Antitumor agents 283. Further elaboration of Desmosdumotin C analogs as potent antitumor agents: Activation of spindle assembly checkpoint as possible mode of action

Article abstract of DOI:10.1016/j.bmc.2011.01.001

In our ongoing study of the desmosdumotin C (1) series, twelve new analogues, 21-32, mainly with structural modifications in ring-A, were prepared and evaluated for in vitro antiproliferative activity against several human tumor cell lines. Among them, th

Full text of DOI:10.1016/j.bmc.2011.01.001

Bioorganic & Medicinal Chemistry 19 (2011) 1816–1822
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitumor agents 283. Further elaboration of Desmosdumotin C analogs
as potent antitumor agents: Activation of spindle assembly checkpoint
as possible mode of action
Kyoko Nakagawa-Goto a, , Pei-Chi Wu a, Kenneth F. Bastow b, Shuenn-Chen Yang c, Sung-Liang Yu d,
⇑
Hsuan-Yu Chen e, Jau-Chen Lin f, Masuo Goto g, Susan L. Morris-Natschke a, Pan-Chyr Yang c,d,h
,
Kuo-Hsiung Lee a,i,
⇑
a Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
b Division of Medicinal Chemistry & Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
c Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan
d Department of Clinical and Laboratory Sciences and Medical Biotechnology, National Taiwan University, College of Medicine, Taipei, Taiwan
e Institute of Statistical Science, Academia Sinica, Taipei, Taiwan
f Department of Respiratory Therapy, Fu-Jen Catholic University, Taipei, Taiwan
g Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7090, USA
h Department of Internal Medicine, National Taiwan University, College of Medicine, Taipei, Taiwan
i Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
In our ongoing study of the desmosdumotin C (1) series, twelve new analogues, 21–32, mainly with struc-
tural modifications in ring-A, were prepared and evaluated for in vitro antiproliferative activity against
several human tumor cell lines. Among them, the 40-iodo-3,3,5-tripropyl-4-methoxy analogue (31)
showed significant antiproliferative activity against multiple human tumor cell lines with ED50 values
Received 15 November 2010
Revised 23 December 2010
Accepted 1 January 2011
Available online 15 January 2011
of 1.1–2.8 lM. Elongation of the C-3 and C-5 carbon chains reduced activity relative to propyl substituted
analogues; however, activity was still better than that of natural compound 1. Among analogues with
various ether groups on C-4, compounds with methyl (2) and propyl (26) ethers inhibited cell growth
of multiple tumor cells lines, while 28 with an isobutyl ether showed selective antiproliferative activity
against lung cancer A549 cells (ED50 1.7 lM). The gene expression profiles showed that 3 may modulate
the spindle assembly checkpoint (SAC) and chromosome separation, and thus, arrest cells at the
Keywords:
Desmosdumotin C
Antiproliferative activity
Human tumor cell lines
Microarray
G2/M-phase.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
was modified with triethyl and tripropyl groups at C-3 and -5 posi-
tions and various substituted aromatic B-rings were also incorpo-
Desmosdumotin C (1), isolated from the roots of Desmos
dumosus,1 has a distinctive chalcone skeleton with an unusual
non-aromatic A-ring possessing a gem-dimethyl group on C-3
and methyl group on C-5 (Fig. 1). This compound showed signifi-
cant and selective antiproliferative activity against 1A9 (ovarian
cancer) and A549 (human lung carcinoma) cell lines with ED50 val-
rated.3 From the preliminary data, analogues with tripropyl
substitution at the C-3 and C-5 positions (i.e., 2) showed better
activity than analogues with triethyl and trimethyl groups.
Furthermore, addition of a bromophenyl B-ring (bromide at C-40)
enhanced cell growth inhibition against all tested tumor cell lines.
ues of 3.5 lg/mL (11.2 l
M).1 In addition, it was more active against
X
KB-VIN [vincristine-resistant KB, overexpressing P-glycoprotein
(P-gp)] cells than against the parent KB (epidermoid nasopharyn-
geal carcinoma) cell line. We previously established the first total
synthesis of 1.2 Based on our synthetic methodology, the A-ring
4'
Pr
Pr
Me Me
B
MeO
Pr
O
MeO
Me
O
3
A
1'
4
1
5
O
OH
O
OH
⇑
2:
3:
X = H
X = Br
Corresponding authors. Tel.: +1 919 843 6325; fax: +1 919 966 3893 (K.N.G.);
1
: Desmosdumotin C
tel.: +919 962 0066; fax: +919 966 3893 (K.H.L.).
E-mail addresses: goto@email.unc.edu (K. Nakagawa-Goto), khlee@unc.edu
(K.-H. Lee).
Figure 1. Desmosdumotin C and its analogs.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.001

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
1817
As a result, 3,5,5-tripropyl-40-bromo analogue 3 (Fig. 1) possessed
3. Results and discussion
the most potent activity against A549, HCT-8 (colon adenocarci-
noma), 1A9, PC-3 (prostate cancer), KB and KB-VIN with ED50 val-
Newly synthesized analogues 21–32 were evaluated for in vitro
ues of 0.87–2.25 lg/mL (1.8–2.6 lM).
antiproliferative activity against the cell line panel described
Subsequent modifications of 1 focused on the A-ring and in-
cluded further elongation of the C-3 and C-5 carbon side chains
as well as introduction of various alkoxy groups at C-4. Naturally
occurring C-prenylated flavonoids, including chalcones,4 have
shown interesting bioactivities, such as antimalarial, antitumor,
anti-HIV and anti-oxidant effects.4,5 Thus, the insertion of prenyl
groups at the C-3 and -5 positions was another design target. Be-
cause we did not include C-40 iodinated compounds in our prior
study, 4’-iodo analogues were also prepared. Herein, we describe
the synthesis of 1-derivatives and evaluation of newly synthesized
analogues against eight human tumor cell lines, A549, HCT-8,
MCF-7 (breast cancer), 1A9, PC-3, HepG2 (liver cancer), KB and
KB-VIN.
above. The average ED50 values (lM) are listed in Table 1, together
with those of 1–3 as references. All analogs, except for 23, inhibited
tumor cell growth, with greater or similar potency to that of the
parent compound (1).
