Antitumor agents. 284. new desmosdumotin B analogues with bicyclic B-ring as cytotoxic and antitubulin agents

Article abstract of DOI:10.1021/jm1011947

We previously reported that the biological acti-vity of analogues of desmosdumotin B (1) was dramatically changed depending on the B-ring system. A naphthalene B-ring analogue 3 exerted potent in vitro activity against a diverse panel of human tumor cell lines with GI₅₀ values of 0.8-2.1 μM. In contrast, 1 analogues with a phenyl B-ring showed unique selective activity against P-glycoprotein (P-gp) overexpressing multidrug resistant cell line. We have now prepared and evaluated 1 analogues with bicyclic or tricyclic aromatic B-ring systems as in vitro inhibitors of human cancer cell line proliferation. Among all synthesized derivatives, 21 with a benzo[b]thiophenyl B-ring was highly active, with GI₅₀ values of 0.06-0.16 μM, and this activity was not influenced by overexpression of P-gp. Furthermore, 21 inhibited tubulin assembly in vitro with an IC₅₀ value of 2.0 μM and colchicine binding by 78% as well as cellular microtubule polymerization and spindle formation.

Full text of DOI:10.1021/jm1011947

ARTICLE
pubs.acs.org/jmc
Antitumor Agents. 284. New Desmosdumotin B Analogues with
Bicyclic B-Ring as Cytotoxic and Antitubulin Agents
Kyoko Nakagawa-Goto,*,† Pei-Chi Wu,† Chin-Yu Lai,† Ernest Hamel,‡ Hao Zhu,§ Liying Zhang,§
Takashi Kozaka,† Emika Ohkoshi,† Masuo Goto,|| Kenneth F. Bastow,*,^ and Kuo-Hsiung Lee*,†,#
†Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599, United States
‡Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
§Laboratory for Molecular Modeling, Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, United States
^Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
North Carolina 27599, United States
#Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
S Supporting Information
b
ABSTRACT: We previously reported that the biological acti-
vity of analogues of desmosdumotin B (1) was dramatically
changed depending on the B-ring system. A naphthalene
B-ring analogue 3 exerted potent in vitro activity against a
diverse panel of human tumor cell lines with GI50 values of
0.8-2.1 μM. In contrast, 1analogues with a phenyl B-ring showed
unique selective activity against P-glycoprotein (P-gp) over-
expressing multidrug resistant cell line. We have now prepared
and evaluated 1 analogues with bicyclic or tricyclic aromatic
B-ring systems as in vitro inhibitors of human cancer cell line proliferation. Among all synthesized derivatives, 21 with a
benzo[b]thiophenyl B-ring was highly active, with GI50 values of 0.06-0.16 μM, and this activity was not influenced by
overexpression of P-gp. Furthermore, 21 inhibited tubulin assembly in vitro with an IC50 value of 2.0 μM and colchicine binding by
78% as well as cellular microtubule polymerization and spindle formation.
’ INTRODUCTION
cell rounding without immediate detachment, leading us to
hypothesize antitubulin activity as a mechanism of action, which was
tested and confirmed using biochemical assays. Compound 3,
therefore, represents a new scaffold for targeting tubulin assembly.
Design and synthesis of compounds targeting the microtubule
constitute an attractive strategy for the discovery of new anti-
tumor agents.3 The microtubule network is an essential compo-
nent of the cytoskeleton, and its timely depolymerization and
repolymerization are critical for the cell to construct a functional
mitotic spindle. Typically, cells arrested in apparent mitosis even-
tually undergo apoptosis. Antimitotic agents targeting tubulin are
generally classified into two groups, compounds that either stimu-
late or inhibit microtubule assembly, depending on their effects
on the tubulin-microtubule equilibrium. Taxoids and epothi-
lones are well-known enhancers of microtubule polymerization.
We previously reported that desmosdumotin B (1, Figure 1)
exerted selective inhibition of a P-glycoprotein (P-gp) over-
expressing multidrug resistant (MDR) tumor cell line with
significantly lower activity against non-MDR tumor cells.1 The
observed selectivity index [collateral sensitivity (CS),2 activity
ratio of MDR line versus non-MDR line] was greater than 20.
This selective in vitro antitumor activity was further enhanced by
replacing the three methyl groups at C-6 and C-8 with ethyl
groups (2, Figure 1) and also by adding an alkyl group at the C-40
position. During this study, we also found that analogues in
which the phenyl B-ring was replaced with a naphthyl moiety (3,
Figure 1) had dramatically different activity profiles, displaying
strong cytotoxicity against multiple cancer cells, regardless of
MDR expression, with GI50 values of 0.8-2.1 μM. Thus, placing
a larger, more electron-rich aromatic B-ring at C-2 resulted in
broader antiproliferative activity and loss of specific activity
against the MDR cell line. These compounds also induced rapid
Received: September 13, 2010
Published: February 01, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of new analogues of 1.
Colchicine and the vinca alkaloids are the best known inhibitors
of microtubule assembly. All of these agents bind to β-tubulin to
exert their antimitotic activity. The best characterized drug
binding domains are known as the colchicine site, the vinca
domain, and the taxoid site.
The vinca alkaloids and the taxoids are among the most useful
drugs for cancer therapy, and numerous compounds targeting
drug binding sites on tubulin have been developed. Unfortu-
nately, most antitubulin agents that have entered clinical trials
have failed because of adverse effects, such as limited therapeutic
effects at maximally tolerated doses, perhaps because of drug
resistance, high toxicity, or poor physicochemical properties
involved in the absorption, distribution, metabolism, and excre-
tion profile. Interestingly, thus far, no colchicine site drug has
proved useful in cancer treatment. Despite its lack of utility in
cancer therapy, colchicine is used for the treatment of
gout, familial Mediterranean fever, secondary amyloidosis, and
scleroderma. Moreover, colchicine has long been an important
tool for the study of microtubule structure/function4 and a key
molecular model for structure-activity relationship studies.
Previous research revealed that a two-ring aromatic system with
the rings either directly bonded or separated by a one to three
atom bridge is a common structural feature for binding to
the colchicine site.5 Combretastatin A-4 (CA-4) is an attractive
natural product that targets tubulin polymerization via the
colchicine site. CA-4 exerts strong cytotoxicity against multi-
ple human tumor cell lines.6 The discovery of CA-4 and its simple
molecular structure stimulated research to identify new chemo-
types that interact with the colchicine site.
Our interesting discovery of 2-naphthyldesmosdumotin B (3)
as an antitubulin agent prompted us to prepare additional 3
analogues with bicyclic and tricyclic aromatic substituents at C-2.
In the work presented here, we describe the syntheses and
bioactivities of this compound series.
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ARTICLE
’ CHEMISTRY
were also tested for inhibitory effects on tubulin assembly and
inhibition of binding of [3H]colchicine to tubulin, together with
2 for comparison. Table 2 lists the antiproliferative data for 1-48
and vincristine, the positive control. The tubulin study data are
presented in Table 3.
Monophenyl analogues 1 and 2 were clearly “nonactive”
against all non-MDR tumor cells (2 was also inactive in the
tubulin assembly assay), while they were “active” against the P-gp
overexpressing MDR tumor cell line KB-VIN. In comparison,
most analogues with a bicyclic substituent at C-2 (groups I and II)
showed in vitro activity against all tested cell lines, with GI50
values ranging from 0.1 to 20 μM, while three of the five
analogues (30-32) with a tricyclic substituent at C-2 (group III)
were inactive.
Thirty-four newly synthesized analogues, 4-37, are depicted
in Figure 1. They were synthesized from 38 (R = Et for 6-34,
R = Me for 4 and 35-37, and R = Pr for 5)1 through the related
chalcones (39) as shown in Scheme 1.1,7 The chalcones (39)
were prepared by the Claisen-Schmidt condensation of 38 with
various commercially available aromatic aldehydes (ArCHO)
except for 55 and 56. In most cases, the condensation was carried
out in the presence of 50% aqueous KOH in EtOH. However,
under these conditions, the reaction with 3-quinoline aldehyde
resulted in extremely low yields of product. Finally, the reactions
were run with good yields in the presence of piperidine or
Ba(OH)2 H2O. The iodine-catalyzed cyclization of 39, followed
3
by demethylation, provided 3-37. The treatment of 3 (R = Et,
Ar = naphthyl) with excess Lawesson’s reagent8 resulted in
replacement of oxygen with sulfur to yield both the monothio-
ketone 41 and the dithioketone 42. The structure of 41 was
determined from the 1H NMR and MS spectra, which indicated
the insertion of a sulfur atom. Compared to the 1H NMR of the
related compound 3, the spectra of 41 showed a 0.79 ppm high
field shift of a proton at the C-3 position and no shift for the ethyl
protons at the C-6 and C-8 positions. There was, however, a high
field shift for the ethyl protons of dithioketone 42 (Table 1).
Bromination of 40a (R = Et, Ar = naphthyl) with n-Bu4NBr in the
presence of PhI(OAc)29 afforded 3-bromo derivative 43, which
was converted to 44 by demethylation with BBr3. Meanwhile,
flavanone analogue 48 was prepared by the treatment of 39 with
HI in HOAc.
Aldehydes 55 and 56 were prepared as shown in Scheme 2.
