Synthesis of labeled curcumin derivatives as tools for in vitro blood brain barrier trafficking studies

Article abstract of DOI:10.1002/jlcr.1907

Successful drug delivery to the brain is crucial for the therapy of many neurological disorders, including Alzheimer's disease. A stable tritiated pyrazole curcumin derivative, exhibiting ability to interact with Aβ peptide, has been synthesized to allow

Full text of DOI:10.1002/jlcr.1907

Research Article
Received 7 March 2011,
Revised 2 May 2011,
Accepted 23 May 2011
Published online 19 July 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1907
Synthesis of labeled curcumin derivatives as
tools for in vitro blood brain barrier trafficking
studies
*
Cristiano Zona and Barbara La Ferla
Successful drug delivery to the brain is crucial for the therapy of many neurological disorders, including Alzheimer’s disease.
A stable tritiated pyrazole curcumin derivative, exhibiting ability to interact with Ab peptide, has been synthesized to allow
tracking studies in an in vitro model of blood brain barrier.
Keywords: blood brain barrier (BBB); trafficking studies; curcumin pyrazole derivatives; synthesis
labeled compound was synthesized, involving a deprotonation reac-
tion in 85% yield, with a specific activity high enough for the
purpose.
Introduction
Alzheimer’s disease is the most common cause of dementia
among the elderly population.1–5 This pathology is characterized
The synthesis of the deuterium- and tritium-labeled com-
by an overproduction of amyloid beta (Ab) peptides and by their
pounds 4 and 5, respectively, is shown in Scheme 1. Curcumin
aggregation to form oligomer fibrils and plaques within the brain.
(1,1-diferuloylmethane) is present in a keto–enol tautomerism
in water, with enol as the active component.12 With this informa-
One important aspect in the treatment of neurological disorders is
the successful delivery of drugs to the brain. To achieve this task,
the drug must be able to pass or to be carried through the blood
brain barrier (BBB). Among all the natural Ab ligands, curcumin, a
major component of the yellow curry spice turmeric, was selected
tion, we synthesize an enol-form analog, treating the natural com-
pound with ethyl 2-hydrazinylacetate hydrochloride in refluxing
toluene, with trifluoroacetic acid (TFA) as catalyst. This reaction
afforded the pyrazole derivative 1 in good yield (87%). Carboxylic
acid 2 was obtained from 1 by basic hydrolysis of the ethyl ester
in a quantitative yield. This compound is the starting material for
the following functionalization with standard amide coupling pro-
for its known ability to inhibit Ab peptide aggregation both in vitro
and in vivo.6 However, curcumin has very high instability and very
low solubility in physiological conditions.7–10 These problems
could be addressed by using different strategies, such as employ-
cedure. Derivative 3 was obtained from 2 through coupling with
ing stable derivatives. In a project devoted to the generation of
propargylamine in 68% yield. In a project dedicated to the design
nanoparticles for the diagnosis and therapy of Alzheimer’s disease,
of different types of nanoparticles, compound 3 is very important
we synthesized different stable pyrazole curcumin derivatives for
because, in addition to being incorporated into particles such as
both the surface decoration and the inclusion inside these nanode-
liposomes, it may be linked to their surface by chemoselective
ligation procedures, such as Huisgen 1,3 cycloaddition.11 With
vices. We studied the ability of these stable derivatives to interact
with Ab peptides (data not published). Among them, derivative 3
this possibility, we set up the tritium labeling of this compound,
setting up the experimental protocol with its non-radioactive iso-
tope, which allowed us to evaluate both the isotopic exchange
(Scheme 1) was selected as a compound suitable for the surface
decoration of preformed nanoliposomes11 or for their incorpora-
tion into nanoparticles. In order to track these compounds both
and the yield of the reaction. Compound 3 was treated with
in vitro and in vivo, we synthesized labeled compounds 4 and 5.
freshly prepared lithium di-iso-propyl amide to obtain the
Radiolabeled compound 5 was incorporated into nanoliposomes
removal of both the protons in a to the amide position and
and used to investigate the ability of the nanoparticles to over-
the acetylene proton; we ran this reaction in anhydrous solvent
come the BBB in an in vitro model. Promising preliminary results
were obtained.
under inert atmosphere. The reaction mixture containing the dia-
nionic species was treated with a 20-fold excess of [2H2]water,
obtaining in 88% yield the desired isotopic exchange product
Result and discussion
[3H2]Curcumin derivative 5 was used for the preparation of a
device aimed at the study of molecule BBB trafficking in an
in vitro model of BBB. For this purpose, the radiolabeled molecule
was used as such or incorporated in different types of nanolipo-
somes, and the ability of the different devices to overcome the
BBB was measured, affording interesting preliminary results. The
Department of Biotechnology and Bioscience, University of Milano-Bicocca,
Piazza della Scienza 2, I-20126, Milano, Italy
*Correspondence to: Barbara la Ferla, Department of Biotechnology and
Bioscience, University of Milano-Bicocca, Milan, Italy.