Consistent with our prior findings,3 2 (3,3,5-tripropyl) was
again more potent than 1 (3,3,5-trimethyl). Further elongation of
the C-3 and C-5 alkyl chains to Bu, iBu, iPen, and prenyl decreased
the potency. The rank order of potency based on C-3 and -5
alkyl substituents was Pr (2) > iBu (22) ꢀ Bu (21) > Et3b > Me
(1) ꢀ Prenyl (24) > iPen (23).
Our next interest was to determine how the alkoxy group (OR0)
at C-4 affected activity. From comparison of data in Table 1, the
rank order of activity was OPr (26) > OMe (2) > OEt (25) > OBu
(27) > OiBu (28) > OiPen (29). Analogue 26 showed good in vitro
antiproliferative activity against all tested cell lines with ED50 val-
2. Chemistry
ues of 1.9–8.6
line selectivity, isobutoxy analogue 28 displayed selective activity
against A549 with an ED50 value of 1.7 M. Isopentoxy analogue
lM. While 2 and 25–27 did not show significant cell
Trialkylated intermediates 5–8 with propyl, butyl, isobutyl, and
isopentyl (R = Pr, Bu, iBu, and iPen, respectively) at the C-3 and C-5
positions were obtained by reacting 2,4,6-trihydroxyacetophenone
(4) with the appropriate alkyl halide and sodium methoxide at
reflux temperature as shown in Scheme 1. However, attempted
tri-prenylation of 4 under the same conditions gave a complex
mixture. Triprenylated 9 (R = prenyl) was finally obtained by treat-
ment of 4 with prenyl bromide and KOH in water.6 Target com-
pounds 21–24 were produced by methylation of the 4-OH of 5–9
using TMSCHN2 followed by Claisen–Schmidt condensation of
the resulting 10–14 with benzaldehyde.
Regioselective mono-alkylation of the 4-OH group of tripropyl 5
(R = Pr) was accomplished by using various alkyl halides (EtI, PrI,
BuI, iBuI, and iPenI) in the presence of potassium carbonate, rather
than TMSCHN2, to obtain 15–19 (R = Pr; R0 = Et, Pr, Bu, iBu, and
iPen). Under the same reaction conditions, use of prenyl bromide
resulted in 20, in which the prenyl is attached to the carbon at
the C-5 position rather than on the hydroxy group. Compounds
15–20 were condensed with benzaldehyde to obtain the target
compounds 25–30. Claisen–Schmidt condensation of 10 (R = Pr;
R0 = Me) and 12 (R = Pr; R0 = Pr) with 4-iodobenzaldehyde produced
31, and 32, respectively. Compounds 1–3 were previously synthe-
sized.2,3 All final compounds exist as a mixture of two tautomeric
isomers, as discussed in our prior papers.3
l
29 exhibited the lowest activity against all tested cell lines.
Analogue 30, which has a tetraalkyl, rather than trialkyl, substi-
tution pattern at C-3 (gem-propyl) and C-5 (propyl and prenyl),
showed significant tumor cell growth inhibition, while tetramethyl
analogue 333b (Fig. 2) did not show cytotoxic activity. These results
suggested that the alkyl or alkoxy groups on ring-A can strongly af-
fect the activity.
As previously reported,3 analogue 3 with tripropyl substitution
on ring-A and bromide at C-40 on ring-B showed 5- to 7-fold great-
er activity than 1. Replacement of bromide with iodide led to
slightly higher potency. Thus, the 40-iodo-3,3,5-tripropyl substi-
tuted analogue 31 exhibited significant antiproliferative activity
against all tested cell lines (ED50 1.1–2.8
more potent than 1. Interestingly, 31 was active even against PC-3
and HepG2 (ED50 2.8 and 2.4 M, respectively) cell lines, which
lM), and was 6- to 13-fold
l
were generally less sensitive than the other cell lines to 1-ana-
logues. We postulated that analogue 32, which has tripropyl sub-
stitution at C-3 and C-5 as well as propoxy at C-4 and iodide at
C-40, would exhibit greater inhibitory potency against tumor cell
growth. Unexpectedly, 32 was less active than 31 (methoxy at
C-4) or 26 (hydrogen at C-40).
The cell growth of KB-VIN cancer cells, which express P-gp and
selected for resistance to vincristine, was inhibited by analogues at
R
R
R
R
R
R
HO
OH
Ac
HO
R
OH
Ac
R'O
R
OH
Ac
R'O
R
O
Ar
a
c
b
OH
4
O
5-9
O
10-19
O
OH
1-3, 21-29, 31, 32
(see Table 1)
R
Cmpd
15
Cmpd
Cmpd
R
R'
R
R'
Pr
10
11
12
13
14
5
6
7
8
9
Pr
Me
Me
Me
Me
Me
Pr Et
d
Bu
Bu
iBu
iPen
Prenyl
16
Pr Pr
Pr Bu
Pr iBu
Pr iPen
iBu
iPen
Prenyl
17
18
19
Pr
Pr
Pr
Pr
O
OH
O
OH
Ac
c
Pr
Pr
Ph
O
O
O
20
30
Scheme 1. Syntheses of Desmosdumotin C derivatives. Reagents and conditions: (a) prenyl Br, KOH, water for R = prenyl; Rl, NaOMe, MeOH, reflux for others; (b) TMSCHN2
for R0 = Me; R0l, K2CO3, acetone, reflux for others; (c) 50% aq KOH, EtOH, ArCHO, rt; (d) prenyl Br, K2C03, acetone reflux.