We originally expected that 5-methoxybenzothiophene and
5-methylbenzothiophene, 5310 and 54,11 could be obtained from
the related 3-carboxyaldehydes through formylation.12 While
Rieche formylation of 5-methylbenzothiophene (54) using
SnCl4 provided the desired 3-carboxaldehyde 56 in good yield,
the same treatment of 5-methoxybenzothiophene (53) unexpec-
tively produced 4-formyl product 55. The structure of 55 was
identified by 1H NMR, which showed two doublet signals with a
coupling constant of 5.6 Hz at 8.39 and 7.65 ppm for the C-3 and
C-2 protons, respectively. Furthermore, NOE cross-peaks were
observed between the aldehyde and methoxy protons, the
aldehyde and C-3 protons, as well as the methoxy and C-6
protons. The interesting difference in reactivity between the
5-methyl and 5-methoxy groups might be caused by a chelation
effect of the Lewis acid with the oxygen atom of the methoxy
group.
With respect to the alkyl groups at C-6 and C-8, the 6,8,8-tri-
Me (4) and tri-Et (3) analogues showed similar growth inhibi-
tory activity against the tested tumor cell lines, while the tripropyl
analogue 5 was much less active.
Group II compound 21, with a tri-Et A-ring and a benzo-
[b]thiophene B-ring at C-2, had dramatically increased antipro-
liferative activity, exhibiting GI50 values of 0.06-0.16 μM against
the human tumor cell lines. Although the activity of 37, with a tri-
Me A-ring and a benzo[b]thiophene B-ring at C-2, was less than
that of 21, it still showed potent activity with GI50 values of
0.09-1.08 μM. In comparison, the related analogue 17, with the
chromene skeleton attached at the 20-position of the benzo-
[b]thiophene system rather than at the 30-position, showed
considerally lower activity against all cell lines (GI50
=
3.8-19.0 μM), with significant activity only against PC-3. The
replacement of sulfur (21) with oxygen (19) or N-methyl (20)
led to reduced activity. Other relatively active analogues, with
mostly submicromolar GI50 values, were tri-Et compounds 3, 6,
8, 11, and 15, as well as the related tri-Me analogues 4 and
35-37. All of these compounds had a naphthyl B-ring at C-2
except for 15 and 36, which had a 2,3-dihydrobenzo[1,4]dioxine
group at C-2. Quinoline 13, benzofurans 16 and 19, and indole
20, each bearing a bicyclic heteroaromatic B-ring system, dis-
played only moderate activity against all tested cell lines.
Analogue 14, with a benzo[1,3]dioxolyl B-ring system, also
moderately inhibited tumor cell growth, although it was about
10 times less active than 21. These data demonstrated that
although a 10π-electron B-ring system was optimal, it was not
essential for activity. Among the group III compounds, those
with the three rings arranged linearly were inactive while
compound 29 (C-2 dihydroacenaphthyl) showed significant
antiproliferative activity. Compounds 33 (C-2 phenanthryl)
and 34 (C-2 biphenyl) showed only moderate potency.
When the ketone oxygen at C-7 was replaced with a methoxy
group (3 vs 40a), there was a substantial loss of activity (GI50
values of 0.8-2.1 μM for 3 and 9.7-20.9 μM for 40a). The latter
compounds would lose the possibility of a hydrogen bond
between the C-4 ketone carbonyl and the C-5 hydroxy group,
which might account for the difference in potency. Activity also
decreased when a bromide group was inserted at the C-3 position
(3 vs 44), although the bromine atom had no effect when a C-7
methoxy group was present (cf. 40a and 43). Reduced activity
also occurred when both oxygens in the C-4 and C-7 ketones
were changed to sulfur (3 vs 42). However, monothioketone 41
retained relatively good activity, compared with 3, against A549
and KB cells, with GI50 values of 1.86 and 1.41 μM, respectively.
The saturation of the double bond between C-2 and C-3 also
decreased activity (3 vs 48). Finally, it is pointed out that MDR
Newly synthesized analogues could be classified into the
following five groups according to the C-2 substituent: (I)
naphthalene group (3-12 and 35), (II) heterobicyclic aromatic
group (13-28, 36, and 37), (III) tricyclic aromatic group
(29-33), (IV) biphenyl group (34), and (V) naphthalane group
with A- and/or C-ring modifications (40a-44 and 48).
’ RESULTS AND DISCUSSION
All new analogues were evaluated against seven human tumor
cell lines, specifically, HCT-8 (colon adenocarcinoma), PC-3
(prostate cancer), A549 (lung carcinoma), MCF-7 (breast
cancer) or DU145 (prostate cancer), HepG2 (hepatocellular
carcinoma), KB (epidermoid carcinoma of the nasopharynx),
and KB-VIN, which is an MDR (P-gp-overexpressing) KB sub-
line selected using increasing concentrations of vincristine. The
selected active compounds 3, 6, 8, 11, 15, 21, 22, 26, and 35-37
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ARTICLE
Scheme 1. Syntheses of New Analogues of 1a
a Reagents and conditions: (a) ArCHO, base/solvent [piperidine/EtOH for 3-quinolinecarboxaldehyde, Ba(OH)2 8H2O/MeOH for 4-dimethyla-
3
mino-1-naphthaldehyde, and KOH/EtOH for others]; (b) I2 (cat.), DMSO, H2SO4 (cat.), 90-95 ꢀC, 1 h; (c) BBr3, 0ꢀ C to room temp; (d) Lawesson’s
reagent, toluene, reflux; (e) PhI(OAc)2, n-Bu4NBr, room temp; (f) 45% HI, HOAc.
1
Table 1. H NMR Chemical Shifts (ppm) of 3, 41, and 42
C-6
C-8
C-3
H
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
3
6.82 (s, 1H)
7.62 (s, 1H)
7.62 (s, 1H)
2.49 (q, 2H)
1.07 (t, 3H)
2.26-2.12 (m, 2H)
1.98-1.84 (m, 2H)
2.31-2.07 (m, 2H)
2.01-1.86 (m, 2H)
2.58-2.42 (m, 2H)
2.30-2.16 (m, 2H)
0.73 (t, 6H)
41
42
2.52 (q, 2H)
2.99 (q, 2H)
1.07 (t, 3H)
1.10 (t, 3H)
0.74 (t, 6H)
0.64 (t, 6H)
Scheme 2. Syntheses of 5-Substituted Benzothiophenecarboxaldehydea
a Reagents and conditions: (a) 2-chloro-1,1-dimethoxyethane, NaOMe, MeOH, xylene, 110 ꢀC; (b) H3PO4, xylene, 110 ꢀC; (c) Cl2CHOMe, SnCl4,
CH2Cl2, room temp, 75% for 55, 82% for 56.
KB-VIN cells were as sensitive as the parental KB cells to all
active compounds.
and 50-Br (24) analogues lost activity. (3) Activity varied with the
position of attachment of the benzo[b]thiophene system to the
chromene skeleton. The rank order of overall activity was 30 (21)
> 70 (27) > 20 (17) >50 (28). (4) Compound 28 displayed
selective activity against the DU145 and KB cell lines. (5)
Compound 26 (50-OH) exhibited potent cytotoxicity against
all tested cell lines, while 25 (50-OMe) lost activity against most
cell lines. (6) Compounds 24 and 27 possessed “collateral
Because compound 21, containing a benzo[b]thiophene
system, showed potent antiproliferative activity, we investigated
additional related derivatives 22-28. While none of these
compounds showed greater activity than 21, some interesting
facts were revealed. (1) The 20-Me derivative (22) retained
activity against all tested cell lines except PC-3. (2) 50-Me (23)
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Table 2. Antiproliferative Activity of 1 and Analogues of 1a
GI50 (μM)b
Cmpd
HCT-8
PC-3
A549
DU145
MCF-7
HepG2
KB
KB-VIN
Group I
NT
1
NA
NA
NA
NA
NA
NA
13.51
1.07
0.77
0.58
17.44
0.27
19.90
1.56
NA
2
NA
NA
NA
NT
NA
NA
NA
3
1.03
1.16
23.26
0.62
17.41
3.35
NA
1.03
1.45
17.44
2.49
12.44
2.39
NA
1.55
1.73
23.26
0.50
17.41
1.48
NA
NT
1.55
1.73
NA
2.06
NT
1.55
0.29
18.60
0.47
19.90
1.67
NA
4
NT
5
NT
NA
6
NT
0.75
13.68
3.59
NA
1.24
16.67
1.48
NA
7
NT
8
NT
9
NT
10
11
12
35
NA
NT
NA
NT
NT
NT
NA
35.71
1.16
9.57
0.72
1.29
11.96
2.56
2.58
4.78
13.33
1.16
16.75
1.25
NT
1.42
8.37
NT
1.55
13.40
1.33
1.03
10.77
0.61
NT
NT
Group II
NT
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
36
37
12.85
13.09
2.27
12.85
5.24
12.85
26.18
1.52
17.20
13.96
>25
19.28
11.26
1.89
NA
14.91
11.78
1.52
15.42
18.32
1.26
14.91
17.02
1.14
14.55
9.90
>25
NT
1.52
NT
5.82
5.82
NT
15.08
15.74
>25
14.81
11.17
>25
19.04
>25
3.81
NT
15.23
NT
>25
>25
15.61
15.86
0.13
28.04
20.46
0.16
9.79
5.63
0.06
1.35
>98
NT
NT
17.72
21.74
0.13
4.50
2.09
2.81
0.07
0.76
>98
NT
17.39
0.11
NT
18.67
0.08
NT
2.70
90.69
>98
3.92
>98
2.03
0.61
93.14
42.19
19.58
3.66
NT
>98
53.92
26.37
11.32
0.76
37.97
51.89
21.46
3.81
42.19
66.04
3.17
5.84
>25
31.65
21.46
1.12
0.74
10.41
NT
NT
30.59
24.76
1.02
10.55
11.08
0.78
0.94
>25
NT
NT
11.17
>25
NT
1.90
2.49
>25
NT
>25
11.22
0.76
1.30
1.50
0.76
0.23
NT
23.73
0.57
0.65
0.09
1.08
0.91
NT
NT
0.11
Group III
NT
29
30
31
32
33
2.42
NA
6.04
NA
6.04
NA
3.62
NA
NA
NA
9.13
4.59
NA
NA
NA
NT
2.17
NA
1.93
NA
NA
NA
9.82
NT
NA
NA
NA
NT
NA
NA
NA
NA
NT
NA
12.79
13.70
18.26
NT
11.42
Group IV
NT
34
NA
8.45
18.12
15.70
19.32
18.12
12.08
Group V
NT
40a
41
11.69
8.17
14.68
16.58
24.05
10.79
NA
16.67
1.86
20.90
3.22
20.65
8.42
24.05
14.52
NA
11.19
1.41
7.38
9.13
6.41
5.93
0.004
9.70
2.70
5.95
9.96
11.97
3.87
9.091
NT
42
13.33
7.68
5.95
NT
14.05
11.00
24.57
NT
43
4.77
NT
44
13.89
9.28
25.64
7.99
NT
48
12.63
0.018
NT
9.02
NT
VCR
0.036
0.007
0.018
0.018
a NA: not active; test compound (20 μg/mL) did not reach 50% inhibition. NT: not tested. b Antiproliferative activity as GI50 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
colon adenocarcinoma (HCT-8), prostate cancer (PC-3 and DU145), lung carcinoma (A549), breast cancer (MCF-7), hepatocellular carcinoma
(HepG2), epidermoid carcinoma of the nasopharynx (KB), and MDR line overexpressing P-glycoprotein (KB-VIN).
