Hydrazinocurcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation

Article abstract of DOI:10.1016/S0968-0896(02)00129-3

Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel curcumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC₅₀=520 nM) without cytotoxicity. In vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent.

Full text of DOI:10.1016/S0968-0896(02)00129-3

Bioorganic & Medicinal Chemistry 10 (2002) 2987–2992
Hydrazinocurcumin, a Novel Synthetic Curcumin Derivative, is a
Potent Inhibitor of Endothelial Cell Proliferation
a
a
a
a
b
Joong Sup Shim, Dong Hoon Kim, Hye Jin Jung, Jin Hee Kim, Dongyeol Lim,
c
c
d
e
f
Seok-Ki Lee, Kyu-Won Kim, Jong Woong Ahn, Jong-Shin Yoo, Jung-Rae Rho,
f
a,
Jongheon Shin and Ho Jeong Kwon *
aDepartment of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu,
Seoul 143-747, South Korea
b
Department of Applied Chemistry, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, South Korea
cResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
dKorea Research Institute of Chemical Technology, PO Box 107, Yusong, Daejon, 305-600, South Korea
eKorea Basic Science Institute, PO Box 41, Yusong, Daejon, 305-333, South Korea
fMarine Natural Products Laboratory, Korea Ocean Research and Development Institute, Ansan 425-600, South Korea
Received 12 February 2002; accepted 6 April 2002
Abstract—Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel cur-
cumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited
the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC50=520 nM) without cytotoxicity. In
vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent. # 2002 Published by Elsevier
Science Ltd.
Introduction
Curcumin, a natural product found in the rhizome of
Curcuma longa, is a potent chemopreventive agent that
has been entered into the phase I clinical trials for
Angiogenesis is the process of new blood vessel forma-
1
7
tion by endothelial cells. The process is complex and
chemoprevention by National Cancer Institute. Cur-
cumin showed a potent anti-carcinogenic activity
against a broad range of tumor types including skin,
involves several distinct steps such as membrane degra-
dation by proteolytic enzymes secreted by the endothe-
lial cells, chemotactic migration toward the stimulus,
proliferation of these cells, and formation of vascular
8À11
forestomach, duodenal, and colon carcinogenesis.
It
has been postulated that the broad spectrum of anti-
carcinogenic activity of curcumin may be due in part to
2
loops. Each step of the process is tightly controlled by
1
2,13
regulatory factors that stimulate or inhibit angiogenesis.
However, these elaborated control mechanisms are
often disordered in several pathologic diseases including
angiogenesis inhibition.
Curcumin and some of its
derivatives including demethoxycurcumin (DC) and
tetrahydrocurcumin (THC) were known as potent inhi-
3
14
cancer. Extensive studies have shown that angiogenesis
bitors of angiogenesis. DC as well as THC, a murine
is a crucial process for the outgrowth of cancer cells and
their spreading into other tissues. Therefore, the specific
inhibition of angiogenesis is considered as a powerful
way to inhibit tumor growth and metastasis. Several
angiogenesis inhibitors from natural products and che-
mical libraries have been developed for this purpose and
some of them, including TNP-470, are undergoing clin-
metabolite of curcumin, showed a significant inhibition
of corneal neovascularization, but the potential of the
activity was relatively weaker than that of curcumin.
Extensive studies on the structure–activity relationship
of curcuminoids have revealed that phenol ring struc-
tures of the compound may be essential for the anti-
ical trials for cancer therapy.4
À6
oxidant activity, and diketone moiety of THC also
1
15
6
involves antioxidative mechanism of the compound.
In addition, phenolic hydroxyl or methoxyl groups were
modified to enhance the potency of other biological
activities of curcumin including the induction of Phase 2
*
3
Corresponding author. Tel.: +82-2-3408-3640; fax: +82-2-3408-
334; e-mail: kwonhj@sejong.ac.kr
0
968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00129-3

2988
J. S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2987–2992
ꢀ
detoxification enzymes and the inhibition of HIV-1
1
by HRMS, thus 13 of unsaturation. The structure of
this compound was determined by combined 2-D NMR
experiments as well as comparison of spectral data with
those obtained for curcumin (Table 1). All of the signals
of protons and carbons of the benzene ring were readily
7,18
integrase.
These studies suggest that diketone moi-
ety of curcumin may be essentially involved in its broad-
range biological activities.
