Design, synthesis and biological evaluation of dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in cancer chemotherapy

Article abstract of DOI:10.1016/j.bmc.2008.07.050

A novel series of dihydronaphthalene and benzosuberene analogs bearing structural similarity to the combretastatins in terms of 1,2-diarylethene, trimethoxyphenyl, and biaryl functionality has been synthesized. The compounds have been evaluated in regard

Full text of DOI:10.1016/j.bmc.2008.07.050

Bioorganic & Medicinal Chemistry 16 (2008) 8161–8171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of dihydronaphthalene
and benzosuberene analogs of the combretastatins as inhibitors
of tubulin polymerization in cancer chemotherapy
Madhavi Sriram, John J. Hall, Nathan C. Grohmann, Tracy E. Strecker, Taylor Wootton,
*
Andreas Franken, Mary Lynn Trawick, Kevin G. Pinney
Department of Chemistry and Biochemistry, Baylor University, One Bear Place, Waco, TX 76798, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of dihydronaphthalene and benzosuberene analogs bearing structural similarity to the
combretastatins in terms of 1,2-diarylethene, trimethoxyphenyl, and biaryl functionality has been syn-
thesized. The compounds have been evaluated in regard to their ability to inhibit tubulin assembly
and for their cytotoxicity against selected human cancer cell lines. From this series of compounds, ben-
zosuberene analogs 2 and 4 inhibited tubulin assembly at concentrations comparable to that of combre-
tastatin A-4 (CA4) and combretastatin A-1 (CA1). Furthermore, analog 4 demonstrated remarkable
Received 11 June 2008
Revised 17 July 2008
Accepted 17 July 2008
Available online 24 July 2008
Keywords:
cytotoxicity against the three human cancer cell lines evaluated (for example GI50 = 0.0000032
against DU-145 prostate carcinoma).
lM
Benzosuberenes
Dihydronaphthalenes
Combretastatins
Tubulin
Ó 2008 Elsevier Ltd. All rights reserved.
Vascular disrupting agents
Cancer Chemotherapy
1. Introduction
phosphate (CA1P also known as Oxi4503).12–14 In addition,
AVE8062,15,16 a serinamide prodrug is currently in phase I/II hu-
man clinical development as a VDA, while ZD6126,12,17,18 a colchi-
cine analog, has recently been halted in human clinical trials
(Fig. 1). The combretastatins (CSAs), discovered by Pettit and co-
workers in the late 1970s, were originally isolated from the African
Cape Bushwillow tree Combretum caffrum Kuntze.10,19,20
VDAs, such as CA4P, undergo de-phosphorylation and subse-
quently function by binding to an internal system of cross-linked
proteins called the tubulin cytoskeleton (microtubules; polymers
Solid tumors larger than 1–3 mm in diameter depend exten-
sively on their own vasculature for the supply of nutrients and oxy-
gen.1,2 Since the discovery of the concept that blocking blood
supply to solid tumors led to tumor regression in mice by
Denekamp,3,4 compounds that target well-established tumor vas-
culature have gained considerable significance over the past two
decades. Tumor blood vessels, unlike the normal arteries and veins,
lack the hierarchical branching patterns and are often chaotic, di-
lated, and tortuous, and have poorly developed endothelial mono-
layers.5–7 In addition, the increased rate of proliferation renders the
tumor vascular endothelial cells as a specific target for anti-cancer
agents.6 Molecules that specifically interact with the tumor vascu-
lature are known as vascular targeting agents (VTAs). VTAs are fur-
ther sub-divided into two classes:8–10 (a) anti-angiogenic drugs
that prevent neovascularization in tumors and (b) vascular dis-
rupting agents (VDAs) that target already established tumor ves-
sels.10 VDAs that are currently in clinical development are either
ligand-directed biological agents or small molecule compounds
that include flavonoids such as DMXAA11 and the microtubule
depolymerizing agents represented by combretastatin A-4 phos-
phate (CA4P also known as ZybrestatTM), and combretastatin A-1
of a,b-tubulin heterodimers) in the endothelial cells lining the in-
ner layer of tumor vasculature.21,22 This results in microtubule
depolymerization, which is accompanied by cell signaling path-
ways, followed by cytoskeletal rearrangement and ultimately irre-
versible microvessel damage selectively in the tumor
microenvironment.5,22 Colchicine was the first drug reported to
function through
a tubulin binding mechanism in the late
1930s.23,24 However, colchicine was eventually withdrawn from
clinical use in cancer treatment due to significant in vivo toxic-
ity.24,25 Nonetheless, colchicine has been used in the treatment of
gout at very low doses since the late 1950s.26
Numerous structure–activity relationship (SAR) studies have fo-
cused on compounds that bind to tubulin at the colchicine site.
Collectively, these studies suggest important pharmacophore com-
ponents that include the trimethoxyphenyl unit, p-methoxyphenyl
unit, presence of the aromatic rings in a cis-configuration, and a
* Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710 4272.
E-mail address: Kevin_Pinney@baylor.edu (K.G. Pinney).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.050

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M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
R1
R2
H3CO
O
R2
R3
H3CO
H3CO
n
H3CO
R1
OCH3
5, R1 = R2 = OH, n = 0
6, R1 = R2 = OH, n = 1
7, R1 = OH, R2 = H, n = 0
8, R1 = OH, R2 = H, n = 1
1, R1 = R2 = H, R3 = -3′,4′,5′-trimethoxyphenyl
2, R1 = R3 = H, R2 = -3′,4′,5′-trimethoxyphenyl
3, R1 = OH, R2 = H, R3 = -3′,4′,5′-trimethoxyphenyl
4, R1 = OH, R2 = -3′,4′,5′-trimethoxyphenyl, R3 = H
Class I
Class II
Figure 2. Class I and class II compounds.
ketone 19 through a Clemmensen reduction followed by chro-
mium trioxide oxidation.34–37 Some interesting observations were
made during the synthesis of the compounds described in scheme
1. Initial attempts to protect the hydroxy functionality in tetralin
13a with an isopropyl group in the presence of potassium carbon-
ate38 or triethylamine as base were unsuccessful. However, use of
cesium carbonate led successfully to compound 14.39 To avoid par-
tial deprotection of the isopropyl group observed during the
Clemmensen reduction of ketone 17, a Wolff-Kishner reduction40
was attempted; however, these conditions did not yield the de-
sired product 18. Finally, an effort to oxidize 18 at the benzylic
position using DDQ (following a protocol similar to that used for
the benzylic oxidation of compound 14)35 was also unsuccessful.
The benzosuberones (11,37 12,37 17, and 19) were then coupled
to lithiated 3,4,5-trimethoxybenzene (Schemes 2 and 3) to obtain
tertiary alcohols 20, 23, 21, and 24, respectively.
Figure 1. Representative inhibitors of tubulin assembly and vascular disrupting
agents (VDAs).
distance of approximately 4–5 Å between the two aryl rings.10,27,28
A variety of combretastatin analogs have been synthesized in an ef-
fort to optimize the distance between the two aryl rings while
maintaining them in a cis or quasi cis-configuration. Representative
analogs include the phenstatins synthesized by Pettit and co-work-
ers,29 and the dihydronaphthalenes (ex. Oxi6196)30 and indole
analogs synthesized by both Pinney and co-workers (Fig. 1),31,32
and later by Flynn and co-workers (in the case of indole analogs).33
In this paper, the design and synthesis of a series of molecules
containing the dihydronaphthalene and benzosuberene scaffolds
structurally comparable to CA4, CA1, and colchicine are reported.
Preliminary biochemical evaluation of the compounds in terms of
inhibition of tubulin polymerization along with cytotoxicity stud-
ies against NCI-H460 (non-small cell lung carcinoma), DU-145
(prostate carcinoma), and SK-OV-3 (ovarian adenocarcinoma) hu-
man cancer cell lines is presented.
The alcohols were subjected to acid catalyzed elimination of
water followed by deprotection (in the case of compounds 22
and 25) to afford benzosuberenes 1–4 in moderate yields.
The synthesis of compounds 5 through 8 was achieved via a
modified Shapiro coupling reaction (Scheme 4).41 5,6,7-Trimeth-
oxytetralone 26 and 6,7,8-trimethoxybenzosuberone 27 were pre-
pared according to the reported procedures by Gardner42 and
Khanolkar and co-workers.43 The ketones were then converted to
their respective hydrazones 28 and 29.41 Formation of the corre-
sponding vinyl lithium adducts followed by treatment with alde-
hydes 3844 and 3945 gave secondary alcohols 30 through 33. The
alcohols were converted to the requisite ketones 34 through 37,
respectively, via Dess–Martin oxidation.46 Ketones 34 and 35 were
selectively debenzylated without affecting the a,b-unsaturated ke-
tone moiety using 1,4-cyclohexadiene in the presence of Pd–C,47 to
obtain the desired dihydronaphthalene 5 and benzosuberene 6 in
46% and 63% yields, respectively. The isopropyl groups in ketones
36 and 37 were removed using anhydrous AlCl3 at reduced tem-
perature to yield the final products 7 and 8 in yields of 44% and
66%, respectively.33
2. Results and discussions
2.1. Chemistry
On one occasion involving the deprotection of the benzyl
groups from ketone 35, H2 gas was unintentionally used as a
source of hydrogen instead of 1,4-cyclohexadiene (Scheme 5). This
resulted in not only removal of the benzyl groups, but also in
reduction of the double bond and the ketone to afford benzocyclic
analog 9 in 23% yield. The structure of compound 9 was confirmed
by X-ray crystallographic analysis (Fig. 3).48
The designed molecules fall into two classes34 (Fig. 2): class I in-
cludes compounds 1 through 4 that are structurally analogous to
the dihydronaphthalene Oxi6196,30 (Fig. 1); while class II includes
compounds 5 through 8 in which the aryl rings are connected via a
carbonyl bridge. Compounds
serendipitously.
