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

Article abstract of DOI:10.1016/S0968-0896(02)00116-5

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. 2002 Elsevier Science. All rights reserved.

Full text of DOI:10.1016/S0968-0896(02)00116-5

Bioorganic & Medicinal Chemistry 10 (2002) 2439–2444
Hydrazinocurcumin, a Novel Synthetic Curcumin Derivative, Is a
Potent Inhibitor of Endothelial Cell Proliferation
Joong Sup Shim,a Dong Hoon Kim,a Hye Jin Jung,a Jin Hee Kim,a Dongyeol Lim,b
Seok-Ki Lee,c Kyu-Won Kim,c Jong Woong Ahn,d Jong-Shin Yoo,e Jung-Rae Rho,f
Jongheon Shinf and Ho Jeong Kwona,*
aDepartment of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu,
Seoul 143-747, Republic of Korea
bDepartment of Applied Chemistry, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu,
Seoul 143-747, Republic of Korea
cResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
dKorea Research Institute of Chemical Technology, PO Box 41, Yusong, Daejon, 305-600, Republic of Korea
eKorea Basic Science Institute, PO Box 107, Yusong, Daejon, 305-333, Republic of Korea
fMarine Natural Products Laboratory, Korea Ocean Research and Development Institute, Ansan 425-600, Republic of Korea
Received 12 February 2002; accepted 8 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 Elsevier Science. All
rights reserved. # 2002 Published by Elsevier Science Ltd.
Introduction
some of them, including TNP-470, are undergoing clin-
ical trials for cancer therapy.4À6
Angiogenesis is the process of new blood vessel forma-
tion by endothelial cells.1 The process is complex and
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
loops.2 Each step of the process is tightly controlled by
regulatory factors that stimulate or inhibit angiogenesis.
However, these elaborated control mechanisms are
often disordered in several pathologic diseases including
cancer.3 Extensive studies have shown that angiogenesis
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
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
chemoprevention by National Cancer Institute.7 Cur-
cumin showed
a potent anti-carcinogenic activity
against a broad range of tumor types including skin,
forestomach, duodenal, and colon carcinogenesis.8À11 It
has been postulated that the broad spectrum of anti-
carcinogenic activity of curcumin may be due in part to
angiogenesis inhibition.12,13 Curcumin and some of its
derivatives including demethoxycurcumin (DC) and
tetrahydrocurcumin (THC) were known as potent inhi-
bitors of angiogenesis.14 DC as well as THC, a murine
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 structure–activity relationship of
curcuminoids have revealed that phenol ring structures
of the compound may be essential for the anti-oxidant
activity,15 and diketone moiety of THC also involves
*Corresponding author. Tel.: +82-2-3408-3640; fax: +82-2-3408-
3334; e-mail: kwonhj@sejong.ac.kr
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00116-5

2440
J.S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2439–2444
antioxidative mechanism of the compound.16 In addi-
tion, phenolic hydroxyl or methoxyl groups were mod-
ified to enhance the potency of other biological activities
of curcumin including the induction of Phase 2 detox-
ification enzymes and the inhibition of HIV-1 inte-
grase.17,18 These studies suggest that diketone moiety of
curcumin may be essentially involved in its broad-range
biological activities.
considerable differences were observed for the NMR
signals corresponding to the protons and carbons at the
side chain. The 13C 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
data revealed that these protons were coupled to each
other with very large coupling constant (J=16.1 Hz).
1
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.
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.
The NMR data of HC contained another 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 signal of the C-
9 carbon was not observed due to the rapid tautomer-
ization between two forms of a pyrazole moiety, the
upfield shift of the C-10 methine carbon in the 13C
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 2D 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.
Results and Discussion
Chemistry
The general procedures for synthesis of derivatives are
shown in Schemes 1 and 2. Curcumin (1a, R1, R2=
OCH3) and its natural analogues including demethox-
ycurcumin (1b, R1=OCH3; R2=H or vice versa) and
bisdemethoxycurcumin (1c, R1=H; R2=H) were pre-
pared from the purification of commercially available
curcuminoids using preparative thin layer chromato-
graphy. Diketone moieties of each curcumin analogue
were replaced with hydrazine derivatives in the presence
of acetic acid. One-step coupling of 1 with hydrazi-
niumdihydrochloride and hydrazinobenzoic acid gave
compounds 2 to 3. Each compound was purified with
preparative TLC and HPLC, and was assayed for anti-
angiogenic activity.
Anti-angiogenic activity of HC
To evaluate anti-angiogenic activities of synthetic cur-
cumin derivatives, we first examined the effect of com-
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.
Structure determination of HC
HC (2a), a synthetic analogue of curcumin was obtained
as pale yellow gum which analyzed for C21H20N2O4 by
HRMS, thus 13 degrees of unsaturation. The structure
of this compound was determined by combined 2D
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
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-
1
were readily assigned on the basis of the results of H
COSY, gHSQC, and gHMBC experiments. However,
Scheme 1.

