Structure Guided Design, Synthesis, and Biological Evaluation of Novel Benzosuberene Analogues as Inhibitors of Tubulin Polymerization

Article abstract of DOI:10.1021/acs.jmedchem.9b00551

A promising design paradigm for small-molecule inhibitors of tubulin polymerization that bind to the colchicine site draws structural inspiration from the natural products colchicine and combretastatin A-4 (CA4). Our previous studies with benzocycloalkeny

Full text of DOI:10.1021/acs.jmedchem.9b00551

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Article
Structure Guided Design, Synthesis, and Biological Evaluation of Novel
Benzosuberene Analogues as Inhibitors of Tubulin Polymerization
Haichan Niu, Tracy E. Strecker, Jeni L. Gerberich, James Campbell, Debabrata Saha, Deboprosad
Mondal, Ernest Hamel, David J. Chaplin, Ralph P. Mason, Mary Lynn Trawick, and Kevin G. Pinney
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00551 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 6, 2019
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Structure Guided Design, Synthesis and Biological
Evaluation of Novel Benzosuberene Analogues as
Inhibitors of Tubulin Polymerization
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a
a
b
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Haichan Niu , Tracy E. Strecker , Jeni L. Gerberich , James W. Campbell III , Debabrata Saha ,
a
d
a,e
b
Deboprosad Mondal , Ernest Hamel , David J. Chaplin , Ralph P. Mason , Mary Lynn
a
a
Trawick , and Kevin G. Pinney *
a
Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798-7348,
United States
b
Department of Radiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, Texas 75390-9058, United States
c
Department of Radiology Oncology, Division of Molecular Radiation Biology, The University of Texas
Southwestern Medical Center, 2201 Inwood Road, Dallas, Texas 75390-9187, United States
d
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Frederick National Laboratory for Cancer Research, National Institutes of
Health, Frederick, Maryland 21702, United States
e
Mateon Therapeutics, 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).
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Keywords: inhibitors of tubulin polymerization; benzosuberene analogues; vascular disrupting
agents; small-molecule synthesis
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Abstract
A promising design paradigm for small-molecule inhibitors of tubulin polymerization that bind to
the colchicine site draws structural inspiration from the natural products colchicine and
combretastatin A-4 (CA4). Our previous studies with benzocycloalkenyl and heteroaromatic ring
systems yielded promising inhibitors with dihydronaphthalene and benzosuberene analogues
featuring phenolic (KGP03 and KGP18) and aniline (KGP05 and KGP156) congeners emerging
as lead agents. These molecules demonstrated dual mechanism of action, functioning as both
potent vascular disrupting agents (VDAs) and as highly cytotoxic anticancer agents. A further
series of analogues was designed to extend functional group diversity and investigate
regioisomeric tolerance. Ten new molecules were effective inhibitors of tubulin polymerization
(IC50 < 5 µM) with seven of these exhibiting highly potent activity comparable to CA4, KGP18,
and KGP03. For one of the most effective agents, dose-dependent vascular shutdown was
demonstrated using dynamic bioluminescence imaging in a human prostate tumor xenograft
growing in a rat.