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Figure 2. (A) Reversal of MDR selectivity of 24 by verapamil cotreat-
ment. (B) Reversal of MDR selectivity of 27 by verapamil co-treatment.
sensitivity” (CS),2 exhibiting over 2-fold greater cytotoxicity
against the MDR line (KB-VIN) than against the parental line
(KB). Figure 2 shows the reversal effects of co-treatment of 24 or
27 together with a nontoxic concentration of verapamil
(VERAP), a known P-gp modulator. Co-treatment of 24 and
27 with VERAP partially reversed the cytotoxic activity, showing
that the MDR selectivity was dependent in part on P-gp function
and consistent with the effect on P-gp activity measured using the
co-incubation treatment protocol. This result is consistent with
data obtained with analogues of 1 containing a phenyl B-ring.13
We observed that cells treated with cytotoxic concentrations
of these agents displayed a characteristic rounded shape that was
morphologically similar to the cells treated with colchicine, when
examined microscopically. We explored microtubules and spin-
dles in A549 cells treated with 21. The A549 cells were treated
with 21, colchicine, pacritaxel, doxorubicine, or DMSO for 24 h
followed by the immunofluorescence staining of tubulin using
monoclonal antibody to R-tubulin. As we expected, microtubule
polymerization and spindle formation were significantly inpaired by
treatment with 21, which was similar to the effects of colchicine and
clearly different from pacritaxel or doxorubicine (Figure 3). The
antimicrotubule activity of 21 was seen within 24 h at 0.1 nM that
was 1000-fold less than that of colchicine (100 nM).
Figure 3. Effect of compound 21 on microtubule assembly. A549 cells
were cultured and treated with agent for 24 h as indicated. Cells were
labeled immunocytochemically using antibody to R-tubulin (left panels)
and DAPI for DNA (right panels). The mitotic spindles were clearly
seen in control (DMSO) and pacritaxel (Tax) treated cells (arrow
heads) while undetectable in the cells treated with 21, colchicine (Col),
or doxorubicine (Dox). The aggregations of tubulin were seen in the
prophase cells treated with 21 or colchicine (arrow). In addition,
microtubules were undetectable in the cells treated with 21 or colchicine
but detectable in cells treated with DMSO, pacritaxel, or doxorubicine.
These observations clearly indicate that 21 inhibits tubulin polymeriza-
tion in the human tumor cells. Bar, 10 μm.
tubulin assembly with an IC50 value of 2.0 μM. It was also the
most active of these compounds as an inhibitor of colchicine
binding to tubulin, inhibiting the binding reaction by 78%. Its
trimethyl analogues 35-37 and cytotoxic analogues 3, 6, 8, 21,
22, and 26 also inhibited tubulin assembly with IC50 values of
2.3-3.7 μM, although these compounds were less active than 21
as inhibitors of colchicine binding. Compounds 11 and 15 were
We therefore evaluated selected active compounds for anti-
tubulin activities, and the results are shown in Table 3. All
cytotoxic compounds (GI50 values of less than 1 μg/mL) also
exhibited potent inhibition of tubulin assembly. Analogue 21,
which showed the most potent cytotoxicity, strongly inhibited
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Table 3. Inhibition of Tubulin Assemblya and Colchicine
Bindingb
compd
IC50 ( SD (μM)
inhibition of colchicine binding ( SD (%)
Group I
2
NA
3
2.3 ( 0.08
2.7 ( 0.1
2.7 ( 0.3
not obtainablec
2.5 ( 0.2
43 ( 10
50 ( 0.4
31 ( 3
6
8
11
35
29 ( 0.4
37 ( 5
Group II
33 ( 2
78 ( 5
38 ( 4
52 ( 0.6
36 ( 4
69 ( 3
99 ( 0.7
15
6.5 ( 0.7
2.0 ( 0.1
3.4 ( 0.3
3.4 ( 0.2
3.7 ( 0.09
2.4 ( 0.007
1.1 ( 0.1
Figure 4. Common pharmacophores of colchicine and CA-4. (A) Five
point pharmacophores, H2 (hydrophobic center), R1 (planar group),
A1, A2 (hydrogen bond acceptors), and D1 (hydrogen bond donor), for
colchicine and CA-4 by Nguyen.14 (B) Structures of colchicine and CA-
4 onto a 3D picture of the five pharmacophores by MOE. R1 is shown as
a hydrophobic/aromatic center in this model.
21
22
26
36
37
CA-4
the other three pharmacophore features, A2, A3, and H1, could
increase the potency of these tubulin-destabilizing agents target-
ing the colchicine site.
a The tubulin assembly assay measured the extent of assembly of 10 μM
tubulin after 20 min at 30 ꢀC. b Tubulin: 1 μM. [3H]colchicine: 5 μM.
Inhibitor: 5 μM. Incubation was for 10 min at 37 ꢀC. c Partial inhibition
observed and was maximal with 4 μM compound. Higher compound
concentrations resulted in the same amount of inhibition observed with
4 μM, suggesting poor solubility of 11 in the reaction mixture.
In summary, we discovered that analogues of 1 with bicyclic
aryl substituents at C-2 possessed promising antitumor activity,
with significant antiproliferative effects against HCT-8, PC-3,
A549, MCF-7, HepG2, KB, and KB-VIN tumor cells. Benzo-
[b]thiophene analogue 21 displayed the most potent inhibitory
effects on tumor cell growth, with GI50 values of 0.06-0.16 μM.
The activity of the compounds, including 21, was not influenced
by overexpression of P-gp (GI50 = 0.07 μM against KB-VIN vs
0.08 μM for the parental KB line). Furthermore, cytotoxicic
analogues 3, 6, 8, 11, 15, 21, 22, 26, and 35-37, which all possess
a bicyclic B-ring at C-2, displayed significant antitubulin activities
with 30-80% inhibition of binding to the colchicine site.
Compounds 3, 21, and 26 overlapped with four out of seven
pharmacophoric points that were derived from the binding
models of 15 selected CSIs. Thus, they are a new class of CSIs,
and further modifications would be useful to develop more
potent antitubulin agents. However, analogues of 1 with a phenyl
B-ring, including the natural product 1 itself, showed selective
cytotoxicity against KB-VIN cells relative to KB cells. Thus, the
results presented here indicate that the B-ring structure is
critically important in the interaction of this compound class
with P-gp. This distinction may well prove valuable in designing
more effective chemotherapeutic agents.
even less active, and 2 had no significant interaction with tubulin
at the highest concentration evaluated (40 μM). Thus, we
concluded that 21 is a potent tubulin polymerization inhibitor.
In 2005, Nguyen et al. studied 15 colchicine site inhibitiors
(CSIs) and proposed seven pharmacophoric points based on
consistent structural features and recurring ligand interactions.14
The seven points were three hydrogen bond acceptors (A1, A2,
and A3), one hydrogen bond donor (D1), two hydrophobic
centers (H1 and H2), and one planar group (R1). The H2 and
R1 points represented the rigid portion of the molecular scaffold,
while the other five points were important for binding specificity.
Nguyen et al.14 also mentioned that A2, H1, H2, and R1 emerged
as essential features for inhibitory activity at the colchicines site.
Most of the 15 CSIs contained five to six of the seven pharma-
cophoric points. For example, Figure 4A shows that the well-
known tubulin polymerization inhibitors, colchicine and CA-4,
map to five pharmacophoric points, A1, A2, D1, H2, and R1.