1
We have developed several new synthetic derivatives of
curcumin to enhance the biological activity, especially,
anti-angiogenic activity of the compound. The potencies
of newly synthesized curcumin derivatives were eval-
uated using several angiogenesis assays including endo-
thelial cell proliferation, chemoinvasion, capillary tube
formation, and in vivo angiogenesis of chorioallantoic
membrane of chick embryo. Among the synthetic deri-
vatives, hydrazinocurcumin (HC) showed the most
potent inhibitory activity against the proliferation of
BAECs. The synthesis, structure determination, and
anti-angiogenic activity of HC will be described.
assigned on the basis of the results of H COSY,
gHSQC, and gHMBC experiments. However, consider-
able differences were observed for the NMR signals
corresponding to the protons and carbons at the side
chain. The C NMR spectra showed a broad signal at d
131.1 (d) which correlated to both of the protons at d
7.09 and 6.94 in the gHSQC spectra. The H COSY
1
3
1
data revealed that these protons were coupled to each
other with very large coupling constant (J=16.1 Hz).
Therefore, these were thought to be the olefinic protons
of a trans-double bond. This interpretation was sup-
ported by ROESY experiments in which cross peaks
were observed between both of the olefinic protons and
aromatic ones at d 7.16 and 6.97.
Results and Discussion
Chemistry
The NMR data of HC contained other signals of an
additional methine group; dH 6.64, dC 99.9. Since the
intensity of the proton signal was almost half of others,
this methine must be corresponded to the C-10 sym-
metric center of the molecule. Although the signal of the
C-9 carbon was not observed due to the rapid tauto-
merization between two forms of a pyrazole moiety, the
The general procedures for synthesis of derivatives are
shown in Schemes 1 and 2. Curcumin (1a, R1,
R =OCH ) and its natural analogues including deme-
2
3
thoxycurcumin (1b, R =OCH ; R =H or vice versa)
1
3
2
and bisdemethoxycurcumin (1c, R =H; R =H) were
1
2
1
3
prepared from the purification of commercially avail-
able curcuminoids using preparative thin layer chroma-
tography. Diketone moieties of each curcumin analogue
were replaced with hydrazine derivatives in the presence
of acetic acid. One step coupling of 1 with hydra-
ziniumdihydrochloride and hydrazinobenzoic acid gave
compounds 2 to 3. Each compound was purified with
preparative TLC and HPLC, and was assayed for anti-
angiogenic activity.
upfield shift of the C-10 methine carbon in the
C
NMR data indicated the placement of an olefinic car-
bon bearing an electronegative atom at adjacent loca-
tion. Data supporting this interpretation were provided
by 2-D NMR experiments in which the gHMBC corre-
lation between H-8 and C-10 as well as the ROESY
cross peaks between H-8 and H-10 were observed. Thus,
the structure of HC was defined as an analogue of cur-
cumin bearing a pyrazole moiety in the middle of chain.
Structure determination of HC
Anti-angiogenic activity of HC
HC (2a), a synthetic analogue of curcumin was obtained
as pale yellow gum which was analyzed for C H N O
4
To evaluate anti-angiogenic activities of synthetic cur-
cumin derivatives, we first examined the effect of com-
2
1
20
2
Scheme 1.
Scheme 2.

J. S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2987–2992
2989
pounds on endothelial cell proliferation using MTT
colorimetric assay. Among the six derivatives tested,
HC showed the most potent growth inhibitory activity
against BAECs with an IC50 of 0.52 mM (Table 2). The
inhibitory potency of HC against BAECs proliferation
was over 30-fold higher than that of curcumin. Hydra-
zine derivative with bulky benzoic acid (3) also showed
an enhanced anti-proliferative activity against BAECs.
However, the potency of 3 was relatively weaker than
that of HC.
different sensitivity to HC with BAECs. In contrast to
HC, curcumin, the parental compound, inhibited the
proliferation of these cells in relatively non-selective
manner (data not shown). These data suggest that HC
inhibits cellular proliferation with an endothelial cell
specificity.
Endothelial cell invasion and the formation of tubular
structures as well as cellular proliferation are essential
steps for angiogenic process. Thus, we next investigated
the effect of HC on BAECs invasion and tube formation
using well established in vitro assays. Basic fibroblast
growth factor (bFGF) was used as a chemoattractant or
an angiogenic factor. HC potently inhibited bFGF-
induced BAECs invasion and tube formation at nano-
molar concentrations (Fig. 2A–C). Trypan blue staining
was performed in parallel with in vitro angiogenesis
assays and cytotoxicity was not shown at concentrations
used in this study (Fig. 2D).