1818
K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
Table 1
R
R
Pr
Pr
3
R'O
R
O
X
O
OH
4
4'
A
B
1
Pr
Ph
5
O
OH
O
O
1-3, 21-29, 31, 32
30
a
Compound
ED50
1A9
(lM)
R
R0
X
A549
HCT-8
MCF-7
PC-3
HepG2
KB
KB-V
1
2
3
Me
Pr
Pr
Bu
iBu
iPen
Prenyl
Pr
Pr
Pr
Pr
Me
Me
Me
Me
Me
Me
Me
Et
H
H
Br
H
H
H
H
H
H
H
H
H
—
I
11.2
3.6
2.0
6.4
4.4
18.4
10.3
4.6
1.9
7.1
1.7
11.3
7.1
1.4
8.2
11.8
3.5
2.6
7.8
7.5
13.0
13.7
5.6
2.6
8.7
8.0
15.3
6.7
1.1
7.8
NTb
NT
NT
11.2
3.7
1.9
5.9
8.0
16.3
NT
4.3
2.0
NT
6.9
NT
NT
1.8
NT
2.09
NT
35.6
8.2
4.7
13.0
12.4
NAc
12.2
12.5
8.6
NT
NT
NT
23.9
14.5
NA
11.2
11.2
8.3
12.8
4.6
1.8
7.0
7.9
17.5
13.3
6.4
2.7
8.0
4.8
10.6
8.2
1.5
6.4
9.6
3.4
1.8
8.6
8.2
20.3
8.9
3.5
2.4
8.9
4.1
19.2
7.3
1.4
6.2
>100
>100
21
22
23
24
25
26
27
28
29
30
31
32
4.4
3.7
25.2
8.2
2.6
1.6
5.3
4.4
9.1
5.8
1.8
5.1
>100
12.1
Pr
Bu
9.6
8.0
iBu
iPen
—
Me
Pr
12.2
17.0
8.4
2.8
9.6
13.4
15.5
6.4
2.4
7.3
Pr
—
Pr
Pr
I
Paclitaxel (nM)
Vincristine (nM)
2.56
4.8
>100
24.2
8.87
12.1
>100
2.9
2.87
2.4
a
Antiproliferative activity as ED50 values for each cell line, the concentration of compound that caused 50% reduction in absorbance at 562 nm relative to untreated cells
using the sulforhodamine B assay. Human lung carcinoma (A549), colon adenocarcinoma (HCT-8), breast cancer (MCF-7), ovarian carcinoma (1A9), prostate cancer (PC-3),
liver cancer (HepG2), epidermoid carcinoma of the nasopharynx (KB), and MDR expressing P-glycoprotein (KB-VIN).
b
NT, not tested.
NA, not active. Test compound (20
c
lg/mL) did not reach 50% inhibition.
down-regulated 1726 genes. The differentially expressed genes
were analyzed for GeneGo canonical pathway maps by using Meta-
Core Analytical Suite (GeneGo Inc., St Joseph, MI). The top ten path-
ways involved in analog 3 affected genes were shown in Table 2.
Seven pathways are cell cycle-related pathways and three are
DNA damage-related. For example, in the spindle assembly check-
point (SAC) or chromosome segregation pathway, the genes altered
by the treatment with 3 encoded mitotic kinases (e.g., CDK1-cyclin
B, Aurora A, Aurora B, and NEK2A), SAC proteins (e.g., MAD1,
MAD2, securin, and separase), and motor proteins (e.g., dynein1,
dynein activator complex dynactin, and KNSL1) (Fig. 3). The cyclin
dependent kinases, Aurora kinases, and NEK2A kinase are critical
for mitotic progression, through phosphorylation of their numer-
ous substrates. NEK2A or Aurora kinases are required for spindle
formation at the onset of mitosis or chromosome segregation and
cytokinesis, respectively. The SAC proteins, such as MAD2, activate
spindle checkpoint and inhibit securin degradation, until all chro-
mosomes are aligned at the metaphase plate. When chromosomes
are aligned correctly, the E3 ubiquitin ligase anaphase-promoting
complex/cyclosome (APC/C) inhibitor MAD2 is dissociated from
the APC/C and removed from the attached kinetochore by dynein.
O
OH
Ph
O
O
33
Figure 2.
the same or greater level than the parent KB cells, supporting the
idea that all analogues are not affected by P-gp-related multidrug
resistance (MDR).
Analogue 3 also showed potent antiproliferative activity against
the highly invasive non-small-cell lung cancer cell line CL1-5 with
an ED50 value of 0.11 lM. To determine which genes were differen-
tially expressed upon CL1-5 treatment with analogue 3, the gen-
ome-wide mRNA expression profiles of 3-treated cells and
control cells were determined using Affymetrix human genome
U133 plus 2.0 GeneChip according to the Manufacturer’s protocols
(Santa Clara, CA, http://www.affymetrix.com) by the Microarray
Core Facility of National Research Program for Genomic Medicine
of National Science Council in Taiwan as previously described.7
This Affymetrix GeneChip contains 54,675 probe sets to analyze
the expression levels of 47,400 transcripts and variants, including
38,500 well-characterized human genes. GeneChips from the
hybridization experiments were read by the Affymetrix GeneChip
scanner 3000 7G, and raw data were processed using GC-RMA
algorithm. The raw data were then analyzed by GeneSpring GX
software version 11.01.8 2.5 ꢁ 105 CL1-5 cells were treated for
Table 2
Statistically significant pathways
p Value
Cell cycle_The metaphase checkpoint
Cell cycle_Role of APC in cell cycle regulation
1.28Eꢂ23
2.09Eꢂ19
1.62Eꢂ16
3.87Eꢂ16
1.39Eꢂ15
1.35Eꢂ12
2.08Eꢂ11
1.05Eꢂ10
3.56Eꢂ09
1.51Eꢂ08
Cell cycle_Start of DNA replication in early S phase
Cell cycle_Spindle assembly and chromosome separation
Cell cycle_Chromosome condensation in prometaphase
Cell cycle_Transition and termination of DNA replication
Cell cycle_Role of Nek in cell cycle regulation
DNA damage_ATM/ATR regulation of G2/M checkpoint
DNA damage_ATM/ATR regulation of G1/S checkpoint
DNA damage_Role of Brca1 and Brca2 in DNA repair
24 h with 3 at a concentration of 0.05 lg/mL, and then total RNA
was extracted by TRIzol (Life Technologies, Gaithersburg, MD).
RNA from non-treated CL1-5 cells was used as a control. A total
of 2838 genes showed at least two-fold changes in expression lev-
els between the CL1-5 treated with 3 and CL1-5 DMSO control.