Figure 4B overlays the structures of colchicine and CA-4 onto a
3D picture of the five pharmacophores. We used Molecular
Operating Environment (MOE) software to search for the best-
fit 3D conformations of three of our active compounds, 3, 21 and
26, with the five pharmacophore features. The superpositions are
shown in Figure 5. The ring systems of 3, 21, and 26 matched
well with the planar center (R1) and hydrophobic center (H2),
which are critical portions for the molecular scaffold. In addition,
the hydrogen acceptor (A1) and hydrogen bond donor (D1)
pharmacophores overlapped with the oxygen at the 4-position
and hydroxyl group at the 5-position, respectively, of 3, 21, and
26. Compound 21 showed a better fit to the pharmacophore
points R1 and H2 than compounds 3 and 26, which could explain
the greater inhibitory activity of 21 against the colchicine binding
site. Another possible reason of its great potency is that the sulfur
atom in 21 might interact positively with the binding site. Further
structural modifications to enhance expression of one or more of
’ EXPERIMENTAL SECTION
All chemicals and solvents were used as purchased. All melting
points were measured on a Fisher-Johns melting point apparatus
1
without correction. H and 13C NMR spectra were recorded on a
Varian Gemini 2000 (300 MHz) or a Varian Inova (400 MHz) NMR
spectrometer with TMS as the internal standard. All chemical shifts
are reported in ppm. NMR spectra were referenced to the residual
solvent peak. Chemical shifts δ are in ppm, and apparent scalar
coupling constants J are in Hz. Mass spectroscopic data were obtained
on a Shimazu LC-MS2010 instrument. Analytical thin-layer chroma-
tography (TLC) was carried out on Merck precoated aluminum silica
gel sheets (Kieselgel 60 F-254). Biotage Flash or Isco Companion
systems were used for flash chromatography. All target compounds were
characterized and determined as at least >95% pure by 1H NMR, MS, and
elemental analyses or analytical HPLC.
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dx.doi.org/10.1021/jm1011947 |J. Med. Chem. 2011, 54, 1244–1255

Journal of Medicinal Chemistry
ARTICLE
Figure 5. Alignment of pharmacophore and compounds 3, 21, and 26: hydrophobic center (H2), planar group (R1), hydrogen bond acceptors (A1 and A2),
hydrogen bond donor (D1).
General Synthetic Procedures for 31. A solution of 30 in
EtOH-50% aqueous KOH (1:1, v/v) and an appropriate aromatic
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 and then extracted with CH2Cl2. The extract was washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
chromatographed on silica gel with CH2Cl2-hexane as eluent to afford
the target compound, which was crystallized from CH2Cl2-hexane.
General Synthetic Procedures for 3-37. Compound 39 was
dissolved separately in 1% H2SO4 in DMSO, then I2 (0.1 equiv mol) was
added and the reaction mixture heated at 90 ꢀC for 1 h. The reaction
mixture was treated in the same manner as described above to afford
compound 40, which was dissolved in anhydrous CH2Cl2. BBr3 (3 equiv
mol, 1.0 M solution in CH2Cl2) was added to the solution at 0 ꢀC, which
was warmed to room temperature spontaneously and stirred overnight.
After addition of water, the reaction mixture was extracted three times with
CH2Cl2. The extracts were combined, washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residues were chromatographed
on silica gel, eluting with EtOAc-hexane (1:4) to obtain analogues 5-37.
2-(Naphthalen-10-yl)-6,8,8-tripropyldesmosdumotin B (5). 1H
NMR (300 MHz, CDCl3): δ 13.07 (s, 1H, chelated-OH), 8.09 (d, 1H,
J = 7.9 Hz, 50-H), 8.01-7.97 (m, 1H, 40- or 80-H), 7.92-7.86 (m, 1H, 40
or 80-H), 7.70-7.58 (m, 4H, J = 7.3 Hz, 20-, 30-, 60-, and 70-H), 6.80 (s,
1H, 3-H), 2.43 (t, 2H, J = 7.5 Hz, 6-CH2CH2CH3), 2.12 (dt, 2H, J = 12.5
and 4.3 Hz, 8-CH2CH2CH3), 1.83 (dt, 2H, J = 12.5 and 4.3 Hz, 8-CH2-
CH2CH3), 1.49 (q, 2H, J = 7.7 Hz, 6-CH2CH2CH3), 1.24-1.11 (m, 2H,
8-CH2CH2CH3), 1.10-0.50 (m, 2H, 8-CH2CH2CH3), 0.96 (t, 3H, J =
7.7 Hz, 6-CH2CH2CH3), 0.82 (t, 6H, J = 7.5 Hz, 8-CH2CH3 ꢀ 2). MS
(ESIþ) m/z: 431 (Mþ þ 1). Anal. (C28H30O4) C, H, O.
CH3), 2.20-2.06 (m, 2H, 8-CH2CH3), 1.88-1.74 (m, 2H, 8-CH2-
CH3), 1.07 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.69 (t, 6H, J = 7.3 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 403 (Mþ þ 1). Anal. (C26H26-
O4 1/4H2O) C, H, O.
3
2-(40-Methoxynaphthalen-10-yl)-6,8,8-triethyldesmosdu-
motin B (8). 1H NMR (300 MHz, CDCl3): δ 13.19 (s, 1H, chelated-
OH), 8.40 (dd, 1H, J = 7.3 and 2.2 Hz, 50-H), 7.94 (dd, 1H, J = 7.3 and
2.2 Hz, 80-H), 7.65 (d, 1H, J = 8.0 Hz, 20-H), 7.65-7.56 (m, 2H, 60 and
70-H), 6.93 (d, 1H, J = 8.0 Hz, 30-H), 6.78 (s, 1H, 3-H), 4.10 (s, 3H,
-OCH3), 2.48 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.24-2.12 (m, 2H,
8-CH2CH3), 1.99-1.84 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.4 Hz,
6-CH2CH3), 0.72 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z:
419 (Mþ þ 1). Anal. (C26H26O5) C, H, O.
2-(40,70-Dimethoxynaphthalen-10-yl)-6,8,8-triethyldes-
mosdumotin B (9). 1H NMR (300 MHz, CDCl3): δ 13.22 (s, 1H,
chelated-OH), 8.31 (d, 1H, J = 10.0 Hz, 50-H), 7.59 (d, 1H, J = 8.2 Hz,
20-H), 7.24 (d, 1H, J = 10.0 Hz, 60-H), 7.23 (s, 1H, 20-H), 6.80 (d, 1H, J =
8.2 Hz, 30-H), 6.77 (s, 1H, 3-H), 4.07 (s, 3H, -OCH3), 3.89 (s, 3H,
-OCH3), 2.48 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.27-2.13 (m, 2H,
8-CH2CH3), 2.01-1.87 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.4 Hz,
6-CH2CH3), 0.71 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z:
449 (Mþ þ 1). Anal. (C27H28O6 1/4H2O) C, H, O.
3
2-(20,30-Dimethoxynaphthalen-10-yl)-6,8,8-triethyldes-
mosdumotin B (10). 1H NMR (300 MHz, CDCl3): δ 13.09 (s, 1H,
chelated-OH), 7.81 (d, 1H, J = 8.2 Hz, 50-H), 7.54-7.38 (m, 3H, 60,70
and 80-H), 7.49 (s, 1H, 40-H), 6.67 (s, 1H, 3-H), 4.05 (s, 3H, -OCH3),
3.89 (s, 3H, -OCH3), 2.49 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.20-2.06
(m, 2H, 8-CH2CH3), 1.92-1.78 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J =
7.4 Hz, 6-CH2CH3), 0.69 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS
(ESIþ) m/z: 449 (Mþ þ 1). Anal. (C27H28O6 H2O) C, H, O.
2-(40-Methylnaphthalen-10-yl)-6,8,8-triethyldesmosdumo-
tin B (6). 1H NMR (300 MHz, CDCl3): δ 13.11 (s, 1H, chelated-OH),
8.17-8.12 (m, 1H, 50-H), 7.97-7.92 (m, 1H, 80-H), 7.70-7.56 (m, 2H,
60- and 70-H), 7.58 (d, 1H, J = 7.3 Hz, 20-H), 7.46 (d, 1H, J = 7.3 Hz,
30-H), 6.79 (s, 1H, 3-H), 2.80 (s, 3H, 40-CH3), 2.48 (q, 2H, J = 7.3 Hz,
6-CH2CH3), 2.26-2.12 (m, 2H, 8-CH2CH3), 1.96-1.84 (m, 2H,
8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.72 (t, 6H, J = 7.3
3
2-(Naphthalen-20-yl)-6,8,8-triethyldesmosdumotin B (11). 1H
NMR (300 MHz, CDCl3):δ13.10 (s, 1H, chelated-OH),8.33(d, 1H,J=1.8
Hz, 10-H), 8.04-7.96 (m, 2H, 50- and 80-H), 7.96-7.90 (m, 1H, 40-H), 7.80
(dd, 1H, J = 1.8 and 8.5 Hz, 30-H), 7.69-7.60 (m, 2H, 60- and 70-H), 7.04 (s,
1H, 3-H), 2.48 (q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.38-2.24 (m, 2H,
8-CH2CH3), 2.15-2.01 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz,
6-CH2CH3), 0.71 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 389
(Mþ þ 1). Anal. (C25H24O4) C, H, O.
Hz, 8-CH2CH3 ꢀ 2). Anal. (C26H26O4 1/4H2O) C, H, O.
3
2-(20-Methylnaphthalen-10-yl)-6,8,8-triethyldesmosdu-
motin B (7). 1H NMR (300 MHz, CDCl3): δ 13.05 (s, 1H, chelated-
OH), 7.96 (d, 1H, J = 8.7 Hz, 40-H), 7.94-7.88 (m, 1H, 50-H),
7.58-7.48 (m, 3H, 60-, 70-, and 80-H), 7.46 (d, 1H, J = 8.7 Hz, 30-H),
6.64 (s, 1H, 3-H), 2.49 (q, 2H, J = 7.5 Hz, 6-CH2CH3), 2.46 (s, 3H, 20
2-(60-Methoxynaphthalen-20-yl)-6,8,8-triethyldesmosdumo-
tin B (12). 1H NMR (300 MHz, CDCl3): δ 13.18 (s, 1H, chelated-
OH), 8.24 (d, 1H, J = 1.8 Hz, 10-H), 7.88 (d, 2H, J = 8.7 Hz, 40 and 80-H),
7.76 (dd, 1H, J = 8.7 and 1.8 Hz, 30-H), 7.27 (dd, 1H, J = 8.7 and 1.8 Hz,
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Journal of Medicinal Chemistry
ARTICLE
70-H), 7.19 (d, 1H, J= 1.8 Hz, 50-H), 6.99 (s, 1H, 3-H), 3.98(s, 3H, -OCH3),
2.47 (q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.38-2.23 (m, 2H, 8-CH2CH3),
2.14-2.00 (m, 2H, 8-CH2CH3), 1.05 (t, 3H, J=7.3Hz,6-CH2CH3),0.70(t,
6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). Anal. (C26H26O5) C, H, O.
