Synthesis of structurally diverse benzosuberene analogues and their biological evaluation as anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2013.08.035

Diversely functionalized, fused aryl-alkyl ring systems hold a prominent position as well-established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7 fused, also referred to as dihydro-5H-benzo[7] annulene and benzocycloheptene) ring system has emerged as a valuable molecular core component for the development of inhibitors of tubulin assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39) and its corresponding amine-based congener (referred to as KGP156, compound 45), which demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy examples of such benzosuberene-based compounds. In order to extend the structure-activity relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied. The synthetic approach to these compounds featured a sequential Wittig olefination, reduction, Eaton's reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate, and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against tubulin assembly (IC₅₀ approximately 1.0 μM) and strongly cytotoxic against selected human cancer cell lines (for example, GI₅₀ = 5.47 nM against NCI-H460 cells with fluoro-benzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were also synthesized and evaluated in this study.

Full text of DOI:10.1016/j.bmc.2013.08.035

Accepted Manuscript
Synthesis of structurally diverse benzosuberene analogues and their biological
evaluation as anti-cancer agents
Rajendra P. Tanpure, Clinton S. George, Tracy E. Strecker, Laxman Devkota,
Justin K. Tidmore, Chen-Ming Lin, Christine A. Herdman, Matthew T.
MacDonough, Madhavi Sriram, David J. Chaplin, Mary Lynn Trawick, Kevin
G. Pinney
PII:
S0968-0896(13)00729-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.035
BMC 11051
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 June 2013
10 August 2013
18 August 2013
Please cite this article as: Tanpure, R.P., George, C.S., Strecker, T.E., Devkota, L., Tidmore, J.K., Lin, C-M.,
Herdman, C.A., MacDonough, M.T., Sriram, M., Chaplin, D.J., Trawick, M.L., Pinney, K.G., Synthesis of
structurally diverse benzosuberene analogues and their biological evaluation as anti-cancer agents, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.035
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Synthesis of structurally diverse benzosuberene
analogues and their biological evaluation as anti-
cancer agents
a
a
a
a
Rajendra P. Tanpure , Clinton S. George , Tracy E. Strecker , Laxman Devkota , Justin K.
a
a
a
a
a
Tidmore , Chen-Ming Lin , Christine A. Herdman , Matthew T. MacDonough , Madhavi Sriram ,
b
a
a
David J. Chaplin , Mary Lynn Trawick , and Kevin G. Pinney *
a
Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco,
Texas 76798-7348, United States
b
OXiGENE Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, California 94080,
United States
*
Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710 4272.
E-mail address: Kevin_Pinney@baylor.edu (K.G. Pinney).
1

Abstract
Diversely functionalized, fused aryl-alkyl ring systems hold a prominent position as well-
established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7
fused, also referred to as dihydro-5H-benzo[7]annulene and benzocycloheptene) ring system has
emerged as a valuable molecular core component for the development of inhibitors of tubulin
assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular
disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39)
and its corresponding amine-based congener (referred to as KGP156, compound 45), which
demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent
cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy
examples of such benzosuberene-based compounds. In order to extend the structure-activity
relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of
eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the
pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied.
The synthetic approach to these compounds featured a sequential Wittig olefination, reduction,
Eaton’s reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate,
and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a
ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the
fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl
ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against
tubulin assembly (IC approximately 1.0 M) and strongly cytotoxic against selected human
5
0
cancer cell lines (for example, GI50 = 5.47 nM against NCI-H460 cells with fluoro-
benzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as
2

KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were
also synthesized and evaluated in this study.
Keywords: inhibitors of tubulin assembly, benzosuberene-based anti-cancer agents, vascular
disrupting agents (VDAs), combretastatin analogues.
3

1
. Introduction
The discovery and development of small-molecule, anti-cancer agents that demonstrate
pronounced cytotoxicity against human cancer cell lines remains an important goal in the search
for new cancer treatment agents and related therapeutic strategies. An established approach
involves the selective targeting of tumor vasculature and more specifically the tubulin-
microtubule protein system. Research efforts in this area have led to a class of therapeutics
1
,2
known as vascular targeting agents (VTAs) that is further sub-divided into angiogenic
3
,4,5
inhibiting agents (AIAs),
which interfere with tumor neovascularization and vascular
including both small-molecules and biologics, which selectively
3,4,6-9
disrupting agents (VDAs),
damage existing tumor vasculature. Tubulin-binding VDAs function through the inhibition of
tubulin polymerization within endothelial cells lining tumor microvessels. A subsequent series
of cell signaling events leads to morphological transformations (flat to round) of these
endothelial cells and results in microvessel occlusion and vascular shutdown, which ultimately
3
,6,10,11
starves the tumor of necessary nutrients and oxygen.
While still an active area of research
inquiry, there is evidence indicating that activated endothelial cells (such as those lining the
vasculature supplying tumors) are affected by VDAs to a greater extent than quiescent
12-14
endothelial cells found in vasculature feeding normal tissue.
A significant focal point centers
1
5,16
on small-molecule VDAs that bind to the colchicine site on the -tubulin heterodimer. It is
important to note that AIAs and VDAs function biologically through mechanistically distinct
9
,17
pathways.
A progressive research agenda in our laboratory, focused on the design and synthesis of
inhibitors of tubulin assembly, led to the establishment of functionalized benzosuberene
18-20
analogues as promising compounds for further evaluation as anti-cancer agents.
This
4

exploration was guided, in part, by the molecular structures of colchicine and certain members of
the combretastatin family of natural products along with our previously discovered
18,21,22
dihydronaphthalene-based analogues
(Fig. 1).
Figure 1. Representative Small-Molecule Inhibitors of Tubulin Assembly That Bind to the
1
8
19
Colchicine Site; Including: Benzosuberene Analogues (KGP18, KGP156 ),
1
8,21-23
22,23
Dihydronaphthalene Analogues (KGP03,
(
KGP05 ), and Combretastatin Analogues
2
4,25
26
27,28
10,29
30
CA4, CA1, Ajinomoto Amine, KGP06, KGP08 ).
2
4,25
26
Specifically, combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are among the most
potent colchicine site tubulin-binding agents and they were each further developed into their
31-33
corresponding phosphate prodrug salts to improve aqueous solubility.
Efforts to mimic the
combretastatin molecular-framework and optimize a wide-variety of biological parameters,
3
4,35
resulted in a cadre of structural modifications. A very limited sub-set of these molecules that
3
6,37
18,21-23,38,39
dihydronaphthalene,
were inspired by the combretastatins include benzophenone,
40-43
indole,
18,19,20,44
2
and benzosuberene
analogues in which a single sp hybridized carbon atom
4
5
bridges the two aromatic rings and maintains the pseudo cis-orientation that is important for
5

enhanced biological activity. Two such benzosuberene-based compounds, referred to as KGP18
1
8,20,46
19
(
phenol-based)
and KGP156 (amine-based), have emerged as potential pre-clinical
candidates (Fig. 1). In addition to their robust in vitro cytotoxicity against human cancer cell
lines (picomolar for KGP18 and nanomolar for KGP156), preliminary studies have shown that
1
9,20,47
these benzosuberene analogues function as vascular disrupting agents (VDAs).
Our original
synthetic route to these benzosuberene analogues included a ring expansion, reduction, selective
1
8
oxidation sequence that while reliable, was somewhat limiting due to low reaction yields.
A
revised synthetic methodology that relies on an efficient ring-closing cyclization step was
19
utilized in our synthesis of the amine analogue, KGP156, and a recent publication by Maderna
4
4
and co-workers describes an efficient ring closing metathesis (RCM) step to assemble the
benzosuberene molecular core followed by a Suzuki coupling reaction. In order to advance the
known structure activity relationship (SAR) data, a collection of eleven benzosuberene
analogues, selected primarily to explore functional group modification at the C-1 position, were
prepared by chemical synthesis and evaluated for their cytotoxicity against selected human
cancer cell lines, and for their ability to inhibit tubulin assembly.
2
2
. Results and Discussions
.1 Chemistry
A series of eleven functionalized benzosuberene-based analogues were prepared by chemical
synthesis. The synthetic strategy relied on an intramolecular Fridel-Crafts annulation with
4
8,49
Eaton’s reagent
to install the benzosuberone ring system (Scheme 1). The prerequisite
carboxylic acid derivatives were prepared through a sequential Wittig olefination followed by
catalytic hydrogenation sequence, which, overall, is nearly identical to the synthetic
5
0
methodology described by Negoro et al. This synthetic strategy to prepare benzosuberone
6