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Introduction
Solid tumors require a functional vasculature to supply oxygen and nutrients to their cells when
3
1,2
they exceed 1 mm in size. Unlike normal vasculature, the tumor-associated vascular network
tends to expand irregularly, incorporating fragile and chaotic bulges and blind ends.3–5 The
primitive character and inherent fragility of tumor-associated vasculature, along with the seminal
observations by Denekamp and co-workers that blocking established tumor-associated blood flow
leads to tumor regression in mice, positioned tumor-associated vasculature as a promising target
for cancer therapy.6–8 Two categories of small-molecule, vascular-targeted therapies have been
developed: angiogenesis inhibiting agents (AIAs) that inhibit neovascularization in developing
tumors; and, separately, vascular disrupting agents (VDAs) that irreversibly damage established
tumor-associated vasculature.9–13 The two major sub-divisions of VDAs include biologics and
small-molecule anticancer agents. The majority of small-molecule VDAs function as inhibitors of
tubulin polymerization, which destabilize the tubulin-microtubule protein system by binding to the
colchicine site on β-tubulin in close proximity to the α,β-tubulin heterodimer interface. The
endothelial cells lining microvessels undergo rapid cytoskeletal disruption, manifested by
morphological changes (flat to round) in response to inhibition of their tubulin-microtubule protein
system cytoskeleton triggered by VDA binding to the colchicine site. This rapid endothelial cell
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cytoskeletal rearrangement leads to irreversible damage to the tumor-associated vasculature,
culminating in tumor necrosis.4,14–17
The natural products colchicine, combretastatin A-4 (CA4), and combretastatin A-1 (CA1), along
with the synthetic analogue phenstatin (Fig. 1), are potent colchicine site inhibitors of tubulin
polymerization that function as promising VDAs.18–24 These molecules have provided structural
inspiration and guidance for the design, synthesis, and biological evaluation of many second-
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generation (and beyond) molecules in a world-wide effort to identify a small-molecule colchicine
site agent with the necessary efficacy coupled with safety to be utilized as a cancer therapeutic in
humans. To date, no small-molecule therapeutic agent that interacts with the colchicine site and
functions as either an antiproliferative agent or a VDA (or demonstrates a dual mechanism of
action) has reached FDA approval. Structural similarities between these natural products include
a trimethoxy phenyl ring, a separate hydroxylated p-methoxy aryl moiety, and a bridging
functionality connecting the two rings with a comparable centroid-to-centroid distance. Our long-
standing program focused on molecular recognition for the colchicine site has led to the discovery
of promising synthetic analogues and derivatives,25–28 including stilbenoid,14,29–31
benzo[b]thiophene,32–34 benzofuran,34 dihydronaphthalene,8,35,36 benzosuberene,8,25,35–38 and
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indole-based molecules (Fig. 1). Numerous other studies have investigated a myriad of structural
and functional group modifications to the A-ring, the B-ring, and the ethylene bridge of
combretastatin A-4; the results of which are conveyed in extensive publications (too numerous to
cite herein) and summarized in various review articles, yet none are comprehensive.40–49
Two benzosuberene-based analogues (referred to as KGP188,25,37 and its amino congener
KGP15635,38) emerged from our previous studies as molecules with high relevance as potential
preclinical candidates due, in part, to their potent inhibition of tubulin polymerization, pronounced
cytotoxicity against human cancer cell lines, and promising activity as VDAs.8,25,38
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Figure 1. Representative small-molecule inhibitors of tubulin polymerization: colchicine, combretastatins (CA4,
CA1),15,23 phenstatin, dihydronaphthalene analogues (KGP03, KGP05),35,36 benzosuberene analogues (KGP18,
KGP156),8,25,37,38 indole analogue (OXi8006),39,50 and benzo[b]furan analogue (BNC105).51–53
Studies by us and others have investigated a variety of functional group modifications on both the
fused and pendant aryl rings of tubulin-binding benzosuberene and dihydronaphthalene molecular
frameworks (Fig. 2).8,25,35,36,54,55 In particular, Maderna and co-workers contributed efficient
methodology towards the benzosuberene structural motif and presented a number of important B-
ring modified benzosuberene analogues (Fig. 2) along with an evaluation of their biological
potency and their drugability characteristics.56,57 Interestingly, a benzosuberene B-ring diene
analogue was identified as one of the most potent cytotoxic agents amongst their synthesized series
5
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of eleven members, and this same molecule (Maderna compound, listed as 68 herein) was
obtained during our synthetic campaign as an unexpected product. It is also noteworthy that a new
class of benzodiazepines has been reported as inhibitors of tubulin polymerization.58
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Figure 2. Selected KGP18 derivatives as inhibitors of tubulin polymerization with modifications at the C-4 position
of the A ring and the C-6, 7, 8 positions of the B ring.25,57
Herein we designed, synthesized and biologically evaluated a series of structurally varied
analogues using KGP18 and KGP05 as lead compounds and further adopted our methodology to
investigate functional group modifications on the A-ring (C-4 position) and the B-ring (C-6, 7, 8,
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positions), along with regiochemical translocation of the pendant aryl ring (C-ring) in regard to
their influence on inhibition of tubulin polymerization and cytotoxicity against several human
cancer cell lines.