Hz, 6-CH2CH3), 2.36-2.21 (m, 2H, 8-CH2CH3), 2.10-1.96 (m, 2H,
8-CH2CH3), 1.05 (t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.4 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 392 (Mþ þ 1). Anal. (C24H25O4N)
C, H, O.
6,8,8-Triethyl-2-(quinolin-30-yl)desmosdumotin B (13). 1H
NMR (300MHz, CDCl3): δ 12.88 (s, 1H, chelated-OH), 9.29(d, 1H, J =
2.2 Hz, 20-H), 8.56 (d, 1H, J = 2.2 Hz, 40-H), 8.21 (d, 1H, J = 8.5 Hz,
50-H), 8.00 (d, 1H, J = 7.4 Hz, 80-H), 7.94-7.86 (m, 1H, 70-H), 7.72 (dd,
1H, J = 8.5 and 7.4 Hz, 60-H), 7.09 (s, 1H, 3-H), 2.48 (q, 2H, J = 7.3 Hz,
6-CH2CH3), 2.39-2.25 (m, 2H, 8-CH2CH3), 2.12-1.99 (m, 2H,
8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.71 (t, 6H, J = 7.3
Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 390 (Mþ þ 1). Anal.
2-(Benzo[b]thiophen-30-yl)-6,8,8-triethyldesmosdumotin
B (21). 1H NMR (300 MHz, CDCl3): δ 13.08 (s, 1H, chelated-OH),
8.12-8.06 (m, 1H, 40-H), 8.07 (s, 1H, 20-H), 8.02-7.96 (m, 1H, 70-H),
7.61-7.48 (m, 2H, 50- and 60-H), 6.94 (s, 1H, 3-H), 2.47 (q, 2H, J = 7.3
Hz, 6-CH2CH3), 2.36-2.08 (m, 2H, 8-CH2CH3), 2.07-1.93 (m, 2H,
8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.71 (t, 6H, J = 7.3 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 395 (Mþ þ 1). Anal. (C23H22
O4S 1/8H2O) C, H, O.
3
(C24H23O4N 1/8H2O) C, H, O.
2-(20-Methylbenzo[b]thiophen-30-yl)-6,8,8-triethyldesmos-
dumotin B (22). 1H NMR (400 MHz, CDCl3): δ 13.08 (1H,
chelated-OH), 7.86-7.82 (m, 1H, 40-H), 7.72-7.68 (m, 1H, 70-H),
7.48-7.38 (m, 2H, 50- and 60-H), 6.67 (s, 1H, 3-H), 2.97 (s, 3H, 20-
CH3), 2.48 (q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.28-2.16 (m, 2H,
8-CH2CH3), 1.98-1.86 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz,
6-CH2CH3), 0.70 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z
409 (Mþ þ 1). HPLC.
3
2-(Benzo[d][10,30]dioxol-50-yl)-6,8,8-triethyldesmosdumotin
B (14). 1H NMR (300 MHz, CDCl3): δ 13.13 (s, 1H, chelated-OH), 7.37
(dd, 1H, J = 8.2 and 1.8 Hz, 60-H), 7.20 (d, 1H, J = 1.8 Hz, 40-H), 6.97 (d,
1H, J = 8.2 Hz, 70-H), 6.76 (s, 1H, 3-H), 6.12 (s, 2H, 20-CH2-), 2.45 (q, 2H,
J = 7.3 Hz, 6-CH2CH3), 2.32-2.17 (m, 2H, 8-CH2CH3), 2.03-1.90 (m,
2H, 8-CH2CH3), 1.04 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.67 (t, 6H, J = 7.3
Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 383 (Mþ þ 1). Anal.
(C22H22O6 1/4H2O) C, H, O.
2-(50-Methylbenzo[b]thiophen-30-yl)-6,8,8-triethyldesmos-
dumotin B (23). 1H NMR (400 MHz, CDCl3): δ 13.09 (1H,
chelated-OH), 8.03 (s, 1H, 20-H), 7.89 (br s, 1H, 40-H), 7.84 (d, 1H,
J = 8.4 Hz, 70-H), 7.34 (dd, 1H, J = 0.98 and 8.4 Hz, 60-H), 6.93 (s, 1H,
3-H), 2.54 (s, 3H, 50-CH3), 2.48 (q, 2H, J = 7.4 Hz, 6-CH2CH3),
2.34-2.22 (m, 2H, 8-CH2CH3), 2.06-1.95 (m, 2H, 8-CH2CH3), 1.06
(t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.72 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2).
MS (ESIþ) m/z 409 (Mþ þ 1). Anal. (C24H24O4S) C, H, O.
2-(50-Bromobenzo[b]thiophen-30-yl)-6,8,8-triethyldesmos-
dumotin B (24). 1H NMR (400 MHz, CDCl3): δ 12.97 (1H,
chelated-OH), 8.25 (d, 1H, J = 1.9 Hz, 40-H), 8.07 (s, 1H, 20-H), 7.84
(d, 1H, J = 8.8 Hz, 70-H), 7.61 (dd, 1H, J = 1.9 and 8.8 Hz, 60-H), 6.88 (s,
1H, 3-H), 2.47 (q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.37-2.24 (m, 2H,
8-CH2CH3), 2.05-1.94 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.3 Hz,
6-CH2CH3), 0.71 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z
473 and 475 (Mþ þ 1). Anal. (C23H21BrO4S) C, H, O.
0 3
2-(20,3 -Dihydrobenzo[d][10,40]dioxin-60-yl)-6,8,8-triethyl-
desmosdumotin B (15). 1H NMR (300 MHz, CDCl3): δ 13.17 (s,
1H, chelated-OH), 7.34-7.28 (m, 2H, 50- and 70-H), 7.02 (d, 1H, J = 9.2
Hz, 80-H), 6.77 (s, 1H, 3-H), 4.40-4.30 (m, 4H, -OCH2CH2O-), 2.45
(q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.31-2.16 (m, 2H, 8-CH2CH3),
2.05-1.91 (m, 2H, 8-CH2CH3), 1.04 (t, 3H, J = 7.3 Hz, 6-CH2CH3),
0.66 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 397 (Mþ þ
1). Anal. (C23H24O6 1/4H2O) C, H, O.
30
2-(Benzofuran-2 -yl)-6,8,8-triethyldesmosdumotin B (16).
1H NMR (300 MHz, CDCl3): δ 12.98 (1H, chelated-OH), 7.71 (d, 1H,
J = 7.7 Hz, 40-H), 7.60 (d, 1H, J = 7.9 Hz, 70-H), 7.49 (dd, 1H, J = 7.9 and
7.2 Hz, 60-H), 7.43 (s, 1H, 30-H), 7.36 (dd, 1H, J = 7.7 and 7.4 Hz, 50-H),
7.01 (s, 1H, 3-H), 2.46 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.34-2.18 (m,
2H, 8-CH2CH3), 2.06-1.92 (m, 2H, 8-CH2CH3), 1.04 (t, 3H, J = 7.4 Hz,
6-CH2CH3), 0.69 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS m/z 351 (Mþ
- 1). MS (ESIþ) m/z: 379 (Mþ þ 1). Anal. (C23H22O5) C, H, O.
2-(Benzo[b]thiophen-20-yl)-6,8,8-triethyldesmosdumotin
B (17). 1H NMR (300 MHz, CDCl3): δ 12.96 (s, 1H, chelated-OH),
7.94-7.88 (m, 2H, 40- and 70-H), 7.91 (s, 1H, 30-H), 7.52-7.46 (m, 2H,
50- and 60-H), 6.83 (s, 1H, 3-H), 2.46 (q, 2H, J = 7.4 Hz, 6-CH2CH3),
2.34-2.21 (m, 2H, 8-CH2CH3), 2.10-1.96 (m, 2H, 8-CH2CH3), 1.05
(t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2).
MS (ESIþ) m/z: 395 (Mþ þ 1). Anal. (C23H22O4S) C, H, O.
2-(30-Methylbenzo[b]thiophen-20-yl)-6,8,8-triethyldesmos-
dumotin B (18). 1H NMR (400 MHz, CDCl3): δ 13.00 (1H,
chelated-OH), 7.91-7.82 (m, 2H, Ar-H), 7.55-7.45 (m, 2H, Ar-H),
6.82 (s, 1H, 3-H), 2.72 (s, 3H, 30-CH3), 2.46 (q, 2H, J = 7.4 Hz,
6-CH2CH3), 2.32-2.20 (m, 2H, 8-CH2CH3), 2.07-1.95 (m, 2H,
8-CH2CH3), 1.05 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.3
Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z 409 (Mþ þ 1). Anal.
(C24H24O4S) C, H, O.