19,46,51
derivatives has proved to be highly proficient within our laboratory.
Protecting group
strategies were included when necessary. Additional modifications of benzosuberones 16 and 18
5
2
were achieved through selective demethylation with ionic liquid [TMAH][Al Cl ] resulting in
2
7
phenolic
Scheme 1. Synthetic route to benzosuberone intermediates 16-20.
benzosuberones 21-22 which were subsequently converted to their corresponding silyl ethers 23-
4 with TBSCl (Scheme 2). Confirmation of the regioselective demethylation to form
benzosuberone intermediate 22 was provided by X-ray crystallographic analysis of the final
2
Scheme 2. Synthetic modifications affording benzosuberone intermediates 23-24.
7

compound KGP18 (compound 39) that resulted from further synthetic manipulation of
intermediate 22 (see Supplementary Data). Similarly, the regioselective demethylation to form
intermediate 21 was confirmed by HSQC and HMBC analysis, along with X-ray crystallography,
of final compound 38 (see Supplementary Data). The functionalized pendant aryl ring was
incorporated, in each case, through the addition of an appropriately functionalized aryllithium
intermediate (prepared in situ from the corresponding aryl bromide) to the requisite
benzosuberone derivative to generate the corresponding tertiary alcohol that underwent
elimination to form the benzosuberene core structure (Scheme 3). This overall synthetic
sequence proved to be quite robust and is complementary to other known synthetic routes toward
1
8,44,53,54
55
benzosuberene ring systems
(including Friedel-Crafts cyclization ). An alternative
synthetic strategy for the preparation of KGP18 (compound 39) that utilized an intramolecular
acid chloride mediated cyclization strategy was also successful (see Supplementary Data for
details).
Li
H
3
CO
H
3
CO
R
4
R4
H
3
CO
H CO
3
O
H
3
CO
R
4
OH
R
R
3
2
R3
OCH
3
AcOH, reflux
52-97%
R
R
3
2
-78 oC, THF
11-84%
R2
R
1
R1
= OCH
R
1
1
1
1
2
2
2
2
7: R
8: R
9: R
0: R
3: R
4: R
5: R
1
1
1
1
1
1
1
= OTs, R
= R = OCH
= Cl, R = OCH
= F, R
= R = OCH
= OTBS, R
= R = R = H
2
= R
3
= OCH
= H
, R
, R
3
26: R
1
1
1
1
1
1
1
1
= OTs, R
= R = OCH
= Cl, R
= F, R
= R = OCH
2
= R
3
3
, R
4
= OCH
3
34: R
1
1
1
1
1
1
1
1
= OTs, R
= R = OCH
= Cl, R
= F, R
= R = OCH
= OH, R
= OTBS, R
= R = R = H, R
2
= R
3
= OCH
= H, R
, R = H, R
, R = H, R
= OH, R
, R = H, R
= OCH
= OCH
3
, R
4
= OCH
3
2
3
, R
3
27: R
28: R
29: R
30: R
31: R
32: R
2
3
, R
3
= H, R
4
= H
35: R
36: R
37: R
38: R
39: R
40: R
41: R
2
3
, R
3
4
= H
2
3
3
= H
2
= OCH
3
, R
3
= H, R
= H, R
= OTBS, R
4
= OCH
3
2
= OCH
3
3
4
= OCH
3
2
= OCH
3
3
= H
= OTBS
, R = H
2
= OCH
3
, R
3
4
= OCH
= OCH
3
2
= OCH
3
3
4
= OCH
= OCH
= OCH
3
3
3
3
, R
2
3
3
, R
2
4
3
3
3
, R
2
4
3
2
= OCH
3
3
= OTBS, R
= OTBS, R
2
= OCH
= OCH
3
, R
, R
3
= H, R
= H, R
4
4
= OCH
3
2
= OCH
3
3
4
2
3
2
3
3
= H
2
3
, R
3
= H, R
4
= H
3
3: R
= R
2
= R
3
= H, R
4
= OCH
3
2
3
4
3
Scheme 3. Synthetic route to benzosuberene analogues 34-41.
The preparation of benzosuberene analogues 42-44 required further synthetic manipulation.
Analogue 42 was achieved through desilylation with TBAF and analogue 43 was obtained by
removal of the tosyl protecting group upon treatment with NaOH (Scheme 4). In order to
8

facilitate a variety of planned in vivo studies, the hydrophobic benzosuberene analogue 39
(
(
KGP18) was converted to its corresponding water-soluble, disodium phosphate prodrug salt 44
KGP265) through phosphorylation with POCl followed by treatment with NaOH. This
3
phosphate prodrug strategy has proved to be highly effective for both combretastatin A-4P
3
3
31
(
CA4P) and combretastatin A-1P (CA1P).
H
3
CO
H
3
CO
H
3
CO
H
3
CO
TBAF, THF
6%
8
H
3
CO
3
H CO
OTBS
OH
42
OCH
40
3
H CO
OCH
3
H
3
CO
H
3
CO
3
H CO
H
3
CO
OCH
3
OCH
3
3
H
3
CO
H
3
CO
3
H CO
H
H
3
3
CO
CO
NaOH (2M)
μW, 100 o
4%
H
H
3
3
CO
CO
1. POCl , CH Cl , Py
3 2 2
H
3
CO
C
2
. NaOH (2M)
8%
O
O
Na
Na
H
3
CO
P
8
5
OTs
OH
43
O
O
OH
39
34
44
Scheme 4. Synthetic route to benzosuberene analogues 42-44.
1
9
Previously described hydrophobic aniline analogue 45 (KGP156) was converted to its
corresponding water-soluble, serinamide salt 48 through initial amide bond formation between
benzosuberene 45 and acetyl-Fmoc protected serinamide to form serinamide 46 and subsequent
treatment with NaOH to yield serinamide benzosuberene analogue 47 (Scheme 5). Treatment of
serinamide 47 with HCl led to the corresponding hydrochloride serinamide salt 48. This
5
6
chemistry is reminiscent of the synthetic strategy employed by Ohsumi and co-workers for the
preparation of water-soluble amino acid (and related) prodrug salts of 3-amino-combretastatin
5
7,58
(
serinamide analogue referred to as AVE8062) along with our later studies of 2-amino-
1
0,29
30
combretastatin and 2,3-diamino-combretastatin serinamide salts.
9

Scheme 5. Synthetic route to benzosuberene analogues 47-48.
.2 Biological Evaluation
2
The batch of KGP18 (prepared using the synthetic strategy described herein) along with ten new
analogues (structures depicted in Fig. 2) incorporating functional group modifications were
evaluated (Table 1) for their ability to inhibit tubulin assembly (cell free assay) and for their
cytotoxicity against three human cancer cell lines (SK-OV-3, ovarian; NCI-H460, lung; and DU-
1
45, prostate). As anticipated, the potent cytotoxicity (GI < 100 pM) observed for KGP18
50
(
compound 39, bearing a 1-hydroxy group) in this study mirrors (within error-limits inherent to
the assay) our previously reported data for KGP18 (prepared by a separate synthetic
1
8
methodology).
Replacement of the 1-hydroxy moiety with a fluorine atom resulted in a
benzosuberene analogue 37 that was both strongly inhibitory against tubulin assembly (IC =
5
0
0
.89 M) and potently cytotoxic (GI = 5.47 nM against NCI-H460 cells, for example). The
5
0
chlorine atom congener 36 was equally active as an inhibitor of tubulin assembly and only
slightly less cytotoxic. Fluorine atom substitution has been a productive strategy in certain
5
9-60
structurally related combretastatin analogues.
An analogue of KGP18 that replaced the
pendant trimethoxyaryl ring with a dimethoxyaryl ring (compound 42) was also active against
tubulin assembly and as a cytotoxic agent (GI = 33.4 nM against SK-OV-3 cells). Other
5
0
structural modifications (compounds 35, 38, 41, and 43) around the pendant aryl ring resulted in
benzosuberene analogues that were inactive (IC > 40 M) as inhibitors of tubulin assembly and
5
0
1
0

