derivatives for development of Fꢀ
5

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labeled radioligands for tumor imaging (Fig. 1). HC derivatives (1ꢀ6) were readily
7,12
synthesized by hydrazination of curcumin derivatives (7ꢀ12) (Scheme 1).
Curcumin
derivatives (7-12) were synthesized using a previously reported method with slight
18,19
modifications;
sequential aldol condensation of 1,3ꢀdiketone with one equivalent of
vanillin and then with one equivalent of fluoroalkyl vanillin (17ꢀ19) was carried out via
coupling of a boron complex of the 1,3ꢀdiketone moiety with aldehyde in the presence of
amine, followed by acid treatment of the coupling product. Fluoroalkyl vanillin (15 and 18-
19) was synthesized by Oꢀalkylation of vanillin using 2ꢀ(2ꢀchloroethoxy)ethanol or
o
20
2 3
fluoroalkyl tosylate in the presence of K CO at 100ꢀ110 C. The hydroxyl group of 15 was
converted into a methanesulfonyl group, which was then reacted with CsF in tꢀBuOH to give
2
1
the fluoropegylated vanillin 17. The E,Eꢀconfigurations of the 2,6ꢀdimethyl groups of 2
were confirmed by twoꢀdimensional rotatingꢀframe nuclear Overhauser effect spectroscopy
22ꢀ24
(ROESY) (see 2D ROESY spectra, Supplementary Information).
These experiments
showed no crossꢀpeaks between methyl protons at C2 and C6 positions (2.30 ppm) and
olefinic protons at C1 and C7 positions (6.98 and 6.99 ppm), indicating that methyl protons
do not exist in close proximity (< 5 Å) in space with both olefinic protons. However, crossꢀ
peaks were observed between methyl protons at C2 and C6 positions and phenyl protons
(6.93, 6.92, and 6.90 ppm) and between the methyl protons and a proton at C4 position (6.58
6

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ppm), indicating that methyl protons at C2 and C6 are on the same side as proton at C4.
Moreover, the intensities of crossꢀpeaks between the methyl protons and aromatic protons
2
3,25
were dependent on the distance between the two protons.
These data demonstrated that
the dimethyl groups of 2 are in E,Eꢀconfigurations. Based on these results, the 2,6ꢀdimethyl
HC derivatives synthesized in this study (2, 4, and 6) were assigned to have E,Eꢀ
configurations.
18
18
For radiochemical synthesis of [ F]1 and [ F]2, the tosylate precursors 26 and 27 were
synthesized (Scheme 2). Compound 20 was synthesized from 13 and pegylated vanillin 15; in
this reaction, recrystallization after flash column chromatography was required because
compounds 15 and 20 were not separable on flash column chromatography. Subsequent
hydrazination of the 1,3ꢀdiketone moiety of 20 was readily carried out to yield 22. The bisꢀ
1
8
methoxymethyl (MOM) protected precursor, 26 was then used for preparation of [ F]1.
18
Precursor 27 for synthesis of [ F]2 was prepared as described for synthesis of 26.
Synthesis of radioligands
18
18
[
F]1 and [ F]2 were synthesized by reaction of the corresponding precursors (26 and 27)
18
4
with nꢀBu N[ F]F at 110 °C for 10 min followed by removal of the protecting groups in 6 N
18
HCl at 120 °C for 10 min (Scheme 2). Fꢀlabeling of the precursors was readily carried out
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by protecting the aromatic OH and pyrazole NH groups, unlike the multiꢀstep synthesis of
18
18
[
F]fluoropropylcurcumin from 4ꢀ(3ꢀ[ F]fluoropropoxy)ꢀ3ꢀmethoxybenzaldehyde and 5ꢀ
6
hydroxyꢀ1ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ1,4ꢀhexadienꢀ3ꢀone.
Subsequent
HPLC
18
18
purification gave [ F]1 and [ F]2 in overall decayꢀcorrected radiochemical yields of 25ꢀ35%
and with specific activity of 51.06 GBq/ꢁmol and 44.5 GBq/ꢁmol, respectively.
Measurement of partition coefficient
18
18
The partition coefficients of [ F]1 and [ F]2 were measured and expressed as log P values
18
of 2.00 ± 0.01 and 2.11 ± 0.01, respectively. As predicted, lipophilicity of [ F]2 was higher
18
than that of [ F]1, due to the presence of two methyl groups at C2 and C6 positions.
Sulforhodamine B (SRB) assay
Cytotoxicity of HC derivatives was measured by carrying out the SRB assay for two cell
lines, human umbilical vascular endothelial cells (HUVEC) and rat C6 glioma cells, 72 h
after treatment with the compounds (Table 1). All of the compounds showed a similar trend
in cytotoxicity for the cell lines studied. HC had superior cytotoxicity to the other compounds
including curcumin, which was consistent with results in the literature for bovine aortic
12
endothelial cells. The HC derivatives substituted with a miniꢀPEG moiety (1 and 2) showed
8

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more potent cytotoxicity than the other HC derivatives against both HUVECs and C6 glioma
cells, and furthermore, 2,6ꢀdimethyl derivative (2) was more potent than the corresponding
18
2
,6ꢀunsubstituted derivative (1). Thus, the two compounds, 1 and 2, were selected for Fꢀ
labeling.
Reduction with alcohol dehydrogenase
Reduction of 1 and 2 was performed using alcohol dehydrogenase, an enzyme known to be
26,27
involved in the reductive metabolism of curcumin.
Compound 1 was eluted at the
retention time between 37.9 and 38.9 min (Fig. 2A) and remained intact under complete
incubation conditions in the presence of alcohol dehydrogenase and NADPH (Fig. 2B). The
peak from 33.0 to 33.8 min was identified as an impurity derived from alcohol
dehydrogenase, which was also present in incubation of the enzyme itself (Fig. 2C).