2-(50-Methoxybenzo[b]thiophen-40-yl)-6,8,8-triethyldesmo-
sdumotin B (25). 1H NMR (400 MHz, CDCl3): δ 13.19 (1H,
chelated-OH), 8.00 (d, 1H, J = 8.8 Hz, Ar-H), 7.63 (d, 1H, J = 5.6 Hz,
Ar-H), 7.25 (d, 1H, J = 5.6 Hz, Ar-H), 7.16 (d, 1H, J = 8.8 Hz, Ar-H), 6.75
(s, 1H, 3-H), 3.92 (s, 3H, 50-OCH3), 2.47 (q, 2H, J = 7.4 Hz, 6-CH2CH3),
2.20-2.10 (m, 2H, 8-CH2CH3), 1.94-1.85 (m, 2H, 8-CH2CH3), 1.06 (t,
3H, J = 7.4 Hz, 6-CH2CH3), 0.69 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS
(ESIþ) m/z 425 (Mþ þ 1). Anal. (C24H24O5S) C, H, O.
2-(50-Hydroxybenzo[b]thiophen-40-yl)-6,8,8-triethyldesmo-
sdumotin B (26). 1H NMR (400 MHz, CDCl3): δ 13.11 (1H,
chelated-OH), 7.90 (d, 1H, J = 8.8 Hz, Ar-H), 7.82 (br s, 1H, 50-OH),
7.63 (d, 1H, J = 5.6 Hz, Ar-H), 7.32 (d, 1H, J = 5.6 Hz, Ar-H), 7.14 (d, 1H,
J = 8.8 Hz, Ar-H), 7.12 (s, 1H, 3-H), 2.49 (q, 2H, J = 7.4 Hz, 6-CH2CH3),
2.25-2.14 (m, 2H, 8-CH2CH3), 2.02-1.91 (m, 2H, 8-CH2CH3), 1.07 (t,
3H, J = 7.4 Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS
(ESIþ) m/z 411 (Mþ þ 1). Anal. (C23H22O5S) C, H, O.
2-(Benzofuran-30-yl)-6,8,8-triethyldesmosdumotin B (19).
1H NMR (400 MHz, CDCl3): δ 13.03 (s, 1H, chelated-OH), 8.23 (s,
1H, 20-H), 7.90-7.84 (m, 1H, 40- or 70-H), 7.68-7.63 (m, 1H, 40- or
70-H), 7.53-7.42 (m, 2H, 50- and 60-H), 6.89 (s, 1H, 3-H), 2.5-2.42 (m,
2H, 6-CH2CH3), 2.38-2.24 (m, 2H, 8-CH2CH3), 2.06-1.94 (m, 2H,
8-CH2CH3), 1.05 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.71 (t, 6H, J = 7.3 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 379 (Mþ þ 1). Elemental analysis
results are not available because of limited quantity.
2-(Benzo[b]thiophen-70-yl)-6,8,8-triethyldesmosdumotin
B (27). 1H NMR (400 MHz, CDCl3): δ 13.10 (1H, chelated-OH),
8.06 (dd, 1H, J = 7.8 and 0.8 Hz, 40-H), 7.79 (d, 1H, J = 7.6 Hz 60-H),
7.62 (d, 1H, J = 5.6 Hz, 20-H), 7.57 (dd, 1H, J = 7.8 and 7.6 Hz, 50-H),
7.50 (d, 1H, J = 5.6 Hz, 30-H), 7.12 (s, 1H, 3-H), 2.48 (q, 2H, J = 7.3 Hz,
6-CH2CH3), 2.34-2.18 (m, 4H, 8-CH2CH3 ꢀ 2), 1.06 (t, 3H, J = 7.3
Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ)
m/z 395 (Mþ þ 1). HPLC
2-(10-Methyl-1H-indol-30-yl)-6,8,8-triethyldesmosdumo-
tin B (20). 1H NMR (300 MHz, CDCl3): δ 13.61 (1H, chelated-OH),
8.00-7.93 (m, 1H, 40-H), 7.70 (s, 1H, 20-H), 7.48-7.34 (m, 3H, 50-, 60-,
and 70-H), 6.79 (s, 1H, 3-H), 3.94 (s, 3H, N-CH3), 2.46 (q, 2H, J = 7.4
2-(Benzo[b]thiophen-50-yl)-6,8,8-triethyldesmosdumotin
B (28). 1H NMR (400 MHz, CDCl3): δ 13.10 (1H, chelated-OH),
8.26 (d, 1H, J = 1.9 Hz, 40-H), 8.05 (d, 1H, J = 8.5 Hz, 70-H), 7.72 (dd,
1252
dx.doi.org/10.1021/jm1011947 |J. Med. Chem. 2011, 54, 1244–1255

Journal of Medicinal Chemistry
ARTICLE
1H, J = 1.9 and 8.5 Hz, 60-H), 7.62 (d, 1H, J = 5.4 Hz, 20-H), 7.49 (d, 1H,
J = 5.4 Hz, 30-H), 6.98 (s, 1H, 3-H), 2.46 (q, 2H, J = 7.3 Hz, 6-CH2CH3),
2.36-2.24 (m, 2H, 8-CH2CH3), 2.10-2.00 (m, 2H, 8-CH2CH3), 1.05
(t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.70 (t, 6H, J = 7.3 Hz, 8-CH2CH3 ꢀ 2).
MS (ESIþ) m/z 395 (Mþ þ 1).
(s, 1H, chelated-OH), 7.34-7.30 (m, 2H, Ar-H), 7.03-7.00 (m, 1H,
Ar-H), 6.77 (s, 1H, 3-H), 4.40-4.30 (m, 4H, -OCH2CH2O-), 1.86 (s,
3H, 6-CH3), 1.56 (s, 6H, 8-CH3 ꢀ 2). MS (ESIþ) m/z: 355 (Mþ þ 1).
Anal. (C20H18O6 1/2H2O) C, H, O.
3
2-(Benzo[b]thiophen-30-yl)-6,8,8-trimethyldesmosdumo-
tin B (37). 1H NMR (400 MHz, CDCl3): δ 13.08 (s, 1H, chelated-
OH), 8.11 (d, 1H, J = 8.1 Hz, 40- or 70-H), 8.07 (s, 1H, 20-H), 7.98 (d, 1H,
J = 8.1 Hz, 40- or 70-H), 7.60-7.48 (m, 2H, 50- and 60-H), 6.92 (s, 1H,
3-H), 1.90 (s, 3H, 6-CH3), 1.60 (s, 6H, 8-CH3 ꢀ 2). MS (ESIþ) m/z:
2-(10,20-Dihydroacenaphthylen-50-yl)-6,8,8-triethyldesmos-
dumotin B (29). 1H NMR (300 MHz, CDCl3): δ 13.21 (1H,
chelated-OH), 7.81 (d, 1H, J = 8.2 Hz, 80-H), 7.72 (d, 1H, J = 7.5 Hz,
40-H), 7.61 (dd, 1H, J = 8.2 and 7.5 Hz, 70-H), 7.46 (d, 1H, J = 7.5 Hz, 30
or 60-H), 7.42 (d, 1H, J = 7.5 Hz, 30- or 60-H), 6.88 (s, 1H, 3-H), 3.49 (s,
4H, -CH2- ꢀ 2), 2.48 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.30-2.15 (m,
2H, 8-CH2CH3), 2.04-1.90 (m, 2H, 8-CH2CH3), 1.06 (t, 3H, J = 7.4
Hz, 6-CH2CH3), 0.72 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ)
m/z: 415 (Mþ þ 1). Anal. (C27H26O4) C, H, O.
351 (Mþ - 1). Anal. (C20H16O4S 1/4H2O) C, H, O.
3
6,8,8-Triethyl-7-methoxy-2-(naphthalen-10-yl)-4H-chro-
mene-4,5(8H)-dione (40a). 40a was prepared according to the
previously reported procedure.1b 1H NMR (300 MHz, CCDCl3): δ
8.04 (d, 1H, J = 8.2 Hz, Ar-H), 8.00-7.92 (m, 2H, Ar-H), 7.67-7.54 (m,
4H, Ar-H), 6.72 (s, 1H, 3-H), 4.00 (s, 3H, OCH3), 2.60 (q, 2H, J = 7.3
Hz, 6-CH2CH3), 2.16-2.00 (m, 2H, 8-CH2CH3), 2.24-1.89 (m, 2H,
8-CH2CH3), 1.18 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.76 (t, 6H, J = 7.3 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 403 (Mþ þ 1). Anal. (C26H26O4) C,
H, O.
2-(90-Ethyl-90H-carbazol-20-yl)-6,8,8-triethyldesmosdumotin
B (30). 1H NMR (300 MHz, CDCl3): δ 13.31 (1H, chelated-OH), 8.47
(d, 1H, J = 1.8 Hz), 8.29 (d, 1H, J = 1.8 Hz), 7.91 (dd, 1H, J = 1.8 and 8.7
Hz), 7.65 (dd, 1H, J = 1.8 and 8.7 Hz), 7.55 (d, 1H, J = 8.7 Hz), 7.37 (d, 1H,
J=8.7 Hz), 6.97 (s, 1H, 3-H), 4.42 (q, 2H, J=7.3 Hz, N-CH2CH3), 2.46 (q,
2H, J = 7.4 Hz, 6-CH2CH3), 2.40-2.27 (m, 2H, 8-CH2CH3), 2.16-2.03
(m, 2H, 8-CH2CH3), 1.48 (t, 3H, J = 7.3 Hz, N-CH2CH3), 1.06 (t, 3H, J =
7.4 Hz, 6-CH2CH3), 0.72 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ)
m/z: 455 (Mþ þ 1). Elemental analysis results are not available because of
limited quantity.