Results and Discussion
Molecular Design and Synthesis. Eighteen benzosuberene and dihydronaphthalene analogues
(Fig. 3) were prepared by chemical synthesis and evaluated for their cytotoxicity against selected
human cancer cell lines and their ability to inhibit tubulin polymerization. Structural modifications
included: 1) functional group (R) modifications on the fused aryl ring including the installation of
alcohol, aldehyde, nitrile, and ester groups along with ether linkages to facilitate extension of the
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polar alcohol moiety away from the fused six-seven ring system; 2) R
2
and R incorporation at the
3
olefinic and allylic positions of the seven-membered ring introduced -Br, -OH, and -NHAc groups;
3) modification (R position) of the fused aliphatic ring adjacent to the tertiary alcohol site; 4)
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olefination and pendant trimethoxy phenyl ring regiochemistry on the fused non-aromatic ring.
The synthesis of analogues 3, 6, 8, 11, 13, 15 was initiated from a common intermediate ketone 1
2
5
that was readily available utilizing our previous methodolgy. Treatment of benzosuberone 1 with
trimethoxyphenyllithium (prepared from the corresponding bromide) generated tertiary alcohol 2,
which was subsequently converted to diol 3 upon removal of the phenolic TBS protecting group.
Separately, tertiary alcohol 2 was converted to its corresponding benzosuberene 4, which
underwent treatment with a series of oxidants (m-CPBA, NBS, OsO ) to facilitate epoxidation
4
followed by ring opening and oxidation, bromination, and Upjohn dihydroxylation.59,60 Following
the removal of protecting groups, target compounds 6, 13, and 15 were obtained. Similarly,
2
5
reaction of 4-methylbenzosuberene 12 (prepared by us previously ) with NBS/AIBN afforded
vinylbromide 13. Lead compound 7 (referred to as KGP18), also available through this
methodology, was directly converted to its corresponding ether analogues 8 and 11 (Scheme 1).25,61
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Li
H CO
3
H CO
3
OCH
3
OCH
3
H
3
CO
3
H CO
O
H
3
CO
OCH
3
OH
OH
OCH
3
TBAF
THF, RT, 4 h
H
3
CO
THF, -78 ºC to RT, 12 h
H
3
CO
3
H CO
OTBS
OTBS
2 (89%)
OH
3 (35%)
1
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AcOH, r.t., 6h
Br
H
3
CO
H
3
CO
H
CO
H
3
CO
3
H CO
3
OCH
3
OCH
3
OCH
OCH
3
O
OCH3
3
H
3
CO
H
3
CO
H
3
CO
H
3
CO
3
H CO
OH
OH
O
TBAF
O
mCPBA
Cl , -5 °C to RT,
TBAF
K
2
CO
3
,
3
H CO
THF, 0 ºC, 1 h
CO
CH
THF, RT, 1 h
CO
2
2
DMF, RT, 15 h, 150 ºC, 3 h
H CO
H
3
CO
H
3
14 h
H
CO
H
3
3
3
OH
OTBS
OTBS
OH
O
6
(96%)
5 (47%)
4 (66%)
7 (81%)
DMF, reflux, 24 h
O
NBS, AIBN, CCl
reflux, 2 h
4
,
5% mol OsO
ACN/H O (7:3), RT, 12 h
, NMO,
O
O
8 (18%)
4
2
3
H CO
OCH
3
O
OCH
3
TBAB
3
H CO
H
3
CO
3
H CO
OCH
Br
3
OCH
3
H
3
CO
3
H CO
OH
OH
H
3
CO
O
H
3
CO
3
H CO
HO
OTBS
OTBS
11 (68%)
9
(quantitative)
10 (33%)
TBAF, THF, 0 ºC, 1 h
TBAF, THF, 0 ºC, 1 h
H
3
CO
H
3
CO
H
CO
3
CO
OCH
3
OCH
Br
3
OCH
3
H
3
CO
3
H CO
H
3
NBS, AIBN
CCl , reflux, 2 h
CO
OH
OH
4
H
3
CO
H
3
3
H CO
CH
3
R
OH
1
1
3 (quantitative), R = OH
4 (80%), R = CH
1
2
15 (51%)
3
Scheme 1. Synthesis of benzosuberene and benzosuberane intermediates and analogues.