Compound 2 was eluted at the retention time between 40.4 and 41.6 min (Fig. 2D) and
remained intact under complete incubation conditions (Fig. 2D and E). Both compounds were
also unchanged under conditions omitting NADPH (Fig. 2F). On the other hand, curcumin
was mostly converted to tetrahydrocurcumin (Fig. 2G and H), which was confirmed by the
17
same retention time as the authentic tetrahydrocurcumin (Fig. 2I).
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In vivo metabolism studies in ICR mice
HPLC chromatograms of the blood and liver samples obtained from mice injected with [ F]1
18
18
18
and [ F]2 showed the same pattern of radiometabolite formation (Fig. 3). Radioligand [ F]2
18
was metabolically more stable than [ F]1 in blood 5 min after injection, probably due to its
higher lipophilicity (Fig. 3a). However, the two radioligands were below detection limit at 30
28
min except a polar peak at 4.8 min. This rapid metabolism were similar to that of curcumin.
18
On the other hand, two radiometabolites were detected in the liver samples of [ F]1 and
18
[
F]2 5 and 30 min after injection (Fig. 3c and d), and there were no radiometabolites
detected at 60 min (data not shown). In order to identify whether one of the radiometabolites
18
is a glucuronide conjugate, the 5ꢀmin liver samples of [ F]1 were incubated with βꢀ
glucuronidase. The results demonstrated that the radiometabolite, m1 (t = 24.2 min), was
R
18
18
hydrolyzed to [ F]1 (t
R
= 40.1 min) (Fig 3Aꢀe), suggesting that m1 is [ F]1ꢀglucuronide.
Based on the HPLC data of the known metabolites of curcumin, it is likely that m2 is its
28
sulfate conjugate.
Cellular uptake
18
18
We carried out the cellular uptake studies of [ F]1 and [ F]2 using two different cell lines,
18
HUVECs and C6 glioma cells. HUVEC uptake of [ F]1 increased in a timeꢀdependent
10

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manner from 100% (2.2% ID) at 5 min to 160% (3.5% ID) at 120 min (P <0.01). Blocking
studies demonstrated inhibition of radioligand uptake in the presence of 1, curcumin, and HC
by 56%, 64%, and 55%, respectively (P <0.001) (Fig. 4A and B). Similar results were
obtained using C6 cells; uptake increased from 100% (2.8% ID) at 5 min to 163% (4.6% ID)
at 120 min (P <0.01) and radioligand uptake was reduced in the presence of 1, curcumin, and
HC by 52%, 52%, and 52%, respectively (P <0.001) (Fig. 4C and D).
18
HUVEC uptake of [ F]2 increased in a timeꢀdependent manner from 100% (2.6% ID) at 5
min to 165% (4.2% ID) at 120 min (P <0.001). Blocking studies showed reduction of the
radioligand uptake in the presence of 2, curcumin, and HC by 60%, 58%, and 58%,
respectively (P <0.001) (Fig. 4E and F). Similar results were obtained using C6 cells; uptake
from 100% (3.2% ID) at 5 min to 153% (4.9% ID) at 120 min (P <0.01) and reduction of the
radioligand uptake in the presence of 2, curcumin, and HC by 61%, 52%, and 49%,
respectively (P <0.001) (Fig. 4G and H).
MicroPET imaging
18
MicroPET images of C6 glioma xenografted mice were acquired after injection with [ F]1
18
and [ F]2 dissolved in 0.2% polysorbate 80 in saline, which is a FDAꢀapproved inactive
ingredient and is compatible for clinical use. The images revealed accumulation of high
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levels of radioactivity in the intestines at 30 min postꢀinjection with slow clearance over time.
18
Tumor was not clearly detected in mice injected with [ F]1 (Fig. 5A), whereas it was clearly
18
detected at 30 min after injection of [ F]2 and uptake was retained at 60 min (Fig. 5B).
18
Region of interest (ROI) analysis of tumors in mice injected with [ F]1 revealed 1.10 ±
0.10% ID/g at 30 min and 0.97 ± 0.12% ID/g at 60 min. In contrast, significantly higher ROI
18
values were obtained in tumors of mice injected with [ F]2: 3.30 ± 0.07% ID/g at 30 min (P
<
0.01) and 3.90 ± 0.70% ID/g at 60 min (P < 0.001) (Fig. 5C). Tumor to muscle uptake
18
18
ratios of [ F]1 and [ F]2 were 1.46 and 2.02 at 30 min and 1.28 and 2.28 at 60 min,
respectively.
Biodistribution
Biodistribution studies carried out immediately after microPET imaging of tumorꢀbearing
mice demonstrated accumulation of high levels of radioactivity in the intestines. Low levels
18
18
of femur uptake by both [ F]1 and [ F]2 indicated that the radioligands do not undergo in
1
8
vivo defluorination. Tumor uptake of [ F]1 at 65 min after injection was 0.98 ± 0.31% ID/g,
1
8
18
whereas that of [ F]2 was 3.20 ± 0.35% ID/g (Fig. 6). Although tumor uptake of [ F]2 was
18
significantly higher than that of [ F]1 (P < 0.01), tumorꢀtoꢀbackground uptake ratios of the
two radioligands were within the same range (tumor/muscle and tumor/blood: 2.30 and 0.87
12

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vs. 2.21 and 0.86, respectively). However, it is interesting to note that small intestine uptake
18
18
was markedly higher for [ F]1 than for [ F]2 (P < 0.05) (Fig. 6).
