Synthesis and anti-proliferative activity of novel azazerumbone conjugates with chalcones

Article abstract of DOI:10.1016/j.bmcl.2015.09.069

The conjugation of azazerumbone ((3E,7E,11E)-5,5,8,12-tetramethylazacyclododeca-3,7,11-trien-2-one (7)) and 2,4-dihydroxychalcones was carried out for the preparation of novel target compounds 9a-g with 1-ethylene-4-methylene-1,2,3-triazole linker and 10a

Full text of DOI:10.1016/j.bmcl.2015.09.069

Accepted Manuscript
Synthesis and Anti-proliferative Activity of Novel Azazerumbone Conjugates
with Chalcones
Vuong Van Truong, Tran Duy Nam, Truong Ngoc Hung, Nguyen Thi Nga,
Pham Minh Quan, Luu Van Chinh, Sang-Hun Jung
PII:
S0960-894X(15)30103-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.069
BMCL 23150
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 September 2015
25 September 2015
29 September 2015
Please cite this article as: Truong, V.V., Nam, T.D., Hung, T.N., Nga, N.T., Quan, P.M., Chinh, L.V., Jung, S-H.,
Synthesis and Anti-proliferative Activity of Novel Azazerumbone Conjugates with Chalcones, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.09.069
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Synthesis and Anti-proliferative Activity of Novel Azazerumbone Conjugates with
Chalcones
Vuong Van Truonga, Tran Duy Nama, Truong Ngoc Hungb, Nguyen Thi Ngab, Pham Minh
Quanb, Luu Van Chinhb* and Sang-Hun Jungc*
aVietnam – Russia Tropical Centre, 2-Nguyen Van Huyen-Cau Giay-Hanoi-Vietnam
bInstitute of Natural Products Chemistry, Vietnam academy of Science and Technology, 18-
Hoang Quoc Viet-Cau Giay-Hanoi-Vietnam. Email: chinhluuvan@gmail.com
cCollege of Pharmacy, Chungnam National University, Daejeon, Korea. jungshh@cnu.ac.kr
Abstract
The conjugation of azazerumbone ((3E,7E,11E)-5,5,8,12-tetramethylazacyclododeca-3,7,11-
trien-2-one (7)) and 2,4-dihydroxychalcones was carried out for the preparation of novel target
compounds 9a-g with 1-ethylene-4-methylene-1,2,3-triazole linker and 10a-f with propylene
linker between amide nitrogen of azazerumbone and 4-hydroxy group of chalcone. The anti-
proliferative activity of these compounds against the LU-1, Hep-G2, MCF-7 and SW480 human
cancer cell lines were significantly improved compared to those of azazerumbone or zerumbone.
The
methoxyphenyl)acryloyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-5,5,8,12-tetramethyl
azacyclododeca-3,7,11-trien-2-one (9b) and (3E,7E,11E)-1-(3-(4-((E)-3-(3,4,5-
anti-proliferative
activities
of
(3E,7E,11E)-1-((1-(2-(3-hydroxy-4-((E)-3-(3-
trimethoxyphenyl)acryloyl)phenoxy)propyl)-5,5,8,12-tetramethylazacyclododeca-3,7,11-trien-2-
one (10d) are nearly comparable to those of ellipticine.
Keywords: Zerumbone, Azazerumbone, Chalcone, Conjugate, Anti-proliferative Activity.

*Corresponding author: jungshh@cnu.ac.kr. Tel.; +82 42 821 5939; Fax +82 42 823 6566;
Present address: College of Pharmacy and Institute of Drug Research and development,
Chungnam National University, Daejeon 305-764, Korea

A number of natural products1-5 (Figure 1) has recently recognized to show anticancer activity
through inhibition of NF-κB activity. Specially, compounds like curcumin (1) 1, zerumbone (2)2
and thiacremonone (3)3,4 from garlic expressed anti-cancer activity through the inhibition of NF-
κB. The compound SK-2009 (4)5 (Figure 1) was most potent in suppressing NF-κB activation in
KBM-5 leukemic cells. Recently, chalcones such as 5 (figure 1) was reported to have cytotoxic
activity against various human cancer cell lines as well as NF-κB inhibitory activity.6
O
O
OH
O
OH
HO
HO
OH
OCH3
O
S
O
O
OCH3
(1)
curcumin
(3)
thiacremonone
(4)
(2)
SK-2009
Zerumbone
OH
O
O
N
N
N
O
H
HO
O
N
OH
N
O
H3CO
OCH3
O
6
Chalcone pyrrolobenzodiazepine conjugate
5
2,4'-dihydroxy-6-alkoxychalcone
Figure 1: The structures of some compounds exhibiting NF-κB inhibitor activity
Zerumbone7 (2, (2E,6E,10E)-2,6,9,9-tetramethylcycloundeca-2,6,10-trienone, Figure 1) has been
focused as a promising cytotoxic sesquiterpene ketone abundantly occurring in the rhizome of Z.
zerumbet. This unique sesquiterpene is a eleven membered ketone containing pentadienone
moiety that is reported to be the center of bioactivity and considered as an active Michael
acceptor with preferential activity towards thiol group in certain protein8. In 2006, Giang and
colleagues9 studied the inhibition of NF-κB activity by zerumbone and also pointed out that the