Analogue
3 up-regulated the expression of 1112 genes and

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
1819
Figure 3.
Subsequently, APC/C is activated by CDC20 or CDH1.9 APC/C-
CDC20 or -CDH1 recognizes substrates such as cyclins, NEK2A,
and securin or Aurora kinases and cyclins, respectively. At the on-
set of anaphase, the separase inhibitor securin is poly-ubiquitinat-
ed by activated APC/C followed by digestion by the proteasome.
Subsequently, activated separase cleaves the cohesin complex,
resulting in separation of the sister chromatids. All of these pro-
teins are expressed in a cell cycle-dependent manner.10 In our oli-
gonucleotide microarray studies, genes encoding these proteins
were up-regulated by the treatment with 3. The up-regulation of
MAD2L1 transcript was confirmed by semi-quantitative RT-PCR
(Fig. 4). Therefore, we assume that 3 may modulate SAC and chro-
mosome separation, and conclude that 3 induces cell cycle arrest
mainly in the G2/M-phase. Because oligonucleotide microarray
data are quite complicated and can be contradictory, we will need
to conduct additional experiments, such as real-time qPCR, to ver-
ify our results.
40-iodo-phenyl B-ring (3 and 31) led to the greatest tumor cell
growth inhibition. Isobutoxy analog 28 selectively inhibited the
A549 lung tumor cell line. Oligonucleotide microarray studies
showed that 3 may modulate SAC and chromosome separation
and arrest cells mainly in the G2/M-phase. Further modifications
of 28 as selective anti-lung tumor agents as well as further inves-
tigations to verify oligonucleotide microarray data are currently
undergoing, and will be reported in the future.
4. Experimental section
All chemicals and solvents were used as purchased. All melting
points were measured on a Fisher–Johns melting point apparatus
without correction. 1H NMR spectra were recorded on a Varian
Gemini 300 (300 MHz) spectrometer with TMS as the internal
standard. All chemical shifts are reported in ppm. 13C NMR spectra
were recorded on a Varian Inova 400 (400 MHz) spectrometer,
referenced to the residual solvent peak. Mass spectroscopic data
were obtained on a TRIO 1000 mass spectrometer. Analytical
thin-layer chromatography (TLC) was carried out on Merck pre-
coated aluminum silica gel sheets (Kieselgel 60 F-254). Final target
compounds were characterized by 1H NMR and HRMS analyses,
and others were characterized by 1H NMR. The purities of the final
targets were >90% determined by 1H NMR and HPLC analyses.
In summary, among the tested compounds, tripropyl substitu-
tion at C-3 and -5 (R = Pr) was optimal for tumor cell growth inhi-
bition. A methoxy or propoxy group at C-4 (OR0 = OMe or OPr) was
generally preferred over other alkyl ether groups. Finally, the com-
bination of a 3,3,5-tripropyl-4-methoxy A-ring and a 40-bromo- or
4.1. 2-Acetyl-4,4,6-tributyl-3,5-dihydroxycyclohexa-2,5-dienone
(6)
A
solution of 2,4,6-trihydroxyacetophenone (4, 595 mg,
3.5 mmol), sodium methoxide (2.5 mL, 11.6 mmol, 25% MeOH
solution) and butyl iodide (1.2 mL, 10.6 mmol) in anhydrous MeOH
(3 mL) was refluxed overnight. The reaction mixture was cooled to
0 °C and acidified with 1 N aqueous HCl solution, then extracted
three times with EtOAc. The combined organic layers were dried
over Na2SO4 and concentrated under vacuum. The residue was
chromatographed on silica gel with EtOAc–hexane (1:9 to 1:4,
Figure 4. CL1-5 cells were treated for 24 h with DMSO (w/o) or 0.05 lg/mL of 3,
followed by RNA isolation and RT-PCR for MAD2L1 (arrow in upper panel). The Gb-
like was used as control (lower panel). Asterisk in upper panel shows non-specific
amplification by primer-dimer.

1820
K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
v/v) as an eluent to obtain 6 (516 mg, 44%) as colorless solid, which
was used directly in the next reaction without recrystallization. 1H
NMR (300 MHz, CDCl3): d 19.03 and 18.40 (2:1, each s, 1H,
chelated-OH), 5.95 and 5.38 (2:1, each s, 1H, OH), 2.72 and 2.62
(1:2, each s, 3H, COCH3), 2.48–2.38 (m, 2H), 2.04–1.87 (m, 2H),
1.80–1.67 (m, 2H), 1.52–1.32 (m, 4H), 1.29–1.13 (m, 4H), 1.06–
0.90 (m, 8H), 0.86–0.78 (m, 6H). MS (ESI, m/z) 337 [M+H]+.
EtOAc–hexane as an eluent to obtain 15 (50 mg, 44%) as brown
oil, along with recovered starting material (39 mg, 38%). 1H NMR
(300 MHz, CDCl3): d 19.00, 18.31 and 18.14 (2:1:1, each s, 1H, che-
lated-OH), 4.19 and 4.07 (2:1, each q, 2H, J = 7.0 Hz, OCH2CH3), 2.69
and 2.60 (1:2, each s, 3H, COCH3), 2.55–2.37 (m, 2H), 1.93–1.64 (m,
8H), 1.58–1.34 (m, 3H), 1.30–1.10 (m, 2H), 1.10–0.92 (m, 3H),
0.90–0.73 (m, 6H). MS (ESI, m/z) 323 [M+H]+.
