Glycosylated nordihydroguaiaretic acids as anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.10.137

Three perglycosylated nordihydroguaiaretic acids (NDGA) were synthesized through the Huiseng 1,3-dipolar cycloaddition reaction. These sugar-NDGA conjugates containing triazole-linkages possessed good solubility in water. NDGA-(triazol-galactose)₄

Full text of DOI:10.1016/j.bmcl.2010.10.137

Bioorganic & Medicinal Chemistry Letters 21 (2011) 380–382
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Glycosylated nordihydroguaiaretic acids as anti-cancer agents
Jih Ru Hwu a,b, , Chuan-I Hsu a,c, Ming-Hua Hsu a, Yu-Chuan Liang d, Ru Chih C. Huang c, , Yuan C. Lee c,
⇑
⇑
⇑
a Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC
b Department of Chemistry, National Central University, Jhongli 32001, Taiwan, ROC
c Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA
d Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Three perglycosylated nordihydroguaiaretic acids (NDGA) were synthesized through the Huiseng 1,3-
dipolar cycloaddition reaction. These sugar–NDGA conjugates containing triazole-linkages possessed
good solubility in water. NDGA-(triazol-galactose)4 (12b) and NDGA-(triazol-glucose)4 (12c) were found
to act as inhibitors against human hepatocellular carcinoma Hep3B cells in culture.
Ó 2010 Published by Elsevier Ltd.
Received 19 May 2010
Revised 23 October 2010
Accepted 28 October 2010
Available online 31 October 2010
Keywords:
Nordihydroguaiaretic acid
NDGA
Hepatocellular carcinoma
Triazol-glucose
Triazol-galactose
Nordihydroguaiaretic acid (NDGA, 1), a naturally occurring lig-
nan, is isolated from a desert medicinal plant in the Creosote bush
(Larrea tridentate). It is known as the inhibitor of lipoxygenase1 and
an anti-oxidation agent.2 Huang and co-workers3 reported that
30-O-methylated-NDGA (i.e., Mal.4, 2) can inhibit HIV transcription
by interfering with binding of transcription factor Sp1 to the pro-
moter of the HIV proviral template. Meanwhile, in vitro, NDGA
shows no inhibitory effect on HIV transcription and no effect on
Sp1 binding. Thus, a series of NDGA derivatives with modification
at the four phenolic hydroxyl groups therein were developed for
these purposes.4–7
Bioactivities of the O-methylated derivatives of NDGA, espe-
cially tetra-O-methyl-NDGA (M4N, 3), have been examined exten-
sively.4 M4N can inhibit human immunodeficiency virus (HIV) Tat
transactivation, replication of herpes simplex virus type 1 (HSV-
1),8,9 and human papilloma virus (HPV) E6/E7 promoter activity.10
It can also arrest growth and induce apoptosis of cancer cells,
including mouse C3 tumor cell and five different human xenograph
tumors.11 Moreover, Sp1-dependent transcription of many pro-
moters is inhibited by M4N, such as the HIV long transcribed
region and genes related to code for the cell cycle protein cyclin-
dependent kinase (Cdc2) and apoptosis protein survivin.3,12,13 In
addition, M4N is able to reverse the MDR phenotype of the tumor
cells in a xenograft model system; the combination therapy with
M4N and paclitaxel is effective at inhibiting growth of tumors in
nude mice.14 Although the methylated NDGA analogs are poten-
tially good candidates for antiviral and anti-tumor drugs, their
water solubility is poor.
HO
RO
OR
OR
OH
OH
OR
OR
Conjugation of carbohydrates with drugs becomes increasingly
important in bio-organic chemistry and chemical biology. In addi-
tion to facilitation of the active transport of the modified drugs
across biological membranes, sugar-conjugation may allow to
modify the physico-chemical properties of the conjugated drugs,
including polarity, solubility, and stability at physiological pH.15
In some examples, sugar-conjugated drugs are used in enzyme-
specific activation,16 selective transport of O6-methylguanine-
DNA methyltransferase (MGMT) inhibitors,17 specific targeted
delivery,18 and providing amphipathic characteristics.19 There is
some precedence of glycosylated drugs, of which physico-chemical
3. R = Me (M4N)
1. R = H (NDGA)
2. R = Me (Mal.4)
N
N
N
4. R =
CH2
sugar
O
n
⇑
Corresponding authors.
E-mail address: jrhwu@mx.nthu.edu.tw (J.R. Hwu).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.10.137