9
and 10 were prepared
Another interesting observation was made when the attempted
deprotection of the isopropyl group in ketone 36 (with anhydrous
AlCl3 at room temperature rather than ꢀ78 °C as in Scheme 4) re-
sulted in an unexpected cyclization to afford the isopropyl pro-
tected benzo[a]fluorene 40 in a 51% yield (Scheme 6). X-ray
crystallographic analysis confirmed the structure of compound
Key intermediates 17 and 19 were prepared from 5-hydroxy-6-
methoxytetralin 13 (Scheme 1).30,35 Following introduction of an
isopropyl protecting group, compound 14 was oxidized selectively
at the benzylic position with DDQ to afford tetralone 15 in good
yield.35 A Wittig reaction followed by a cyanogen azide ring expan-
sion reaction36,37 gave ketone 17, which was converted to isomeric

M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
8163
1. TMEDA, sec-BuLi / 0 o
2. B(OMe)3 / 0 oC to r.t.
C
HO
+
H3CO
3. H2O2 / HOAc / 0 oC to r.t
H3CO
H3CO
OH
12 h
13b (63%)
13a (23%)
O
Cs2CO3 / Acetone
2 -bromopropane
DDQ /
Dioxane:H2O (95:5)
r.t. 12 h, 71% yield
reflux, 12 h
H3CO
H3CO
H3CO
93% yield
OH
13a
O
15
O
14
CH2
O
NaH / DMSO
CH3PPh3I
65-75 oC, 8 h
68% yield
CNN3 / CH3CN
MeOH / 48 h, r.t.
33% yield
Zn(Hg), Conc.
HCl reflux, 3 h,
19% yield
H3CO
H3CO
O
O
16
17
O
CrO3 / HOAc-H2O,
r.t. 24 h
H3CO
H3CO
O
13% yield
O
18
19
Scheme 1. Synthesis of benzosuberones 17 and 19.
H3CO
OCH3
OCH3
Br
Li
HO
n
-BuLi / Et2O
-78 oC
OCH3
OCH3
O
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
R
R
20, R = H (30% yield)
1137, R = H
21, R = O
i-Pr (66% yield)
17, R = O -Pr
i
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
HOAc, reflux, 12 h
AlCl3 / CH2Cl2
r.t. 10 min,
41% yield
H3CO
H3CO
R
1, R = H (13% yield)
22, R = O -Pr (quantitative)
OH
3
i
Scheme 2. Synthesis of benzosuberene analogs 1 and 3.
4048 (Fig. 4), the formation of which may have taken place through
a Nazarov type of cyclization.49 Deprotection of the isopropyl
group at reduced temperature in the benzo[a]fluorene 40 afforded
phenol 10.
(with a hydrogen at C-6) and benzosuberene 4 (with a hydroxy
at C-6) were both strong inhibitors of tubulin assembly, compound
4 demonstrated remarkable cytotoxicity against NCI-H460, DU-
145, and SK-OV-3 cancer cell lines compared to compound 2. This
suggests the possibility that benzosuberene 4 may demonstrate an
additional mechanism of inhibition of cancer cell growth beyond
that attributable to the tubulin-based mechanism alone.
2.2. Biology
Vascular disrupting agents such as CA4P are, in general, potent
inhibitors of tubulin assembly in parent compound form (CA4,
Compounds 1 and 3 in which the trimethoxy aryl ring is located
at C-2 are not inhibitors of tubulin polymerization (IC50 > 40 lM);
IC50 = 1.2
l
M). Therefore, the benzocyclic compounds in this series
however, these compounds do maintain considerable cytotoxicity
against the three human cancer cell lines studied. Introduction of
a carbonyl moiety along with positioning the trimethoxy motif
on the A-ring of the fused ring system resulted in compounds 6
and 8 (a di-phenol and mono-phenol, respectively) that were inac-
tive both as inhibitors of tubulin assembly and in regard to
were evaluated for their ability to inhibit tubulin assembly. Com-
pounds 2 and 4 inhibited tubulin polymerization with IC50 values
of 1.4
comparable with those of CA4 and CA1, which are between 1.2
and 1.9
M.50 It is intriguing to note that while benzosuberene 2
lM and 1.7 lM, respectively (Table 1). These results are
l

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M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
H3CO
H3CO
OCH3
Br
Li
n
-BuLi / Et2O
-78 oC
OCH3
OCH3
OH
O
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
R
R
1237, R = H
19, R = O -Pr
23, R = H (50% yield)
24, R = O -Pr (14% yield)
i
i
H3CO
H3CO
H3CO
H3CO
OCH3
OCH3
HOAc, reflux, 12 h
AlCl3 / CH2Cl2
r.t. 10 min
56% yield
H3CO
H3CO
R
2, R = H (91% yield)
25, R = O -Pr (quantitative)
OH
4
i
Scheme 3. Synthesis of benzosuberene analogs 2 and 4.
cytotoxicity toward the cancer cell lines. Compound 9, the chemi-
cally reduced analog of 6, was similarly inactive against human
cancer cell lines; however, it demonstrated moderate activity in re-
gard to inhibition of tubulin polymerization.
The analogous tetralin compounds (5 and 7) were also inactive
or minimally active as inhibitors of tubulin assembly but main-
tained limited cytotoxicity. The tetracyclic analog 10, which is
essentially a ring-closed variant of compound 7, was inactive as
an inhibitor of tubulin assembly; however, the two compounds
(10 and 7) showed disparate cytotoxicity.
was used as common solvent for recording the NMR. All the chem-
ical shifts are expressed in ppm (d), coupling constants (J, Hz), and
peak patterns are reported as broad (b), singlets (s), doublets (d),
triplets (t), quartets (q), pentets (p), septets (sep), and multiplets
(m). Elemental analysis was performed by Atlantic Microlab, Nor-
cross, GA. High resolution mass spectra (HRMS) were obtained in
the Baylor University Mass Spectrometry Core Facility on a VG
Prospec Micromass spectrometer using EI.
4.1. Chemistry
3. Conclusions
4.1.1. 5-Hydroxy-6-methoxy-1,2,3,4-tetrahydronaphthalene
(13a)35
A novel series of ten benzocyclic compounds bearing structural
similarity to the combretastatins, colchicine, and Oxi6196 have
been synthesized and assessed for their preliminary biochemical
and biological activity. Benzosuberenes 2 and 4 are potent inhibi-
tors of tubulin polymerization, and compound 4 exhibited out-
standing cytotoxicity against all three cancer cell lines evaluated.
While compound 1, the regioisomer of benzosuberene 2, was not
active as an inhibitor of tubulin assembly, it was cytotoxic against
the cancer cell lines. The remarkable activity of benzosuberene 4
suggests that a suitable prodrug formulation of this compound
may lead to a new vascular disrupting agent for the potential treat-
ment of certain solid tumor cancers.
To a well-stirred solution of 6-methoxy-1,2,3,4-tetrahydronap-
thalene (14.15 g, 86.75 mmol) in sec-BuLi (100 mL, 110 mmol) at
0 °C was added freshly distilled TMEDA (13.6 mL) dropwise. After
the addition was complete, the reaction mixture was stirred at rt
for 1 h. The reaction mixture was cooled again to 0 °C and
B(OMe)3 (12.5 mL, 110 mmol) was added dropwise. Then the reac-
tion mixture was stirred for 1 h at rt. The reaction mixture was
cooled back to 0 °C and glacial HOAc (7 mL) was added dropwise,
followed by the dropwise addition of 35% wt. H2O2 (15 mL). Finally,
the reaction mixture was allowed to stir at rt for 12 h. Saturated
aqueous NH4Cl (100 mL) was added and product was extracted
with Et2O (3ꢁ 400 mL). The combined organic phases were washed
with brine and dried over Na2SO4, filtered, and concentrated in va-
cuo. Purification by flash column chromatography (silica gel,
EtOAc/hexanes, gradient 2:98 to 5:95) yielded 5-hydroxy-6-meth-
oxy-1,2,3,4-tetrahydronaphthalene 13a (3.50 g, 19.6 mmol, 23%
yield) as an off-white solid and 7-hydroxy-6-methoxy-1,2,3,4-tet-
rahydronaphthalene 13b (9.70 g, 54.4 mmol, 63%) as colorless
crystals, Rf13a = 0.48, Rf13b = 0.37 (15:85, EtOAc/hexanes).
4. Experimental51
All reactions were performed under inert atmosphere using
either nitrogen or argon gas unless specified differently. Chemical
reagents used in the synthetic procedures were obtained from var-
ious chemical suppliers (Sigma–Aldrich Chem. Co., Acros Chem.
Co., Alfa Aesar, Fisher Scientific, EMD Chemicals, and VWR). Silica
gel (200–400 mesh, 60 Å) used for column chromatography was
obtained from either Silicycle Inc. or VWR. TLC plates (pre-coated
glass plates with silica gel 60 F254, 0.25 mm thickness, EMD chem-
icals, VWR) were used to monitor reactions. Intermediates and
products synthesized were characterized based on 1H NMR (Bruker
DPX operating at 300 MHz or Varian operating at 500 MHz), and
13C NMR (Bruker DPX operating at 75 MHz or Varian operating at
125 MHz). Deuterated CDCl3 (with 0.03% TMS as internal standard)
1H NMR for 13a (CDCl3, 500 MHz) d 6.67 (d, J = 8.5 Hz, 1H), 6.23
(d, J = 8.9 Hz, 1H), 5.63 (s, 1H), 3.86 (s, 3H), 2.70 (t, J = 6.0 Hz, 4H),
1.71 (m, 4H); 13C NMR for 13a (CDCl3, 125 MHz) d 143.8, 143.0,
130.8, 123.7, 119.4, 108.1, 56.2, 29.1, 23.1, 23.0, 22.6.
4.1.2. 5-Isopropoxy-6-methoxy-1,2,3,4-tetrahydronaphthalene
(14)
Anhydrous Cs2CO3 (66.55 g, 204.3 mmol) was added to a well-
stirred solution of 13a35 (4.55 g, 25.5 mmol) in anhydrous acetone

M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
8165
CH3
O
O
S
NH
O
N
1. TMEDA, n-BuLi
p
-toluenesulfone
H3CO
H3CO
-50 oC to r.t.
H3CO
H3CO
hydrazide
2.