Discussion
A few radiolabeled curcumin and hydrazinocurcumin derivatives have been developed to date
nat
for βꢀamyloid plaque imaging. Among these radioligands, Gaꢀlabeled homodimers of
curcumin derivatives were tested for cancer cellular uptake using their intrinsic fluorescence.
However, none of these radioligands have been studied for tumor imaging. Our study as well
as other reported studies showed that HC has more potent cytotoxicity than curcumin for
11
various cancer cell lines (Table 1). Furthermore, 2,6ꢀdimethylcurcumin was shown to
undergo significantly diminished reductive metabolism on olefinic double bonds compared
with curcumin, possibly by creating steric hindrance against reductive metabolizing
17
enzymes. In this study, therefore, fluorineꢀsubstituted HC derivatives (1, 3, and 5) and their
,6ꢀdimethyl substituted compounds (2, 4, and 6) were synthesized. In compounds 1 and 2,
miniꢀpolyethylene glycol (PEG) moiety was added between 4’ꢀOH group of HC backbone
2
18
18
and F to improve their in vivo properties. FꢀPEG has been successfully used to adjust
lipophilicity of compounds while retaining their biological activity, as shown in the studies of
1
8
29,30
FꢀPEGꢀstilbene and styrylpyridines for βꢀamyloid plaque imaging.
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1
2
As HC is known to have inhibitory activity on endothelial cell proliferation, cytotoxicity
of HC derivatives was measured for HUVECs in addition to C6 glioma cells using the SRB
18
assay (Table 1). Compounds 1 and 2 were selected for Fꢀlabeling because of their potent
18
18
cytotoxicity against the two cells studied. Synthesis of radioligands ([ F]1 and [ F]2) was
18
carried out by labeling bisꢀMOM protected precursors (26 and 27) with F, followed by
1
8
18
deprotecting MOM groups. Attempts to prepare [ F]1 by [ F]fluorination of unprotected or
’ꢀMOM protected tosylate precursor yielded the product in very low yield (< 3% based on
radioꢀTLC). Therefore, aromatic OH and pyrazole NH groups of 22 and 23 were selectively
4
®
31,32
protected with MOM groups in the presence of catalytic amounts of Adogen 464 ,
and
the residual aliphatic OH group was subsequently converted into tosylate ester. The bisꢀ
MOM protected precursors were then used for the oneꢀpot synthesis of radioligands in
relatively high yields and with high specific activity.
Metabolism of HC derivatives was assessed in vitro and in vivo to determine whether
dimethyl substitution on HC could prevent reduction of olefinic double bonds. In vitro
reductive metabolism study of 1 and 2 using alcohol dehydrogenase demonstrated that the
26,27,33
olefinic double bonds of 1 as well as 2 were not reduced, unlike curcumin.
In accord
with these results, attempts to synthesize tetrahydroꢀ and hexahydroꢀforms of compounds 1
and 2 using a previously reported method were not successful, whereas tetrahydroꢀ and
14

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hexahydroꢀcurcumin, which are metabolite standards of curcumin, were readily synthesized
2
6,33
using the same method.
In vivo metabolism study was performed using the blood and
liver samples obtained from mice injected with the radioligands, because a host of
endogenous molecules would be also detected by HPLC with UV absorption at 280 nm if
28
nonꢀradioactive compounds 1 and 2 were used instead of radiologands.
HPLC
1
8
18
chromatograms of the liver samples showed that [ F]1 and [ F]2 were mostly
biotransformed to two radiometabolites, both of which are most likely to be the conjugation
products of the radioligands (Fig. 3). This result was consistent with that obtained from in
vitro reduction of 1 and 2 with alcohol dehydrogenase (Fig. 2), suggesting that the pyrazole
ring of HC derivatives is responsible for resistance to reductive metabolism because
28,34
curcumin is known to be a substrate for reductive metabolism.
It was reported that HC has inhibitory activity on endothelial cell proliferation and high
1
2,13
antiangiogenesis activity,
and furthermore, compounds 1 and 2 showed cytotoxicity
comparable to HC for HUVECs (Table 1). Therefore, we carried out the cellular uptake
18
18
studies of [ F]1 and [ F]2 using two different cell lines, HUVECs and C6 glioma cells.
Blocking studies were carried out using nonꢀradioactive compound (1 or 2), curcumin, and
18
18
HC. The results demonstrate that cellular uptake of [ F]2 is slightly higher than that of [ F]1,
18
probably because of the presence of two methyl groups in [ F]2, which confer higher
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18
lipophilicity than [ F]1. Similar levels of blocking of radioligand uptake by nonꢀradioactive
compounds, curcumin, and HC were obtained, suggesting that the radioligands may share the
same binding sites as HC and curcumin.
MicroPET images acquired in C6 glioma xenografted mice after injection with
radioligands revealed significantly higher radioactivity in the tumor of mice injected with
18
18
[
F]2 than with [ F]1 (Fig. 5). However, the two radioligands showed similar tumorꢀtoꢀ
1
8
background uptake ratios, except for the predominantly higher uptake of [ F]1 by the small
intestines, indicating its more rapid clearance. Furthermore, the high levels of radioactivity in
the intestines of the two radioligands are similar to the known distribution of curcumin in
5
rodents. These results suggest that the differences in the in vivo uptake may be attributable to
18
18
the higher lipophilicity of [ F]2 than [ F]1.
It has been shown that HC has diverse biological activity probably resulting from its
7
ꢀ14
interaction with multiple targets.
Our microPET and biodistribution studies demonstrated
high retention of radioactivity in tumors of C6 tumorꢀbearing mice. However, the molecular
targets of these radioligands for the tumor uptake have not been identified. Therefore, further
investigation may be necessary, not only to enhance the tumorꢀtoꢀbackground uptake ratios of
these radioligands through structural modifications, but also to elucidate their specific
molecular targets.