J. R. Hwu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 380–382
381
R1 OAc
properties and therapeutic effects are improved. For examples,
R1
a. OAc
:
R2
H
:
7
O
R2
AcO
carbohydrate–geldanamycin conjugates for enzyme-specific acti-
vation increase tumor selectivity.16,20 Glucose-conjugated MGMT
and O6-methylguanine-DNA methyltransferase are developed as
antagonists for selective transport of MGMT inhibitors in order to
target glucose transporters that are often over expressed in tumor
cells.17,21 Accordingly, we planned to synthesize new water-
soluble NDGA analogs containing sugar units.
We applied the Huisgen Cu-catalyzed 1,3-dipolar cycloaddition
between alkynyl NDGA and (azido)glycosides to form triazole link-
ages. This method was reported by Tornøe,22 Rostovtsev,23 and
their co-workers. Our targets included five new NDGA derivatives:
three of them contained different lengths of triazole-linkage with
four galactose or glucose moieties (i.e., 4); the remaining two were
not attached by any sugar moiety. We found that NDGA derivatives
with sugar units can be easily dissolved in an aqueous solution
or other polar solvents. More important is that NDGA-(triazol-
galactose)4 and NDGA-(triazol-glucose)4 exhibited inhibited cell
proliferation toward Hep3B cell line.
OAc
OAc
b.
H
OAc
7
+
HO
OH
8
a.
b.
n :
2
6
HO
Br
n
OH
OH
1
8
.
+
BF3 Et2O
CH2Cl2
Br
R1
:
R2
:
n :
2
R1
R2
AcO
9
5
OAc
O
a. OAc
H
H
Br
n
O
K2CO3
b.
c.
OAc
H
6
6
OAc
18-crown-6
acetone, reflux
OAc
9
NaOMe
MeOH
R1
a. OH
b. OH
:
R2
H
:
n :
2
10
R1 OH
O
R2
HO
H
6
Br
n
O
c.
H
OH
6
OH
10
NaN3
DMF, 60 ¢XC
O
O
O
O
R1 OH
O
R2
HO
N3
n
O
To obtain perglycosylated NDGA targets 12a–c, we first treated
commercially available NDGA (1) with 6.0 equiv of propargyl bro-
mide (5), a catalytic amount of 18-crown-6, and 5.5 equiv of potas-
sium carbonate in acetone (Scheme 1).24 After the solution was
heated at reflux, peralkynyl NDGA 6 was generated in 71% yield.
OH
6
11
R1
:
R2
:
n :
11 and 12
.
CuSO4 5H2O
sodium ascorbate
a.
b.
c.
OH
H
H
OH
2
6
6
Second, we prepared
mercially available penta-O-acetyl-
x
-(azidoalkyl)glycosides 11a–c from com-
-galactose and -glucose (i.e.,
OH
H
D
EtOH (95%)
7a and 7b) by utilizing several published procedures.25,26 Reactions
of 7a,b in dichloromethane with a bromoalkanol (i.e., 8a or 8b) in
the presence of boron trifluoride etherate25 produced the corre-
sponding (bromoalkyl)glycosides 9a–c, respectively, in 31–45%
yields. These products were assigned as the b-anomers because
of the large coupling constants (J1,2 = 7.2–7.6 Hz) associated with
their anomeric protons. Subsequent deacetylation of 9a–c gave
the unprotected glycosides 10a–c. Treatment of these bromides
N
O
N
N
O
N
N
O
O
O
N
N
n
n
with sodium azide in DMF26 produced
x
-(azidoalkyl)glycosides
N
OH
O
O
R1
N
N
N
N
11a–c in 71–90% yields.
OH
OH
n
O
n
O
R2
Coupling of peralkynyl NDGA 6 with an excess of (azido)glyco-
sides 11a–c (6.7–8.4 equiv) was accomplished by the use of CuSO4/
sodium ascorbate23 in 95% EtOH as described previously27 (Scheme
1). The triazole-linked conjugated products 12a (57%), 12b (56%),
and 12c (81%) were obtained with purity >99.5% after purification
with Sephadex LH-20 and Sephadex G-15 chromatography.
Furthermore, we applied the same conditions to couple 6 with
azides 13 (see Scheme 2). The tetratriazole alcohols 14a and 14b
were obtained and used as reference compounds in bioassays.
The NDGA derivatives M4N (3) and 14a,b were found insoluble