O
H
n
p-TSA H2O / EtOH
n
r.t. 12 h
OCH3
R
2 38, R1 = R2 = OBn
39, R1 = O -Pr, R2 = H
OCH3
26, n = 1
27, n = 2
i
28, n = 1 (92% yield)
29, n = 2 (98% yield)
R1
OCH3
R1
R1
H3CO
R2
H3CO
R2
OH
O
Dess-Martin periodinane
10% Na2S2O3, CH2Cl2
r.t. 5 min
H3CO
H3CO
H3CO
H3CO
n
n
OCH3
OCH3
36-37, R1 = O
i
-Pr, R2 = H
34-35, R1 = R2 = OBn
34, n = 1 (70% yield)
35, n = 2 (63% yield)
32-33, R1 = Oi-Pr, R2 = H
32, n = 1 (69% yield)
33, n = 2 (29% yield)
30-31, R1 = R2 = OBn
30, n = 1 (57% yield)
31, n = 2 (48% yield)
36, n = 1 (60% yield)
37, n = 2 (57% yield)
Pd-C / 1,4-cyclohexadiene
EtOH, r.t. 2 h
AlCl3 / CH2Cl2
-78 oC, 2 h
Deprotection of
benzyl groups
Deprotection of
isopropyl groups
OH
OH
HO
HO
OH
H3CO
OH
H3CO
H3CO
H3CO
O
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
OCH3
OCH3
6 (63% yield)
OCH3
5 (46% yield)
OCH3
7 (44% yield)
8 (66% yield)
Scheme 4. Synthesis of dihydronaphthalene and benzosuberene compounds 5–8.
BnO
HO
OH
OBn
H3CO
H3CO
O
H3CO
H3CO
H3CO
Pd-C / H2
EtOH, r.t.
H3CO
45 min, 23% yield
OCH3
OCH3
35
9
Scheme 5. Preparation of compound 9.
(50 mL). 2-Bromopropane (23.9 mL, 255 mmol) was added and the
reaction mixture was heated at reflux for 12 h at which point the
reaction mixture was filtered and the solvent evaporated in vacuo.
Purification by flash column chromatography (silica gel, 1:99,
EtOAc/hexanes) afforded 5-isopropoxy-6-methoxytetralin 14
(5.23 g, 23.7 mmol, 93% yield) as a pale yellow oil, Rf = 0.61
(15:85, EtOAc/hexanes).
Figure 3. Thermal ellipsoid plot at 50% probability for compound 9.
4.1.3. 5-Isopropoxy-6-methoxy-3,4-dihydro-2H-naphthalen-1-
one (15)
To a well-stirred solution of tetralin 14 (0.12 g, 0.55 mmol) in
H2O/dioxane (5:95, 5 mL) was added a solution of DDQ (0.25 g,
1.09 mmol) in dioxane (5 mL) dropwise. The reaction mixture
was stirred for 12 h at rt. The precipitate was filtered and washed
1H NMR (CDCl3, 300 MHz) d 6.77 (d, J = 8.3 Hz, 1H), 6.71 (d,
J = 8.3 Hz, 1H), 4.46 (sep, J = 6.2 Hz, 1H), 3.80 (s, 3H), 2.71 (m,
4H), 1.72 (m, 4H), 1.28 (d, J = 6.2 Hz, 6H).

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M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
O
O
OH
H3CO
OCH3
OCH3
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
AlCl3 / CH2Cl2
-45 oC, 2.5 h
64% yield
AlCl3 / CH2Cl2
r.t. 1 h
51% yield
OCH3
OCH3
OCH3
36
40
10
Scheme 6. Preparation of compounds 40 and 10.
4.1.4. 5-Isopropoxy-6-methoxy-1-methylene-1,2,3,4-
tetrahydronaphthalene (16)
Anhydrous DMSO (35 mL) was added to NaH (1.14 g,
47.4 mmol) and the reaction mixture was heated to 70–75 °C for
1–2 h, until the evolution of hydrogen ceased. The reaction mixture
was cooled to room temperature and additional DMSO (20 mL)
was added. Methyltriphenylphosphonium iodide (27.02 g,
66.84 mmol) was added in portions over a period of 30 min and
the reaction mixture was subsequently stirred for 20 min at room
temperature. A solution of tetralone 15 (7.83 g, 33.4 mmol) in
anhydrous DMSO (8 mL) was added and the reaction mixture
was stirred at 60–65 °C for 8 h. The reaction mixture was poured
into a 250 mL Erlenmeyer flask containing ice/hexanes (50:50,
100 mL), stirred vigorously for 15 min, and then extracted with
hexanes (2ꢁ 100 mL). The combined organic layers were washed
with DMSO/H2O (1:1), dried over Na2SO4, filtered, and evaporated
under reduced pressure. Purification by flash column chromatogra-
phy (silica gel, 5:95, EtOAc/hexanes) yielded benzosuberone 16
(5.31 g, 22.9 mmol, 68% yield) as a colorless oil, Rf = 0.63 (30:70,
EtOAc/hexanes).
Figure 4. Thermal ellipsoid plot at 50% probability for compound 40.
Table 1
Inhibition of tubulin polymerization and cytotoxicity studies against human cancer
cell lines NCI-H460, DU-145, and SK-OV-3
Compound Inhibition of tubulin
GI50 (lM) SRB assay
polymerization IC50 (lM)
1H NMR (CDCl3, 500 MHz) d 7.37 (d, J = 8.5 Hz, 1H), 6.75 (d,
J = 8.5 Hz, 1H), 5.35 (s, 1H), 4.84 (s, 1H), 4.46 (sep, J = 6.5 Hz, 1H),
3.83 (s, 3H), 2.82 (t, J = 6.5 Hz, 2H), 2.48 (t, J = 6.0 Hz, 2H), 1.82
(p, J = 6.0 Hz, 2H), 1.27 (d, J = 6.5 Hz, 6H); 13C NMR (CDCl3,
125 MHz) d 152.1, 144.1, 143.5, 132.6, 128.6, 119.4, 110.1, 106.1,
74.2, 55.7, 33.1, 25.1, 23.5, 22.6.
NCI-
DU-145
SK-OV-3
H460
CA4
CA1
1
2
3
1.2a
1.9a
>40d
1.4d
>40
1.7
0.002b
0.002b
0.51b
0.55
0.027
0.44
0.00013
ndc
0.44
0.052
0.27
2.2b
0.47
0.051
1.2
4
0.000028 0.0000032 <0.00003
5
6
7
8
9
10
>40
>40
24
>40
7.4
2.8
>10
6.0
>10
>10
>10
3.4
>10
3.1
>10
>10
>10
1.4
nd
nd
nd
nd
nd
4.1.5. 1-Isopropoxy-2-methoxy-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (17)
Preparation of CNN3: finely powdered NaN3 (0.510 g,
7.85 mmol) was added rapidly to a solution of CNBr (0.830 g,
7.85 mmol) in CH3CN (anhydrous, 5 mL) at 0 °C. The reaction mix-
ture was stirred for 4 h at 0 °C, after which the clear supernatant
solution containing CNN3 was drawn into a syringe to be used
for the ring expansion reaction.
>40
a
b
c
See Ref. 50.
See Ref. 29.
nd, not determined in this study.
See Ref. 55
d
Ring expansion reaction: to a stirred solution of exocyclic olefin
16 (0.32 g, 1.3 mmol) in MeOH/CH3CN (1:1, 5 mL) at room temper-
ature was added freshly prepared CNN3, and the reaction mixture
was stirred for 48 h. HCl (6 M, 5 mL) was added and the reaction
mixture was heated at 50 °C for 4 h. The reaction mixture was then
cooled to room temperature, and extracted with Et2O (2ꢁ 25 mL).
The ethereal extracts were washed with H2O until neutral and then
dried over Na2SO4. The organic phase was then percolated through
a column of basic alumina capped with a layer of CeliteTM to remove
the potentially explosive azides. Evaporation of the solvent and
subsequent purification by flash column chromatography (silica
gel, 8:92, EtOAc/hexanes) yielded benzosuberone 17 (110 mg,
0.44 mmol, 33% yield) as an off-white solid, Rf = 0.48 (70:30, hex-
anes/EtOAc).
with EtOAc. The filtrate was concentrated under reduced pressure,
saturated aqueous NaHCO3 solution (10 mL) was added, and the
resulting solution was extracted with Et2O (3ꢁ 25 mL). The com-
bined organic phases were dried over Na2SO4, filtered, and the sol-
vent evaporated in vacuo. Purification by flash column
chromatography (silica gel, 30:70, EtOAc/hexanes) afforded tetra-
lone 15 (0.09 g, 0.38 mmol, 71% yield) as a colorless oil, Rf = 0.458
(40:60, EtOAc/hexanes).
1H NMR (CDCl3, 500 MHz) d 7.84 (d, J = 8.5 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 4.45 (sep, J = 6.0 Hz, 1H), 3.89 (s, 3H), 2.95 (t,
J = 6.0 Hz, 2H), 2.59 (t, J = 6.5 Hz, 2H), 2.06 (p, J = 6.5 Hz, 2H),
1.29 (d, J = 6.0 Hz, 6H); 13C NMR (CDCl3, 125 MHz) d 197.7,
157.0, 143.2, 139.4, 126.7, 124.0, 109.9, 74.7, 55.7, 38.8, 24.2,
23.0, 22.6.
1H NMR (CDCl3, 300 MHz) d 6.84 (d, J = 8.3 Hz, 1H), 6.72 (d,
J = 8.2 Hz, 1H), 4.43 (sep, J = 6.1 Hz, 1H), 3.81 (s, 3H), 3.64 (s, 2H),

M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
8167
3.04 (t, J = 6.9 Hz, 2H), 2.51 (t, J = 6.9 Hz, 2H), 1.94 (p, J = 6.9 Hz,
2H), 1.28 (d, J = 6.1 Hz, 6H); 13C NMR (CDCl3, 75 MHz) 209.5,
152.2, 144.4, 134.6, 126.9, 124.2, 109.9, 74.8, 55.6, 49.7, 43.2,
25.3, 24.4, 22.5.
152.2, 146.0, 143.9, 137.8, 136.3, 128.2, 126.8, 109.1, 101.3, 74.8,
72.2, 60.6, 55.5, 48.2, 45.7, 26.4, 22.9, 22.5, 22.3.
4.1.9. 2-Hydroxy-7-methoxy-2-(30,40,50-trimethoxyphenyl)-
benzosuberan (20)
4.1.6. 1-Isopropoxy-2-methoxy-6,7,8,9-tetrahydro-5H-benzocy-
cloheptane (18)
Following the general procedure for 21, compound 20 (0.52 g,
1.4 mmol, 30% yield) was obtained from ketone 11 as a pale yellow
oil, Rf = 0.25 (35:65, EtOAc/hexanes).