16

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Conclusion
Of six fluorineꢀsubstituted HC derivatives, compounds 1 and 2 were selected for Fꢀlabeling
1
8
18
18
because of their potent cytotoxicity. Although in vitro cellular uptake of [ F]1 and [ F]2 was
comparable, in vivo properties of the radioligands in C6 glioma xenografted mice differed,
18
18
such that [ F]2 had significantly lower small intestine uptake than [ F]1 but higher uptake in
tumors and in other major organs, resulting in similar tumor to muscle and tumor to blood
18
18
uptake ratios. Furthermore, [ F]1 and [ F]2 were resistant to reductive metabolism in vitro
and in vivo, probably because of the presence of a pyrazole ring. To the best of our
knowledge, these are the first microPET studies of radiolabeled HC derivatives in tumor
xenografted mice.
Experimental
Materials and equipment
Chemicals, sulforhodamineꢀB, equine alcohol dehydrogenase, βꢀglucuronidase, and
polysorbate 80 (10% in water) were purchased from SigmaꢀAldrich (St. Louis, MO, USA).
1
H NMR spectra were obtained using a Bruker Avance 500 (500 MHz) spectrometer
(Rheinstetten, Germany), and chemical shifts (δ) were reported as the ppm downfield of the
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3 6
internal tetramethylsilane. All NMR samples were prepared in CDCl or DMSOꢀd at 20 mM,
1
3
and under these conditions, the NH and OH protons were not detected in all compounds. C
NMR spectra were obtained on a Bruker Avance 600 (150 MHz) spectrometer. Twoꢀ
dimensional ROESY experiments were carried out on a Bruker Avance 600 (600 MHz)
spectrometer at National Center for InterꢀUniversity Research Facilities, Seoul National
University (Seoul, Korea). The spectra were acquired using the statesꢀTPPI mode with
mixing times of 700 ms and acquisition times of 0.14 s. Electron impact (EI) and fast atom
bombardment (FAB) mass spectra were obtained using a JMSꢀ700 Mstation (JEOL Ltd,
Tokyo, Japan). Purification and analysis of radioligands were performed using HPLC
(Thermo Scientific, Waltham, MA, USA) equipped with a semiꢀpreparative column (YMCꢀ
Pack Si, 5 µm, 10 × 250 mm) or an analytical column (YMCꢀPack C18, 5 µm, 4.6 × 250
mm). The eluent was monitored simultaneously, using UV (254 nm) and NaI(T1)
radioactivity detectors. TLC was performed on Merck F254 silica plates and analyzed on a
Bioscan radioꢀTLC scanner (Washington, D.C., USA).
18
18
18
[
F]Fluoride was produced by the O(p,n) F reaction using a GE Healthcare PETtrace
cyclotron (Uppsala, Sweden). Radioactivity was measured in a dose calibrator (Biodex
2
Medical Systems, Shirley, NY, USA) and tissue radioactivity was measured in a Wizard
automatic gamma counter (PerkinElmer, Waltham, MA, USA). MicroPET images were
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acquired at the Center for Molecular and Cellular Imaging, Samsung Biomedical Research
Institute (SBRI, Seoul, Korea) using an Inveon microPET/CT scanner (Siemens Medical
Solutions, Malvern, PA, USA).
Synthesis of fluorine-substituted HC derivatives (1, 3, and 5)
Hydrazine hydrate (3 eq) and catalytic amounts of acetic acid (0.5 eq) were added to a
solution of compound 7 (50 mg, 0.10 mmol), 9 (50 mg, 0.11 mmol), or 11 (50 mg, 0.12
o
mmol) in ethanol (500 ꢁL), and the reaction solution was heated at 80 C for 18 h. At the end
of the reaction, the reaction mixture was extracted twice with ethyl acetate (30 mL × 2), and
3
the organic layer was washed with a saturated NaHCO solution and with brine, and then
2 4
dried over Na SO . The product was purified using flash column chromatography (1:2
hexaneꢀethyl acetate) to yield 1 (26.8 mg, 53%), 3 (20.3 mg, 45%), or 5 (37.6 mg, 74%) as a
light yellow solid.
1
1
3
2
: Purity (HPLC), 99.0%; H NMR (CDCl
3
) δ 3.84 (dt, J = 26.5 and 4.0 Hz, 2H), 3.88(s, 3H),
.91 (s, 3H), 3.94 (t, J = 5.0 Hz, 2H), 4.23 (t, J = 5.0 Hz, 2H), 4.59 (dt, J = 47.5 and 4.0 Hz,
13
H), 6.59 (s, 1H), 6.90 (d, J = 14.0 Hz, 4H), 6.93 (d, J = 4.0 Hz, 4H), 6.97ꢀ6.98 (m, 2H); C
3
NMR (CDCl ) δ 149.49, 147.52, 146.82, 145.82, 144.53, 135.29, 131.26, 129.80, 121.66,
114.60, 113.59, 112.23, 110.98, 109.19, 108.10, 101.07, 83.20 (d, J = 169.1 Hz), 70.65 (d, J =
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+
1
9.4 Hz), 69.90, 68.74, 55.88, 55.85; MS (FAB) m/z 455 (M + H); HRMS calcd for
C
25
H
28FN
2
O
5
, 455.1982; found, 455.1990.