decidedly less cytotoxic (against these three cell lines) thus underscoring the limited structural
6
1
variation that is tolerated in these molecules. Data for both combretastatin A-4 (CA4) and our
1
9
previously reported 6-aminobenzosuberene analogue (KGP156, compound 45) are included in
Table 1 for comparative reference. The water-soluble phosphate prodrug salt of KGP18,
referred to as KGP265 (compound 44) was inactive (IC > 40 M) against tubulin as anticipated
5
0
in this cell-free assay (which is devoid of enzymes necessary to cleave the inactive prodrug to its
active parent compound (KGP18)), however it was active in terms of cytotoxicity (GI = 9.51
5
0
nM against DU-145 cells, for example). It is well-established that human cancer cell lines have
6
2
one or more phosphatase enzyme at their cell surface, thus it was expected that the prodrug
would be very effectively cleaved to yield the parent compound under these conditions. A
water-soluble serinamide prodrug salt 48 of KGP156 (compound 45) proved to be inactive
against tubulin assembly (as anticipated in this type of cell free assay) and also surprisingly
inactive in terms of cytotoxicity. It is possible that either the level of requisite aminopeptidase
enzymes secreted from these cells is not sufficient to cleave the prodrug construct, or that this
particular benzosuberene prodrug (compound 48) itself is not a good substrate for the enzyme,
since other (structurally non-related) serinamide prodrug salts do show cytotoxicity in this type
6
3
of assay and some have been evaluated in the presence of exogenous peptidase enzymes to
5
6,64
demonstrate enzyme-mediated hydrolysis with release of the parent amino-drug.
Further
2
7,61
study is necessary in this regard. It is well documented throughout the literature that the most
active small-molecule inhibitors of tubulin assembly are typically in the low micromolar range
(
in terms of IC ) while the same compounds demonstrate in vitro cytotoxicity with GI values in
5
0
50
the nanomolar to sub-nanomolar range. This activity difference (cell-free tubulin assay versus
cell-based in vitro cytotoxicity assay) can be attributed to several possible factors including
stoichiometry (inhibitor to tubulin heterodimer ratio) differences between the cell-free assay and
1
1

what takes place in cells, the cell-based release (during microtubule disassembly) of molecular
components (factors) that increase cytotoxicity through signal transduction pathways, and the
6
5
practical lower limit inherent to this type of inhibition of tubulin assembly assay.
1
2

Table 1. Inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines
SK-OV-3, NCI-H460, and DU-145.
Inhibition of tubulin
polymerization
a
Compound
IC ( M)
GI ( M) SRB assay
5
0
50
SK-OV-3
0.00455
0.00119
>89.5
NCI-H460
DU-145
b
c
c
CA4
1.0
0.00223
0.00194
>136.5
0.0713
0.00547
18.0
0.00327
0.00323
42.5
b
c
c
CA4P
>40
>40
0.93
0.89
>40
3
3
3
3
3
4
4
4
4
4
4
4
5
6
7
8
9
1
2
3
4
5
7
8
0.0444
0.00751
7.13
0.152
0.0189
15.7
d
e
e
e
1.4
0.0000543
19.1
0.0000418
32.5
0.0000249
22.0
>40
0.74
>40
>40
0.0334
18.1
0.262
0.109
25.9
38.4
0.00772
0.00796
0.00951
f
g
g
g
1.2
0.000102
0.00280
0.00223
h
nd
36.8
39.0
53.8
70.5
h
nd
35.0
69.4
a
b
c
d
Average of n > 3 independent determinations. Data from ref. 61 For additional data see ref. 61. Data from ref.
e
f
g
h
1
9. For additional data see ref. 18. Data from ref. 19. For additional data see ref. 19. nd = not determined in
this study.
3
. Conclusion
In summary, we have prepared eleven new benzosuberene-based analogues through an extension
1
9,20
of our previously reported synthetic methodology directed towards these ring-fused systems.
The most active compounds (in terms of inhibition of tubulin assembly and cytotoxicity against
selected human cancer cell lines) feature fluorine atom (compound 37) or chlorine atom
1
3

(
compound 36) incorporation at position-1 of the fused aryl ring along with a dimethoxyaryl ring
modification (compound 42) of the trimethoxyaryl ring bearing-phenolic analogue KGP18
compound 39). Thus the known SAR for benzosuberene derivatives of this type has been
(
extended. The two water-soluble prodrug salts (44 and 48) should prove useful for future in vivo
studies.
4
4
4
. Experimental Section
.1 Chemistry
.1.1 Materials and instrumentation
Methylene chloride (CH Cl ), acetonitrile, methanol (MeOH), ethanol (EtOH),
2
2
dimethylformamide (DMF), and tetrahydrofuran (THF) were used in their anhydrous form as
obtained from the chemical suppliers. Reactions were performed under an inert atmosphere using
nitrogen gas unless specified. Thin-layer chromatography (TLC) plates (pre-coated glass plates
with silica gel 60 F , 0.25 mm thickness) were used to monitor reactions. Reactions carried out
2
54
under microwave irradiation were performed with a Biotage Initiator Microwave Synthesizer.
Purification of intermediates and products was carried out with a Biotage Isolera 1 or 4 flash
purification system using silica gel (200-400 mesh, 60 Å) or RP-18 prepacked columns.
1
Intermediates and products synthesized were characterized on the basis of their H NMR (500
1
3
31
19
MHz), C NMR (125 MHz), P NMR (202 MHz), and F NMR (470 MHz) spectroscopic data.
All the chemical shifts are expressed in ppm (δ), coupling constants (J) are presented in Hz, and
peak patterns are reported as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), septet
(
sept), and multiplet (m). Mass spectrometry was carried out under positive ESI (electrospray
ionization) using a Thermo scientific LTQ Orbitrap Discovery instrument. Purity of the final
compounds was further analyzed at 25 °C using a Agilent 1200 HPLC system with a diode-array
1
4

detector ( = 190-400 nm), a Zorbax XDB-C18 HPLC column (4.6 mm x 150 mm, 5 μm), and a
Zorbax reliance cartridge guard-column; eluents, solvent A: H O, solvent B: acetonitrile;
2
gradient, 90%A / 10%B to 0%A / 100%B over 0 to 40 min; flow rate 1.0 mL/min; injection
volume 20 μL; monitored at wavelengths of 254, 280 and 300 nm. Column volume is
represented by CV.
Experimental Procedures for Final Compound 38
2
0
4
.1.2. (Z)/(E)- 5-(2′,3′,4′-Trimethoxyphenyl)pent-4-enoic acid (6). To a well-stirred solution of
(
3-carboxypropyl)triphenylphosphonium bromide (24.06 g, 56.05 mmol) in THF (400 mL) was
°
added K-OtBu (12.30 g, 109.6 mmol). The reaction mixture was then cooled to 0 C and stirred
for 15 min. A solution of aldehyde 1 (9.84 g, 50.2 mmol) in THF (25 mL) was added dropwise
and the reaction mixture was stirred and allowed to reach room temperature. The reaction
mixture was diluted with H O (50 mL) and extracted with Et O (2 × 200 mL). The aqueous
2
2
phase was acidified with 2 M HCl until the product precipitated making the solution cloudy and
then becoming clear again. The acidified aqueous phase was extracted with EtOAc (3 x 100
mL). The combined organic extract was washed with brine, dried over Na
2 4
SO , filtered,
concentrated under reduced pressure. Purification by flash chromatography using a prepacked
1
2
60 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 20%A / 80%B (1 CV),
0%A / 80%B → 60%A / 40%B (10 CV), 60%A / 40%B (2 CV); flow rate: 50 mL/min;
monitored at 254 and 280 nm] afforded the mixture of E/Z-isomers 6 (9.48 g, 35.6 mmol, 64%)
1
as a pale yellow liquid. H NMR (Mixture of E and Z) (CDCl
, 500 MHz): δ 7.11 (1H, d, J =
3
8
.7 Hz, H-6′), 6.94 (1H, d, J = 8.6 Hz, H-6′), 6.65 (1H, d, J = 8.7 Hz, H-5′), 6.65 (1H, d, J = 8.6
Hz, H-5′), 6.64 (1H, d, J = 16.1 Hz, H-5), 6.52 (1H, dt, J = 11.5, 2 Hz, H-5), 6.11 (1H, m, J =
6.1 Hz, H-4), 5.63 (1H, m, J = 11.5 Hz, H-4), 3.88-3.83 (3 × 3H, s, OCH -2′, -3′, -4′), 3.88-3.83
3 × 3H, s, OCH -2′, -3′, -4′), 2.59 (2H, m, CH -2/3), 2.56-2.54 (4H, m, CH -2, -3), 2.47 (2H, m,
1
3
(
3
2
2
1
5