Amalgamated zinc was prepared by shaking Zn dust (16 g),
HgCl2 (1.6 g), H2O (16 mL), and HCl (concd, 1 mL) in a 100 mL
round-bottomed flask for 10 min. The supernatant liquid was dec-
anted and benzosuberone 17 (0.830 g, 1.13 mmol) was added, fol-
lowed by HCl (concd, 60 mL). After heating at reflux for 3 h, the
reaction mixture was cooled and extracted with Et2O (3ꢁ 30 mL)
and the combined ethereal extracts were washed with H2O until
neutral, then dried over Na2SO4, filtered, and evaporated under re-
duced pressure. Separation by flash column chromatography (neu-
tral alumina, 4:96, EtOAc/hexanes) afforded benzosuberan 18
(150 mg, 0.64 mmol, 19% yield) as a colorless oil, Rf = 0.55 (15:85,
EtOAc/hexanes).
1H NMR (CDCl3, 300 MHz) d 7.03 (d, J = 8.1 Hz, 1H), 6.79 (s, 2H),
6.75 (d, J = 2.6 Hz, 1H), 6.68 (dd, J = 8.1 Hz, 2.6 Hz, 1H), 3.89 (s, 6H),
3.86 (s, 3H), 3.80 (s, 3H), 3.59 (d, J = 15.2 Hz, 1H), 2.88 (m, 3H), 1.98
(m, 4H).
4.1.10. 2-Methoxy-5-(30,40,50-trimethoxyphenyl)-6,7,8,9-tetrahy-
drobenzocyclohepten-5-ol (23)
Following the general procedure for 21, compound 23 (0.81 g,
2.3 mmol, 50% yield) was obtained from ketone 12 as a pale yellow
oil, Rf = 0.16 (30:70, EtOAc/hexanes).
1H NMR (CDCl3, 300 MHz) d 7.42 (d, J = 8.5 Hz, 1H), 6.82 (d,
J = 2.6 Hz, 1H), 6.81 (m, 1H), 6.49 (d, J = 2.6 Hz, 2H), 3.84 (s, 3H),
3.82 (s, 3H), 3.76 (s, 6H), 2.64 (m, 2H), 2.07 (m, 6H).
1H NMR (CDCl3, 300 MHz) d 6.77 (d, J = 8.2 Hz, 1H), 6.60 (d,
J = 8.2 Hz, 1H), 4.31 (sep, J = 6.2 Hz, 1H), 3.78 (s, 3H), 2.88 (m,
2H), 2.73 (m, 2H), 1.76 (m, 2H), 1.61 (m, 4H), 1.27 (d, J = 6.2 Hz,
6H).
4.1.11. 1-Isopropoxy-2-methoxy-5-(30,40,50-trimethoxyphenyl)-
6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (24)
4.1.7. 1-Isopropoxy-2-methoxy-6,7,8,9-tetrahydrobenzocyclo-
hepten-5-one (19)
Following the general procedure for 21, compound 24 (0.012 g,
0.029 mmol, 14% yield) was obtained from ketone 19 as a colorless
oil, Rf = 0.14 (30:70, EtOAc/hexanes).
CrO3 (0.47 g, 4.7 mmol) dissolved in HOAc (2 mL) and H2O
(0.5 mL) was added dropwise to a well-stirred solution of benzo-
suberan 18 (0.37 g, 1.6 mmol) in glacial HOAc (5 mL). The resulting
reaction mixture was then stirred at room temperature for 28 h.
H2O (10 mL) was added and the product was extracted in Et2O
(3ꢁ 25 mL). The combined ethereal extracts were washed with a
5% aqueous NaOH solution until the aqueous phases tested basic
with pH paper. The combined organic phases were first washed
with H2O until neutral, followed by brine, and then dried over
Na2SO4. The solution was filtered and the solvent was removed un-
der reduced pressure to obtain the crude product which was then
subjected to flash column chromatography (neutral alumina, 4:96,
EtOAc/hexanes) to afford benzosuberone 19 (0.05 g, 0.2 mmol, 13%
yield) as colorless crystals, Rf = 0.60 (70:30, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.51 (d, J = 8.6 Hz, 1H), 6.83 (d,
J = 8.6 Hz, 1H), 4.41 (sep, J = 6.2 Hz, 1H), 3.88 (s, 3H), 3.03 (m,
2H), 2.69 (m, 2H), 1.79 (m, 4H), 1.29 (d, J = 6.2 Hz, 6H); 13C NMR
(CDCl3, 75 MHz) d 205.1, 156.2, 143.7, 136.2, 124.9, 109.5, 74.9,
55.6, 40.6, 24.7, 23.8, 22.4, 21.5, 20.9.
1H NMR (CDCl3, 300 MHz) d 7.31 (d, J = 8.7 Hz, 1H), 6.76 (d,
J = 8.7 Hz, 1H), 6.47 (s, 2H), 4.38 (sep, J = 6.2 Hz, 1H), 3.87 (s, 3H),
3.85 (s, 3H), 3.73 (s, 6H), 3.33 (m, 1H), 2.56 (m, 1H), 2.12 (m,
6H), 1.29 (d, J = 6.2 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H).
4.1.12. 4-Isopropoxy-3-methoxy-8-(30,40,50-trimethoxyphenyl)-
6,7-dihydro-5H-benzocycloheptene (22)
A mixture of alcohol 21 (1.15 g, 2.64 mmol) in HOAc (30 mL)
was heated at reflux for 12 h. The reaction mixture was cooled, ex-
tracted with CH2Cl2 (3ꢁ 50 mL), and the combined organic phases
were dried over Na2SO4. The solvent was evaporated and the crude
product was purified by flash column chromatography (silica gel,
10:90, EtOAc/hexanes) to afford trimethoxyphenylbenzocyclohep-
tene 22 (1.04 g, 2.61 mmol, 95% yield) as a white crystalline solid,
Rf = 0.56 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 6.95 (d, J = 8.4 Hz, 1H), 6.76 (d,
J = 8.4 Hz, 1H), 6.71 (s, 1H), 6.69 (s, 2H), 4.44 (sep, J = 6.1 Hz, 1H),
3.90 (s, 6H), 3.87 (s, 3H), 3.84 (s, 3H), 2.93 (t, J = 6.0 Hz, 2H), 2.62
(t, J = 6.7 Hz, 2H), 2.19 (p, J = 6.5 Hz, 2H), 1.32 (d, J = 6.1 Hz, 6H).
4.1.8. 1-Isopropoxy-2-methoxy-6-(30,40,50-trimethoxyphenyl)-
6,7,8,9-tetrahydro-5H-benzocyclohepten-6-ol (21)
4.1.13. 3-Methoxy-8-(30,40,50-trimethoxyphenyl)-6,7-dihydro-
5H-benzocycloheptene (1)
Following the general procedure for compound 22, tertiary
alcohol 20 yielded benzosuberene 1 (0.06 g, 0.18 mmol, 13% yield)
as a colorless oil, Rf = 0.37 (35:65, EtOAc/hexanes).
To a well-stirred solution of 3,4,5-trimethoxybromobenzene
(1.50 g, 6.05 mmol) in dry Et2O (200 mL) at ꢀ78 °C was added n-
BuLi (3.20 mL, 8.06 mmol) dropwise. The reaction mixture was
stirred until the temperature rose to ꢀ30 °C. Ketone 17 (1.05 g,
4.03 mmol) dissolved in dry Et2O (25 mL) was added dropwise.
The reaction mixture was then gradually allowed to warm to room
temperature. At this point, H2O (30 mL) was added and the organic
phase was separated. The aqueous layer was extracted with Et2O
(2ꢁ 100 mL). The combined organic phases were dried over
Na2SO4, filtered, and the solvent was evaporated under reduced
pressure. Purification of the crude product by flash column chro-
matography (silica gel, 30:70, EtOAc/hexanes) yielded alcohol 21
1H NMR (CDCl3, 300 MHz) d 7.17 (d, J = 7.9 Hz, 1H), 6.73 (m, 5H),
3.90 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.80 (t, J = 6.2 Hz, 2H), 2.63 (t,
J = 6.2 Hz, 2H), 2.21 (p, J = 6.2 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d
158.3, 152.9, 142.8, 140.8, 140.4, 137.3, 131.8, 129.8, 127.9, 114.7,
111.0, 103.6, 60.9, 56.1, 55.2, 34.8, 33.1, 29.9; HRMS (EI) Calcd for
340.1675, found 340.1681.
4.1.14. 3-Methoxy-9-(30,40,50-trimethoxyphenyl)-6,7-dihydro-
5H-benzocycloheptene (2)
(1.15 g, 2.76 mmol, 66% yield) as
a white crystalline solid,
Rf = 0.15 (70:30, hexanes/EtOAc).
Following the general procedure for compound 22, tertiary
alcohol 23 yielded benzosuberene 2 (0.690 g, 2.03 mmol, 91%
yield) as a white solid, Rf = 0.43 (30:70, EtOAc/hexanes).
1H NMR (CDCl3, 300 MHz) d 6.99 (d, J = 8.5 Hz, 1H), 6.83 (d,
J = 2.6 Hz, 1H), 6.75 (dd, J = 8.5 Hz, 2.6 Hz, 1H), 6.49 (s, 2H), 6.35
1H NMR (CDCl3, 300 MHz) d 6.83 (d, J = 8.1 Hz, 1H), 6.78 (s, 2H),
6.58 (d, J = 8.1 Hz, 1H), 4.39 (m, 1H), 3.87 (s, 6H), 3.85 (s, 3H), 3.79
(s, 3H), 3.60 (m, 2H), 2.90 (d, J = 14.1 Hz, 1H), 2.49 (m, 1H), 2.16 (m,
2H), 1.55 (s, 2H), 1.29 (m, 6H); 13C NMR (CDCl3, 75 MHz) d 152.7,

8168
M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
(t, J = 7.3 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.80 (s, 6H), 2.64 (t,
J = 7.0 Hz, 2H), 2.18 (p, J = 7.0 Hz, 2H), 1.98 (m, 2H); Anal. Calcd
for C21H24O4: C, 74.09; H, 7.09. Found: C, 73.98; H, 7.09; HRMS
(EI) Calcd for 340.1675, found 340.1672.
4.1.18.2. (6,7,8-Trimethoxy-2,3,4,5-tetrahydrobenzocyclohep-
tene)-1-p-toluenesulfonylhydrazone (29). Reaction of 27
(8.21 g, 32.8 mmol) with p-toluenesulfonylhydrazide (6.11 g,
32.8 mmol) under similar reaction conditions (as described for
compound 28) yielded hydrazone 29 (13.50 g, 32.26 mmol, 98%
yield) as a white solid, Rf = 0.32 (60:40, hexanes/EtOAc).