1
3
: Purity (HPLC), 99.0%; H NMR (CDCl
3
) δ 3.89 (s, 3H), 3.90 (s, 3H), 4.30 (dt, J = 23.0
and 4.0 Hz, 2H), 4.80 (dt, J = 46.0 and 4.5 Hz, 2H), 6.60 (s, 1H), 6.88 (d, J = 9.5 Hz, 2H),
1
3
6
1
1
4
5
3
6
1
1
4
3
.93ꢀ6.97 (m, 6H), 6.99 (d, J = 15.5 Hz, 2H); C NMR (CDCl ) δ 149.28, 147.48, 146.82,
45.81, 144.50, 134.21, 130.88, 129.80, 122.85, 114.18, 113.02, 112.21, 109.97, 108.99,
08.23, 99.84, 82.80 (d, J = 169.0 Hz), 70.38 (d, J = 19.4 Hz), 55.88, 55.83; MS (FAB) m/z
+
11 (M + H); HRMS calcd for C23
H
24FN
2
O
4
, 411.1720; found, 411.1728.
1
: Purity (HPLC), 99.8%; H NMR (CDCl
3
) δ 2.25 (dt, J = 26.5 and 6.0 Hz, 2H), 3.92 (s, 3H),
.95 (s, 3H), 4.20 (q, J = 5.0 Hz, 2H), 4.70 (dt, J = 47.0 and 6.0 Hz, 2H), 6.16 (s, 1H), 6.89ꢀ
13
3
.95 (m, 5H), 7.00ꢀ7.07 (m, 5H); C NMR (CDCl ) δ 149.38, 147.51, 147.02, 146.03,
45.12, 135.36, 131.28, 130.01, 122.56, 115.90, 114.32, 112.78, 111.01, 109.84, 108.14,
01.03, 82.54 (d, J = 170.0 Hz), 70.54 (d, J = 18.8 Hz), 55.88, 55.84, 30.70; MS (FAB) m/z
+
25 (M + H); HRMS calcd for C24
H
26FN
2
O
4
, 425.1877; found, 425.1875.
Synthesis of fluorine-substituted 2,6-dimethyl-HC derivatives (2, 4, and 6)
Compounds 2, 4, and 6 were synthesized as described for syntheses of 1, 3, and 5 using 8 (50
mg, 0.10 mmol), 10 (30 mg, 0.06 mmol), or 12 (50 mg, 0.10 mmol) instead of 7, 9, or 11.
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Flash column chromatography (hexaneꢀethyl acetate) yielded 2 (34.5 mg, 73%), 4 (26.4 mg,
9%), or 6 (34.6 mg, 58%) as a light yellow solid. Configurations of 2,6ꢀdimethyl groups of 2
8
were determined by 2D ROESY.
1
2
: Purity (HPLC), 99.6%; H NMR (CDCl
3
) δ 2.30 (s, 6H), 3.84 (dt, J = 30.5 and 4.0 Hz, 2H),
3.87(s, 3H), 3.91(s, 3H), 3.94 (t, J = 5.0 Hz, 2H), 4.23 (t, J = 4.5 Hz, 2H), 4.59 (dt, J = 47.5
and 4.0 Hz, 2H), 6.58 (s, 1H), 6.90 (d, J = 9.8 Hz, 2H), 6.92 (d, J = 9.5 Hz, 2H), 6.93 (d, J =
13
2
.5 Hz, 2H), 6.98 (s,1H), 6.99 (s, 1H); C NMR (CDCl
3
) δ 149.09, 147.15, 146.25, 144.70,
130.79, 129.54, 126.74, 126.53, 122.55, 121.89, 114.27, 113.33, 112.98, 111.82, 99.37, 83.20
(d, J = 169.0 Hz), 70.61 (d, J = 19.6 Hz), 69.83, 68.57, 55.86, 55.84, 16.07; MS (FAB) m/z
+
4
83 (M + H); HRMS calcd for C27
H
32FN
2
O
5
, 483.2295; found, 483.2290.
1
4
: Purity (HPLC), 98.9%; H NMR (CDCl
3
) δ 2.28 (s, 6H), 3.88 (s, 3H), 3.91 (s, 3H), 4.32
(dt, J = 36.5 and 4.5 Hz, 2H), 4.79 (dt, J = 47.0 and 4.5 Hz, 2H), 6.59 (s, 1H), 6.88 (s, 1H),
1
3
6
1
9
4
6
2
3
.91ꢀ6.94 (m, 5H), 6.98 (s, 1H), 7.00(s, 1H); C NMR (CDCl ) δ 149.28, 146.83, 146.22,
44.71, 131.28, 129.52, 126.77, 126.43, 122.59, 121.86, 114.26, 113.77, 113.15, 111.77,
9.44, 81.96 (d, J = 170.5 Hz), 68.42 (d, J = 19.6 Hz), 55.95, 55.87, 16.03; MS (FAB) m/z
+
39 (M + H); HRMS calcd for C25
H
28FN
2
O
4
, 439.2033; found, 439.2025.
1
: Purity (HPLC), 100%; H NMR (CDCl
3
) δ 2.24 (dt, J = 26.5 and 6.0 Hz, 2H), 2.29 (s, 3H),
.30 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 4.18 (q, J = 6.0 Hz, 2H), 4.68 (dt, J = 47.5 and 6.0 Hz,
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2H), 6.59 (s, 1H), 6.90 (d, J = 9.8 Hz, 2H), 6.94 (d, J = 4.0 Hz, 4H), 6.97 (s,1H), 6.98 (s, 1H);
1
3
3
C NMR (CDCl ) δ 149.33, 146.89, 146.20, 144.75, 131.22, 129.48, 127.01, 126.68, 122.63,
1
5
4
21.88, 114.24, 113.82, 113.18, 111.74, 99.47, 81.95 (d, J = 170.7 Hz), 68.43 (d, J = 20.6 Hz),
+
5.99, 55.90, 31.9, 16.02; MS (FAB) m/z 453 (M + H); HRMS calcd for C26
H
30FN
2
O
4
,
53.2190; found, 453.2191.