E-mail: barbara.laferla@unimib.it
J. Label Compd. Radiopharm 2011, 54 629–632
Copyright © 2011 John Wiley & Sons, Ltd.

C. Zona and B. La Ferla
Scheme 1. Reagents and conditions: (i) ethyl 2-hydrazinyl acetate hydrochloride, TEA, TFA, toluene, reflux, 2.5h; (ii) KOH 1.8M methanolic solution, room temperature,
overnight, 87% (over two steps); (ii) propargylamine, TBTU, HOBT, TEA, DMF dry, room temperature, overnight, 63%; (iii) n-BuLi, iPr2NH, THF dry, 0ꢀC!room temperature,
[2H2]water, 0.5h, 88%; (iv) n-BuLi, iPr2NH, THF dry, 0ꢀC!room temperature, [3H2]water, 0.5h, 85%, 405mCi/mmol.
4, with an isotopic exchange of 57% at the acetylene proton and for at least 24h prior to use. [3H2]Water, 2.5mCi/g, was
33% at the proton a to the amide position, as demonstrated by obtained from Perkin-Elmer, Boston, MA. Thin layer chromato-
mass spectral and NMR analyses. This procedure affords stable graphy (TLC) was performed on silicagel60F254 plates (Merck)
labeling, since in physiological condition no isotopic exchange is with detection under UV light when possible, or by charring with
possible. This deuterated compound was also useful for the incor- a solution of (NH4)6Mo7O24 (21g), Ce(SO4)2 (1g), concentrated
poration of the labeled molecule inside the nanoliposomes with- H2SO4 (31mL) in water (500mL) or with an ethanol solution of
out loss of any amount of radioactive material.
Using this labeling procedure, we performed the synthesis of the chromatography was performed on silica gel 230–400 mesh
ninhydrin or with Dragendorff’ spray reagent.13 Flash-column
1
13
radiolabeled compound 5. After the treatment with freshly prepared (Merck). H and C NMR spectra were recorded at 25ꢀC, unless
lithium di-iso-propyl amide, we added 100 mL of 250mCi [3H2] otherwise stated, with a Varian Mercury 400-MHz instrument.
water to obtain the radiolabeling of the starting curcumin deriva- Chemical shift assignments, reported in parts per million, were
tive 3. After 30min, the desired compound was precipitated by acid- referenced to the corresponding solvent peaks. Mass spectra
ifying the mixture with 5% HCl(aq) to pH<2. The solid was filtered were recorded on a QTRAP system with ESI source, while HRMS
and re-dissolved with 5% NaOH(aq) to remove all the radioisotopes were registered on a QSTAR elite system with a nanospray ion
linked to the phenolic hydroxyl group of the two ferulic moieties. source. Beta counting was done with a Packard Tri-Carb liquid
The acidification with 5% HCl(aq) to pH<2 and the filtration of scintillation counter.
the precipitate provided compound 5 in 85% chemical yield and
6% overall radiochemical yield, which was calculated from the
(E)-Ethyl 2-[3,5-bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-
1-yl]acetate (1)
starting activity of the commercial [3H2]water. The radiolabeling
efficacy was much lower with respect to the deuterium labeling
due to the different isotope enrichment of the labeling reagent
To a solution of curcumin (1.000g, 2.7mmol, 1eq) in dry toluene
(28mL) was added ethyl 2-hydrazinylacetate hydrochloride
(0.839g, 5.4mmol, 2eq), triethylamine (0.753mL, 5.4mmol, 2
eq), and TFA (0.1mL, 1.3mmol, 0.5eq), and the reaction was held
at reflux for 2.5h. The reaction was monitored by TLC. The reac-
tion mixture was then cooled to room temperature, diluted with
CH2Cl2, and washed with 5% HCl(aq) twice. The organic phase
was dried over Na2SO4 and evaporated in vacuum to afford
the crude product, which was further purified by flash chromato-
graphy (CH2Cl2/isopropanol 98:2) to afford the desired product
([2H2]water 99% pure, [3H2]water 2.5mCi/mL),
Further attempts to optimize the reaction were set aside
because of accelerated delivery timeline as requested by the custo-
mer group for the radioactive molecule. The tritium labeling was
acceptable to deliver the target molecule with an activity high
enough for invitro studies. In order to increase the radiochemical
activity of 5, which could be important for other applications
such as in surface decoration of nanoliposomes, [3H2]water with
higher starting activity should be employed.