1H NMR (DMSO-d6, 300 MHz) d 10.33 (s, 1H), 7.83 (s-broad, 2H),
7.51 (s-broad, 2H), 6.38 (s, 1H), 3.73 (s, 3H), 3.69 (s, 6H), 2.73 (m,
7H), 1.60 (s, 2H), 1.47 (s, 2H); 13C NMR (DMSO-d6, 75 MHz) d
160.4, 151.3, 150.7, 143.8, 143.1, 136.5, 133.6, 129.8, 128.1,
124.8, 107.6, 61.7, 60.8, 55.9, 27.8, 25.2, 22.7, 21.4, 21.2.
4.1.15. 1-Isopropoxy-2-methoxy-5-(30,40,50-trimethoxyphenyl)-
6,7,8,9-tetrahydro-5H-benzocycloheptene (25)
Following the general procedure for compound 22, tertiary
alcohol 24 yielded benzosuberene 25 (0.01 g, 0.02 mmol, quantita-
tive yield) as a colorless oil, Rf = 0.46 (70:30, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 6.73 (s, 2H), 6.47 (s, 2H), 6.33 (t,
J = 7.3 Hz, 1H), 4.50 (sep, J = 6.2 Hz, 1H), 3.85 (s, 6H), 3.79 (s, 6H),
2.77 (t, J = 6.9 Hz, 2H), 2.21 (m, 2H), 1.97 (m, 2H), 1.33 (d,
J = 6.2 Hz, 6H).
4.1.19. Modified Shapiro coupling reactions to prepare 30–33—
General synthetic procedure
4.1.19.1. (200,300-Bis-benzyloxy-400-methoxyphenyl)-(50,60,70-tri-
4.1.16. 2-Methoxy-6-(30,40,50-trimethoxyphenyl)-8,9-dihydro-7H-
benzocyclohepten-1-ol (3)
methoxy-30,40-dihydronaphthalen-10-yl)-methanol (30).
n-
BuLi (7.42 mL, 14.8 mmol) was added to freshly distilled TMEDA
(20 mL), and the mixture was cooled to ꢀ50 °C. At this point,
hydrazone 28 (1.49 g, 3.71 mmol) was added, and the reaction
mixture was stirred until the temperature reached 25 °C. Aldehyde
38 (5.18 g, 14.8 mmol) was added, and the reaction mixture was
stirred for 1 h. H2O (25 mL) was added and the product was ex-
tracted in ethyl acetate (2ꢁ 100 mL). The combined organic phases
were washed with 10% aqueous CuSO4 solution (100 mL), followed
by brine, dried over Na2SO4, filtered, and evaporated under re-
duced pressure. Purification of the crude product by flash column
chromatography (silica gel, 16:84, EtOAc/hexanes) yielded alcohol
30 (1.20 g, 2.11 mmol, 57% yield) as a pale yellow oil, Rf = 0.40
(60:40, hexanes/EtOAc).
To a stirred solution of compound 22 (0.22 g, 0.55 mmol) in
anhydrous CH2Cl2 (15 mL) at room temperature was added AlCl3
(0.150 g, 1.10 mmol). After 10 min, H2O (10 mL) was added and
the product was extracted in CH2Cl2 (2ꢁ 20 mL). The combined or-
ganic phases were dried over Na2SO4, evaporated under reduced
pressure, and purified by flash column chromatography (silica
gel, 90:10, hexanes/EtOAc). Phenol 3 (0.08 g, 0.2 mmol, 41% yield)
was obtained as a colorless oil, Rf = 0.54 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 6.78 (d, J = 8.4 Hz, 1H), 6.72 (d,
J = 8.4 Hz, 1H), 6.70 (s, 3H), 5.77 (s, 1H), 3.90 (s, 6H), 3.89 (s, 3H),
3.86 (s, 3H), 2.93 (t, J = 6.1 Hz, 2H), 2.63 (t, J = 6.1 Hz, 2H), 2.23
(p, J = 6.1 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 152.8, 145.0,
142.4, 141.2, 140.2, 137.2, 131.6, 128.1, 126.8, 121.7, 107.6,
103.5, 60.8, 56.1, 55.9, 32.8, 29.8, 24.8; HRMS (EI) Calcd for
356.1624, found 356.1620.
1H NMR (CDCl3, 300 MHz) d 7.38 (m, 10H), 7.03 (d, J = 8.6 Hz,
1H), 6.66 (m, 2H), 6.16 (t, J = 4.6 Hz, 1H), 5.93 (d, J = 3.0 Hz, 1H),
5.19 (m, 2H), 5.03 (m, 2H), 3.84 (s, 6H), 3.80 (s, 3H), 3.65 (s, 3H),
2.87 (m, 2H), 2.68 (m, 2H), 1.77 (d, J = 3.0 Hz, 1H); 13C NMR (CDCl3,
75 MHz) d 153.6, 151.1, 150.2, 150.0, 141.2, 141.0, 129.2, 129.0,
128.7, 128.5, 128.4, 128.34, 128.31, 128.0, 125.0, 122.5, 122.3,
107.9, 104.0, 75.8, 75.4, 67.4, 60.8, 60.7, 56.0, 55.9, 22.5, 20.1.
4.1.17. 2-Methoxy-5-(30,40,50-trimethoxyphenyl)-6,7,8,9-tetrahy-
dro-5H-benzocyclohepten-1-ol (4)
Following the general procedure for compound 3, benzosube-
rene 25 yielded phenol 4 (0.005 g, 0.012 mmol, 56% yield) as a col-
orless oil, Rf = 0.35 (70:30, EtOAc/hexanes).
4.1.19.2. (200,300-Bis-benzyloxy-400-methoxyphenyl)-(10,20,30-tri-
methoxy-80,90-dihydro-70H-benzocyclohepten-50-yl)-methanol
1H NMR (CDCl3, 300 MHz) d 6.71 (d, J = 8.4 Hz, 1H), 6.57 (d,
J = 8.4 Hz, 1H), 6.50 (s, 2H), 6.34 (t, J = 7.3 Hz, 1H), 5.74 (s, 1H),
3.91 (s, 3H), 3.86 (s, 3H), 3.80 (s, 6H), 2.76 (t, J = 7.3 Hz, 2H), 2.14
(p, J = 7.1 Hz, 2H), 1.97 (m, 2H); 13C NMR (CDCl3, 75 MHz) d
152.8, 145.0, 142.7, 142.3, 138.4, 137.3, 134.2, 127.7, 127.2,
120.8, 107.6, 105.3, 60.8, 56.1, 55.9, 33.5, 25.6, 23.5; HRMS (EI)
Calcd for 356.1624, found 356.1624.
(31).
Coupling between hydrazone 29 (1.54 g, 3.68 mmol) and
aldehyde 38 (5.14 g, 14.7 mmol) under similar conditions (as de-
scribed for compound 30) afforded alcohol 31 (1.02 g, 1.75 mmol,
48% yield) as a pale yellow oil, Rf = 0.56 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.38 (m, 10H), 7.13 (d, J = 8.6 Hz,
1H), 6.69 (d, J = 8.6 Hz, 1H), 6.63 (s, 1H), 6.09 (t, J = 7.1 Hz, 1H),
5.86 (d, J = 4.2 Hz, 1H), 5.06 (m, 4H), 3.85 (s, 3H), 3.82 (s, 6H),
3.63 (s, 3H), 3.01 (t, J = 7.0, 2H), 2.06 (t, J = 7.0, 2H), 1.86 (m, 2H).
4.1.18. Synthesis of tosylhydrazones (28 and 29)—General
procedure
4.1.18.1. (5,6,7-Trimethoxy-3,4-dihydro-2H-naphthalen)-1-p-
4.1.19.3. (300-Isopropoxy-400-methoxyphenyl)-(50,60,70-trimeth-
oxy-30,40-dihydronaphthalen-10-yl)-methanol (32).
Coupling
toluenesulfonylhydrazone (28).
To a well-stirred solution of
of hydrazone 28 (0.998 g, 2.47 mmol) with aldehyde 39 (1.93 g,
9.89 mmol) under similar conditions (as described for compound
30) yielded alcohol 32 (0.70 g, 1.7 mmol, 69% yield) as a pale yellow
oil, Rf = 0.31 (60:40, hexanes/EtOAc).
cyclic ketone 26 (2.96 g, 12.5 mmol) in anhydrous EtOH (60 mL)
was added p-toluenesulfonylhydrazide (2.34 g, 12.5 mmol). Solu-
tion was achieved within 5 min at which point p-TSA monohydrate
(0.11 g, 0.63 mmol) was added and the reaction mixture was stir-
red for 12 h at room temperature. Hydrazone 28 precipitated out
as a white solid, which was then filtered, washed with ice-cold
EtOH, and dried under reduced pressure to afford 28 (4.65 g,
11.5 mmol, 92% yield) as a white solid, Rf = 0.31 (60:40, hexanes/
EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.01 (m, 3H), 6.66 (s, 1H), 6.17 (t,
J = 4.5 Hz, 1H), 5.65 (s, 1H), 4.45 (sep, J = 6.0 Hz, 1H), 3.85 (s, 6H),
3.81 (s, 3H), 3.66 (s, 3H), 2.75 (t, J = 7.8 Hz, 2H), 2.34 (m, 2H),
1.32 (m, 6H).
1H NMR (DMSO-d6, 300 MHz) d 10.30 (s, 1H), 7.83 (d, J = 8.2 Hz,
2H), 7.41 (d, J = 8.2 Hz, 2H), 7.14 (s, 1H), 3.77 (s, 3H), 3.74 (s, 3H),
3.70 (s, 3H), 2.57 (t, J = 5.7 Hz, 2H), 2.45 (t, J = 6.3 Hz, 2H), 2.37 (s,
3H), 1.71 (m, 2H); 13C NMR (DMSO-d6, 75 MHz) d 152.6, 151.1,
150.0, 143.3, 143.1, 136.0, 129.2, 127.8, 126.9, 126.6, 102.6, 60.4,
55.4, 25.4, 21.6, 21.0, 20.9.
4.1.19.4. (300-Isopropoxy-400-methoxyphenyl)-(10,20,30-trimeth-
oxy-80,90-dihydro70H-benzocyclohepten-50-yl)-methanol
(33).
Hydrazone 29 (1.05 g, 2.40 mmol), when coupled with al-
de-hyde 39 (1.86 g, 9.56 mmol) under similar Shapiro conditions (as
described for compound 30) afforded alcohol 33 (0.30 g, 0.70 mmol,
29% yield) as a pale yellow oil, Rf = 0.29 (60:40, hexanes/EtOAc).