Next, we investigated to determine the cell line specifi-
city of HC on its anti-proliferative activity. Several epi-
thelial and fibroblast cells including, HT29, colon
carcinoma, NIH3T3, normal fibroblast, Chang, normal
liver cells, and BAECs, were examined and the result
showed that HC inhibited the proliferation of each cell
lines with a different activity spectrum (Fig. 1). Inter-
estingly, there was high endothelial cell specificity on the
anti-proliferative activity of HC. Specificity factors over
20 were obtained versus HT29, colorectal carcinoma
cells. Other normal cell lines also showed a degree of
In vivo experimental angiogenesis was performed using
chorioallantoic membrane (CAM) from growing chick
embryo. HC-loaded thermanox coverslip was placed on
CAM surface and neovascularized zones were observed
under microscope. The inhibition of the angiogenesis of
retinoic acid used as the positive control was 85%
Table 1. The structure of HC
(
n=13), and that of empty coverslips was 0% (n=19).
However, HC effectively inhibited the neovasculariza-
tion of chick embryo (73%, n=15) without affecting
any pre-existing vessels (Table 3). These results indicate
that HC potently inhibits angiogenesis without affecting
endothelial cell viability, and suggest that the compound
can be developed as a novel anti-angiogenic agent.
1H
13C
HMBC
ROESY
1
2
3
4
5
6
7
8
9
1
129.5 s
110.0 d
148.8 s
148.0 s
116.1 d
121.2 d
131.1 d
131.1 d
N.A.a
7.16, 2H, br s
3, 4, 6, 7
7, 8, OMe
6.78, 2H, d(7.8)
6.97, 2H, dd (7.8, 1.5)
7.09, 2H, d (16.1)
6.94, 2H, d (16.1)
1, 3
2, 4, 7
2, 6
6
5, 7
2, 6, 10
2, 10
10
0
6.64, 1H, s
3.86, 6H, s
99.9 d
56.2 d
7, 8
2
OMe
3
aSignal was not observed.
Table 2. IC50 values of curcuminoids and synthetic derivatives for
BAECs
Figure 1. Effect of HC on the growth of various cell lines. Cell growth
was measured using MTT colorimetric assay. Data represent mean-
ÆSE from three independent experiments.
R
1
R
2
BAECs, IC50 (M)a
À5
Curcuminoids, 1
OCH
OCH
H
3
OCH
H
H
3
3
3
1.5Æ0.3Â10
À5
3
2.2Æ0.5Â10
Table 3. Inhibitory effect of HC on in vivo neovascularization of
CAM
À5
5.3Æ0.6Â10
À7
Hydrazino
curcuminoids, 2
OCH
OCH
H
3
OCH
H
H
5.2Æ0.4Â10
À6
Drug
Inhibited
egg/live egg
Inhibition
ratio (%)
3
1.8Æ0.3Â10
À6
5.8Æ0.2Â10
À7
Hydrazinobenzoyl
curcuminoids, 3
OCH
OCH
H
3
OCH
H
H
9.3Æ0.4Â10
Empty
0/19
11/13
11/15
0
85
73
À6
3
2.4Æ0.1Â10
À6
Retinoic acid (1 mg/egg)
8.7Æ0.2Â10
(
10 mg/egg)
aIC50 values are the meanÆSE from three independent experiments.

2990
J. S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2987–2992
Figure 2. Effect of HC on in vitro angiogenesis. (A) Inhibitory activity of HC on endothelial cell invasion. Serum-starved BAECs left in serum-free
medium (Control) or treated with bFGF in the presence or absence of HC were used for invasion assay. Data represent meanÆSE from three
independent experiments. (B) Microscopic observation of invaded cells (Â100 magnification): (a) control; (b) bFGF alone; (c) bFGF+HC (100 nM);
(d) bFGF+HC (300 nM). (C) Effect of HC on tube forming ability of BAECs: (a) control; (b) bFGF alone; (c) bFGF+HC (100 nM); (d)
bFGF+HC (300 nM). (D) Trypan blue staining to evaluate in vitro toxicity of HC on tube-formed endothelial cells. Staurosporin, a cytotoxic
agent, was used as an indicator of in vitro toxicity: (a) bFGF+HC (300 nM); (b) bFGF+staurosporin (10 mM).