1
3
2
CH -3/2). C NMR (CDCl33, 125 MHz) δ 178.9, 178.7, 152.9, 152.8, 151.7, 151.1, 142.3,
1
6
4
42.2, 129.5, 127.5, 125.4, 125.2, 124.4, 124.2, 123.9, 120.7, 107.7, 106.9, 61.1, 61.0, 61.95,
0.89, 56.0, 55.9, 34.0, 33.9, 28.3, 23.9.
2
0,66
.1.3. 5-(2′,3′,4′-Trimethoxyphenyl)pentanoic acid (11).
To a solution of 6 (9.15 g, 34.4
mmol) in MeOH (200 mL) was added 10% Pd-C. The flask was evacuated under vacuum and
gas was introduced via balloon. The reaction was stirred for 24 h and checked for completion
H
2
by filtering a small amount of the reaction mixture through Celite®, evaporating the solvent, and
1
recording the H NMR. On completion, the reaction mixture was filtered through Celite®,
concentrated under reduced pressure, and the resulting pale yellow liquid was subjected to flash
chromatography using a prepacked 50 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10%A / 90%B (1 CV), 10%A / 90%B → 50%A / 50%B (10 CV), 50%A / 50%B (2
CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] affording 11 (8.76 g, 32.6 mmol,
1
9
6
4
5%) as a pale yellow liquid. H NMR (CDCl
3
, 500 MHz): δ 6.81 (1H, d, J = 8.5 Hz, H-6´),
-2′), 3.86 (3H, s, OCH -3′), 3.84 (6H, s, OCH
′), 2.57 (2H, t, J = 7.5 Hz, H-5), 2.39 (2H, t, J = 7.3 Hz, H-2), 1.68 (2H, m, H-3), 1.61 (2H, m,
.60 (1H, d, J = 8.5 Hz, H-5'), 3.87 (3H, s, OCH
3
3
3
-
1
3
H-4). C NMR (CDCl
3
, 125 MHz): δ 179.9 (C=O, C-1), 152.0 (C, C-4′), 151.8 (C, C-2′), 142.3
(
C, C-3′), 128.0 (C, C-1′), 123.7 (CH, C-6′), 107.2 (CH, C-5′), 60.9 (CH , OCH -2′), 60.7 (CH ,
3
3
3
OCH
3
-3′), 56.0 (CH
3
, OCH
3
-4′), 33.9 (CH
2
, C-2), 30.2 (CH
2
, C-4), 29.3 (CH
2
2
, C-5), 24.5 (CH ,
C-3).
1
8,20,66
4
.1.4. 1,2,3-Trimethoxy-benzocycloheptan-5-one (16). Pentanoic acid 11 (2.68 g, 10
mmol) was dissolved in Eaton’s reagent [40.2 mL, P O (7.7 wt%) in methanesulfonic acid] and
2
5
the reaction mixture was stirred for 12 h. The reaction mixture was poured over ice and the ice
was allowed to melt. The aqueous phase was extracted with CH Cl (2 × 100 mL) and the
2
2
1
6

3
combined organic extract was washed with saturated NaHCO (2 × 200 mL). The organic
extract was dried over Na SO , concentrated under reduced pressure, subjected to flash
2
4
chromatography using a prepacked 50 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10%A / 90%B (1 CV), 10%A / 90%B → 50%A / 50%B (10 CV), 50%A / 50%B (2
CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] affording 16 (2.34 g, 9.34 mmol,
1
9
3%) as a pale yellow liquid. H NMR (CDCl
3
, 500 MHz): δ 7.13 (1H, s, H-4), 3.93 (3H, s,
-1), 2.94 (2H, dd, J = 6.9, 5.2 Hz, H-9), 2.72
OCH
3
-2), 3.88 (3H, s, OCH
2H, m, H-6), 1.83 (2H, m, H-8), 1.81 (2H, m, H-7). C NMR (CDCl
C=O, C-5), 151.6 (C, C-3), 151.0 (C, C-1), 145.9 (C, C-2), 134.4 (C, C-10/11), 128.9 (C, C-
0/11), 107.5 (CH, C-4), 61.4 (CH , OCH -1), 60.8 (CH , OCH -2), 56.0 (CH , OCH -3), 40.8
CH , C-6), 25.0 (CH , C-8), 23.0 (CH , C-9), 20.9 (CH , C-7).
.1.5. [TMAH][Al Cl ]. To a suspension of AlCl (26.71 g, 101.4 mmol) in 200 mL CH
3
-3), 3.84 (6H, s, OCH
3
1
3
(
(
3
, 125 MHz): δ 205.0
1
3
3
3
3
3
3
(
2
2
2
2
5
2
4
2
7
3
2 2
Cl
cooled to 0 ºC, trimethylammonium chloride [TMAH] (9.55 g, 50.7 mmol) was added. The
reaction mixture was allowed to warm to room temperature and stirred for 2 h. The transparent
yellow solution of ionic liquid was used as such for the deprotection of methyl ethers of
benzosuberones.
2
0
4
.1.6. 2-Hydroxy-1,3-dimethoxy-benzocycloheptan-5-one (21). To a solution of 16 (5.30 g,
2
1.2 mmol) in CH Cl (50 mL) cooled to 0 ºC, [TMAH][Al Cl ] (36.00 mL, 23.32 mmol, 1.93
2
2
2
7
M in CH Cl ) was added dropwise. The reaction was monitored by TLC and upon completion,
2
2
ice cold water was added to the reaction. The aqueous layer was extracted with CH
2 2
Cl (2 x 100
mL). The organic extract was washed with brine, dried over MgSO , filtered, concentrated, and
4
subjected to flash chromatography using a prepacked 50 g silica column [solvent A: EtOAc;
solvent B: hexanes; gradient: 10%A / 90%B (1 CV), 10%A / 90%B → 40%A / 60%B (10 CV),
1
7

4
0%A / 60%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280 nm]; to afford phenol 21
0.58 g, 2.45 mmol, 12%). NMR characterization took place after the TBS protection (see
compound 23).
.1.7. 2-[(tert-Butyldimethylsilyl)oxy]-1,3-dimethoxy-benzocycloheptan-5-one (23). To a
solution of phenol 21 (0.58 g, 2.45 mmol) and DIPEA (2.00 mL, 11.5 mmol) in DMF (5 mL) at
ºC was added TBSCl (0.82 g, 5.44 mmol) in portions. The reaction mixture was stirred for 6 h,
diluted with H O (5 mL), and extracted with Et O (2 × 20 mL). The organic extract was washed
with brine, dried over MgSO , filtered, concentrated under reduced pressure, and subjected to
(
2
0
4
0
2
2
4
flash chromatography using a prepacked 25 g silica column [solvent A: EtOAc; solvent B:
hexanes; gradient: 3%A / 97%B (1 CV), 3%A / 97%B → 30%A / 70%B (10 CV), 30%A / 70%B
(
2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford ketone 23 (0.82 g, 2.34
1
mmol, 94%) as a white solid. H NMR (CDCl
3
, 500 MHz): δ 7.03 (1H, s, H-4), 3.72 (3H, s,
OCH
.93 (9H, (CH
C, C-3), 149.3 (C, C-1), 142.6 (C, C-2), 131.9 (C, C-10/11), 129.4 (C, C-10/11), 40.8 (CH, C-
), 107.2 (CH, C-4), 60.8 (CH , OCH -2/3), 55.3 (CH , OCH -2/3), 25.7 (CH , (CH ), 25.1
CH , C-8), , 23.1 (CH , C-9), 21.0 (CH , C-7), 18.7 (C, (C(CH ) ), -4.6 (CH , Si(CH ) )
3
3
-1/3), 3.65 (3H, s, OCH -1/3), 2.85 (2H, m, H-9), 2.61 (2H, m, H-6), 1.71 (4H, m, H-7, -8),
13
0
3
)
3
), , 0.07 (6H, Si(CH
3 2 3
) ). C NMR (CDCl , 125 MHz): δ 204.8 (C, C-9), 149.9
(
8
3
3
3
3
3
3 3
)
(
2
2
2
3 3
3
3 2
4
.1.8. 2-[(tert-Butyldimethylsilyl)oxy]-1,3-dimethoxy-5-(3′,4′,5′-trimethoxyphenyl)-
2
0
benzocycloheptan-5-ol (30). To a solution of 3,4,5-trimethoxyphenyl bromide (1.04 g, 4.21
mmol) in THF (50 mL) at -78 ºC, n-BuLi (1.70 mL, 2.5 M) was added and the reaction mixture
was stirred for 30 min. Ketone 23 (0.73 g, 2.08 mmol) in 5 mL THF was added using an
addition funnel over a period of 15 min. The reaction mixture was stirred for 12 h and was
allowed to warm to room temperature. The reaction mixture was diluted with H
2
O (25 mL) and
1
8