α,β-unsaturated ketone group is the key to the activity of this compound. Zerumbone was also
reported to inhibit several human cancer cell lines including Hep-G2 (IC50 =3.15µg/ml)10, HeLa
(IC50: 2.5 µg/ml)11, 12 and P-338D113. In these regards, the chemical modifications of zerumbone
have attracted significant attention of chemists to find out the suitable structures for application
in preventing and treatment of cancer. However, chemical interventions have concentrated on
α,β-unsaturated ketone moiety led to a significant reduction of anticancer activity 14
Therefore, we focused to the exploration of novel zerumbone derivatives as an anticancer agent
by the conjugation with other bioactive component, while maintaining the bioactive center α,β-
unsaturated ketone. This molecular conjugation has been known as the rational design of new
chemical entities by the fusion of both active compounds and/or pharmacophoric units
recognized and derived from known bioactive molecules15,16 and successfully employed for the
enhancement of anti-proliferative activity against various cancer cell lines as shown in 6 (Figure
1) 17,18. However zerumbone itself does not have proper functional group for conjugation. Thus
azazerumbone 7 (Figure 2) was selected for the conjugation as zerumbone motif in this study.
Recently, structural transformation of zerumbone to azazerumbone 7 (Figure 2) was performed
and these compounds showed antimutagenic activity19. Thus zerumbone and azazerumbone are
considered to have same range of bioactivities. Azazerumbone 7 has a lactam unit, which can be
readily used for conjugation to other functionality. The 2,4-dihydroxychalcones (8, Figure 2)
were selected as the other part of conjugate designed since chalcones also showed NF-κB
6,20
inhibitory
and anti-proliferative activity 6, 21,22. With these characteristics of azazerumbone
and chalcones, conjugated structures 9a-g with 1-ethylene-4-methylene-1,2,3-triazole linker and
10a-f with propylene linker between amide nitrogen of azazerumbone and 4-hydroxy group of

chalcone (Figure 2) were designed, prepared and evaluated their anti-proliferative activity
against human cancer cell lines.
R
O
R1
R2
R3
HO
R4
N
O
H
8a;
,
2 4-dihydroychalcones (R=OH)
; 4-hydroychalcones (R=H)
7
Azazerumbone
8b
OH
O
R1
R2
R3
N
N
N
O
N
O
3
1
2
4
R4
a
b
c
d
e
f
: R = OCH3, R = R = R = H
2
1
3
4
: R = OCH3, R = R = R = H
9
Conjugate analog
1
2
3
4
: R = OCH3, R = R = R = H
O
R1
2
3
4
1
: R = R = R = OCH3, R = H
R2
R3
3
1
2
4
: R = CH3, R = R = R = H
3
2
: R = OCH3, R = 3-morpholinopropoxy,
R1 = R4 = H
N
O
3
1
2
4
R4
g
: R = CH(CH3)2, R = R = R = H
O
10
Conjugate analog
Figure 2. Azazerumbones and new designed conjugates.
The syntheses of designed conjugates 9a-g and 10a-f of azazerumbone 7 with chalcones were
outlined in Scheme 1 and 2, respectively. For the preparation 9a-g, azidochalcones 15a-g were
synthesized from 2ꞌ,4ꞌ-dihydroxyacetophenone 11 in three steps (Scheme 1). Firstly, 2ꞌ,4ꞌ-
dihydroxyacetophenone 11 was selectively O-alkylated by treatment with 1,2-dibromoethane in
the presence of K2CO3 in acetonitrile at 50 oC for overnight to give 12 in 63 % yield. Compound
12 was then reacted with sodium azide in DMSO to afford 4'-(2-azidoethoxy)-2'-
hydroxyacetophenone 13 in 90 % yield. Intermediate chalcones 15a-g were obtained in 81-93 %
yields by Claisen-Schmidt condensation in alkaline media of 13 with corresponding aldehydes