M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
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1H NMR (CDCl3, 300 MHz) d 6.95 (m, 2H), 6.80 (d, J = 8.2 Hz, 1H),
6.65 (s, 1H), 6.30 (t, J = 8.0 Hz, 1H), 5.53 (s, 1H), 4.45 (sep, J = 6.0 Hz,
1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H), 2.53 (m, 2H),
2.05 (m, 2H), 1.91 (m, 2H), 1.29 (d, J = 6.0 Hz, 6H).
132.8, 130.5, 127.0, 124.5, 116.1, 110.5, 108.4, 71.2, 61.5, 60.8,
56.0, 55.9, 33.9, 26.0, 23.8, 21.9.
4.1.21. Synthesis of compounds 5 and 6—deprotection of benzyl
groups—General methodology
4.1.20. Synthesis of compounds 34–37—Dess–Martin oxidation—
General methodology
4.1.21.1. (200,300-Dihydroxy-400-methoxyphenyl)-(50,60,70-trimeth-
oxy-30,40-dihydronaphthalen-10-yl)-methanone (5).
To
a
4.1.20.1. (200,300-Bis-benzyloxy-400-methoxyphenyl)-(50,60,70-trime-
well-stirred solution of ketone 34 (0.796 g, 1.41 mmol) in EtOH
(20 mL) was added Pd–C (10% wt., 0.5 g) followed by 1,4-cyclo-
hexadiene (3.37 mL, 35.3 mmol). The reaction mixture was stirred
for 1.75 h. The solution was filtered through CeliteTM, and the resi-
due was washed with EtOAc. The combined filtrates were evapo-
rated under reduced pressure and purified by flash column
chromatography (silica gel, 20:80, EtOAc/hexanes) to obtain diol
5 (0.25 g, 0.65 mmol, 46% yield) as a yellow solid, Rf = 0.37
(50:50, hexanes/EtOAc).
thoxy-30,40-dihydronaphthalen-10-yl)-methanone (34).
To a
well-stirred solution of Dess–Martin periodinane (1.30 g,
2.24 mmol) in dry CH2Cl2 (20 mL) at room temperature was added
a solution of alcohol 30 (1.16 g, 2.04 mmol) in anhydrous CH2Cl2
(20 mL) followed by 10% aqueous Na2S2O3 (0.05 mL). After stirring
for 5 min, a solution of 10% aqueous Na2S2O3 and saturated aque-
ous NaHCO3 (30 mL, 1:1 ratio) was added, and the reaction mixture
was stirred for 5 min. The product was extracted in CH2Cl2 (3ꢁ
50 mL). The combined organic phases were washed with brine
and dried over Na2SO4. The organic phases were filtered, and the
solvent was removed under reduced pressure. The crude product
was purified by flash column chromatography (silica gel, 10:90,
EtOAc/hexanes) to afford ketone 34 (0.80 g, 1.4 mmol, 70% yield)
as a pale yellow oil, Rf = 0.41 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 500 MHz) d 12.44 (s, 1H), 7.27 (d, J = 8.0 Hz,
1H), 6.52 (s, 2H), 6.45 (d, J = 8.0 Hz, 1H), 6.29 (t, J = 5.0 Hz, 1H),
5.53 (s, 1H), 3.96 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H), 3.72 (s, 3H),
2.86 (t, J = 8.5 Hz, 2H), 2.45 (m, 2H); 13C NMR (CDCl3, 125 MHz) d
201.8, 152.6, 151.9, 151.2, 150.8, 142.5, 137.4, 133.6, 127.5,
125.7, 121.9, 114.9, 105.6, 102.7, 61.2, 61.1, 56.5, 56.4, 22.9, 20.0;
HRMS (EI) Calcd for 386.1366, found 386.1364.
1H NMR (CDCl3, 300 MHz) d 7.27 (m, 11H), 7.05 (s, 1H), 6.72 (d,
J = 8.5 Hz, 1H), 6.34 (t, J = 4.5 Hz, 1H), 5.07 (s, 2H), 5.02 (s, 2H), 3.90
(s, 6H), 3.82 (s, 3H), 3.76 (s, 3H), 2.66 (t, J = 7.9 Hz, 2H), 2.28 (m,
2H); 13C NMR (CDCl3, 75 MHz) d 195.9, 156.9, 152.2, 151.5,
150.5, 142.1, 141.5, 139.8, 139.6, 137.3, 137.2, 128.8, 128.6,
128.5, 128.4, 128.3, 128.0, 127.5, 126.2, 122.4, 107.1, 106.5, 76.1,
75.6, 61.1, 56.3, 56.2, 23.4, 19.6.
4.1.21.2. (200,300-Dihydroxy-400-methoxyphenyl)-(10,20,30-trimeth-
oxy-80,90-dihydro-70H-benzocyclohepten-50-yl)-methanone (6).
Debenzylation of ketone 35 (0.995 g, 1.72 mmol) under similar
reaction conditions (as described for compound 5) afforded ben-
zosuberene diol 6 (0.55 g, 1.4 mmol, 80% yield) as a yellow solid,
Rf = 0.40 (70:30, hexanes/EtOAc).
4.1.20.2. (200,300-Bis-benzyloxy-400-methoxyphenyl)-(10,20,30-tri-
methoxy-80,90-dihydro-70H-benzocyclohepten-50-yl)-methanone
1H NMR (CDCl3, 500 MHz) d 12.49 (s, 1H), 7.07 (d, J = 9.0 Hz,
1H), 6.68 (t, J = 6.8 Hz, 1H), 6.41 (s, 1H), 6.39 (d, J = 9.0 Hz, 1H),
5.50 (s, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H), 3.69 (s, 3H),
2.75 (t, J = 6.5 Hz, 2H), 2.18 (m, 4H); 13C NMR (CDCl3, 125 MHz) d
200.7, 151.9, 151.3, 151.0, 141.8, 141.4, 139.1, 133.3, 132.3,
127.2, 125.1, 114.3, 107.8, 102.5, 61.5, 60.7, 56.1, 55.9, 33.8, 26.0,
23.8; Anal. Calcd for C22H24O7: C, 65.99; H, 6.04. Found: C, 66.02;
H, 6.05; HRMS (EI) Calcd for 400.1522, found 400.1509.
(35).
Oxidation of alcohol 31 (1.60 g, 2.75 mmol) under simi-
lar Dess–Martin reaction conditions (as described for compound
34) afforded ketone 35 (1.05 g, 1.80 mmol, 63% yield) as a yellow
oil, Rf = 0.43 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.40 (m, 10H), 7.13 (d, J = 8.4 Hz,
1H), 6.80 (t, J = 7.3 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 6.61 (s, 1H),
5.09 (s, 2H), 5.02 (s, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H),
3.70 (s, 3H), 2.54 (t, J = 6.5 Hz, 2H), 2.10 (m, 4H).
4.1.22. Synthesis of compounds 7 and 8—deprotection of iso-
propyl groups—General methodology
4.1.20.3. (300-Isopropoxy-400-methoxyphenyl)-(50,60,70-trimeth-
4.1.22.1. (300-Hydroxy-400-methoxyphenyl)-(50,60,70-trimethoxy-
oxy-30,40-dihydronaphthalen-10-yl)-methanone (36).
Secon
30,40-dihydronaphthalen-10-yl)-methanone (7).
To a solution
dary- alcohol 32 (0.71 g, 1.7 mmol) was oxidized under similar
Dess–Martin reaction conditions (as described for compound 34)
to yield ketone 36 (0.42 g, 1.0 mmol, 60% yield) as a colorless oil,
Rf = 0.51 (60:40, hexanes/EtOAc).
of ketone 36 (0.11 g, 0.24 mmol) in anhydrous CH2Cl2 (10 mL) at
ꢀ78 °C was added AlCl3 (anhydrous, 0.08 g, 0.6 mmol). The reaction
mixture was stirred for 3 h. NH4Cl (saturated, 5 mL) was added and
the organic phase was separated. The aqueous layer was extracted
with CH2Cl2 and the combined organic phases were dried over
Na2SO4, filtered, and the solvent evaporated under reduced pressure.
The crude product was purified by flash column chromatography (sil-
ica gel, 30:70, EtOAc/hexanes) to yield phenol 7 (0.04 g, 0.11 mmol,
44% yield) as a pale yellow oil, Rf = 0.22 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.49 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H),
6.70 (s, 1H), 6.39 (t, J = 4.6 Hz, 1H), 5.78 (s, 1H), 3.95 (s, 3H), 3.88 (s,
3H), 3.85 (s, 3H), 3.72 (s, 3H), 2.82 (t, J = 7.8 Hz, 2H), 2.42 (m, 2H);
13C NMR (CDCl3, 75 MHz) d 195.9, 151.4, 150.6, 150.4, 145.2, 142.0,
137.94, 135.2, 131.5, 127.5, 123.7, 121.8, 109.7, 105.8, 60.8, 56.1,
56.0, 22.8, 19.7; Anal. Calcd for C21H22O6ꢂH2O: C, 64.94; H, 6.23.
Found: C, 64.96; H, 5.94; HRMS (EI) Calcd for 370.1416, found
370.1416.
1H NMR (CDCl3, 300 MHz) d 7.49 (m, 2H), 6.87 (d, J = 8.3 Hz, 1H),
6.69 (s, 1H), 6.40 (t, J = 4.7 Hz, 1H), 4.61 (sep, J = 6.0 Hz, 1H), 3.92 (s,
3H), 3.88 (s, 3H), 3.87 (s, 3H), 3.73 (s, 3H), 2.84 (t, J = 7.8 Hz, 2H),
2.44 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); 13C NMR (CDCl3, 75 MHz) d
195.9, 154.6, 151.5, 150.4, 147.1, 142.0, 138.2, 134.8, 130.7,
127.7, 125.1, 121.8, 115.7, 110.4, 105.9, 71.4, 60.9, 56.0, 22.8,
21.9, 19.8.
4.1.20.4. (300-Isopropoxy-400-methoxyphenyl)-(10,20,30-trimethoxy-
80,90-dihydro-70H-benzocyclohepten-50-yl)-methanone (37).