4.2. 2-Acetyl-4,4,6-triisobutyl-3,5-dihydroxycyclohexa-2,5-
dienone (7)
4.6. 2-Acetyl-4,4,6-tripropyl-5-propoxy-3-hydroxycyclohexa-
2,5-dienone (16)
Compound 4 (412 mg, 2.5 mmol), sodium methoxide (1.8 mL,
8.3 mmol, 25% MeOH solution), and isobutyl iodide (0.9 mL,
7.8 mmol) in anhydrous MeOH (3 mL) were treated similarly to
the above procedure to obtain 7 (174 mg, 21%) as colorless solid,
which was used directly in the next reaction without recrystalliza-
tion. 1H NMR (300 MHz, CDCl3): d 19.02 and 18.30 (3:1, each s, 1H,
chelated-OH), 6.07 and 5.40 (3:1, each s, 1H, OH), 2.71 and 2.61
(1:3, each s, 3H, COCH3), 2.32 and 2.29 [3:1, each d, 2H,
J = 7.4 Hz, 6-CH2CH(CH3)2], 2.16–1.81 (m, 3H), 1.76–1.67 (m, 2H),
1.48–1.36 (m, 2H, 4-CH2CH(CH3)2 ꢁ 2], 0.96 and 0.95 [3:1, each
d, 6H, J = 6.6 Hz, CH2CH(CH3)2], 0.81 and 0.80 [1:3, each d, 6H,
J = 6.6 Hz, CH2CH(CH3)2], 0.72 and 0.71 [1:3, each d, 6H, J =
6.6 Hz, CH2CH(CH3)2]. MS (ESI, m/z) 337 [M+H]+.
Compound
5
(95 mg, 0.32 mmol), potassium carbonate
(940 mg, 6.8 mmol), and propyl iodide (0.5 mL, 2.1 mmol) were
treated as described above for 15 to obtain 16 (67 mg, 62%) as
brown oil. 1H NMR (300 MHz, CDCl3): d 19.00, 18.38 and 18.14
(2:1:1, each s, 1H, chelated-OH), 4.09 and 3.96 (2:1, each t, 2H,
J = 6.4 Hz, OCH2CH2CH3), 2.69, 2.62 and 2.60 (1:2:1, each s, 3H,
COCH3), 2.50–2.36 (m, 2H), 1.90–1.65 (m, 8H), 1.58–1.38 (m, 2H),
1.30–1.10 (m, 2H), 1.10–0.92 (m, 6H), 0.90–0.74 (m, 6H). MS (ESI,
m/z) 337 [M+H]+.
4.7. 2-Acetyl-4,4,6-tripropyl-5-isobutoxy-3-hydroxycyclohexa-
2,5-dienone (18)
Compound
5 (110 mg, 0.37 mmol), potassium carbonate
4.3. 2-Acetyl-4,4,6-triisopentyl-3,5-dihydroxycyclohexa-2,5-
dienone (8)
(1272 mg, 9.2 mmol) and isobutyl iodide (0.4 mL, 3.5 mmol) were
treated as described above for 15 to obtain 18 (36 mg, 28%) as
brown oil along with the recovery of starting material (77 mg,
70%). 1H NMR (300 MHz, CDCl3): d 19.00, 18.38 and 18.14 (2:1:1,
each s, 1H, chelated-OH), 3.90 and 3.77 [2:1, each d, 2H,
J = 6.4 Hz, OCH2CH(CH3)2], 2.69, 2.62 and 2.61 (1:2:1, each s, 3H,
COCH3), 2.51–2.38 (m, 2H), 2.10–1.65 (m, 8H), 1.56–1.40 (m, 2H),
1.25–1.10 (m, 2H), 1.10–0.92 (m, 9H), 0.90–0.75 (m, 6H). MS (ESI,
m/z) 351 [M+H]+.
Compound 4 (510 mg, 3.0 mmol), sodium methoxide (2.2 mL,
10.2 mmol, 25% MeOH solution), and isobutyl iodide (1.3 mL,
9.9 mmol) in anhydrous MeOH (2 mL) were treated as described
above to obtain 8 (317 mg, 28%) as colorless solid, which was
used directly in the next reaction without recrystallization. 1H
NMR (300 MHz, CDCl3): d 19.01 and 18.30 (3:1, each s, 1H, che-
lated-OH), 5.82 and 5.22 (3:1, each s, 1H, OH), 2.73 and 2.70 (1:3,
each s, 3H, COCH3), 2.48–2.38 (m, 2H), 2.04–1.90 (m, 4H), 1.79–
1.56 (m, 3H), 1.46–1.30 (m, 6H), 0.96 [d, 6H, J = 6.6 Hz,
CH2CH2CH(CH3)2], 0.81 and 0.80 [1:3, each d, 12H, J = 6.6 Hz,
CH2CH2CH(CH3)2 ꢁ 2]. MS (ESI, m/z) 379 [M+H]+.
4.8. 2-Acetyl-4,4,6-tripropyl-5-isopentoxy-3-hydroxycyclohexa-
2,5-dienone (19)
Compound
5 (154 mg, 0.52 mmol), potassium carbonate
(1840 mg, 13.3 mmol) and iso-pentyl iodide (0.7 mL, 5.3 mmol)
were treated as described above for 15 to obtain 19 (94 mg, 26%)
as brown oil.
4.4. 2-Acetyl-4,4,6-triprenyl-3,5-dihydroxycyclohexa-2,5-
dienone (9)
To a solution of 4 (835 mg, 4.5 mmol) and KOH (572 mg,
10.2 mmol) in H2O (5.7 mL) at 0 °C under an argon atmosphere
was added prenyl bromide (1.2 mL, 10.3 mmol) dropwise over five
min. The resulting mixture was stirred at 0 °C for 1 h. Subse-
quently, KOH (255 mg, 4.6 mmol) in H2O (0.25 mL) and prenyl bro-
mide (0.55 mL, 4.7 mmol) were added at 0 °C. After stirring for
15 min at 0 °C, the mixture was allowed to warm to room temper-
ature and stirred for 1 h. The reaction mixture was quenched with
aqueous HCl to pH 1 and extracted three times with EtOAc. The
combined organic layers were washed with brine, dried over anhy-
drous magnesium sulfate, filtered, and concentrated under vac-
4.9. 4-Acetyl-2-(3-methylbut-2-en-1-yl)-2,6,6-tripropyl-5-
hydroxycyclohex-4-ene-1,3-dione (20)
Compound
5 (157 mg, 0.53 mmol), potassium carbonate
(1030 mg, 7.5 mmol) and prenyl bromide (0.15 mL, 1.3 mmol)
were treated as described above for 15 to obtain 20 (84 mg, 44%)
as brown oil. 1H NMR (300 MHz, CDCl3): d 19.00 (s, 1H, chelated-
OH), 6.98 (s, 1H, OH), 5.20–5.09 [m, 1H, CH2CHC(CH3)2],
4.86–4.75 [m, 2H, CH2CHC(CH3)2 ꢁ 2], 3.21–3.13 [m, 2H,
CH2CHC(CH3)2], 2.70–2.46 [m, 4H, CH2CHC(CH3)2 ꢁ 2], 1.82–1.74
(m, 6H), 1.65–1.50 (m, 12H). MS (ESI, m/z) 363 [M+H]+.
uum. Purification on silica gel (hexane/EtOAc) provided
9
(269 mg, 16%) as brown oil, tetraprenyl compound (100 mg, 5%)
and diprenyl compound (327 mg, 24%).