Lead compounds benzosuberene 16 (referred to as KGP156) and its corresponding
dihydronaphthalene analogue 17 (referred to as KGP05), which were readily available from our
previous synthetic studies, were subjected to a Sandmeyer radical-nucleophilic aromatic
substitution protocol to generate nitrile analogues 18 and 19 (Scheme 2).35,38,62,63 The
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benzosuberene aldehyde analogue 20 was obtained after subsequent reduction of the nitrile
analogue (Scheme 2).
H3CO
H3CO
3
H CO
3
H CO
OCH3
OCH3
OCH3
H CO
3
3
H CO
0
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) DIBAL-H,
toluene,0 ºC to RT, 12 h
1) HCl/ MeOH (1:1), NaNO2, 0 ºC, 1 h
2) CuCN, 60 ºC, 2 h
2) HCl (1 M), RT, 30 min
3) Na2CO3, NaCN, RT, overnight
H3CO
3
H CO
3
H CO
n
n
n
NH2
CN
CHO
1
6 (KGP156), n = 1
18 (40%), n = 1
19 (32%), n = 0
20 (78%), n = 1
17 (KGP05), n = 0
Scheme 2. Synthesis of nitrile- and aldehyde- substituted R position analogues 18-20.
As part of a larger program focused on the use of potently cytotoxic benzosuberene and
dihydronaphthalene analogues as payloads in antibody-drug conjugates (ADCs) and as prodrugs
targeted for selective release in regions of profound tumor hypoxia, we were intrigued by the
possibility of replacing the heteroatom [oxygen (phenol) or nitrogen (aniline)] at the 4-position
with a short carbon chain terminating with a primary alcohol (or amino) moiety, thus maintaining
its hydrogen-bond donor nature and serving as a viable position for future attachment of various
linkers. Accordingly, methylation of phenolic bromo-aldehyde 21 (Scheme 3), followed by Wittig
olefination and subsequent hydrogenation (under Ph
2
S mediation to maintain the aryl bromine
group) afforded methyl ester 24. Benzosuberone 25 was obtained through an intramolecular
Friedel-Crafts annulation facilitated by Eaton’s reagent (7.7% weight percent P in CH SO H),
6
4
2
O
5
3
3
and subsequent treatment of compounds 25 with trimethoxyphenyllithium, followed by reaction
work-up under acidic conditions, generated benzosuberene intermediate 26.65,66 We have
2
5
previously evaluated corresponding fluorine and chlorine benzosuberene analogues. Halogen-
lithium exchange, followed by reaction with ethyl chloroformate, afforded ethyl ester 27, which
was reduced (LiAlH ) to generate benzylic alcohol 28. Notably, while it proved possible to obtain
4
analogue 28 directly from intermediate 26, the isolated yield was quite low (≤ 8%) under these
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conditions, which was likely due, in part, to the low solubility of paraformaldehyde at low
temperature in THF and its low reactivity as a polymer (Scheme 3).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of ester analogue 27 and primary alcohol analogue 28.
Secondary allylic alcohol 37 and its corresponding N-acetyl congener 49 (Scheme 4) were
prepared to investigate structure-activity relationship correlations associated with heteroatom
incorporation on the conformationally flexible fused seven-membered ring. Appropriate aldehyde
chain elongations were facilitated by Meldrum’s acid (towards 37) and Wittig-ylide methodology
(towards 49), and subsequent functional group transformations (including installation of
carboxylic acid moieties obtained under saponification conditions) afforded protected alcohol 34
and N-acetamide 45, separately. Lewis acid mediated cyclization to obtain the benzosuberone
molecular core was achieved by treatment of the requisite acyl chloride with either tin tetrachloride
(to obtain ketone 35) or Eaton’s reagent (to obtain ketone 46). In each case, the pendant ring was
installed through reaction with 3,4,5-trimethoxyphenyllithium. The secondary alcohol moiety
(target compound 37) was revealed (by deprotection) prior to the organolithium step, while the
phenolic moiety (target compound 49) was revealed after the organolithium reaction (Scheme 4).