Oxidation of alcohol 31 (0.30 g, 0.70 mmol) under similar Dess–
Martin reaction conditions (as described for compound 34) affor-
ded ketone 37 (0.17 g, 0.39 mmol, 57% yield) as a pale yellow oil,
Rf = 0.62 (40:60, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.43 (dd, J = 8.4 Hz, 2.03 Hz, 1H),
7.31 (d, J = 2.0 Hz, 1H), 6.80 (m, 2H), 6.47 (s, 1H), 4.49 (sep,
J = 6.0 Hz, 1H), 3.89 (s, 3H), 3.88 (s, 6H), 3.69 (s, 3H), 2.77 (t,
J = 6.6 Hz, 2H), 2.22 (m, 4H), 1.33 (d, J = 6.0 Hz, 6H); 13C NMR
(CDCl3, 75 MHz) d 195.4, 154.0, 151.3, 146.7, 142.6, 141.6, 140.4,
4.1.22.2. (300-Hydroxy-400-methoxyphenyl)-(10,20,30-trimethoxy-
80,90-dihydro-70H-benzocyclohepten-50-yl)-methanone
(8).
Deprotection of the isopropyl group in ketone 37 (69 mg,
0.16 mmol) under similar reaction conditions (as described for

8170
M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
compound 7) afforded 8 (35 mg, 0.09 mmol, 56% yield) as a color-
less oil, Rf = 0.23 (60:40, hexanes/EtOAc).
56.1, 56.0, 33.1, 32.2, 29.0, 27.8, 24.9; Anal. Calcd for C22H28O6: C,
68.02; H, 7.27. Found: C, 68.04; H, 7.26; HRMS (EI) Calcd for
388.1886, found 388.1887.
1H NMR (CDCl3, 300 MHz) d 7.41 (d, J = 2.0 Hz, 1H), 7.34 (dd,
J = 8.3 Hz, 2.0 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.79 (t, J = 6.9 Hz,
1H), 6.5 (s, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.72 (s, 3H),
2.72 (t, J = 6.6 Hz, 2H), 2.14 (m, 4H); 13C NMR (CDCl3, 75 MHz) d
195.7, 151.1, 150.1, 145.1, 142.2, 141.6, 141.2, 132.5, 131.6, 127.3,
132.4, 115.9, 109.6, 108.4, 61.5, 60.8, 56.0, 55.9, 33.8, 26.0, 23.7;
Anal. Calcd for C22H24O6ꢂH2O: C, 65.66; H, 6.51. Found: C, 65.59; H,
6.23; HRMS (EI) Calcd for 384.1573, found 384.1570.
4.1.26. 9-Isopropoxy-2,3,4,8-tetramethoxy-5,6-dihydro-benzo
[a]fluoren-11-one (40)
Anhydrous AlCl3 (0.05 g, 0.4 mmol) was added to a well-stirred
solution of ketone 36 (0.14 g, 0.34 mmol) in anhydrous CH2Cl2
(10 mL) at 5 °C under N2". The reaction mixture was stirred for 1 h.
H2O (5 mL) was added and the product was extracted with CH2Cl2
(3 ꢁ 20 mL). The combined organic phases were washed with brine,
dried over Na2SO4, filtered and evaporated under reduced pressure.
The crude product obtained was crystallized using EtOAc/hexanes
(15:85) to afford benzo[a]fluorene 40 (0.07 g, 1.7 mmol, 51% yield)
as white crystals, Rf = 0.32 (60:40, hexanes/EtOAc).
4.1.23. 2,3-Bis-benzyloxy-4-methoxybenzaldehyde (38)
Anhydrous K2CO3 (14.39 g, 104.1 mmol) was added to a well-
stirred solution of 2,3-dihydroxy-4-methoxybenzaldehyde35
(5.31 g, 29.8 mmol) in MeOH (anhydrous, 50 mL). Benzyl bromide
(10.7 mL, 89.2 mmol) was added and the reaction mixture was
heated at reflux for 1.5 h. The reaction mixture was filtered and
the solvent was evaporated under reduced pressure. H2O
(100 mL) and CH2Cl2 (100 mL) were added to the crude reaction
mixture. The organic phases were separated and the aqueous
phase was extracted with additional CH2Cl2 (2ꢁ 150 mL). The com-
bined organic phases were washed with H2O (2ꢁ 200 mL), fol-
lowed by brine, and dried over anhydrous Na2SO4. The organic
phases were filtered and the solvent was evaporated under re-
duced pressure. The crude product obtained was purified by flash
column chromatography (5:95, EtOAc/hexanes) to afford bis-o-
benzyl-aldehyde 38 (10.2 g, 29.3 mmol, 98% yield) as a yellow so-
lid, Rf = 0.51 (70:30, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.17 (s, 1H), 7.07 (s, 1H), 6.99 (s,
1H), 4.57 (sep, J = 6.1 Hz, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.85 (s,
3H), 3.77 (m, 5H), 2.74 (m, 1H), 2.16 (m, 1H), 2.01 (m, 2H), 1.39
(d, J = 6.1 Hz, 3H), 1.37 (d, J = 6.1 Hz, 3H).
4.1.27. 9-Hydroxy-2,3,4,8-tetramethoxy-5,6-dihydro-benzo[a]
fluoren-11-one (10)
To a solution of benzo[a]fluorene 40 (73 mg, 0.17 mmol) in
anhydrous CH2Cl2 (10 mL) at ꢀ45 °C was added anhydrous AlCl3
(64 mg, 0.43 mmol). The reaction mixture was stirred for 2.5 h at
which point H2O (5 mL) was added and the organic phases were
separated. The aqueous layer was extracted with CH2Cl2 and the
combined organic phases were washed with brine, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
crude product was purified by flash column chromatography (silica
gel, 35:65, EtOAc/hexanes) to yield alcohol 10 (0.04 g, 0.1 mmol,
64% yield) as a colorless solid, Rf = 0.14 (60:40, hexanes/EtOAc).
1H NMR (CDCl3, 300 MHz) d 7.24 (s, 1H), 7.06 (s, 1H), 6.99 (s,
1H), 5.70 (s, 1H), 4.04 (s, 3H), 3.94 (s, 3H), 3.85 (s, 3H), 3.77 (m,
5H), 2.70 (m, 1H), 2.11 (m, 3H); 13C NMR (CDCl3, 75 MHz) d
202.7, 153.1, 151.7, 151.2, 150.2, 146.1, 140.8, 130.1, 128.0,
123.6, 109.0, 108.4, 105.7, 60.8, 56.3, 56.0, 51.1, 38.2, 28.6, 19.3;
Anal. Calcd for C21H22O6: C, 68.10; H, 5.99. Found: C, 67.63; H,
6.04; HRMS (EI) Calcd for 370.1416, found 370.1414.
1H NMR (CDCl3, 300 MHz) d 10.10 (s, 1H), 7.60 (d, J = 8.8 Hz,
1H), 7.41 (m, 10H), 6.77 (d, J = 8.8 Hz, 1H), 5.20 (s, 2H), 5.07 (s,
2H), 3.92 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 188.8, 159.4, 155.8,
140.7, 137.0, 136.2, 128.7, 128.5, 128.3, 128.2, 127.6, 126.9,
124.1, 123.9, 107.7, 75.5, 65.3, 56.1.
4.1.24. 3-Isopropoxy-4-methoxybenzaldehyde (39)45
Anhydrous K2CO3 (36.35 g, 262.9 mmol) was added to a solution
of isovanillin (20.34 g, 131.5 mmol) in anhydrous DMF (250 mL) at
50 °C. The reaction mixture was then stirred for 15 min, and 2-bro-
mopropane (24.6 mL, 262 mmol) was added. The reaction mixture
was then stirred for 8 h at 90 °C. H2O (200 mL) was added and the
product was extracted with CH2Cl2 (3ꢁ 300 mL). The combined or-
ganic phases were washed with H2O (2ꢁ 300 mL), followed by
brine, and then dried over Na2SO4. The organic phases were filtered
and concentrated to dryness in vacuo. The crude product was sub-
jected to flash column chromatography (silica gel, 5:95, hexanes/
EtOAc) to obtain product 39 (22.71 g, 116.9 mmol, 89% yield) as pale
yellow crystals, Rf = 0.40 (70:30, hexanes/EtOAc).
4.2. Biology
4.2.1. Tubulin polymerization assay22,52
Tubulin was purified from calf brain following a method re-
ported by Hamel and Lin.52 Polymerization was followed turbidi-
metrically at 350 nm. IC50 values of the various analogs were
determined from the data using nonlinear regression analysis with
Prism software (GraphPad) 3.02 version.
1H NMR (CDCl3, 300 MHz) d 9.84 (s, 1H), 7.43 (m, 2H), 6.84 (d,
J = 8.0 Hz, 1H), 4.65 (sep, J = 6.0 Hz, 1H), 3.93 (s, 3H), 1.40 (d,
J = 6.0 Hz, 6H); 13C NMR (CDCl3, 75 MHz) d 190.8, 155.5, 147.7,
129.9, 126.3, 112.6, 110.9, 71.2, 56.0, 21.8.