Conclusion
Hydrazinocurcumin (2a). Purified 1a (10 mg, 0.027 mmol)
was dissolved in methanol (2 mL), and hydraziniumdi-
hydrochloride (14 mg, 0.135 mmol) and triethylamine
(18.8 mL, 0.135 mmol) were added to the solution. In the
presence of catalytic amount of acetic acid, the reaction
mixture was incubated for 24 h at room temperature
with gentle stirring. The solvent was evaporated in
vacuo and the residue purified by preparative TLC
(CHCl /MeOH=6:1; R =0.4) and HPLC (semi-
Data presented here showed a novel synthetic analogue
of curcumin with altered potency of anti-angiogenic
activity, and its chemical and physical properties were
characterized using several analytical methods. Biologi-
cal activities of HC also were evaluated through in vivo
and in vitro angiogenesis assays. Our results demon-
strate HC as a new candidate for anti-angiogenic agent.
3
f
preparative C18 column; acetonitrile/H O=50:50; flow
2
rate=3 mL/min; retention time: 21 min) gave 2a as pale
yellow gum (6.19 mg, 65%) which analyzed by HRFAB-
MS. Exact mass calcd for C H N O : 364.1423,
Experimental
2
1
20
2
4
General procedures
observed (M+H) 365.1501.
Curcuminoids (mixture of curcumin, demethoxy-
curcumin, and bisdemethoxycurcumin) was obtained by
Sigma (St. Louis, MO, USA). Chromatography pur-
ification: Silica gel column chromatography (Merck,
Darmstadt, Germany), thin-layer chromatography
Hydrazinodemethoxycurcumin (2b). Utilizing the same
protocol as described for the synthesis of 2a, 2b (5.9mg,
61%) was obtained from 1b (10 mg, 0.029 mmol), hydra-
ziniumdihydrochloride (14 mg, 0.135 mmol), and triethy-
lamine (18.8 mL, 0.135 mmol). Preparative TLC (CHCl3/
(
TLC, Merck), and high performance liquid chromato-
MeOH=6:1; R =0.32) gave 2b as a pale yellow gum
f
graphy (HPLC, Shimadzu, Kyoto, Japan) were used to
perform general purification procedures. Spectral ana-
lysis: The HRFAB-MS spectra were obtained using a
Jeol JMS-HX 110 mass spectrometer. The NMR spec-
which was analyzed by HRFAB-MS. Exact mass calcd
for C H N O : 334.1317, observed (M+H) 335.1393.
2
0
18
2
3
Hydrazinobisdemethoxycurcumin (2c). Utilizing the same
protocol as described for the synthesis of 2a, 2c (5.5 mg,
57%) was obtained from 1c (10 mg, 0.032 mmol), hydra-
ziniumdihydrochloride (14 mg, 0.135 mmol), and trie-
thylamine (18.8 mL, 0.135 mmol). Preparative TLC
(CHCl /MeOH=6:1; R =0.26) gave 2c as a pale yellow
tra were recorded in CDCl solutions on a Varian Unity
3
5
00 spectrometer. The proton and carbon NMR spectra
were measured at 500 and 125 MHz, respectively. All
chemical shifts were recorded according to an internal
Me Si. All solvents used were of spectral grade or dis-
4
3
f
tilled from glass prior to use.
gum which was analyzed by HRFAB-MS. Exact mass

J. S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2987–2992
2991
calcd for C H N O : 304.1212, observed (M+H)
1
coated with 10mL of gelatin (1mg/mL), whereas the upper
side was coated with 10mL of Matrigel (3 mg/mL). Expo-
9
16
2
2
3
05.1291.
5
nentially growing cells (1Â10 cells) were placed in the
Hydrazinobenzoylcurcumin (3a). To a solution of 1a
10 mg, 0.027 mmol) in methanol (2 mL) was added
-hydrazinobenzoic acid (20 mg, 0.135 mmol), triethyl-
upper part of the filter and HC was applied to the lower
part for 30min at room temperature before seeding. The
(
4
ꢀ
chamber was then incubated at 37 C for 18h. The cells
amine (18.8 mL, 0.135 mmol), and catalytic amount of
acetic acid. After incubation for 24 h, the solvent was
evaporated in vacuo and the residue purified by pre-
parative TLC (CHCl /MeOH=4:1; R =0.5) and HPLC
were fixed with methanol and stained with hematoxylin/
eosin. The cell invasion was determined by counting
whole cells on the lower side of the filter using an optical
microscope at Â100 magnification.