4.10. General procedures for Aldol reactions
A solution of acetyl compound (10–19) in EtOH–50% aq. KOH
(1:1, v/v) and an appropriate aldehyde (excess) was stirred at
room temperature. After the reaction was complete by TLC
analysis, the mixture was poured into ice-cold 1 N HCl, then
extracted with CH2Cl2. The extract was washed with brine, dried
over Na2SO4, and concentrated under vacuum. The residue was
chromatographed on silica gel with CH2Cl2–hexane as eluent to
afford the target compound, which was crystallized from CH2Cl2–
hexane.
4.5. 2-Acetyl-4,4,6-tripropyl-5-ethoxy-3-hydroxycyclohexa-2,5-
dienone (15)
To a solution of 5 (102 mg, 0.35 mmol) in anhydrous acetone
(2 mL), potassium carbonate (1045 mg, 7.6 mmol) and ethyl iodide
(0.13 mL, 1.6 mmol) were added, and the mixture was stirred for
2 days. After filtration, the solvent was removed under vacuum.
The residue was purified by column chromatography with

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
1821
4.10.1. 3,3,5-Tributyldesmosdumotin C (21)
14.39, 10.63, 10.59. HRMS: calcd for C27H35O4 423.2535 [MꢂH]+,
Yield 56%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.30 and
18.90 (2:1, each s, 1H, chelated-OH), 8.51 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.96 and 7.93 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.73–7.65 (m, 2H, Ar-H), 7.44–7.36 (m, 3H, Ar-H), 4.00
and 3.92 (2:1, each s, 3H, OCH3), 2.59–2.48 (m, 2H), 2.00–1.70
(m, 4H), 1.58–1.37 (m, 4H), 1.30–1.14 (m, 4H), 1.10–0.92 (m,
6H), 0.86–0.78 (m, 6H). HRMS: calcd. for C28H39O4 439.2848
[M+H]+, found 439.2876
found 423.2559.
4.10.7. 4-Butoxy-3,3,5-tripropyldesmosdumotin C (27)
Yield 67%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.33 and
18.90 (2:1, each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.94 and 7.92 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.73–7.64 (m, 2H, Ar-H), 7.44–7.35 (m, 3H, Ar-H), 4.15
and 4.02 [2:1, each t, 2H, J = 6.4 Hz, OCH2(CH)2CH3], 2.00–1.81
(m, 2H), 1.80–1.69 (m, 2H). 1.60–1.42 (m, 6H), 1.15–0.96 (m,
10H), 0.89–0.77 (m, 6H). HRMS: calcd for C28H39O4 439.2848
[M+H]+, found 439.2880.
4.10.2. 3,3,5-Triisobutyldesmosdumotin C (22)
Yield 36%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.36 and
18.86 (2:1, each s, 1H, chelated-OH), 8.48 and 8.44 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.96 and 7.93 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.73–7.64 (m, 2H, Ar-H), 7.43–7.36 (m, 3H, Ar-H), 4.03
and 3.94 (2:1, each s, 3H, OCH3), 2.57–2.50 (m, 2H), 1.97–1.84
(m, 4H), 1.80–1.71 (m, 1H), 1.54–1.39 (m, 2H), 0.94 and 0.93
[2:1, d, 6H, J = 6.6 Hz, CH2CH(CH3)2], 0.83 and 0.82 [1:2, d, 6H,
J = 6.6 Hz, CH2CH(CH3)2], 0.73 and 0.72 [2:1, d, 6H, J = 6.6 Hz,
CH2CH(CH3)2]. HRMS: calcd for C28H39O4 [M+H]+ 439.2848, found
439.2879.
4.10.8. 4-Isobutoxy-3,3,5-tripropyldesmosdumotin C (28)
Yield 32%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.34 and
18.83 (2:1, each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.94 and 7.92 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.73–7.64 (m, 2H, Ar-H), 7.45–7.35 (m, 3H, Ar-H), 3.92
and 3.79 [2:1, each d, 2H, J = 6.4 Hz, OCH2CH(CH3)2], 2.56–2.42
(m, 2H), 2.11–1.70 (m, 6H), 1.60–1.48 (m, 2H), 1.18–0.95 (m,
10H), 0.90–0.78 (m, 6H). HRMS: calcd for C28H39O4 439.2848
[M+H]+, found 439.2878.
4.10.3. 3,3,5-Triisopentyldesmosdumotin C (23)
Yield 43%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.29 and
18.89 (2:1, each s, 1H, chelated-OH), 8.50 and 8.41 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.96 and 7.93 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.74–7.66 (m, 2H, Ar-H), 7.45–7.37 (m, 3H, Ar-H), 4.00
and 3.93 (2:1, each s, 3H, OCH3), 2.60–2.46 (m, 2H), 1.98–1.71
(m, 5H), 1.70–1.60 (m, 1H), 1.51–1.36 (m, 4H), 0.98 and 0.97
[2:1, d, 6H, J = 6.6 Hz, CH2CH(CH3)2], 0.94–0.80 (m, 15H). HRMS:
calcd for C31H45O4 481.3318 [M+H]+, Found 481.3342.
4.10.9. 4-Isopentoxy-3,3,5-tripropyldesmosdumotin C (29)
Yield 52%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.31 and
18.87 (2:1, each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.94 and 7.92 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.74–7.65 (m, 2H, Ar-H), 7.44–7.35 (m, 3H, Ar-H), 4.17
and 4.05 [2:1, each t, 2H, J = 6.4 Hz, OCH2CH2CH (CH3)2], 2.56–
2.42 (m, 2H), 1.98–1.46 (m, 8H), 1.14–0.94 (m, 10H), 0.86–0.78
(m, 6H). HRMS: calcd for C29H39O4 451.2848 [M+H]+, found
451.2886.