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8,67–69
Both compounds 36 and 37 undergo elimination first (allylic alcohol), then demethylation if
treated with a Lewis acid such as AlCl
3
or BCl .
3
1
) Oxalyl chloride, DMF(cat.),
Pd/C, H
2
CH Cl , RT, 2 h
2
2
NaBH4
OCH
3
H
3
CO
COOH
3
H CO
COOH
3
H CO
MeOH, RT, 12 h
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2) Meldrum’s Acid, py, CH
2
2
Cl ,
OCH
31 (56%)
O
O
OCH
3
OCH3
3
MeOH, 0 ºC, 1 h,
RT, 1 h
0
ºC, 30 min, RT, 1 h
2
9
30 (quatitative)
3) MeOH, reflux, 3 h
t-BDPSCl, imidazole,
NaOH (2.5 M)
OCH
3
OCH3
OH
H
3
CO
3
H CO
3
H CO
OCH
3
OH
O
DMF, RT, 12 h
OCH3
O
O
MeOH/ THF, 0 ºC, 1 h,
RT, 13 h
OCH3
O
O
Ph Si Ph
33 (49%)
Ph Si Ph
34 (40%)
3
2 (79%)
Li
3
H CO
OCH
3
O
O
3
H CO
1) Oxalyl chloride, DMF(cat.),
Ph
Si Ph
O
2
CH Cl , 0 ºC, 2 h
2
TBAF
H
3
CO
OCH
3
OH
2) SnCl
4
, CH
2
Cl
2
,
H
3
CO
THF, 0 ºC, 30 min,
RT, 16 h
OCH
3
3
H CO
-10 ºC, 30 min
OCH3
OH
OCH3
6 (49%)
THF, -78 ºC,1 h, 0 ºC, 20 h
HCl (2 M), RT, 10 min
3
H CO
35 (54%)
3
OCH
3
3
7 (39%)
ethyl 3-oxo-4-(triphenylphosphoranylidene) butanoate
TBSCl, DMAP, Et
3
N
O
CH
3
3
H CO
H
3
CO
CHO
CH Cl , RT, 12 h
2
2
3
H CO
CHO THF, reflux, 12 h
OTBS
OTBS
O
O
OH
3
8
39 (57%)
40 (80%)
Pd/C, H
2
NH
4
OAc
2
Ac O, py
O
O
CH
3
O
CH
3
O
CH3
3
H CO
H
3
CO
3
H CO
MeOH, RT, 12 h
OTBS
1 (46%)
O
MeOH, 35 ºC, 16 h
THF, reflux, 48 h
OTBS
NH
2
O
OTBS
HN
O
4
42 (93%)
CH
3
4
3 (49%)O
O
Pd/C, H
2
O
CH
3
KOH (1 M)
OH
Eaton’s Reagent
RT, 12 h
H
3
CO
3
H CO
NHAc
MeOH, RT, 60 h
OTBS
HN
O
O
MeOH, 0 ºC to RT, 3 h
OH
HN
O
O
3
H CO
CH
3
CH
3
OH
6 (57%)
4
4 (44%)
45 (58%)
4
Li
3
H CO
3
H CO
OCH
3
OCH
3
O
3
H CO
3
H CO
TBSCl, DMAP, Et
3
N
H
3
CO
OCH
OCH3
3
TBAF
THF, 0 ºC, 2 h
NHAc
CH Cl , RT, 12 h
2
2
3
H CO
NHAc
NHAc
OTBS
THF, -78 ºC to RT, 12 h,
HCl (2 M), RT, 10 min
H
3
CO
3
H CO
OTBS
48 (57%)
OH
4
7 (68%)
49 (44%)
Scheme 4. Synthesis of allylic alcohol analogue 37 and allylic N-acetamide analogue 49.