1
as a yellow solid (1.058g, 87%). H NMR (400MHz, DMSO d6) d
ppm 7.22–6.70 (m, 11H, CHAr and conjugated double bond),
5.16 (s, 2H CH2C=O), 4.13 (q, J=7.05Hz, 2H, OCH2CH3), 3.82–3.77
Experimental
All commercial chemicals were purchased from Sigma-Aldrich. (m, 6H, OCH3), 1.19 (t, J=7.12Hz, 3H, OCH2CH3). 13C NMR
All chemicals were used without further purification. All re- (100MHz, DMSO d6) d 168.32 (C=O), 150.02, 147.86, 147.27,
quired anhydrous solvents were dried with molecular sieves 146.68, 143.35 (5 CqAr), 132.25, 129.79 (2 CHAr), 128.49, 128.06
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 629–632

C. Zona and B. La Ferla
(2 CqAr), 120.81, 120.01, 117.75, 115.57, 111.66, 110.24, 109.58, min, the solution was acidified to pH<2 with 5% HCl(aq), and a
98.63 (8 CHAr and conjugated double bond), 61.11 (CH2), 55.76, yellow suspension was obtained. The solid product was filtered,
55.59 (2 CH3), 50.30 (CH2), 14.09 (CH3). MS (ESI) calcd for [M+H]+ affording pure compound 4 (0.090g, 88%). 1H NMR (400MHz,
451.48; found [M+H]+ 451.60.
CD3OD) d ppm 7.38–6.73 (m, 11H, CHAr and conjugated double
bond), 5.13 (m, 1.3H CH2C=O), 4.04 (s, 2H, CH2NH), 3.96–3.83
(m, 6H, OCH3), 2.63 (m, 0.43H, CH). 13C (100MHz, CDCl3) d ppm
169.31 (C=O) 152.55, 149.33, 148.03, 145.63 (4 CqAr), 135.08,
132.22 (2 CHAr), 130.75, 130.13 (2 CqAr), 122.07, 121.40, 118.27,
116.46, 112.40, 111.08, 110.70, 100.52 (8 CHAr and conjugated
double bond),80.27 (Cq), 72.42 (CH), 56.64, 56.55 (2 CH3), 52.66
(CH2), 29.71 (CH2). MS (ESI) calcd for [M+H]+ 460.49, [[2H]M+
H]+ 461.49, [[2H2]M+H]+ 462.49; found [M+H]+ 460.4, [[2H]M+
H]+ 461.4, [[2H2]M+H]+ 462.4; the highest peak is the one with
two deuterium atoms at 462.4 (experimental). On the basis of
the 1H NMR and mass spectra, we can assume that we obtained
a 33% isotopic exchange of the proton in a to the amide position
and a 57% isotopic exchange of the acetylene proton.
(E)-2-[3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl]
acetic acid (2)
Compound 1 (3.000g, 6.66mmol, 1eq) was dissolved in a metha-
nolic solution of KOH 1.8M (70ml). The solution was left under
magnetic stirring at room temperature overnight. The reaction
was monitored by TLC. The solvent was evaporated in vacuum
and purified by flash chromatography (eluent: ethyl acetate/
methanol gradient from 9:1 to 7:3) to afford the final product
(2.813g, quant.). 1H NMR (400MHz, CD3OD) d ppm 7.23–6.72
(m, 11H, CHAr and conjugated double bond), 4.93 (s, 2H, CH2C=O),
3.85–3.79 (m, 6H, OCH3). 13C NMR (100MHz, DMSO) d 170.17
(C=O), 147.85, 147.16, 146.58, 142.80 (4 CqAr), 131.54, 129.13
(2 CHAr), 128.62, 128.17 (2 CqAr), 120.61, 119.87, 118.08,
115.58, 112.31, 110.08, 109.53, 98.38 (8 CHAr and conjugated
double bond), 55.74, 55.58 (2 CH3), 51.58 (CH2). MS (ESI) calcd
for [M+H]+ 423.43; found [M+H]+ 423.4.