14a-g in methanol. Propargylazazerumbone 16 was obtained from the sequential treatment of
azazerumbone 7 with sodium hydride and then propargyl bromide. Finally, Click reaction of 16
with corresponding chalcones 15a-g using CuI as a catalyst in DMSO to give target compounds
9a-g in 53-59 % yields.
Scheme 1. Preparation of conjugates 9a-g
o
Reagents and conditions: (i) 1,2-dibromoethane, MeCN, K2CO3, 40 C, 12 h, 63 %; (ii) NaN3,
DMSO, 5 h, rt, 90 %; (iii) KOH, MeOH, rt, 81-93%; (iv) CuI, DMSO, 24 h 53-59%; (v) NaH,
propagyl bromide, THF, 0oC- rt, 87 %.
For the preparation of compounds 10a-f (Scheme 2), chalcones 18a-f was obtained in 67-93 %
yields from the Claisen-Schmidt reactions of 4ꞌ-hydroxyacetophenone 17 with aldehydes 14a-f 23.
The structure of synthesized chalcones 18a-f was examined by NMR, MS spectra in good
agreement with data given by Hieu24, Iwata25, Garg26 and Ducki27. O-alkylations of 18a-f were

performed with 1,3-dibromopropane in the presence of potassium carbonate in acetonitrile at 50
oC to give 4ꞌ-bromopropanoxychalcones 19a-f in 51-62 % yields. Finally N-alkylation reaction
of azazerumbone 7 with appropriate 4ꞌ-bromopropanoxychalcones 19a-f in DMF at room
temperature using NaH gave target compounds 10a-f in 58-73 % yields.
The structures of the intermediates and conjugates 9 and 10 are well agreed with IR, MS 1D- and
2D-NMR data 28(Supplementary data).
Scheme 2. Preparation of conjugates 10a-f
Reagents and conditions: (i) aldehydes 14a-f, C2H5OH, NaOH 10% , rt, 12 h-48 h, 67-93%; (ii)
1,3-dibromopropane, K2CO3, 50 oC, 12 h, 51-62%; (iii) NaH, THF, 0oC- r.t. 58-73%
The evaluation of anti-proliferative activity of the synthesized compounds was performed
according to the described protocol29,30. The IC50 value of the assay was evaluated using five
human cancer cell lines: LU, Hep-G2, MCF7, P338 and SW480. The results are listed in Table 1.

Azazerumbone derivatives 9a-g and 10a-f exhibited anti-proliferative activity against to five
cancer cell lines including LU, Hep-G2, MCF7, 338 and SW480 with IC50 values ranging
from0.61 to 3.24 µg/mL.
Table 1: The anti-proliferative activity of azazerumbone derivatives 9a-g and 10a-g
IC50 (µg/mL) a
No
Compounds
LUb
1.67
0.61
2.40
1.28
0.91
1.56
0.77
2.56
1.06
2.82
1.01
1.18
1.22
17.82
6.05
0.63
Hep-G2b MCF7b
P338b
1.21
0.89
1.10
0.89
0.92
0.96
1.39
1.30
1.10
0.83
0.77
1.29
1.08
6.25
2.63
0.44
SW480b
3.24
0.95
0.90
2.61
1.16
0.91
0.85
2.92
1.34
1.33
0.71
1.57
0.80
6.83
3.15
0.38
1
2.06
1.01
1.71
1.44
0.87
1.59
0.84
1.73
1.38
1.44
0.99
1.53
1.53
34.58
13.34
0.36
2.85
1.12
1.17
2.78
1.21
1.13
0.93
3.17
1.37
1.56
0.58
1.21
1.07
15.64
9.67
0.44
9a
2
9b
3
9c
4
9d
5
9e
6
9f
7
9g
8
10a
9
10b
10
11
12
13
14
15
16
10c
10d
10e
10f
7 (azazerumbone)
Zerumbone
Ellipticine
aIC50 values shown for these compounds are the average of three determinations.