extracted with EtOAc (2 × 25 mL). The organic extract was washed with brine, dried over
MgSO , filtered, concentrated under reduced pressure, subjected to flash chromatography using a
4
prepacked 25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 15%A / 85%B (1
CV), 15%A / 85%B → 50%A / 50%B (10 CV), 50%A / 50%B (2 CV); flow rate: 25 mL/min;
monitored at 254 and 280 nm] resulting in alcohol 30 (0.80 g, 1.54 mmol, 74%) as a white solid.
1
3
H NMR (CDCl , 500 MHz): δ 6.92 (1H, s, H-4), 6.28 (2H, s), 3.70-3.48 (15H, m), 2.97 (1H,
m), 2.48 (1H, m), 2.41 (1H, m), 1.98 (2H, m), 1.77 (2H, m), 1.58 (1H, m), 0.87 (9H, s), 0.00
6H, s).
(
2
0
4
.1.9. 1,3-Dimethoxy-2-hydroxy-5-(3′,4′,5′-trimethoxyphenyl)-benzocyclohepta-5-ene (38).
A
2
solution of 30 (0.77 g, 10.6 mmol) in AcOH (20 mL) and H O (20 mL) was heated to reflux at
1
10 °C for 24 h. The reaction mixture was cooled and the reaction mixture was concentrated
under reduced pressure and subjected to flash chromatography using a prepacked 25 g silica
column [solvent A: EtOAc; solvent B: hexanes; gradient: 10%A / 40%B (1 CV), 10%A / 40%B
→
10%A / 90%B (10 CV), 40%A / 60%B (2 CV); flow rate: 25 mL/min; monitored at 254 and
1
2
80 nm] affording benzosuberene 38 (0.49 g, 5.48 mmol, 52%) as a white solid. H NMR
(CDCl
3
, 500 MHz): δ 6.50 (2H, s, H-2′, H-6′), 6.38 (1H, s, H-4), 6.37 (1H, t, J = 12.0 Hz, H-6),
5
3
=
.62 (1H, s, OH-2), 3.92 (3H, s, OCH -1), 3.88 (3H, s, OCH -4′), 3.81 (6H, s, OCH -3′, -5′),
3
3
3
.75 (3H, s, OCH ‐1), 2.66 (2H, t, J = 7.0 Hz, H-9), 2.15 (2H, p, J = 7.1 Hz, H-8), 1.97 (2H, q, J
3
1
3
3
7.2 Hz, H-7). C NMR (CDCl , 125 MHz): δ 153.1 (C, C-3′, C-5′), 145.4 (C, C-3), 144.6 (C,
C-1), 143.0 (C, C-5), 138.2 (C, C-1′), 137.7 (C, C-2), 137.5 (C, C-4′), 131.7 (C, C-10), 128.4 (C,
C-11), 127.8 (CH, C-4), 108.3 (CH, C-6), 105.3 (CH, C-2′, C-6′), 61.5 (CH , OCH -1), 61.1
3
3
(
CH
3
, OCH
3
-4′), 56.5 (CH , OCH
3
, OCH
3
-3′, -5′), 56.3 (CH
3
3 2 2
-3), 35.3 (CH , C-8), 25.8 (CH , C-
7
), 23.9 (CH
2
, C-9). Analysis: Calculated for C23H O , C 68.38, H 6.78, O 24.84. Found: C
26 6
1
9

+
+
6
8.22, H 6.85. HRMS: m/z: observed 409.1629 [M + Na] , calculated for C22
H
26
O
6
Na ,
4
09.1622. HPLC: 14.68 min.
Experimental Procedures for Final Compounds 39 and 44
2
0
4
.1.10. (Z)/(E)- 5-(2′,3′-Dimethoxyphenyl)pent-4-enoic acid (8). To a well-stirred solution of
(
3-carboxypropyl)triphenylphosphonium bromide (21.65 g, 50.43 mmol) in THF (500 mL) was
o
added K-OtBu (11.3 g, 101 mmol). The reaction mixture was then cooled to 0 C and stirred for
1
5 min. A solution of aldehyde 3 (8.42 g, 50.7 mmol) in THF (60 mL) was added dropwise and
the reaction mixture was allowed to reach room temperature. The reaction mixture was diluted
with H O (50 mL) and extracted with Et O (2 x 200 mL). The aqueous phase was acidified with
M HCl until the product precipitated making the solution cloudy and then becoming clear
again. This acidified aqueous phase was extracted with EtOAc (3 x 100 mL). The organic
extract was washed with brine, dried over Na SO , filtered, concentrated under reduced pressure
2
2
2
2
4
and subjected to flash chromatography using a prepacked 160 g silica column [solvent A:
EtOAc; solvent B: hexanes; gradient: 20%A / 80%B (1 CV), 10%A / 80%B → 60%A / 40%B
(
10 CV), 60%A / 40%B (2 CV); flow rate: 50 mL/min; monitored at 254 and 280 nm] affording
1
a mixture of E/Z-isomers 8 (6.98 g, 29.5 mmol, 58%) as a pale yellow oil. H NMR (E/Z-
isomers) (CDCl , 500 MHz): δ 7.03-6.99 (3H, m), 6.85-6.74 (3H, m), 6.76 (1H, d, J = 15.9 Hz),
3
6
3
.59 (1H, dt, J = 11.5, 1.5 Hz), 6.22 (1H, dt, J = 15.9, 6.0 Hz), 5.70 (1H, dt, J = 11.5, 7.5 Hz),
1
3
.86 (3H, s), 3.85 (3H, s), 3.78 (3H, s), 3.76 (3H, s), 2.60-2.53 (6H, m), 2.46 (2H, m). C NMR
(
E/Z-isomers) (CDCl
3
, 125 MHz): δ 179.14, 179.12, 153.0, 152.8, 146.9, 146.3, 131.5, 131.4,
1
3
30.7, 129.5, 125.7, 125.4, 124.0, 123.6, 121.9, 118.0, 111.3, 111.0, 60.8, 60.6, 55.8, 55.8, 34.0,
3.8, 28.3, 24.0.
2
0

2
0,67
4
.1.11. 5-(2′,3′-Dimethoxyphenyl)pentanoic acid (13).
mmol) in MeOH (100 mL) was added 10% Pd-C (0.70 g). The flask was evacuated under
vacuum and H gas was introduced via balloon and the reaction mixture was stirred for 24 h.
To a solution of 8 (6.78 g, 28.7
2
The reaction was monitored by filtering a small amount of the reaction mixture through Celite®,
1
evaporating the solvent, and recording the H NMR spectrum. Upon completion, the reaction
mixture was filtered through Celite®, concentrated under reduced pressure and subjected to flash
chromatography using a prepacked 50 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10%A / 90%B (1 CV), 10%A / 90%B → 50%A / 50%B (10 CV), 50%A / 50%B (2
CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] affording compound 13 (6.71 g, 28.2
1
mmol, 98%) as a pale yellow oil. H NMR (CDCl , 500 MHz): 6.97 (1H, t, J = 8.6 Hz, H-5′), δ
3
6
7
1
6
.76 (2H, d, J = 8.6 Hz, H-4′, -6′), 3.85 (3H, s, OCH
.0 Hz, H-2), 2.38 (2H t, J = 7.0, H-5), 1.67 (4H, m, H-3, -4). C NMR (CDCl
80.0 (C, C-1), 152.7 (C, C-3′), 147.1 (C, C-2′), 135.9 (C, C-1′), 123.8 (CH, C-5′), 121.9 (CH, C-
3
-3′), 3.81 (3H, s, OCH
3
-2′), 2.65 (2H, t, J =
1
3
3
, 125 MHz): δ
′), 110.2 (CH, C-4′), 60.6 (CH
3
, OCH
3
-2′), 55.7 (CH
3
, OCH
3
-3′), 34.0 (CH
2
2
, C-5), 30.1 (CH ,
+
C-4), 29.3 (CH
2
, C-2), 24.5 (CH
2
, C-3). HRMS: m/z: observed 261.1100 [M+Na] , calculated
+
for C13
H
18
O
4
Na , 261.1097. HPLC: 10.67 min.
.1.12. 1,2-Dimethoxy-benzocycloheptan-5-one (18).
was dissolved in Eaton’s reagent [75 mL, P
for 12 h. The reaction mixture was poured over ice and the ice was allowed to melt. The
aqueous phase was extracted with CH Cl (2 x 100 mL) and the organic extract was washed with
saturated NaHCO (2 x 200 mL). The organic extract was dried over Na SO , concentrated
2
0,68
4
Pentanoic acid 13 (6.76 g, 28.4 mmol)
2
5
O (7.7 wt%) in methanesulfonic acid] and stirred
2
2
3
2
4
under reduced pressure and subjected to flash chromatography using a prepacked 100 g silica
column [solvent A: EtOAc; solvent B: hexanes; gradient: 10%A / 90%B (1 CV), 10%A / 90%B
2
1