Synthesis of fluorine-substituted curcumin derivatives (7, 9, and 11)
B
2
O
3
(0.22 mmol) was added to a solution of 13 (50 mg, 0.21 mmol) in ethyl acetate and the
o
reaction solution was heated at 80 C for 30 min. The reaction mixture was then reacted with
3
an ethyl acetate solution (700 ꢁL) of 17, 18, or 19 (0.21 mmol) and (nꢀBuO) B (0.22 mmol).
o
After stirring at 80 C for 30 min, piperidine (14.1 ꢁL, 0.14 mmol) was added dropwise to the
o
mixture, which was then stirred at 100 C for 2 h. The solution was cooled and treated with
o
0
.4 N HCl (700 ꢁL) at 50 C for 30 min. The reaction mixture was extracted twice with ethyl
2 4
acetate (30 mL × 2), and the organic layer was washed with water and dried over Na SO .
The product was purified using flash column chromatography (hexaneꢀethyl acetate) to give 7
(65.1 mg, 67%), 9 (33.1 mg, 38%), or 11 (54.5 mg, 61%) as a yellow solid.
1
7
: H NMR (CDCl
3
) δ 3.76 (dt, J = 31.5 and 4.0 Hz, 2H), 3.80 (t, J = 5.0 Hz, 2H), 3.84 (s,
6H), 4.24 (t, J = 4.5 Hz, 2H), 4.58 (dt, J = 48.5 and 4.3 Hz, 2H), 6.09 (s, 1H), 6.76 (d, J =
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16.0 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 15.5 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H),
7.24 (dd, J = 8.3 and 1.5 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.35 (d, J = 1.5 Hz, 2H), 7.56 (d,
+
J = 16.0 Hz, 1H), 7.58 (d, J = 16.0 Hz, 1H); MS (FAB) m/z 459 (M + H); HRMS calcd for
25 7
C H28FO , 459.1819; found, 459.1819.
1
9
: H NMR (CDCl
3
) δ 3.93 (s, 3H), 3.95(s, 3H), 4.31 (dt, J =27.0 and 3.5Hz, 2H), 4.78 (dt, J
=
47.5 and 4.3 Hz, 2H), 5.86 (s, 1H), 6.48 (d, J = 16.0 Hz, 1H), 6.51 (d, J = 16.0 Hz, 1H),
6.92 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 1.5 Hz, 1H), 7.09 (d, J = 1.5
+
Hz, 1H), 7.13 (dd, J = 8.0 and 1.5 Hz, 2H), 7.60 (d, J = 16.0 Hz, 2H); MS (FAB) m/z 415 (M
+
6
H); HRMS calcd for C23H24FO , 415.1557; found, 415.1557.
1
1
4
2
1: H NMR (CDCl
3
) δ 2.22ꢀ2.33 (m, 2H), 3.91 (s, 3H), 3.94 (s, 3H), 4.20 (t, J = 6.0 Hz, 2H),
.62 (t, J = 6.0 Hz, 1H), 4.72 (t, J = 5.5 Hz, 1H), 5.81 (s, 1H), 6.47 (dd, J = 15.5 and 7.8 Hz,
H), 6.93 (dd, J = 20.0 and 9.9 Hz, 2H), 7.02ꢀ7.12 (m, 4H), 7.59 (d, J = 15.5 Hz, 2H); MS
+
1
(
6
EI) m/z 428 (M ); HRMS calcd for C24H25FO , 428.1635; found, 428.1638. H NMR data
6
agreed with literature values.
Synthesis of fluorine-substituted 2,6-dimethyl-curcumin derivatives (8, 10, and 12)
Compounds 8, 10, and 12 were synthesized as described for syntheses of 7, 9, and 11 using
14 (100.0 mg, 0.38 mmol) instead of 13. Flash column chromatography (hexaneꢀethyl
acetate) gave 8 (143.2 mg, 78%), 10 (65.8 mg, 39%), or 12 (60.0 mg, 37%) as a light yellow
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solid.
1
8
2
6
1
: H NMR (DMSOꢀd
6
) δ 2.15 (s, 6H), 3.76 (dt, J = 31.5 and 3.5 Hz, 2H), 3.78(t, J = 3.5 Hz,
H), 3.79 (s, 3H), 3.81 (s, 3H), 4.15 (t, J = 4.5 Hz, 2H), 4.60 (dt, J = 47.5 and 4.0 Hz, 2H),
.61 (s, 1H), 6.86 (d, J = 8.5 Hz, 1H), 7.06 (d, J = 8.5 Hz, 2H), 7.11ꢀ7.15 (m, 3H), 7.62 (s,
+
H), 7.63 (s, 1H); MS (FAB) m/z 487 (M + H); HRMS calcd for C27
H32FO
7
, 487.2132;
found, 487.2130.
1
1
2
2
0: H NMR (CDCl
3
) δ 2.29 (s, 6H), 3.93 (s, 3H), 3.97 (s, 3H), 4.31 (dt, J = 25.0 and 3.0 Hz,
H), 4.80 (dt, J = 47.5 and 4.0 Hz, 2H), 6.31 (s, 1H), 6.94 (d, J = 7.0, 2H), 6.97 (d, J = 7.0,
+
H), 7.02 (d, J = 6.5, 2H), 7.55 (s, 2H); MS (FAB) m/z 443 (M + H); HRMS calcd for
C
25
H
28FO
7
, 443.1870; found, 443.1864.