4.10.4. 3,3,5-Triprenyldesmosdumotin C (24)
Yield 63%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 18.82 (s, 1H,
chelated-OH), 8.53 and 8.38 (1:2, each d, 1H, J = 15.6 Hz, olefin),
7.94 and 7.92 (1:2, each d, 1H, J = 15.6 Hz, olefin), 7.74–7.62 (m,
2H, Ar-H), 7.45–7.37 (m, 3H, Ar-H), 5.11–5.02 [m, 1H,
CH2CH@C(CH3)2], 4.85–4.74 [m, 2H, CH2CH@C(CH3)2 ꢁ 2], 3.95
and 3.88 (2:1, each s, 3H, OCH3), 3.28–3.16 [m, 2H,
CH2CH@C(CH3)2], 2.80–2.50 [m, 4H, CH2CH@C(CH3)2 ꢁ 2],
1.76–1.69 (m, 6H), 1.62–1.56 (m, 12H). HRMS: calcd for C31H37O4
473.2692 [M+H]+, found 473.2731.
4.10.10. 3,3,5-Tripropyl-5-prenyldesmosdumotin C (30)
Yield 32%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 18.49
and 18.36 (1:1, each s, 1H, chelated-OH), 8.06–7.94 (m, 2H),
7.71–7.61 (m, 2H), 7.49–7.36 (m, 3H), 5.00–4.87 [m, 1H,
CH2CH@C(CH3)2], 2.70–2.34 (m, 2H), 1.90–1.64 (m, 6H), 1.64–
1.52 (m, 6H), 1.36–1.10 (m, 6H), 0.94–0.80 (m, 9H). HRMS: calcd
for C29H37O4 449.2692 [M+H]+, found 449.2725.
4.10.11. 40-Iodo-3,3,5-tripropyldesmosdumotin C (31)
Yield 23%. Yellow prisms. mp. 123–124 °C (Hexane). 1H NMR
(300 MHz, CDCl3): d 19.27 and 18.82 (2:1, each s, 1H, chelated-
OH), 8.49 and 8.41 (1:2, each d, 1H, J = 15.6 Hz, olefin), 7.84 and
7.81 (1:2, each d, 1H, J = 15.6 Hz, olefin), 7.77–7.70 (m, 2H, Ar-H),
7.44–7.37 (m, 2H, Ar-H), 4.00 and 3.92 (2:1, each s, 3H, OCH3),
2.55–2.43 (m, 2H), 1.99–1.67 (m, 4H), 1.63–1.46 (m, 2H), 1.14–
0.97 (m, 7H), 0.87–0.79 (m, 6H). 13C NMR (400 MHz, CDCl3): d
198.22, 192.85, 186.86, 175.05, 144.03, 143.56, 138.30, 138.25,
135.00, 130.57, 130.50, 126.76, 124.88, 124.70, 121.54, 109.39,
62.35, 59.95, 55.58, 42.15, 40.89, 26.62, 26.14, 23.03, 22.88,
18.46, 18.35, 14.78, 14.64, 14.54, 14.36. HRMS: calcd for
4.10.5. 4-Ethoxy-3,3,5-tripropyldesmosdumotin C (25)
Yield 41%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.32 and
18.89 (2:1, each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.95 and 7.92 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.74–7.64 (m, 2H, Ar-H), 7.45–7.34 (m, 3H, Ar-H), 4.20
and 4.10 (2:1, each q, 2H, J = 6.9 Hz, OCH2CH3), 2.54–2.40 (m,
2H), 2.00–1.66 (m, 4H), 1.61–1.48 (m, 2H), 1.48–1.37 (m,3H),
1.19–0.95 (m, 5H), 0.87–0.79 (m, 6H). HRMS: calcd for C26H35O4
411.2535 [M+H]+, found 411.2573.
4.10.6. 4-Propoxy-3,3,5-tripropyldesmosdumotin C (26)
C
25H32IO4 523.1345 [M+H]+, found 523.1363.
Yield 29%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.33 and
18.84 (2:1, each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.94 and 7.92 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.73–7.64 (m, 2H, Ar-H), 7.44–7.34 (m, 3H, Ar-H), 4.10
and 3.99 (2:1, each t, 2H, J = 6.9 Hz, OCH2CH2CH3), 2.54–2.42 (m,
2H), 2.00–1.64 (m, 6H), 1.52–1.47 (m, 2H), 1.15–0.95 (m, 10H),
0.92–0.78 (m, 6H). 13C NMR (400 MHz, CDCl3): d 200.78, 198.22,
192.81, 189.20, 187.08, 186.47, 174.23, 167.42, 145.40, 144.82,
135.57, 135.47, 130.83, 130.67, 129.24, 129.12, 129.05, 129.02,
124.22, 124.08, 120.66, 112.02, 109.24, 76.15, 75.82, 59.82, 55.48,
42.38, 41.08, 39.04, 38.09, 26.70, 26.23, 24.10, 24.05, 23.17,
23.00, 18.48, 18.42, 18.32, 18.18, 14.75, 14.68, 14.61, 14.56,
4.10.12. 40-Iodo-4-propoxy-3,3,5-tripropyldesmosdumotin C
(32)
Yield 9%. Yellow oil. 1H NMR (300 MHz, CDCl3): d 19.31 and
18.81 (2:1, each s, 1H, chelated-OH), 8.49 and 8.42 (1:2, each d,
1H, J = 15.6 Hz, olefin), 7.84 and 7.80 (1:2, each d, 1H, J = 15.6 Hz,
olefin), 7.76–7.69 (m, 2H, Ar-H), 7.44–7.36 (m, 2H, Ar-H), 4.11
and 3.99 (2:1, each t, 2H, J = 6.9 Hz, OCH2CH2CH3), 2.55–2.42 (m,
2H), 2.00–1.68 (m, 6H), 1.60–1.42 (m, 2H), 1.12–0.95 (m, 10H),
0.86–0.77 (m, 6H). HRMS: calcd for C27H36IO4 551.1658 [M+H]+,
found 551.1691.