3
f
(
semipreparative C18 column; acetonitrile/H O=50:50;
2
flow rate=3 mL/min; retention time: 15 min) gave 3a as
a dark orange powder (7.18 mg, 55%) which was ana-
lyzed by HRFAB-MS. Exact mass calcd for C H
24
Tube formation assay
Matrigel (250 mL, 10 mg/mL) was placed in a 24-well
culture plates and polymerized for 30 min at 37 C. The
2
8
ꢀ
N O : 484.1634, observed (M+H) 485.1715.
2
6
5
BAECs (1Â10 cells) were seeded on the surface of the
Hydrazinobenzoyldemethoxycurcumin (3b). Utilizing the
same protocol as described for the synthesis of 3a, 3b
Matrigel and treated with bFGF (30 ng/mL). Then, HC
was added and incubation was continued for 6–18 h.
The morphological changes in the cells and tubes
formed were observed under a microscope and photo-
graphed at Â100 magnification using JVC digital cam-
era (Victor, Yokohama, Japan).
(
5.52 mg, 42%) was obtained from 1b (10 mg,
0
0
.029 mmol),
4-hydrazinobenzoic
acid
(20 mg,
.135 mmol), and triethylamine (18.8 mL, 0.135 mmol).
Preparative TLC (CHCl /MeOH=4:1; R =0.41) gave
3b as a dark orange powder which was analyzed by
HRFAB-MS. Exact mass calcd for C H N O :
3
f
2
7
22
2
5
Chorioallantoic membrane (CAM) assay
4
54.1529, observed (M+H) 455.1611.
Fertilized chick eggs were kept in a humidified incu-
ꢀ
Hydrazinobenzoylbisdemethoxycurcumin (3c). Utilizing
the same protocol as described for the synthesis of 3a, 3c
bator at 37 C for 3 days. About 2 mL of egg albumin
was then removed with a hypodermic needle allowing
the CAM and yolk sac to drop away from the shell
membrane. On day 3.5, the shell was punched out and
removed and the shell membrane peeled away. At the
stage of a 4.5-day old chick embryo, a HC (10 mg/egg)-
loaded thermanox coverslip was air-dried and applied
to the CAM surface. Two days later, 2 mL of 10% fat
emulsion was injected into the chorioallantois and the
CAM was observed under a microscope. Since retinoic
acid (RA) is known as an anti-angiogenic compound,
(
6.78mg, 50%) was obtained from 1c (10 mg, 0.032 mmol),
-hydrazinobenzoic acid (20 mg, 0.135 mmol), and tri-
ethylamine (18.8 mL, 0.135mmol). Preparative TLC
CHCl /MeOH=4:1; R =0.34) gave 3c as a dark
4
(
3
f
orange powder which was analyzed by HRFAB-MS.
Exact mass calcd for C H N O : 424.1423, observed
2
6
20
2
4
(
M+H) 425.1501.
Cell culture and growth assay
1
mg/egg RA was used as a positive control for anti-
Early passages (5–7 passages) of bovine aortic endothelial
cells (BAECs) were kindly provided by Dr. Jo at the NIH
of Korea. BAECs were grown in MEM supplemented
with 10% fetal bovine serum. HT29 (colon carcinoma),
NIH3T3 (mouse normal fibroblast), and Chang (normal
liver) cells were maintained in RPMI1640 containing 10%
angiogenic responses. When the CAM treated with a
sample showing an avascular zone to a similar degree of
RA-treated CAM that had little vessels compared to
empty coverslip, the response was scored as positive,
and calculated based on the percentage of positive eggs
to the total number of eggs tested.
ꢀ
fetal bovine serum. All cells were grown at 37 C in a
humidified atmosphere of 5% CO . Cell growth assay was
2
carried out using MTT colorimetric assay. Exponentially
3
Acknowledgements
growing cells were seeded at a density of 5Â10 cells/well
in a 96-well plate. The cells were incubated in growth
media for 24h. Various concentrations of curcumin
derivatives were added to each well and incubated for
up to 72 h. After 72 h, 50 mL of MTT (2 mg/mL stock
solution, Sigma) was added and the plate was incubated
for an additional 4 h. After removal of the culture
supernatants, 150 mL of DMSO was added. The plate
was read at 540 nm using a microplate reader (Bio-Tek
Instruments, Inc., Winooski, VT, USA).
This work was supported by a grant to H.J.K. from Korea
Health 21 R & D Project, Ministry of Health & Welfare
(HMP-01-PJ1-PG1–01CH04–0005) and to J.S. from the
Ministry of Science and Technology of Korea (PN 99384).
We are grateful to Dr. Jo at the KNIH for BAECs.
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