1
1
3
6
4
2: H NMR (CDCl
3
) δ 2.18 (s, 6H), 2.24 (dt, J = 26.5 and 6.0 Hz, 2H), 3.89 (s, 3H), 3.92 (s,
H), 4.20 (t, J = 6.5 Hz, 2H), 4.68 (dt, J = 47.0 and 6.0 Hz, 2H), 6.30 (s, 1H), 6.92ꢀ7.03 (m,
+
H), 7.54 (s, 2H); MS (EI) m/z 456 (M ); HRMS calcd for C26
H29FO
6
, 456.1948; found,
56.1946.
Synthesis of 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-hexa-1,4-dien-3-one and its
dimethyl-substituted derivative (13 and 14)
2 3
B O (733.8 mg, 10.54 mmol) was added to a solution of 2,4ꢀpentanedione (600.7 mg, 6.00
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o
mmol) in ethyl acetate, and the solution was stirred at 80 C for 30 min. An ethyl acetate
3
solution (2.7 mL) of vanillin (912.2 mg, 6.00 mmol) and (nꢀBuO) B (0.46 mL, 1.70 mmol)
o
was added to the reaction mixture. After stirring at 80 C for 30 min, nꢀBuNH
2
(0.23 mL,
o
2
.28 mmol) was added dropwise to the mixture, which was then stirred at 80 C for 1 h. The
o
solution was cooled and treated with 1 N HCl (4.5 mL) at 50 C for 30 min. The reaction
mixture was extracted twice with ethyl acetate (200 mL × 2), and the organic layer was
2 4
washed with water and dried over Na SO . Flash column chromatography followed by
recrystallization from ethanol gave 13 (600 mg, 43%) as a yellow solid. Compound 14 was
synthesized as described for synthesis of 13 using 3,5ꢀheptanedione. Flash column
chromatography (3:1 hexaneꢀethyl acetate) yielded 14 (516.9 mg, 33%) as a yellow solid.
1
1
6
2
1
3
3: H NMR (CDCl
3
) δ 2.15 (s, 3H), 3.94 (s, 3H), 5.84 (s, 1H), 6.32 (d, J = 16.0 Hz, 1H),
.93 (d, J = 15.5 Hz, 1H), 7.09 (d, J = 10.5 Hz, 2H), 7.53 (d, J = 16.0 Hz, 1H); MS (EI) m/z
+
34 (M ); HRMS calcd for C13
H
14
4
O , 234.0892; found, 234.0891.
1
4: H NMR (CDCl
3
) δ 1.19 (t, J = 7.5 Hz, 3H), 2.10 (s, 3H), 2.44 (q, J = 7.5 Hz, 2H), 3.91 (s,
H), 5.87 (s, 1H), 6.91 (d, J = 1.5 Hz, 1H), 6.90ꢀ6.97 (m, 2H), 7.50 (s, 1H); MS (EI) m/z 262
+
(
M ); HRMS calcd for C15
H
18
4
O , 262.1205; found,262.1206.
Synthesis of 4-(2-(2-fluoroethoxy)ethoxy)-3-methoxybenzaldehyde (17)
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2ꢀ(2ꢀChloroethoxy)ethanol (1.0 mL, 9.85 mmol) was added to a solution of vanillin (1 g, 6.57
2 3
mmol) in DMF (100 mL) in the presence of K CO (1.18 g, 8.54 mmol), and the reaction
o
mixture was stirred at 100 C for 5 h. At the end of the reaction, the mixture was diluted with
2 2
water, extracted twice with CH Cl (150 mL × 2), and the combined organic layer was
washed with water (50 mL) and with a saturated ammonium chloride solution to remove
2 4
residual DMF and dried over Na SO . Flash column chromatography (1:5 hexaneꢀethyl
acetate) yielded 15 (986.3 mg, 63%) as a white solid.
1
1
5: H NMR (CDCl
3
) δ 3.69 (t, J = 4.4 Hz, 2H), 3.77 (t, J = 5.0 Hz, 2H), 3.94 (s, 3H), 3.96 (t,
J = 4.5 Hz, 2H), 4.26 (t, J = 4.6 Hz, 2H), 6.98 (d, J = 7.9 Hz, 1H), 7.39ꢀ7.45 (m, 2H), 9.85 (s,
+
1
H); MS (EI) m/z 240 (M ); HRMS calcd for C12
H
16O, 240.0998; found, 240.0996.
2 2 3
Compound 15 (500 mg, 2.08 mmol) was dissolved in CH Cl , and Et N (1.74 mL, 12.48
mmol) was added. The mixture was stirred at room temperature for 1 h, and then
o
methanesulfonyl chloride (193.3 mg, 2.49 mmol) was added at 0 C, and the mixture was
stirred at room temperature for 6 h. The reaction was quenched by addition of a saturated
2 2
ammonium chloride solution (50 mL). The reaction mixture was extracted twice with CH Cl
(50 mL × 2), and the combined organic layer was washed with water (50 mL) and dried over
2 4 2 2
Na SO . Flash column chromatography (25:1 CH Cl ꢀmethanol) gave 16 (632 mg, 95%) as a
white solid.
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1
1
2
2
3
3
6 : H NMR (CDCl ) δ 3.05 (s, 3H), 3.86 (t, J = 3.0 Hz, 2H), 3.91 (s, 3H), 3.95 (t, J = 5.0 Hz,
H), 4.27 (t, J = 4.5 Hz, 2H), 4.41 (t, J = 4.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 1H), 7.41ꢀ7.45 (m,
+
H), 9.86 (s, 1H); MS (EI) m/z 318 (M ); HRMS calcd for C13
H
18
O
7
S, 318.0773; found,
18.0796.