in water. In sharp contrast, we found that the glyco-conjugated
NDGA derivatives 12a–c possessed solubility of 503–624 mg/mL
in water. Thus the tetraglycoside moieties attached to NDGA
greatly increased the water solubility of the resultant conjugates.
Moreover, these conjugates were also found soluble in regular
organic solvents, including THF, ether, acetone, ethyl acetate,
DMF, chloroform, etc. They remained intact (>99.5%) on bench at
room temperature for 6 months.
HO
O
HO
R1
O
HO
R1
OH
R2
OH
O
OH
HO
R1
R2 OH
OH
R2
12
Scheme 1. Syntheses of conjugates of sugar–nordihydroguaiaretic acid.
human hepatocellular carcinoma Hep3B cells in culture (Fig. 1
and Table 1). We therefore believe that the glycosidic moieties
are essential to the biological activity associated with the conju-
gated compounds 12b and 12c (cf. 14b). Presence of the ASGP
receptors may allow the galactosyl derivative 12b showing some
preference for hepatocytes cell lines.28 Similarly, the efficacy of
the glucosyl derivative 12c may be due to the glucose trans-
porter.29 These observations, however, require further experiments
to elucidate their mechanisms in detail.
We have previously reported that tetra-O-methyl NDGA (3)
inhibits the growth of human cancer cell lines hepatocellular
carcinoma Hep3B, prostate carcinoma LNCaP, colorectal carcinoma
HT-29, breast carcinoma MCF-7, and ovarian carcinoma
NCI/ADR-RES in culture effectively with similar potency with
In conclusion, three glycosylated NDGA derivatives were
designed and prepared, which possessed good water solubility
ranging from 503 to 624 mg/mL. Two of these glucose- and
galactose-conjugated NDGA derivatives exhibited significant
inhibition activity toward human hepatocellular carcinoma Hep3B
cells.
IC50 6 10 l
M.11,14 We further examined the efficiency of com-
pounds 12a–c and 14a,b in these cell lines. Galactosylated NDGAs
12a,b, glucosylated NDGA 12c, and nor-glycosyl NDGA 14a,b were
found not effective in the inhibition of human tumor cells HT-29,
MCF-7, and NCI/ADR-RES.
In contrast with these results (especially from 14b), we found
that compounds 12b and 12c were able to inhibit the growth of
Results from controlled experiments indicate that these glyco-
syl moieties were essential to their anti-cancer activity associated
with these conjugated compounds. Detailed studies on the uptake
of these two sugar-attached NDGA derivatives 12b and 12c into
the liver cells in targeting human cancer are currently in progress
and will be reported separately.

382
J. R. Hwu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 380–382
Table 1
HO
Br
n
Inhibition of proliferation of human hepatocellular carcinoma cell line by NDGA-
(triazol-galactose)4 12b and NDGA-(triazol-glucose)4 12c
8
NaN3
CH3CN
reflux
a
Compounds
IC50
(
l
M), Hep3B
O
O
NDGA-(triazol-galactose)4 12b
NDGA-(triazol-glucose)4 12c
88 3.01
83 2.17
O
O
HO
N
n
3
a
Data are shown as mean values S.D. of three independent determinations.
6
13
.
CuSO4 5H2O
8, 13, 14
n :
2
6
Acknowledgments
a.
b.
sodium ascorbate
EtOH (95%)
This work was partially supported by grants and contributions
from the National Science Council of Republic of China to J.R.H.
and from Erimos Pharmaceutical, LLC to R.C.C.H.
N
O
N
Supplementary data
O
N
N
O
O
N
N
N
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.137.
n
n
N
N
OH
OH
N
N N
n
n
OH
HO
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