→
50%A / 50%B (10 CV), 50%A / 50%B (2 CV); flow rate: 40 mL/min; monitored at 254 and
1
2
80 nm] affording compound 18 (5.39 g, 24.5 mmol, 86%) as a pale yellow oil. H NMR
(
CDCl
3
, 500 MHz): δ 7.54 (1H, d, J = 8.5 Hz, H-4), 6.84 (1H, d, J = 8.5 Hz, H-3), 3.91 (3H, s,
-1), 3.80 (3H, s, OCH -2), 3.01 (2H, m, H-9), 2.70 (2H, m, H-6), 1.85 (2H, m, H-7), 1.81
, 125 MHz): δ 205.0 (C=O, C-5), 156.1 (C, C-1), 145.9 (C, C-
), 135.7 (CH, C-10/11), 132.8 (C, C-10/11), 125.5 (CH, C-4), 109.7 (CH, C-3), 61.1 (CH3,
-1/2), 55.8 (CH , OCH -1/2), 40.6 (CH , C-6), 24.9 (CH , C-7), 23.3 (CH , C-9), 20.9
, C-8). Analysis: Calculated for C13 : C 70.89, H 7.32, O 21.79. Found: C 70.94, H
.26. HPLC: 10.55 min.
.1.13. 1-Hydroxy-2-methoxy-benzocycloheptan-5-one (22).
Cl (5 mL) was added [TMAH][Al
) and the reaction mixture was subjected to microwave irradiation at 80 °C for 1h.
After the reaction was complete, water was added. The reaction mixture was stirred vigorously
for 2 min and the organic layer was extracted with CH Cl (2 x 25 mL). The organic extract was
washed with brine, dried over MgSO , filtered, concentrated under reduced pressure, and
OCH
3
3
1
3
(2H, m, H-8). C NMR (CDCl
3
2
OCH
3
3
3
2
2
2
(CH
2
16 3
H O
7
2
0,37,69
4
To a solution of methyl aryl
Cl ] (13.00 mL, 1.93 M
ether 18 (2.22 g, 10.1 mmol) in CH
in CH Cl
2
2
2
7
2
2
2
2
4
subjected to flash chromatography using a prepacked 100 g silica column [solvent A: EtOAc;
solvent B: hexanes; gradient: 10%A / 90%B (1 CV), 10%A / 90%B → 50%A / 50%B (10 CV),
5
0%A / 50%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] affording phenol 22
1
(
(
1.59 g, 7.70 mmol, 77%). H NMR (CDCl
3
, 500 MHz): δ 7.34 (1H, d, J = 8.6 Hz, H-4), 6.79
-2), 3.02 (2H, m, H-9), 2.71 (2H, t,
1H, d, J = 8.6 Hz, H-3), 5.79 (1H, s, OH), 3.94 (3H, s, OCH
3
1
3
J = 12.0 Hz, H-6), 1.85 (2H, m, H-8), 1.80 (2H, m, H-7). C NMR (CDCl
3
, 125 MHz): δ 205.0
(C, C-5), 149.2 (C, C-2), 142.4 (C, C-1), 133.3 (C, C-10/11), 127.7 (C, C-10/11), 120.8 (CH, C-
4
), 107.9 (CH, C-3), 56.1 (CH , OCH -2), 40.8 (CH , C-6), 24.5 (CH , C-8), 23.1 (CH , C-9),
3 3 2 2 2
2
2

2
1.3 (CH
HPLC: 7.08 min.
.1.14. 1-[(tert-Butyldimethylsilyl)oxy]-2-methoxy-benzocycloheptan-5-one (24). To a
2 14 3
, C-7). Analysis: Calculated for C12H O : C 69.88, H 6.84. Found: C 69.93, H 6.86.
2
0
4
solution of phenol 22 (6.36 g, 30.8 mmol) and DIPEA (5.75 g, 44.5 mmol) in DMF (25 mL) at 0
ºC was added TBSCl (7.01 g, 46.5 mmol) in portions. The reaction mixture was stirred for 6 h
and diluted with H
the organic extract was washed with brine, dried over MgSO
reduced pressure, and subjected to flash chromatography using a prepacked 50 g silica column
solvent A: EtOAc; solvent B: hexanes; gradient: 0%A / 100%B (1 CV), 0%A / 100%B →
2
O (50 mL). The reaction mixture was extracted with Et
2
O (2 × 100 mL) and
4
, filtered, concentrated under
[
3
0%A / 70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280
1
nm] affording aldehyde 24 (9.80 g, 30.6 mmol, 99%) as a white solid. H NMR (CDCl
3
, 500
-2), 3.01
2H, dd, J = 7.5, 5 Hz, H-9), 2.70 (2H, t, J = 12.0 Hz, H-6), 1.82 (2H, m, H-7), 1.80 (2H, m, H-
MHz): δ 7.38 (1H, d, J = 8.6 Hz, H-4), 6.78 (1H, d, J = 8.6 Hz, H-3), 3.84 (3H, s, OCH
3
(
1
3
8
1
1
2
), 1.02 (9H, (CH
53.2 (C, C-2), 141.8 (C, C-1), 133.10 (CH, C-10/11), 133.08 (C, C-10/11), 122.3 (CH, C-4),
08.8 (CH, C-3), 54.8 (CH , OCH -2), 40.7 (CH , C-6), 26.1 (CH , (CH ), 24.7 (CH ,C-7),
4.0 (CH ,C-9), 21.2 (CH ,C-8), 18.9 (C, (C(CH ) ), -3.90 (CH , Si(CH ) ). Analysis:
3 3 3 2 3
) ), 0.19 (6H, Si(CH ) ). C NMR (CDCl , 125 MHz): δ 205.3 (C, C-5),
3
3
2
3
3
)
3
2
2
2
3 3
3
3 2
Calculated for C18
28 3
H O Si: C 67.46, H 8.81. Found: C 67.70, H 8.82. HPLC: 20.96 min.
4
.1.15. 1-[(tert-Butyldimethylsilyl)oxy]-2-methoxy-5-(3′,4′,5′-trimethoxyphenyl)-
2
0
benzocycloheptan-5-ol (31). To a solution 3,4,5-trimethoxyphenyl bromide (16.8 g, 68.0
mmol) in THF (400 mL) at -78 ºC was added n-BuLi (27.2 mL, 2.5 M in hexanes) and the
reaction mixture was stirred for 30 min. Benzosuberone 24 (9.80 g, 30.6 mmol) in 25 mL THF
was added using an addition funnel over a period of 15 min. The reaction mixture was allowed
2
3

to warm to room temperature over 12 h. Upon completion, the reaction mixture was diluted with
O (50 mL) and extracted with EtOAc (2 × 100 mL). The organic extract was washed with
brine, dried over MgSO , filtered, concentrated under reduced pressure, and subjected to flash
H
2
4
chromatography using a prepacked 100 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 5%A / 95%B (1 CV), 5%A / 95%B → 15%A / 85%B (10 CV), 15%A / 85%B (2 CV);
flow rate: 40 mL/min; monitored at 254 and 280 nm] affording alcohol 31 (11.6 g, 17.4 mmol,
1
5
8
7%) as a white solid. H NMR (CDCl
3
, 500 MHz) δ 7.16 (1H, d, J = 8.7 Hz), 6.70 (1H, d, J =
.7 Hz), 6.50 (2H, s), 3.84 (3H, s), 3.80 (3H, s), 3.75 (6H, s), 3.37 – 3.23 (1H, m), 2.62 – 2.49
(
1H, m), 2.30 – 2.22 (1H, m), 2.18 – 2.05 (1H, m), 1.99 – 1.85 (1H, m), 1.83 – 1.65 (2H, m),
1
3
1
1
2
4
3
.47 – 1.34 (1H, m), 0.99 (9H, s), 0.17 (3H, s), 0.15 (3H, s). C NMR (CDCl , 125 MHz): δ
52.4, 148.6, 145.2, 141.5, 136.5, 135.1, 130.0, 121.3, 109.4, 104.0, 75.5, 60.8, 56.1, 54.7, 41.0,
6.2, 24.8, 19.2, 19.0, -3.6, -3.8.
1
8,20,44
.1.16. 1-Hydroxy-2-methoxy-5-(3′,4′,5′-trimethoxyphenyl)-benzocyclohepta-5-ene (39).
A solution of 31 (11.6 g, 10.6 mmol) in AcOH (150 mL) and H
2
O (100 mL) was heated to reflux
at 110 °C for 12 h. The reaction mixture was cooled to room temperature, concentrated under
reduced pressure and subjected to flash chromatography using a prepacked 100 g silica column
[
solvent A: EtOAc; solvent B: hexanes; gradient: 5%A / 95%B (1 CV), 5%A / 95%B → 15%A /
8
5%B over (10 CV), 15%A / 85%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280
1
nm] affording benzosuberene 39 (6.20 g, 17.4 mmol, 57%) as a white solid. H NMR (CDCl
3
,
5
6
3
00 MHz): δ 6.71 (1H, d, J = 8.6 Hz, H-3), 6.57 (1H, d, J = 8.6 Hz, H-4), 6.50 (2H, s, H-2′, H-
′), 6.33 (1H, t, J = 7.0 Hz, H-6), 5.74 (1H, s, OH), 3.91 (3H, s, OCH -2), 3.86 (3H, s, OCH -4),
.80 (6H, s, OCH -3′, -5′), 2.76 (2H, t, J = 7.0 Hz, H-9), 2.14 (2H, p, J = 7.0 Hz, H-8), 1.96 (2H,
3
3
3
1
3
3
q, J = 7.0 Hz, H-7). C NMR (CDCl , 125 MHz): δ 153.0 (C, C-3′, C-5′), 145.2 (C, C-2), 142.9
2
4