bCell lines: LU (lung adenocarcinoma, ATCC-HTB-57), Hep-G2 (liver hepatocellular carcinoma,
ATCC-HB-8065), MCF7 (breast cancer, ATCC-HTB-22), P338 (leukemia, ATCC-CCl-46) and
SW 480 (colon adenocarcinoma, ATCC-CCL-228) cell lines.
The level of activities of 9a-g and 10a-f indicated that the azazerumbone conjugates showed
approximately two to twenty fold stronger activity against cancer cell lines than those of
azazerumbone 7 or zerumbone.
Compounds 9a-g include the 1-ethylene-4-methylene-1,2,3-triazole linker (Figure 2), which
provides more rigidity and space between azazerumbone and chalcone components compared to
the flexible propylene linker in 10a-f (Figure2) and therefore the entire structure of 9a-f is likely
to be stretched conformation. Although 9a-f exhibited the slightly better activity compared to
10a-f, these significant differences between these linkers did not make any clear discrepancy in
anti-proliferative activity in cancer cell lines tested.
For each series, the anti-proliferative activity of the target compounds were varied along with
the nature and position of substituents attached to the phenyl moiety of the chalcone components.
In series of 9 composed by conjugation of chalcones and azazerumbone with 1-ethylene-4-
methylene-1,2,3-triazole linker, the observed anti-proliferative activity indicated that all
derivatives 9 showed similar growth inhibition of five human cancer cell lines tested with IC50
values ranging from 0.61 to 3.24 µg/mL as shown in Table 1. Among those, 9b containing
methoxy group at position 3 of phenyl in chalcone component showed the best activity against
LU cell lines with IC50 value of 0.61 µg/mL, followed by the anti-proliferative activity of
compound 9g and 9e with a bulky isopropyl and a methyl group at position 4 with IC50 values
of 0.77 and 0.91 µg/mL, respectively. A significant contribution of these groups for anti-

proliferative activity of compounds 9b, 9e and 9g against Hep-G2, MCF7, P388 and SW480 cell
lines was also clearly observed.
In series of 10 formed by the conjugation between the chalcones and azazerumbone via
propylene linker, the observed anti-proliferative activity indicated that all analogs 10 suppressed
the growth of five human cancer cell lines tested with IC50 values ranging from 0.58 to 3.17
µg/mL as shown in Table 1. In particular, compound 10d exhibited good activity against the
tested cancer cell lines MCF7, SW480, P338, Hep-G2 and LU with IC50 values of 0.58, 0.71,
0.77, 0.99, and 1.01µg/mL, respectively. The simultaneous presence of three methoxy groups at
positions 3, 4 and 5 in the phenyl moiety of chalcone component as shown in 10d seems to be
beneficial for anti-proliferative activity. This judge has been further clarified when anti-
proliferative activity of compounds 10a, 10b or 10c containing a methoxy group at position 2, 3,
or 4 has been reduced markedly compared to activity of 10d.
The anti-proliferative activity of the conjugates 9a-g and 10a-f of azazerumbone and 2,4-
dihydroxychalcones against the LU-1, Hep-G2, MCF-7 and SW480 human cancer cell lines were
significantly improved compared to those of azazerumbone or zerumbone. In series of 9
containing 1-ethylene-4-methylene-1,2,3-triazole linker, a methoxy group at position 3 of phenyl
moiety in chalcone component enhances the activity. In series of 10 with propylene linker, the
simultaneous presence of three methoxy groups at positions 3, 4 and 5 in the phenyl moiety of
chalcone component increases the activity. Finally, the anti-proliferative activities of conjugate
derivatives 9b, 9g and 10d are nearly comparable to those of ellipticine as shown in Table 1.
Thus, these good anti-proliferative activities of these conjugates of azazerumbone with chalcones
obviously implicate good orientation to exploit the pharmacological potential of zerumbone in
finding new applications for the treatment of cancer.

Acknowledgment
This work was supported by VAST (VAST04.02/15-16) of Vietnam and Priority Research Centers
Program through the National Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (2009-0093815).
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Synthesis and Anti-proliferative Activity of Novel Azazerumbone Conjugates with
Chalcones
Vuong Van Truonga, Tran Duy Nama, Truong Ngoc Hungb, Nguyen Thi Ngab, Pham Minh
Quanb, Luu Van Chinhb* and Sang-Hun Jungc*
O
OH
O
OCH3
OCH3
N
OCH3
N
N
O
N
N
O
OCH3
O
O
10d; IC50: 1.01(LU), 0.99(Hep-G2), 0.58(MCF7),
0.77(P388), 0.71(SW480) µg/mL
9b; IC50: 0.61(LU), 1.01(Hep-G2), 1.12(MCF7),
0.89(P388), 0.95(SW480) µg/mL