Compound 16 (500 mg, 1.57 mmol) and cesium fluoride (715 mg, 4.71 mmol) were
o
dissolved in tꢀBuOH (20 mL), and the reaction mixture was stirred at 100 C for 19 h. At the
end of the reaction, the mixture was diluted with water, extracted twice with ethyl acetate
(100 mL × 2), and the combined organic layer was washed with water (100 mL) and dried
2 4
over Na SO . Flash column chromatography (1:1 hexaneꢀethyl acetate) yielded 17 (323.8 mg,
8
1
4
5%) as a white solid.
1
7: H NMR (CDCl ) δ 3.84 (dt, J = 25.5 and 5Hz, 2H), 3.92 (s, 3H), 3.97 (t, J = 5.0 Hz, 2H),
3
.29 (t, J = 4.5 Hz, 2H), 4.58 (dt, J = 47.5 and 4.5 Hz, 2H), 7.01 (d, J = 7.5 Hz, 1H), 7.41 (d,
+
J = 2.0 Hz, 1H), 7.01 (dd, J = 8.5 and 1.5 Hz, 1H), 9,86 (s, 1H); MS (EI) m/z 242 (M );
4
HRMS calcd for C12H15FO , 242.0954; found, 242.0956.
Synthesis of 4-(2-fluoroethoxy)-3-methoxybenzaldehyde (18) and 4-(3-fluoropropoxy)-3-
methoxy benzaldehyde (19)
2ꢀFluoroethyl or 3ꢀfluoropropyl tosylate (4.27 mmol) was added to a solution of vanillin (500
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3 2 3
mg, 3.28 mmol) in CH CN (50 mL) in the presence of K CO (590 mg, 4.27 mmol), and the
o
reaction mixture was stirred at 110 C for 3 h. At the end of the reaction, the mixture was
diluted with water, extracted with ethyl acetate (100 mL), and the combined organic layer
2 4
was washed with water (100 mL) and dried over Na SO . Flash column chromatography
(hexaneꢀethyl acetate) afforded 18 (428.2 mg, 67%) or 19 (595.5 mg, 86%) as a white solid.
1
1
4
1
1
1
4
3
8: H NMR (CDCl ) δ 3.94 (s, 3H), 4.36 (dt, J = 27.0 and 4.5 Hz, 2H), 4.82 (dt, J = 47.5 and
.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 1.5 Hz, 1H), 7.45 (dd, J = 8.3 and 2.0 Hz,
+
H), 9,87 (s, 1H); MS (EI) m/z 198 (M ); HRMS calcd for C10
H
11FO
3
, 198.0692; found,
98.0694.
1
9: H NMR (CDCl
3
) δ 2.26 (dt, J = 26.5 and 5.5 Hz, 2H), 3.93 (s, 3H), 4.23ꢀ4.26 (m, 2H),
.68 (dt, J = 47.0 and 6.0 Hz, 2H), 7.00 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 1.5 Hz, 1H), 7.45 (dd,
+
J = 8.5 and 2.0 Hz, 1H), 9.86 (s, 1H); MS (EI) m/z 212 (M ); HRMS calcd for C11
H13FO
3
,
212.0849; found, 212.0845.
Synthesis of curcumin derivatives (20 and 21)
B
2
O
3
(128.10 mg, 1.84 mmol) was added to a solution of 13 or 14 (1.84 mmol) in ethyl
o
acetate (2 mL), and stirred at 80 C for 30 min. An ethyl acetate solution (2 mL) of 15 (300
3
mg, 1.53 mmol) and (nꢀBuO) B (453 ꢁL, 1.68 mmol) was added to the reaction mixture.
28

Page 29 of 54
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DOI: 10.1039/C5RA15380H
o
After stirring at 80 C for 30 min, piperidine (75 ꢁL, 0.76 mmol) was added to the mixture,
o
which was then stirred at 80 C for 1 h. The solution was cooled and treated with 0.4 N HCl
o
(
2 mL) at 50 C for 30 min. The reaction mixture was extracted twice with ethyl acetate (100
mL × 2), and the combined organic layer was washed with water (100 mL) and dried over
2 4
Na SO . Flash column chromatography followed by recrystallization from acetone gave 20
(238.5 mg, 34%) or 21 (317.2 mg, 42%) as a yellow solid.
1
2
0: H NMR (CDCl
3
) δ 3.69 (t, J = 4.0 Hz, 2H), 3.77 (t, J = 3.0 Hz, 2H), 3.92 (s, 3H), 3.93 (t,
J = 4.0 Hz, 2H), 3.95 (s, 3H), 4.22 (t, J = 4.5 Hz, 2H), 6.30 (s, 1H), 6.47 (d, J = 16.0 Hz, 1H),
.49 (d, J = 15.5 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 2.0
6
Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 16.0 Hz, 2H); MS
+
(
FAB) m/z 457 (M + H); HRMS calcd for C25
H
29
O
8
, 457.1862; found, 457.1868.
1
2
3
9
1: H NMR (CDCl
3
) δ 2.18 (s, 6H), 3.69 (t, J = 4.5 Hz, 2H), 3.77 (t, J = 4.5 Hz, 2H), 3.89 (s,
H), 3.92 (s, 3H), 3.93 (t, J = 5.0 Hz, 2H), 4.23 (t, J = 4.5 Hz, 2H), 6.30 (s, 1H), 6.94 (d, J =
.0 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.96 (d, J = 9.5 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 7.02
+
(
d, J = 8.0 Hz, 2H), 7.54 (s, 2H); MS (FAB) m/z 485 (M + H); HRMS calcd for
27 33 8
C H O ,485.2175; found, 485.2173.
Synthesis of HC derivatives (22 and 23)
29