1822
K. Nakagawa-Goto et al. / Bioorg. Med. Chem. 19 (2011) 1816–1822
4.11. Antiproliferative activity assay
Gb-like was amplified from same cDNA pool by PCR (25 cycles)
using forward primer 50-GTATGGAACCTGGCTAACTG-30 and re-
verse primer 50-TACTGATAACTTCTTGCTTC-30. The PCR products
were separated by agarose gel and stained by ethidium bromide.
All stock cultures were grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtitre plates at densi-
ties of 1500–7500 cells per well with compounds added from
DMSO-diluted stock. After three days in culture, attached cells
were fixed with cold 50% trichloroacetic acid and then stained with
0.4% sulforhodamine B (SRB). The absorbency at 562 nm was mea-
sured using a microplate reader after solubilizing the bound dye.
The mean ED50 is the concentration of agent that reduces cell
growth by 50% under the experimental conditions and is the aver-
age from at least three independent determinations that were
reproducible and statistically significant. The following human
tumor cell lines were used in the assay: A549 (human lung
carcinoma), 1A9 (human ovarian carcinoma), HCT-8 (colon adeno-
carcinoma), PC-3 (prostate cancer), KB (nasopharyngeal carci-
noma), KB-VIN (vincristine resistant KB subline), HUVEC (human
umbilical vein endothelial cell). All cell lines were obtained from
the Lineberger Cancer Center (UNC-CH) or from ATCC (Rockville,
MD) and were cultured in RPMI-1640 medium supplemented with
25 mM HEPES, 0.25% sodium bicarbonate, 10% fetal bovine serum,
Acknowledgments
This study was supported by grant CA-17625 from the National
Cancer Institute, NIH, awarded to K.H.L. and by a grant from the
University Research Council, awarded to K.N.G. We thank the tech-
nical assistance by the research assistants at Microarray Core Facil-
ity of National Research Program for Genomic Medicine of National
Science Council in Taiwan.
References and notes
1. Wu, J. H.; McPhail, A. T.; Bastow, K. F.; Shiraki, H.; Ito, J.; Lee, K. H. Tetrahedron
Lett. 2002, 43, 1391.
2. Nakagawa-Goto, K.; Wu, J. H.; Lee, K. H. Synth. Commun. 2005, 35, 1735.
3. (a) Nakagawa-Goto, K.; Wu, J. H.; Bastow, K. F.; Wu, C. C.; Lee, K. H. Bioorg. Med.
Chem. 2005, 13, 2325; (b) Nakagawa-Goto, K.; Chen, T.-H.; Peng, C.-Y.; Bastow,
K. F.; Wu, J. H.; Lee, K. H. J. Med. Chem. 2007, 50, 3354.
and 100 lg/mL kanamycin.
4. For review: (a) Barron, D.; Ibrahim, R. K. Phytochemistry 1996, 43, 921; (b)
Zanoli, P.; Zavatti, M. J. Ethnopharmacol. 2008, 116, 383.
5. Recent reports: (a) Harikumar, K.; Kunnumakkara, A. B.; Ahn, K. S.;
Anand, P.; Krishnan, S.; Guha, S.; Aggarwal, B. B. Blood 2009, 113, 2003; (b)
Rao, G. V.; Swamy, B. N.; Chandregowda, V.; Reddy, G. C. Eur. J. Med. Chem.
2009, 44, 2239; (c) Jamil, S.; Sirat, H. M.; Jantan, I.; Aimi, N.; Kitajima, M. J. Nat.
Med. 2008, 62, 321; (d) Vogel, S.; Heilmann, J. J. Nat. Prod. 2008, 71, 1237; (e)
Vogel, S.; Ohmayer, S.; Brunner, G.; Heilmann, J. Bioorg. Med. Chem. 2008, 16,
4286.
6. (a) Xiao, L.; Tan, W.; Li, Y. Synth. Commun. 1998, 28, 2861; (b) Qi, J.; Porco, J. A.,
Jr. J. Am. Chem. Soc. 2007, 129, 12682.
7. Chao, Y. C.; Pan, S. H.; Yang, S. C.; Yu, S. L.; Che, T. F.; Lin, C. W.; Tsai, M. S.;
Chang, G. C.; Wu, C. H.; Wu, Y. Y.; Lee, Y. C.; Hong, T. M.; Yang, P. C. Am. J. Respir.
Crit. Care Med. 2009, 179, 123.
4.12. RT-PCR
Freshly trypsinized CL1-5 cell suspensions were seeded in
60 mm cell culture dishes at density of 2.5 ꢁ 105 and cultured for
48 h in RPMI-164 medium supplemented with 10% fetal bovine
serum (Gibco-BRL).11 Cells were incubated in 5% CO2 and 95% air
at 37 °C. Cells were treated with compound and continued cultiva-
tion for 24 h followed by the total RNA extraction by TRIzol (Invir-
togen). The cDNAs were synthesized from 1 lg total RNA using
random hexamer primers and Superscript III reverse transcriptase
(Invitrogen). The MAD2L1 was amplified from cDNA pool (1:10
diluted) by PCR (30 cycles) using DyNAzyme II DNA polymerase
(Finnzymes) with forward primer 50-AGGCAGCGCTGAGCTTGT
GG-30 and reverse primer 50- AGGCAGTCTCCAGCAGGGGT-30. The
8. Silicon Genetics, Redwood City, CA. The original data have been deposited to
NCBI database, and the GEO series number is GSE24584.
9. Zich, J.; Hardwick, K. G. Trends Biochem. Sci. 2010, 35, 18.
10. Manchado, E.; Eguren, M.; Malumbres, M. Biochem. Soc. Trans. 2010, 38, 65.
11. Chu, Y. W.; Yang, P. C.; Yang, S. C.; Shyu, Y. C.; Hendrix, M. J.; Wu, R.; Wu, C. W.
Am. J. Respir. Cell. Mol. Biol. 1997, 17, 353.