4.2.2. SRB assay50,53,54
Inhibition of human cancer growth was assessed using the Na-
tional Cancer Institute’s standard sulforhodamine B assay, as previ-
ously described.54 Briefly, cells were distributed into 96-well
plates, followed by treatment with study compounds and doxoru-
4.1.25. 3-Methoxy-6-((10,20,30-trimethoxy-60,70,80,90-tetrahydro-
50H-benzo[7]annulen-50-yl)benzene-1,2-diol (9)
To a solution of ketone 35 (0.98 g, 1.7 mmol) in anhydrous EtOH
(100 mL) was added Pd–C (10% wt., 1.0 g). The reaction mixture
was de-gassed under vacuum and then stirred for 1 h under H2
(using a balloon filled with H2 gas). The reaction mixture was fil-
tered through CeliteTM. The solvent was evaporated under reduced
pressure. The crude product was recrystallized with EtOAc/hex-
anes to obtain diol 9 (0.15 g, 0.38 mmol, 23% yield) as white crys-
tals, Rf = 0.31 (50:50, hexanes/EtOAc).
bicin as a control at concentrations between 0.000005 and 50.0 lg/
mL at 37 °C for 48 h. Due to its extreme cytotoxicity, compound 4
was screened at concentrations between 0.5 ꢁ 10ꢀ13
lg/mL and
50.0 lg/mL at 37 °C for 48 h. A growth inhibition of 50% in compar-
ison to untreated controls (GI50 or the drug concentrations causing
a 50% reduction in net protein increase) was calculated by nonlin-
ear regression analysis.50,53,54
1H NMR (CDCl3, 300 MHz) d 6.55 (m, 2H), 6.37 (d, J = 8.4 Hz, 1H),
5.38 (s, 1H), 5.37 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.79 (s, 6H), 3.05
(m, 5H), 1.68 (m, 6H); 13C NMR (CDCl3, 75 MHz) d 150.8, 150.4,
145.0, 142.2, 141.7, 140.1, 132.1, 128.5, 120.7, 102.3, 61.3, 60.8,
Acknowledgments
The authors are grateful to Oxigene Inc. (Waltham, MA, grants to
K.G.P. and M.L.T.), the Welch Foundation (Grant No. AA-1278 to

M. Sriram et al. / Bioorg. Med. Chem. 16 (2008) 8161–8171
23. Boyland, E.; Boyland, M. E. Biochem. J. 1937, 31, 454.
8171
K.G.P.), and the National Science Foundation for funding both the
Varian 500 MHz NMR spectrometer (Award CHE-0420802) and the
Bruker X8 APEX diffractometer (Grant CHE-0321214). The authors
are thankful to Dr. Benon E. Mugabe (former Baylor Graduate stu-
dent) for performing the tubulin assay for compounds 1 and 2. The
authors would like to thank Dr. Alejandro Ramirez (Mass Spectrom-
etry Core Facility, Baylor University) for HRMS analysis. The authors
also thank H&B Packing (Waco, TX) for providing calf brain, and are
grateful to Ms. Beverly Herrington (Undergraduate student, Baylor
University, 2003) and Mr. Ankur Gupta [Baylor high school summer
science research program (HSSSRP), 2003] for their valuable assis-
tance with chemical synthesis.
24. Ludford, R. J. J. Natl. Cancer Res. 1946, 6, 89.
25. Chaplin, D. J.; Hill, S. A. Int. J. Radiat. Oncol. Biol. Phys. 2002, 54, 1491.
26. Falasca, G. F. Clin. Dermatol. 2006, 24, 498.
27. Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Curr. Pharm. Des. 2005, 11, 1655.
28. Tron, G. C.; Pirali, P.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazani, A. A. J. Med.
Chem. 2006, 49, 1.
29. (a) Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.; Boyd, M. R.; Hamel, E.;
Pettit, R. K. J. Med. Chem. 1998, 41, 1688; (b) Pettit, G. R.; Grealish, M. P.; Herald,
D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 2000, 43, 2731.
30. Pinney, K. G.; Mocharla, V. P.; Chen, Z.; Garner, C. M.; Ghatak, A.; Hadimani, M.;
Kessler, J.; Dorsey, J. M.; Edvardsen, K.; Chaplin, D. J.; Prezioso, J.; Ghatak, U. R.
U.S. Patent Appl. Publ. 20040043969 A1, 2004.
31. Pinney, K. G.; Wang, F.; Hadimani, M.; Mejia, P. M. U.S. Patent Appl. Publ.
20070082872 A1, 2007.
32. Hadimani, M. B.; Kessler, R. J.; Kautz, J. A.; Ghatak, A.; Shirali, A. R.; O’Dell, H.;
Garner, C. M.; Pinney, K. G. Acta Crystallogr., Sect. C 2002, C58, 330.
33. Flynn, B. L.; Hamel, E.; Jung, M. K. J. Med. Chem. 2002, 45, 2670.
34. Pinney, K. G.; Sriram, M. U.S. PCT Appl. WO 2006138427, 2006.
35. Ghatak, A.; Dorsey, J. M.; Garner, C. M.; Pinney, K. G. Tetrahedron Lett. 2003, 44,
4145.
36. McMurry, J. E.; Coppolino, A. P. J. Org. Chem. 1973, 38, 2821.
37. Miller, R. B.; Gutierrez, C. G. J. Org. Chem. 1978, 43, 1569.
38. de Koning, C. B.; Michael, J. P.; Rousseau, A. L. Tetrahedron Lett. 1997, 38, 893.
39. Bringmann, G.; Menche, D.; Kraus, J.; Muhlbacher, J.; Peters, K.; Peters, E.; Brun,
R.; Bezabih, M.; Abegaz, B. M. J. Org. Chem. 2002, 67, 5595.
40. Mazzocchi, P. H.; Kim, C. H. J. Med. Chem. 1982, 25, 1473.
41. Pinney, K. G.; Carlson, K. E.; Katzenellenbogen, J. A. Steroids 1992, 57, 222.
42. Gardner, P. D. J. Am. Chem. Soc. 1954, 76, 4550.
43. Khanolkar, A. D.; Lu, D.; Fan, P.; Tian, X.; Makriyannis, A. Bioorg. Med. Chem. Lett.
1999, 9, 2119.
44. Pettit, G. R.; Lippert, J. W., III Anti-Cancer Drug Des. 2000, 15, 203.
45. Ridley, C. P.; Reddy, M. V.; Rocha, G.; Bushman, F.; Faulker, D. J. Biorg. Med.
Chem. 2002, 10, 3285.
46. Meyer, S. D.; Schrieber, S. L. J. Org. Chem. 1994, 59, 7549.
47. Felix, A. M.; Heimer, E. P.; Lambros, T. J.; Tzougraki, C.; Meienhofer, J. J. Org.
Chem. 1978, 43, 4194.
48. Crystallographic data for structure 9 (deposition number CCDC-690726) and
structure 40 (deposition number CCDC-690727) reported in this paper have
been deposited with the Cambridge Crystallographic Data Centre. Copies of the
data can be obtained, free of charge, on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +41 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
49. He, W.; Sun, X.; Frontier, A. J. J. Am. Chem. Soc. 2003, 125, 14278.
50. Siles, R.; Ackley, F. J.; Hadimani, M. B.; Hall, J. J.; Mugabe, B. E.;
Guddneppanavar, R.; Monk, K. A.; Chapius, J. C.; Pettit, G. R.; Chaplin, D. J.;
Edvardsen, K.; Trawick, M. L.; Garner, C. M.; Pinney, K. G. J. Nat. Prod. 2008, 71,
313.
References and notes
1. Salmon, B. A.; Siemann, D. W. Int. J. Radiat. Oncol. Biol. Phys. 2007, 68, 211.
2. Siemann, D. W.; Chaplin, D. J.; Horsman, M. R. Cancer 2004, 100, 2491.
3. Denekamp, J. Br. J. Cancer 1982, 45, 136.
4. Denekamp, J. Br. J. Radiol. 1993, 66, 181.
5. Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev. Cancer 2005, 5, 423.
6. Hida, K.; Hida, Y.; Shindoh, M. Cancer Sci. 2008, 99, 459.
7. Kanthou, C.; Tozer, G. M. Exp. Opin. Ther. Targets 2007, 11, 1443.
8. Siemann, D. W.; Bibby, M. C.; Dark, G. G.; Dicker, A. P.; Eskens, F. A. L. M.;
Horsman, M. R.; Marme, D.; LoRusso, P. M. Clin. Cancer Res. 2005, 11, 416.
9. Eichhorn, M. E.; Strieth, S.; Dellian, M. Drug Res. Updates 2004, 7, 125.
10. Pinney, K. G.; Jelinek, C.; Edvardsen, K.; Chaplin, D. J.; Pettit, G. R. The Discovery
and Development of the Combretastatins. In Anticancer Agents from Natural
Products; Cragg, G. M., Kingston, D. G. I., Newman, D. J., Eds.; Taylor and Francis,
2005; pp 23–46.
11. Pili, R.; Rosenthal, M. J. Clin. Oncol. (2007 ASCO Annual Meeting Proceedings Part
I) 2007, 25, 18S 5115 (abstract).
12. (a) Lippert, J. W. Biorg. Med. Chem. 2007, 15, 605; (b) Patterson, D. M.; Rustin, G.
J. S. Clin. Oncol. 2007, 19, 443.
13. Akerley, W. L.; Schabel, G. M.; Horvath, E.; Yu, M.; Johnsson, B.; Arbogast, K.
J. Clin. Oncol. (2007 ASCO Annual Meeting Proceedings Part I) 2007, 25, 18S 14060
(abstract).
14. Patterson, D. M.; Ross, P.; Koetz, B.; Saleem, A.; Stratford, M.; Stirling, J.;
Padhani, A.; Assalin, M.; Price, P.; Rustin, G. J. J. Clin. Oncol. (2007 ASCO Annual
Meeting Proceedings Part I) 2007, 25, 18S 14146 (abstract).
15. Demers, B.; Vrignaus, P.; Bissery, M. J. Clin. Oncol. (2006 ASCO Annual Meeting
Proceedings Part I) 2006, 24, 18S 13074 (abstract).
16. Oshumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.;
Nihei, Y.; Ohishi, K.; Akiyama, Y.; Tsuji, T. Anti-Cancer Drug Des. 1999, 14, 539.
17. Blakey, D. C.; Ashton, S. E.; Westwood, F. R.; Walker, M.; Ryan, A. J. Int. J. Radiat.
Oncol. Biol. Phys. 2002, 54, 1497.
18. http://www.clinicaltrials.gov/ct2/show/record?term=ZD6126&rank=1
19. Pettit, G. R.; Singh, S. B.; Margaret, N. L.; Hamel, E.; Schmidt, J. M. J. Nat. Prod.
1987, 50, 119.
20. Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.;
Hogan, F. J. Med. Chem. 1995, 38, 1666.
51. Portions of this work have been reported in the following dissertation: Sriram,
M. Design, synthesis, biochemical, and biological evaluation of benzocyclic and
enediyne analogs of combretastatins as potential tubulin binding ligands in the
treatment of cancer. Ph.D. Dissertation, Baylor University, Waco, TX, 2007.
52. Hamel, E.; Lin, C. M. Biochemistry 1984, 23, 4173.
53. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolf, A. J. Natl. Cancer Inst. 1991, 83, 757.
54. Vichai, V.; Kirtikara, K. Nat. Protocols 2006, 1, 1112.
21. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
22. Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley, J. F.; Studerus, S.
W.; Edvardsen, K.; Trawick, M. L.; Garner, C. M.; Rhodes, M. R.; Pettit, G. R.;
Pinney, K. G. Bioorg. Med. Chem. 2006, 14, 3231.
55. Mugabe, B. E. Structure–Activity Relationships and Thermodynamics of
Combretastatin A-4 and A-1 Derivatives as Potential Inhibitors of Tubulin
Polymerization. Ph.D. Dissertation, Baylor University, Waco, TX, 2005.