J.S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2439–2444
2441
Scheme 2.
Table 1. The structure of HC
1H
13C
HMBC
ROESY
1
2
3
4
5
6
7
8
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
99.9 d
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
9
10
OMe
6.64, 1H, s
3.86, 6H, s
7, 8
2
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.
56.2 d
3
aSignal was not observed.
Table 3. Inhibitory effect of HC on in vivo neovascularization of
CAM
Table 2. IC50 values of curcuminoids and synthetic derivatives for
BAECs
Drug
Inhibited egg/live egg Inhibition ratio (%)
R1
R2 BAECs, IC50 (M)a
Empty
Retinoic acid (1 mg/egg)
HC (10 mg/egg)
0/19
11/13
11/15
0
85
73
Curcuminoids, 1
OCH3 OCH3 1.5Æ0.3Â10À5
OCH3
H
H
H
2.2Æ0.5Â10À5
5.3Æ0.6Â10À5
Hydrazino curcuminoids, 2
OCH3 OCH3 5.2Æ0.4Â10À7
OCH3
H
H
H
1.8Æ0.3Â10À6
5.8Æ0.2Â10À6
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).
Hydrazinobenzoyl curcuminoids, 3 OCH3 OCH3 9.3Æ0.4Â10À7
OCH3
H
H
H
2.4Æ0.1Â10À6
8.7Æ0.2Â10À6
aIC50 values are the meanÆSE from three independent experiments.
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
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.
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

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J.S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2439–2444
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 mean ÆSE 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).
under microscope. The inhibition of the angiogenesis of
retinoic acid used as the positive control was 85%
(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.
by Sigma (St. Louis, MO). Chromatography purifica-
tion: Silica gel column chromatography (Merck, Darm-
stadt, Germany), thin-layer chromatography (TLC,
Merck), and high performance liquid chromatography
(HPLC, Shimadzu, Kyoto, Japan) were used to perform
general purification procedures. Spectral analysis: The
HRFAB-MS spectra were obtained using a Jeol JMS-
HX 110 mass spectrometer. The NMR spectra were
recorded in CDCl3 solutions on a Varian Unity 500
spectrometer. The proton and carbon NMR spectra
were measured at 500 and 125 MHz, respectively. All
chemical shifts were recorded according to an internal
Me4Si. All solvents used were of spectral grade or dis-
tilled from glass prior to use.
Conclusion
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.
Hydrazinocurcumin (2a). Purified 1a (10 mg, 0.027
mmol) was dissolved in methanol (2 mL), and hydrazi-
niumdihydrochloride (14 mg, 0.135 mmol) and triethy-
lamine (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 pre-
parative TLC (CHCl3/MeOH=6:1; Rf=0.4) and HPLC
(semipreparative C18 column; acetonitrile:H2O=50:50;
flow 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 C21H20N2O4:
364.1423, observed (M+H) 365.1501.
Experimental
General procedures
Curcuminoids (mixture of curcumin, demethox-
ycurcumin, and bisdemethoxycurcumin) were obtained