2-(Naphthalen-1-yl)-4-thioxo-6,8,8-triethyldesmosdumo-
tin B (41). Lawesson’s reagent (48 mg, 0.12 mmol) was added to a
solution of 3 (40 mg, 0.10 mmol) in toluene (1.5 mL). After the mixture
was refluxed for 6.5 h, the volatile solvent was removed in vacuo. The
residue was purified by SiO2 column chromatography (EtOAc/hexane,
gradient) to obtain 41 (20 mg, 50%) and 42 (3 mg, 7%) along with
starting material (8 mg, 20%). 1H NMR (300 MHz, CDCl3): δ 13.50
(1H, chelated-OH), 8.10 (d, 1H, J = 7.9 Hz, 50-H), 8.02-7.95 (m, 2H,
30- and 80-H), 7.73 (dd, 1H, J = 7.2 and 1.1 Hz, 20-H), 7.66-7.60 (m, 3H,
40-, 60-, and 70-H), 7.62 (s, 1H, 3-H), 2.52 (q, 2H, J = 7.4 Hz,
6-CH2CH3), 2.31-2.07 (m, 2H, 8-CH2CH3), 2.01-1.86 (m, 2H,
8-CH2CH3), 1.07 (t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.74 (t, 6H, J = 7.4
Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z 405 (Mþ þ 1). Anal.
(C25H24O3S) C, H, O.
2-(Dibenzo[b,d]furan-40-yl)-6,8,8-triethyldesmosdumotin B
(31). 1H NMR (300 MHz, CDCl3): δ 13.14 (s, 1H, chelated-OH),
8.18 (dd, 1H, J = 7.8 and 1.4 Hz, 90-H), 8.03 (dd, 1H, J = 6.9 and 1.4 Hz,
10-H), 7.94 (dd, 1H, J = 7.8 and 1.4 Hz, 60-H), 7.73 (s, 1H, 3-H), 7.70 (d,
1H, J = 8.2 Hz, 30-H), 7.62-7.42 (m, 3H, 20-, 70-, and 80-H), 2.48 (q, 2H,
J = 7.3 Hz, 6-CH2CH3), 2.38-2.24 (m, 2H, 8-CH2CH3), 2.18-2.04 (m,
2H, 8-CH2CH3), 1.07 (t, 3H, J = 7.3 Hz, 6-CH2CH3), 0.72 (t, 6H, J = 7.3
Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 429 (Mþ þ 1). Anal. (C27H24-
O5 1/2H2O) C, H, O.
3
2-(Anthracen-9-yl)- 6,8,8-triethyldesmosdumotin B (32). 1H
NMR (300 MHz, CDCl3): δ 13.06 (1H, chelated-OH), 8.70 (s, 1H, 100-
H), 8.17-8.09 (m, 2H, 50- and 80-H, or 10- and 30-H), 7.83-7.75 (m, 2H,
50- and 80-H, or 10- and 30-H), 7.61-7.54 (m, 4H, 20-, 30-, 60-, and 70-H),
6.83 (s, 1H, 3-H), 2.51 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.18-2.04 (m, 2H,
8-CH2CH3), 1.84-1.70 (m, 2H, 8-CH2CH3), 0.88 (t, 3H, J = 7.4 Hz,
6-CH2CH3), 0.74 (t, 6H, J=7.4Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z:439
4,7-Dithioxo-2-(naphthalen-1-yl)-6,8,8-triethyldesmosdu-
motin B (42). 1H NMR (300 MHz, CDCl3): δ 14.30 (1H, chelated-
OH), 8.11 (d, 1H, J = 8.2 Hz, 50-H), 8.03-7.94 (m, 2H, 30- and 80-H),
7.76 (dd, 1H, J = 7.4 and 1.3 Hz, 20-H), 7.67-7.57 (m, 3H, 40-, 60-,
and 70-H), 7.62 (s, 1H, 3-H), 2.99 (q, 2H, J = 7.4 Hz, 6-CH2CH3),
2.58-2.42 (m, 2H, 8-CH2CH3), 2.30-2.16 (m, 2H, 8-CH2CH3),
1.10 (t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.64 (t, 6H, J = 7.4 Hz,
8-CH2CH3 ꢀ 2). MS (ESIþ) m/z 421 (Mþ þ 1). Anal. (C25H24-
(Mþ þ 1). Anal. (C29H26O4 1/2H2O) C, H, O.
3
2-(Phenanthren-90-yl)-6,8,8-triethyldesmosdumotin B
(33). 1H NMR (300 MHz, CDCl3): δ 13.06 (1H, chelated-OH),
8.86-8.72 (m, 2H, 40- and 50-H), 8.04-7.94 (m, 2H, 10- and 80-H),
7.92-7.62 (m, 5H, 20-, 30-, 60-, 70-, and 100-H), 6.89 (s, 1H, 3-H), 2.49 (q,
2H, J = 7.4 Hz, 6-CH2CH3), 2.24-2.11 (m, 2H, 8-CH2CH3),
1.98-1.83 (m, 2H, 8-CH2CH3), 1.07 (t, 3H, J = 7.4 Hz, 6-CH2CH3),
0.75 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z: 439 (Mþ þ 1).
Anal. (C29H26O4 1/2H2O) C, H, O.
O2S2 H2O) C, H, O.
3
3-Bromo-6,8,8-triethyl-7-methoxy-2-(naphthalen-10-yl)-4H-
chromene-4,5(8H)-dione (43). PhI(OAc)2 (280 mg, 0.87 mmol)
was suspended in anhydrous CH2Cl2 (1.5 mL) under argon at room
temperature. Bu4NBr (281 mg, 0.87 mmol) was added, and the mixture
was stirred for 30 min. 40a (64 mg, 0.16 mmol) in anhydrous CH2Cl2
(1.0 mL) was added, and the mixture was stirred at room temperature for
4 days. The reaction was quenched with saturated aqueous NH4Cl and
extracted with CH2Cl2. The organic layer was dried over Na2SO4, and
the solvent was evaporated in vacuo. The residue was purified by SiO2
column chromatography (EtOAc/hexane, gradient) to obtain 43 (22
mg, 29%). 1H NMR (300 MHz, CCDCl3): δ 8.07 (dd, 1H, J = 2.1 and
7.4 Hz, 50-H), 8.01-7.96 (m, 1H, Ar-H), 7.68-7.52 (m, 5H, Ar-H), 3.99
(s, 3H, OCH3), 2.61 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.10-1.95 (m, 2H,
8-CH2CH3), 1.90-1.78 (m, 2H, 8-CH2CH3), 1.17 (t, 3H, J = 7.3 Hz,
6-CH2CH3), 0.73 (br s, 6H, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z 481 and
483 (Mþ þ 1). Anal. (C26H25 BrO4) C, H, O.
0 3
2-(Biphen-4 -yl)-6,8,8-triethyldesmosdumotin B (34). 1H
NMR (300 MHz, CDCl3): δ 13.08 (1H, chelated-OH), 7.88 (d, 2H, J =
7.8 Hz, 20- and 60-H), 7.78 (d, 2H, J = 7.8 Hz), 7.64 (d, 2H, J = 8.2 Hz),
7.55-7.43 (m, 3H, 80-, 90-, and 100-H), 6.95 (s, 1H, 3-H), 2.46 (q, 2H, J =
7.4 Hz, 6-CH2CH3), 2.36-2.21 (m, 2H, 8-CH2CH3), 2.10-1.96
(m, 2H, 8-CH2CH3), 1.05 (t, 3H, J = 7.4 Hz, 6-CH2CH3), 0.69 (t, 6H,
J = 7.4 Hz, 8-CH2CH3 ꢀ 2). Anal. (C27H26O4) C, H, O.
2-(40-Methylnaphthalen-10-yl)-6,8,8-trimethyldesmosdu-
motin B (35). 1H NMR (400 MHz, CDCl3): δ 13.12 (s, 1H, chelated-
OH), 8.16-8.12 (m, 1H, 50-H), 8.00-7.96 (m, 1H, naphthyl-H),
7.68-7.57 (m, 3H, naphthyl-H), 7.45 (d, 1H, J = 7.3 Hz, naphthyl-H),
6.79 (s, 1H, 3-H), 2.80 (s, 3H, 40-CH3), 1.90 (s, 3H, 6-CH3), 1.53 (s, 6H,
8-CH3 ꢀ 2). Anal. (C23H20O4) C, H, O.
3-Bromo-2-(naphthalen-10-yl)-6,8,8-triethyldesmosdumotin
B (44). To a solution of 43 (18 mg, 0.038 mmol) in anhydrous CH2Cl2
(1.0 mL), BBr3 (0.1 mL, 0.1 mmol, 1.0 M solution in CH2Cl2) was
added at -78 ꢀC under N2. The mixture was stirred overnight at -78 to
0 ꢀC. The reaction mixture was quenched with water and extracted with
CH2Cl2. The organic phase was dried over Na2SO4, and concentrated.
2-(20,30-Dihydrobenzo[d][10,40]dioxin-60-yl)-6,8,8-trimethyl-
desmosdumotin B (36). 1H NMR (400 MHz, CDCl3): δ 13.22
1253
dx.doi.org/10.1021/jm1011947 |J. Med. Chem. 2011, 54, 1244–1255

Journal of Medicinal Chemistry
ARTICLE
The residue was purified by SiO2 column chromatography (EtOAc/
hexane, gradient) to obtain 44 (13 mg, 73%). H NMR (300 MHz,
carcinoma), MCF-7 (breast cancer), HCT-8 (colon adenocarcinoma),
PC-3 (prostate cancer), KB (nasopharyngeal carcinoma), and KB-VIN (vinc-
ristine-resistant KB subline). All cell lines were obtained from the Lineberger
Cancer Center (UNC-CH) or from ATCC (Manassas, VA) except KB-VIN,
which was a generous gift of Professor Y.-C. Cheng, Yale University, CT. Cells
were cultured in RPMI-1640 medium supplemented with 25 mM HEPES,
0.25% sodium bicarbonate, 10% fetal bovine serum, and 100 μg/mL
kanamycin.