[3H2](E)-2-[3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-
yl]-N-(prop-2-yn-1-yl)acetamide (5)
A 1.6M (in hexane) solution of n-BuLi (0.275mL, 0.44mmol, 10eq)
was added to a solution of iPr2NH (0.062mL, 0.044g, 0.44mmol,
10eq) in dry THF (0.5mL) at 0ꢀC. After 30min, a solution of com-
pound 3 (0.020g, 0.044mmol, 1eq) in dry THF (1.0mL) was
added. The reaction was then warmed to room temperature
and monitored by TLC. [3H2]H2O (2.5mCi/mL, 0.100mL, 250mCi)
was added for radiolabeling. The solution was acidified to pH<
2 with 5% HCl(aq), and a pale yellow suspension was obtained.
The solid was filtered and dissolved with 5% NaOH(aq) to remove
all the residues of mobile tritium atoms. After 30min, the solu-
tion was acidified to pH<2 with 5% HCl(aq), obtaining a yellow
suspension. The solid product was filtered, obtaining pure com-
pound 5 (0.017g, 85%) with an activity of 15mCi (0.88mCi/mg
or 405mCi/mmol).
(E- 2-[3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl]-
N-(prop-2-yn-1-yl)acetamide (3)
HObt (0.017g, 0.123mmol, 1.23eq) and TBTU (0.039g, 0.12mmol,
1.2eq) were added to a solution of compound 2 (0.042g, 0.1
mmol, 1eq) in dry DMF (1mL). The reaction mixture was left in
the dark at room temperature under magnetic stirring for 15
min. Propargylamine (0.013mL, 0.2mmol, 2eq) and triethylamine
(0.07mL, 0.5mmol, 5eq) were added, and the reaction was left in
the dark at room temperature under magnetic stirring overnight.
The reaction was monitored by TLC. The solvent was evaporated
in vacuum. The crude product was diluted with CH2Cl2 and
washed three times with water. The organic phase was dried
over Na2SO4 and evaporated in vacuum to afford the crude
product, which was further purified by flash chromatography
(eluent: petroleum ether/ethyl acetate gradient from 3:7 to ethyl
acetate) to give the desired product as an amorphous powder
Conclusions
To achieve our goal of generating a radiolabeled pyrazole curcu-
min analog, we evaluated a synthetic route employing a cold
starting material. We worked with [2H2]water as source of deute
rium atoms for the isotopic exchange in compound 3. After
some good preliminary results were obtained for both the synth-
esis and the incorporation in nanoliposomes, labeled [3H2]curcu-
min derivative 5 was synthesized in 50% overall yield, using [3H2]
1
(0.031g, 68%). H NMR (400MHz, CD3OD) d ppm 7.38–6.73 (m,
11H, CHAr and conjugated double bond), 5.12 (s, 2H CH2C=O),
4.04 (s, 2H, CH2NH), 3.96–3.83 (m, 6H, OCH3), 2.63 (s, 1H, CH).
13C (100MHz, CDCl3) d ppm 169.31 (C=O) 152.55, 149.33,
148.03, 145.63 (4 CqAr), 135.08, 132.22 (2 CHAr), 130.75, 130.13
(2 CqAr), 122.07, 121.40, 118.27, 116.46, 112.40, 111.08, 110.70,
100.52 (8 CHAr and conjugated double bond), 80.27 (Cq), 72.42
(CH), 56.64, 56.55 (2 CH3), 52.66 (CH2), 29.71 (CH2). MS (ESI) calcd
for [M+H]+ 460.49; found [M+H]+ 460.4.
3
water as source of radioactivity. The incorporation of H atoms
was effective, and the resulting specific activity was sufficient
for the trafficking studies carried out using an in vitro BBB model.
The present sequence is a cost-effective and efficient route to
label this stable and highly functionalizable Ab ligand.
[2H2](E)-2-[3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-
yl]-N-(prop-2-yn-1-yl)acetamide (4)
Acknowledgment
A 1.6M (in hexane) solution of n-BuLi (0.830mL, 1.33mmol, 6eq)
was added to a solution of iPr2NH (0.188mL, 0.136g, 1.33mmol,
6eq) in dry THF (0.5mL) at 0ꢀC. After 30min, a solution of com-
pound 3 (0.102g, 0.23mmol, 1eq) in dry THF (3.0mL) was added.
The reaction was then warmed to room temperature and moni-
tored by TLC. [2H2]Water (0.092mL, 0.092g, 4.6mmol, 20eq) was
added to obtain the isotopic exchange. The solution was acidi-
fied to pH<2 with 5% HCl(aq), obtaining a pale yellow suspen-
sion. The solid was filtered and dissolved with 5% NaOH(aq) to
remove all the residues of mobile deuterium atoms. After 30
The research leading to these results has received funding from
the European Community’s Seventh Framework Program (FP7/
2007-2013) under grant agreement no. 212043.
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J. Label Compd. Radiopharm 2011, 54 629–632