J.S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2439–2444
2443
Hydrazinodemethoxycurcumin (2b). Utilizing the same
protocol as described for the synthesis of 2a, 2b (5.9
mg, 61%) was obtained from 1b (10 mg, 0.029 mmol),
hydraziniumdihydrochloride (14 mg, 0.135 mmol), and
triethylamine (18.8 mL, 0.135 mmol). Preparative TLC
(CHCl3/MeOH=6:1; Rf=0.32) gave 2b as a pale yel-
low gum which analyzed by HRFAB-MS. Exact mass
calcd for C20H18N2O3: 334.1317, observed (M+H)
335.1393.
CO2. Cell growth assay was carried out using MTT
colorimetric assay. Exponentially growing cells were
seeded at a density of 5Â103 cells/well in a 96-well plate.
The cells were incubated in growth media for 24 h.
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 540nm
using a microplate reader (Bio-Tek Instruments, Inc.,
Winooski, VT, USA).
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), hydraziniumdihydrochloride (14 mg, 0.135
mmol), and triethylamine (18.8 mL, 0.135 mmol). Pre-
parative TLC (CHCl3/MeOH=6:1; Rf=0.26) gave 2c
as a pale yellow gum which analyzed by HRFAB-MS.
Exact mass calcd for C19H16N2O2: 304.1212, observed
(M+H) 305.1291.
Chemoinvasion assay
The invasiveness of the endothelial cells was performed
in vitro using a Transwell chamber system with poly-
carbonate filter inserts. The lower side of the filter was
coated with 10 mL of gelatin (1 mg/mL), whereas the
upper side was coated with 10 mL of Matrigel (3 mg/
mL). Exponentially growing cells (1Â105 cells) were
placed in the upper part of the filter and HC was
applied to the lower part for 30 min at room tempera-
ture before seeding. The chamber was then incubated at
37 ꢀC for 18 h. The cells 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 magnifi-
cation.
Hydrazinobenzoylcurcumin (3a). To a solution of 1a (10
mg, 0.027 mmol) in methanol (2 mL) was added 4-
hydrazinobenzoic acid (20 mg, 0.135 mmol), triethyla-
mine (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 (CHCl3/MeOH=4:1; Rf,=0.5) and
HPLC (semipreparative C18 column; acetonitrile/
H2O=50:50; flow rate=3 mL/min; retention time: 15
min) gave 3a as a dark orange powder (7.18 mg, 55%)
which analyzed by HRFAB-MS. Exact mass calcd for
C28H24N2O6: 484.1634, observed (M+H) 485.1715.
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
BAECs (1Â105 cells) were seeded on the surface of the
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).
Hydrazinobenzoyldemethoxycurcumin (3b). Utilizing the
same protocol as described for the synthesis of 3a, 3b
(5.52 mg, 42%) was obtained from 1b (10 mg, 0.029
mmol), 4-hydrazinobenzoic acid (20 mg, 0.135 mmol),
and triethylamine (18.8 mL, 0.135 mmol). Preparative
TLC (CHCl3/MeOH=4:1; Rf,=0.41) gave 3b as a dark
orange powder which analyzed by HRFAB-MS. Exact
mass calcd for C27H22N2O5: 454.1529, observed
(M+H) 455.1611.
Chorioallantoic membrane (CAM) assay
Hydrazinobenzoylbisdemethoxycurcumin (3c). Utilizing
the same protocol as described for the synthesis of 3a,
3c (6.78 mg, 50%) was obtained from 1c (10 mg, 0.032
mmol), 4-hydrazinobenzoic acid (20 mg, 0.135 mmol),
and triethylamine (18.8 mL, 0.135 mmol). Preparative
TLC (CHCl3/MeOH=4:1; Rf,=0.34) gave 3c as a dark
orange powder which analyzed by HRFAB-MS. Exact
mass calcd for C26H20N2O4: 424.1423, observed
(M+H) 425.1501.
Fertilized chick eggs were kept in a humidified incu-
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, 1
mg/egg RA was used as a positive control for anti-
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.
Cell culture and growth assay
Early passages (5–7 passages) of bovine aortic endothe-
lial cells (BAECs) were kindly provided by Dr. Jo at the
NIH of Korea. BAECs were grown in MEM supple-
mented with 10% fetal bovine serum. HT29 (colon car-
cinoma), NIH3T3 (mouse normal fibroblast), and
Chang (normal liver) cells were maintained in
RPMI1640 containing 10% fetal bovine serum. All cells
were grown at 37 ꢀC in a humidified atmosphere of 5%

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J.S. Shim et al. / Bioorg. Med. Chem. 10 (2002) 2439–2444
D. G.; Malone, W. F.; Nayfield, S. G.; Seifried, H. E.; Swall,
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This work was supported by a grant to HJK from
Korea Health 21 R&D Project, Ministry of Health &
Welfare (HMP-01-PJ1-PG1–01CH04–0005) and to JS
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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