1
CDCl3): δ 12.63 (1H, chelated-OH), 8.14-8.08 (m, 1H, 50-H),
8.03-7.98 (m, 1H, 80-H), 7.71-7.52 (m, 5H, 20-, 30-, 40-, 60-, and
70-H), 2.49 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.20-2.07 (m, 2H,
8-CH2CH3), 1.87-1.72 (m, 2H, 8-CH2CH3), 1.07 (t, 3H, J = 7.4 Hz,
6-CH2CH3), 0.74 (t, 6H, J = 7.4 Hz, 8-CH2CH3 ꢀ 2). MS (ESIþ) m/z
467 and 469 (Mþ þ 1). Anal. (C22H23BrO4) C, H, O.
2,3-Dihydro-2-(naphthalen-10-yl)-6,8,8-triethyldesmosdu-
motin B (48). Compound 39 (R = Et, Ar = naphthyl,1b 58 mg, 0.14
mmol) was dissolved in HOAc (2.0 mL). Then 45% HI (1.6 mL) was
added to the mixture, which was refluxed for 20 h. The reaction mixture
was cooled to room temperature, and the solvent was removed in vacuo.
Water was added to the residue. The whole mixture was neutralized
to pH 7 with saturated aqueous NaHCO3 and extracted with CH2Cl2.
The organic phase was dried over Na2SO4 and concentrated. The
residue was purified by SiO2 column chromatography (EtOAc/hexane,
gradient) to obtain 48 (20 mg, 37%). 1H NMR (400 MHz, CDCl3): δ
11.64 (1H, chelated-OH), 8.01-7.92 (m, 3H, naphthyl-H), 7.64-7.52 (m,
4H, naphthyl-H), 6.37 (dd, 1H, J = 12.3 and 4.1 Hz, 2-H), 3.19 (dd, 1H, J =
12.3 and 17.3 Hz, 3-Hax), 3.14 (dd, 1H, J = 4.1 and 17.3 Hz, 3-Heq), 2.40 (q,
2H, J = 7.4 Hz, 6-CH2CH3), 2.12-1.98 (m, 2H, 8-CH2CH3), 1.90-1.66
(m, 2H, 8-CH2CH3), 1.00 (t, 3H, J=7.4 Hz, 6-CH2CH3), 0.77(t, 3H, J=7.4
Hz, 8-CH2CH3), 0.50 (t, 3H, J = 7.4 Hz, 8-CH2CH3). MS (ESIþ) m/z 389
(Mþ - 1). Anal. (C25H26O4) C, H, O
Tubulin Assays. Tubulin assembly was measured by turbidimetry at
350 nm as described previously.15 Assay mixtures contained 1.0 mg/mL (10
μM) tubulin, and varying compound concentrations and were preincubated
15 min at 30 ꢀC without guanosine 50-triphosphate (GTP). The samples
were placed on ice, and 0.4 mM GTP was added. Reaction mixtures were
transferred to 0 ꢀC cuvettes, and turbidity development was followed for 20
min at 30 ꢀC following a rapid temperature jump. Compound concentra-
tions that inhibited increase in turbidity by 50% relative to a control sample
were determined.
Inhibition of the binding of [3H]colchicine to tubulin was measured
as described previously.16 Incubation of 1.0 μM tubulin with 5.0 μM
[3H]colchicine and 5.0 μM inhibitor was for 10 min at 37 ꢀC, when
about 40-60% of maximum colchicine binding occurs in control
samples.
Immunofluorescence Staining of Tubulin. A549 tumor cells
were maintained in four-well chamber slides (Lab-Tech) for 12 h prior
to treatment with DMSO, 0.1 nM compound 21, 100 nM colchicine, 1.5
nM pacritaxel, or 500 nM doxorubicin for 24 h at 37 ꢀC. Cells were fixed
with 4% paraformaldehyde in phospate buffered saline (PBS), permea-
bilized with 0.5% Triton X-100 in PBS, and then tubulin was immuno-
stained with monoclonal antibody to R-tubulin (B5-1-2, Sigma) fol-
lowed by fluorescein 5-isothiocyanate (FITC) conjugated secondary
antibody. Nuclei were labeled with 40,6-diamidino-2-phenylindole
(DAPI).17 Fluorescence labeled tubulin and nuclei were observed using
a Zeiss Axioplan fluorescence microscope, and images were captured
by a XL16 Excel cooled digital camera controlled by the Dage Exponent
software (Dage-MTI). Final images were prepared using Adobe
Photoshop.
5-Methoxybenzothiophene-3-carboxaldehyde (55). To a
solution of 53 (122 mg, 0.74 mmol) in anhydrous CH2Cl2 (1.5 mL),
SnCl4 (1.5 mL, 1.5 mmol, 1.0 M solution in CH2Cl2) was added
dropwise at 0 ꢀC under inert gas. Subsquently, dichloromethyl methyl
ether (0.1 mL, 1.11 mmol) was added dropwise at 0 ꢀC. The mixture was
allowed to warm to room temperature and stirred overnight. The
mixture was quenched with saturated NaHCO3 at 0 ꢀC and stirred for
2 h at room temperature. After extraction with CH2Cl2, the organic layer
was washed with brine, dried over Na2SO4, and concentrated. The
residue was chromatographed on SiO2 with EtOAc-hexane gradient to
1
give 55 (107 mg, 0.56 mmol, 75%). H NMR (400 MHz, CDCl3): δ
Pharmacophore Analysis. Pharmacophore analysis was con-
ducted using Molecular Operating Environment (MOE) software
(version 2009.10, Chemical Computing Group, Inc.). First, low-energy
3D conformations were calculated for compounds 3, 21, and 26, as well as
two known antitubulin compounds, colchicine and combretastatin-A4
(CA-4). The pharmacophore was determined by aligning the 3D structures
of colchicine and CA-4. This resulting pharmacophore was later used to
search the best-fit 3D confirmations of compounds 3, 21, and 26.
10.71 (1H, s, CHO), 8.39 (d, 1H, J = 5.6 Hz, 3-H), 7.97 (d, 1H, J = 9.0
Hz, 7-H), 7.65 (d, 1H, J = 5.6 Hz, 2-H), 7.04 (d, 1H, J = 9.0 Hz, 6-H) 3.96
(s, 3H, OCH3). MS (ESIþ) m/z 193 (Mþ þ 1).
5-Methylbenzothiophene-3-carboxaldehyde (56). The ti-
tle compound was prepared in 82% yield following the same procedure as
55 starting from 54 (119.5 g, 0.81 mmol), SnCl4 (2.4 mL, 2.4 mmol, 1.0 M
solution in CH2Cl2), and dichloromethyl methyl ether (0.11 mL, 1.21
mmol). 1H NMR (400 MHz, CDCl3): δ 10.13 (1H, s, CHO), 8.51-8.48
(m, 1H, 4-H), 8.32 (s, 1H, 2-H), 7.86 (d, 1H, J =8.2 Hz, 7-H), 7.30 (dd, 1H,
J = 8.2 and 1.6 Hz, 6-H) 2.5 (s, 3H, CH3). MS (ESIþ) m/z 177 (Mþ þ 1).
Antiproliferative Activity Assay. All stock cultures were grown
in T-25 flasks. Freshly trypsinized cell suspensions were seeded in 96-
well microtiter plates at densities of 1500-7500 cells per well, with
compounds added from DMSO stock solutions and then successively
diluted into medium. The highest concentration of DMSO in the
cultures (0.5% v/v) was without effect on cell replication under
the culture conditions used. After 3 days in culture, attached cells were
fixed with cold 50% trichloroacetic acid and then stained with 0.4%
sulforhodamine B. The absorbency at 562 nm was measured using a
microplate reader after solubilizing the bound dye. The mean GI50 is the
concentration of agent that reduced cell growth by 50% under the
experimental conditions and is the average from at least three indepen-
dent and similar determinations. All values presented in Table 2 are
statistically significant, and standard deviations are shown in the
Supporting Information. For the verapamil reversal experiments, cells
were co-treated with verapamil (1 μg/mL). Control experiments showed
this concentration had no effect on the replication of KB-VIN cells. The
following human tumor cell lines were used in the assay: A549 (lung
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis data for
b
compounds 5-36 and HPLC analysis results for 46-53. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For K.N.-G.: phone, 919-843-5209; fax, 919-966-3893; e-mail,
goto@email.unc.edu. For K.F.B.: phone, 919-966-7633; fax, 919-
966-3893; e-mail, Ken_Bastow@unc.edu. For K.-H.L.: phone,
919-962-0066; fax, 919-966-3893; e-mail, khlee@unc.edu.
’ ACKNOWLEDGMENT
This study was supported by Grant CA-17625 from the
National Cancer Institute, NIH, awarded to K.-H.L., and by a
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dx.doi.org/10.1021/jm1011947 |J. Med. Chem. 2011, 54, 1244–1255

Journal of Medicinal Chemistry
ARTICLE
grant from the University Research Council, awarded to K.N.-G.
We thank Dr. Mitch Eddy (NIEHS/NIH) for providing a
fluorescence microscope.
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’ ABBREVIATIONS USED
CS, collateral sensitivity; CA-4, combretastatin A-4; VERAP,
verapamil; CSI, colchicine site inhibitior; MOE, Molecular Op-
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