(
C, C-1), 142.5 (C, C-5), 138.6 (C, C-1′), 137.4 (C, C-4′), 134.4 (C, C-10/11), 127.9 (C, C-
0/11), 127.3 (CH, C-6), 121.0 (CH, C-4), 107.8 (CH, C-3), 105.4 (CH, C-2′, C-6′), 61.1 (CH
OCH -4′), 56.3 (CH , OCH -3′, -5′), 56.1 (CH , OCH -2), 33.7 (CH , C-8), 25.8 (CH , C-7), 23.7
, C-9). Analysis: Calculated for C21 : C 70.77, H 6.79, O 22.45. Found: C 71.05, H
1
3
,
3
3
3
3
3
2
2
(CH
2
24 5
H O
+
+
6
1
4
24 5
.77. HRMS: m/z: observed 379.1565 [M+Na] , calculated for C21H O Na , 379.1516. HPLC:
5.59 min.
.1.17. Disodium 2-Methoxy-5-(3′,4′,5′-trimethoxyphenyl)-benzocyclohepta-5-ene-1-phosphate
2
0
(
44). To a solution of 39 (0.32 g, 0.83 mmol) in CH Cl was added POCl (0.3 mL, 3.3 mmol)
2 2 3
and pyridine (0.25 mL, 3.01 mmol) and the reaction mixture was stirred for 8 h. NaOH (5 mL,
M) was added dropwise to the reaction mixture and the reaction was stirred for 5 min. The
reaction mixture was extracted with CH Cl (2 × 25 mL) and concentrated under reduced
2
2
2
pressure. NaOH (5 mL, 2 M) was added to the viscous liquid obtained and the solution was
stirred at 60 °C for 15 min. The aqueous phase was concentrated under reduced pressure and
subjected to flash chromatography using a prepacked 25 g RP-18 silica column [solvent A:
3
water; solvent B: CH CN; gradient: 100%A / 0%B (1 CV), 100%A / 0%B → 60%A / 40%B (10
CV), 0%A / 100%B (3 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] affording 44
1
(
0.245 g, 0.478 mmol, 58%) as white solid. H NMR (D
2
O, 500 MHz) δ 6.85 (1H, d, J = 8.5
Hz), 6.70 (3H, d, J = 8.5 Hz), 6.66 (2H, s), 6.46 (1H, t, J = 7.3 Hz), 3.84 (3H, s), 3.80 (6H, s),
1
3
3
.77 (3H, s), 2.85 (2H, t, J = 10 Hz), 2.16 (2H, m), 1.92 (q, J = 7.2 Hz, 2H). C NMR (D
2
O, 125
MHz): δ 152.1 (C, C-3′, C-5′), 151.0 (C, C-2), 141.8 (C, C-5), 140.5 (C, C-1), 139.4 (C, C-1′),
1
36.5 (C, C-10/11), 135.9 (C, C-4′), 133.4 (C, C-10/11), 128.7 (CH, C-6), 124.1 (CH, C-4),
09.4 (CH, C-3), 105.5 (CH, C-2′, C-6′), 60.9 (CH , OCH -4′), 55.9 (CH , OCH -3′, -5′), 55.5
, OCH -2), 33.3 (CH , C-8), 25.1 (CH , C-7), 24.6 (CH , C-9). P NMR (D O, 202 MHz):
1
3
3
3
3
3
1
(CH
3
3
2
2
2
2
2
5

+
+
δ 2.95. HRMS: m/z: observed 481.0999 [M+H] , calculated for C21
H
24
O
8
Na
2
P , 481.0999.
HPLC: 3.66 min.
Experimental Procedures for Final Compound 43
.1.18. (Z)/(E)- 5-(3′,4′-Dimethoxy-2´-(tosyloxy)phenyl)pent-4-enoic acid (7). To a well-stirred
2
0
4
solution of (3-carboxypropyl)triphenylphosphonium bromide (3.82 g, 8.90 mmol) in THF (200
mL) at -50 °C was added n-BuLi (5.4 mL, 2.5 M in hexanes). The reaction mixture was allowed
to warm to room temperature and stirred for 15 min and then cooled to -78 ºC. Aldehyde 2 (2.01
g, 5.97 mmol) dissolved in THF (15 mL) was added dropwise and the reaction mixture was
allowed to reach room temperature. H
extracted with EtOAc (3 x 200 mL). The organic extract was washed with brine, dried with
MgSO , concentrated under reduced pressure, and subjected to flash chromatography [silica gel,
0% EtOAc, 60% Hexanes] to obtain a mixture of E/Z-isomers 7 (1.03 g, 2.53 mmol, 42%) as an
2
O (50 mL) was added and the aqueous phase was
4
4
1
off-white solid. H NMR (Mixture of E and Z) (CDCl
3
, 500 MHz): δ 7.89 (2H, d, J = 8.2 Hz, H-
′′, -6′′), 7.87 (2H, d, J = 8.2 Hz, H-2′′, -6′′), 7.35 (2H, d, J = 8.2 Hz, H-3′′, -5′′), 7.32 (2H, d, J =
.2 Hz, H-3′′, -5′′), 7.16 (1H, d, J = 8.8 Hz, H-4′/5′), 6.96 (1H, d, J = 8.6 Hz, H-4′/5′), 6.81 (1H,
2
8
d, J = 8.6 Hz, H-4′/5′), 6.79 (1H, d, J = 8.8 Hz, H-4′/5′), 6.35 (1H, d, J = 16.0 Hz, H-5), 6.35
1H, dt, J = 11.5 Hz, H-5), 6.04 (1H, m, J = 16.0 Hz, H-4), 5.55 (1H, m, J = 11.5 Hz, H-4), 3.88-
.83 (2 × 3H, s, OCH -2′, -3′), 3.88-3.83 (2 × 3H, s, OCH -2′, -3′), 2.59 (2H, m, H-2/3), 2.56-
(
3
2
1
1
2
3
3
1
3
.54 (4H, m, H-2, -3), 2.47 (2H, m, H-2/3). C NMR δ 152.7, 152.7, 144.9, 144.8, 142.5, 141.1,
34.7, 134.6, 131.1, 129.5, 129.4, 128.9, 128.4, 128.3, 128.3, 128.3, 125.3, 125.1, 124.7, 124.4,
24.2, 120.3, 111.1, 110.4, 110.4, 105.0, 60.7, 56.2, 56.1, 34.7, 33.6, 33.3, 27.9, 23.8, 21.7,
+
+
22 7
1.7.HRMS: m/z: observed 429.0977 [M+Na] , calculated for C20H O NaS , 429.0978.
HPLC: 13.53 min.
2
6

2
0
4
.1.19. 5-(3′,4′-Dimethoxy-2´-(tosyloxy)phenyl)pentanoic acid (12). To a solution of pentanoic
acid 7 (1.25 g, 20.2 mmol) in MeOH (40 mL) and EtOH (15 mL) was added 10% Pd-C (400
mg). The flask was evacuated and H gas was introduced via balloons. The reaction mixture
2
was stirred for 12 h and was checked for completion by filtering a small amount of the reaction
1
mixture through Celite®, concentrating under reduced pressure, and recording the H NMR
spectrum. Upon completion, the reaction mixture was filtered through Celite® and concentrated
1
under reduced pressure to obtain 12 (0.94 g, 2.3 mmol, 75%) as an off-white solid. H NMR
(
CDCl3, 500 MHz): δ 7.93 (2H, d, J = 8.2 Hz, H-2′′, -6′′), 7.35 (2H, d, J = 8.2 Hz, H-3′′, -5′′),
.89 (1H, d, J = 8.6 Hz, H-6′), 6.77 (1H, d, J = 8.6 Hz, H-5′), 3.82 (3H, s, OCH -4′), 3.51 (3H, s,
OCH -3′), 2.58 (2H, t, H-5), 2.46 (3H, s, CH -4′′), 2.34 (2H, m, H-2), 1.61 (4H, m, H-3,-4).
NMR (CDCl3, 125 MHz): δ 179.0 (C, C-1), 151.8 (C, C-4′), 144.7 (C, C-4′′), 142.3 (C, C-2′),
6
3
1
3
3
3
C
1
1
2
4
2
42.1 (C, C-3′), 134.9 (C, C-1′′), 129.5 (CH, C-3′′,-5′′), 129.0 (C, C-1′), 128.1 (C, C-2′′,-6′′),
23.9 (CH, C-6′), 110.8 (CH, C-5′), 60.5 (CH , OCH -3′), 56.1 (CH , OCH -4′), 33.7 (CH , C-2),
9.6 (CH , C-5), 29.5 (CH , C-4), 24.4 (CH , C-3), 21.7 (CH , CH -4′′).
.1.20. 1-Tosyloxy-2,3-dimethoxy-benzocycloheptan-5-one (17). Pentanoic acid 12 (0.90 g,
.2 mmol) was dissolved in Eaton’s reagent [14 mL, P (7.7 wt%) in methanesulfonic acid]
3
3
3
3
2
2
2
2
3
3
2
0
2
O
5
and the solution was stirred for 12 h. The reaction mixture was poured over ice and the ice was
allowed to melt. The aqueous phase was extracted with CH Cl (3 × 100 mL) and NaHCO
powder was added in small amounts until neutralized. The organic extract was washed with
brine, dried over Na SO , filtered, concentrated under reduced pressure, and subjected to flash
2
2
3
2
4
chromatography using a prepacked 25 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 25%A / 75%B (1 CV), 25%A / 75%B → 60%A / 40%B (10 CV), 60%A / 40%B (2
CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] affording 17, (0.70 g, 1.8 mmol,
2
7

1
8
1%) as a white solid. H NMR (CDCl3, 500 MHz) δ 7.93 (2H, d, J = 8.0 Hz, H-2′, -6′), 7.37
2H, d, J = 8.0 Hz, H-3′, -5′), 7.29 (1H, s, H-4), 3.87 (3H, s, OCH -2), 3.58 (3H, s, OCH -3),
.95 (2H, dd, J = 4.9, 6.9 Hz, H-9), 2.73 (3H, m, H-6), 2.48 (3H, s, CH -4′′), 1.84 (2H, m, H-8),
(
3
3
2
1
2
1
3
1
3
.81 (2H, m, H-7). C NMR (CDCl3, 125 MHz) δ 204.0 (C, C-5), 151.3 (C, C-3), 145.4 (C, C-
), 145.0 (C, C-4′), 141.2 (C, C-1), 134.32 (C, C-10/11), 134.28 (CH, C-1′), 129.9 (C, C-10/11),
29.5 (CH, C-3′,-5′), 128.2 (CH, C-2′,-6′), 110.9 (CH, C-4), 60.5 (CH
3
, OCH
3
-3), 56.0 (CH
3
,
OCH
3
-2), 40.7 (CH , C-6), 24.7 (CH , C-8), 24.5 (CH , C-9), 21.7 (CH
2
2
2
3
, CH
3
-4′), 20.8 (CH , C-
2
7
).
.1.21. 1-Tosyloxy-2,3-dimethoxy-5-(3′,4′,5′-trimethoxyphenyl-benzocycloheptan-5-ol (26). To
2
0
4
a solution of 3,4,5-triemethoxyphenyl bromide (0.85 g, 3.4 mmol) in THF (100 mL) at -78 ºC
was added n-BuLi (1.4 mL, 2.5 M in hexanes) and the reaction mixture was stirred for 30 min.
Benzosuberone 17 (0.67 g, 1.7 mmol) in THF (15 mL) was added using an addition funnel over a
period of 15 min. The reaction mixture was allowed to reach room temperature over 12 h. Upon
completion, the reaction mixture was extracted with Et
2
O (150 mL) and EtOAc (15 mL). The
, filtered, concentrated under reduced
organic extract was washed with brine, dried over MgSO
4
pressure, and subjected to flash chromatography using a prepacked 25 g silica column [solvent
A: EtOAc, solvent B: hexanes; gradient: 25%A / 75%B (1 CV), 25%A / 75%B → 80%A / 20%B
(
10 CV), 80%A / 20%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] afforded
1
alcohol 26 (0.61 g, 1.1 mmol, 64%) as a white solid. H NMR (CDCl
3
, 500 MHz): δ 7.91 (2H,
d, J = 8.3 Hz, H-2′′, -6′′), 7.35 (2H, d, J = 8.3 Hz, H-3′′, -5′′), 7.37 (1H, s, H-4), 6.46 (2H, s, H-2′,
H-6′), 3.844 (3H, s, OCH -4′), 3.840 (3H, s, OCH -3), 3.78 (6H, s, OCH -3′, -5′), 3.54 (3H, s,
OCH -2), 3.12 (1H, m, H-9), 2.62 (1H, m, H-6), 2.46 (3H, s, CH -4′′), 2.23 (1H, m, H-9), 2.13
3
3
3
3
3
1
3
(
3
1H, m, H-6), 1.88 (1H, s, H-7/8), 1.72 (3H, m, H-7/8). C NMR (CDCl , 125 MHz): δ 153.2
2
8

(C, C-3′, C-5′), 150.5 (C, C-3), 144.7 (C, C-4′′), 141.7 (C, C-1), 141.4 (C, C-10/11), 140.7 (C, C-
2
1
4
), 139.9 (C, C-1′), 137.5 (CH, C-4′), 134.7 (C, C-1′′), 129.4 (C, C-3′′, C-5′′), 128.4 (C, C-10/11),
28.1 (C, C-2′′, C-6′′), 110.2 (CH, C-4), 104.2 (CH, C-2′, C-6′), 80.2 (C, C-5), 60.8 (CH , OCH
3
3
-
′), 60.5 (CH
, C-7/8), 26.6 (CH
.1.22. 1-Tosyloxy-2,3-dimethoxy-5-(3′,4′,5′-trimethoxyphenyl)-benzocycloheptan-5-ene (34).
O (30 mL) and was
3
, OCH
3
-2), 56.2 (CH
3
, OCH
3
-3′, -5′), 56.0 (CH
3
, OCH
3
-3), 41.1 (CH
2
, C-6), 26.7
(CH
2
2
, C-9), 26.3 (CH
2
, C-7/8), 21.7 (CH
3
, CH
3
-4′′).
2
0
4
Alcohol 26 (0.54 g, 0.97 mmol) was dissolved in AcOH (20 mL) and H
2
heated to reflux at 180 °C for 12 h. The reaction mixture was cooled, concentrated under
reduced pressure, subjected to flash chromatography using a prepacked 25 g silica column
[
solvent A: EtOAc; solvent B: hexanes; gradient: 15%A / 85%B (1 CV), 15%A / 85%B →
5
0%A / 50%B (10 CV), 50%A / 50%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280
1
nm] affording benzosuberene 34 (0.41 g, 0.75 mmol, 78%) as a white solid. H NMR (CDCl
3
,
5
00 MHz): δ 7.99 (2H, d, J = 8.3 Hz, H-2′′, -6′′), 7.37 (2H, d, J = 8.3 Hz, H-3′′, -5′′), 6.54 (1H, s,
3
H-4), 6.48 (2H, s, H-2′, H-6′), 6.44 (1H, t, J = 7.4 Hz, H-6), 3.87 (3H, s, OCH -4′), 3.82 (6H, s,
OCH
3
3 3
-3′, -5′), 3.69 (3H, s, OCH -3), 3.54 (3H, s, OCH -2), 2.71 (2H, t, J = 6.5 Hz, H-9), 2.48
1
3
(
3H, s, CH -4′′), 2.21 (2H, p, J = 7.0 Hz, H-8), 1.99 (2H, q, J = 7.1 Hz, H-7). C NMR (CDCl
3
3
,
1
1
25 MHz): δ 153.0 (C, C-3′, C-5′), 151.0 (C, C-3), 144.7 (C, C-4′′), 141.9 (C, C-5), 141.6 (C, C-
), 140.8 (C, C-2), 137.6 (C, C-1′), 137.5 (C, C-4′), 136.1 (C, C-10/11), 134.8 (C, C-1′′), 129.6
(C, C-10/11), 129.5 (CH, C-3′′, C-5′′), 129.4 (CH, C-6), 128.2 (CH, C-2′′, C-6′′), 112.0 (CH, C-
4
5
4
), 105.2 (CH, C-2′, C-6′), 60.94 (CH
′), 56.2 (CH , OCH -3) 34.6 (CH , C-8), 25.5 (CH
.1.23. 1-Hydroxy-2,3-dimethoxy-5-(3′,4′,5′-trimethoxyphenyl)-benzocycloheptan-5-ene (43).
3
, OCH
3
-4′), 60.93 (CH
3
, OCH
3
-2), 60.5 (CH
3
, OCH
3
-3′, -
3
3
2
2
, C-7), 25.1 (CH
2
, C-9), 21.7 (CH
3
, CH
3
-4′′).
2
0
A solution of sulfonate ester 34 (0.250 g, 0.462 mmol) dissolved in NaOH (1 mL, 2 M) and
2
9