Translocation of the trimethoxyphenyl group (Scheme 5) was achieved by initial cyclization
Eaton’s reagent) of carboxylic acid 54 with concomitant elimination to afford a,b-unsaturated
(
ketone 55. With the α,β-unsaturated ketone in hand, 1,2- and 1,4- addition reactions were
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conducted using the appropriate aryl-lithium and Gilman reagents to provide tertiary alcohol
analogue 56 (with unsaturated 7-membered ring to maintain rigidity), and separately the Michael
adduct, trimethoxy pendant phenyl ring shifted analogue 57 (Scheme 5).70
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ethyl 3-oxo-4-(triphenylphosphoranylidene) butanoate
CHO
Pd/C, H2
O
CH
3
O
CH
3
H
3
CO
MeOH, RT, 12 h
H
3
CO
3
H CO
t
KO Bu, THF, RT, 12 h
OCH
3
OCH
3
O
O
OCH
3
O
O
5
0
51
5
2 (81%, two steps)
O
NaBH
4
NaOH (2.5 M)
MeOH/ THF, 0 ºC, 1 h, RT,12 h
Eaton’s reagent
RT, 14 h
O
CH
3
OH
MeOH, 0 ºC, 1 h, RT, 3 h
H
3
CO
3
H CO
OCH
3
OH
O
OCH
3
OH O
3
H CO
53 (82%)
54 (77%)
OCH
3
55 (50%)
Li
H
3
CO
3 3 2
[(CH O) Ph] CuLi
OCH
3
-78 ºC to -10 ºC,
3
H CO
THF, 9 h
H
3
CO
OCH
3
OH
O
OCH
OCH
OCH
3
OCH
3
THF, -78 ºC to RT, 12 h,
HCl (2 M), RT, 10 min
3
3
H CO
3
H CO
OCH
3
OCH
3
3
5
6 (74%)
5
7 (44%)
Scheme 5. Synthesis of α-unsaturated tertiary alcohol analogue 56 and pendant ring shifted analogue 57.
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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3
4
5
6
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9
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Synthesis of benzosuberdiene 68 and phosphate salt 69.
During the course of an investigation centered on variability in phenolic moiety protecting groups
for a subset of benzosuberene analogues, we were surprised by the formation of diene 67, obtained
upon 1,2-addition of trimethoxyphenyllithium to ketone 66, followed by reaction work-up. In this
case, the secondary alcohol demonstrated a propensity to undergo elimination even under mild
acidic or basic conditions such as TBAF deprotection or BCl cleavage at lowered temperature.
3
We attempted various combinations of 4-position (phenolic moiety on the fused aryl ring) and
allylic alcohol protecting group strategies, which eventually led to the unanticipated formation of
diene 68. It is important to note that this diene (68) was previously obtained by Maderna and co-
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57
workers. Having this compound in hand, and noting its exceptional biological activity (inhibition
5
7
of tubulin polymerization and cytotoxicity against human cancer cell lines, Table 1), motivated
us to prepare the corresponding water-soluble phosphate prodrug disodium salt 69 to facilitate in
vivo studies in a mouse model of prostate cancer to evaluate the efficacy of this compound as a
VDA, as evidenced by bioluminescence imaging (BLI) (Scheme 6).8,25,57,68,69
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Compilation of synthesized benzosuberene, benzosuberane, benzosuberone, and dihydronaphthalene
analogues
Biological Evaluation. Each of the eighteen compounds (Fig. 3) was evaluated for its cytotoxicity
against human cancer cell lines [SK-OV-3 (ovarian), NCI-H460 (lung), DU-145 (prostate)] and
for the ability to inhibit tubulin polymerization (Table 1). Ten of the evaluated molecules were
identified as strong inhibitors of tubulin assembly (IC50 < 5 μM, cell-free assay), while seven of
the ten were highly active (IC50 ≤ 1.2 μM). CA4 (IC50 ≈ 1 μM) and KGP18 (compound 7, IC50
≈
0
.85 μM) were utilized as comparative compounds. Three of the benzosuberene and
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dihydronaphthalene analogues (compounds 13, 19), along with the Maderna compound (68), were
more potent against tubulin in comparison to our dihydronaphthalene lead compound KGP03
(IC50 ≤ 0.5 μM). Excellent IC50 values for inhibition of tubulin polymerization were retained upon
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
alteration of the 4-position phenolic moiety into nitrile, ethyl ester, CH
2
OH groups (18, 19, 27,
2
8), along with modifications to the double bond on seven-member ring that included replacement
with ketone and tertiary alcohol groups, substitution with a bromine group, and increased
unsaturation (6, 13, 68). Extension of the alkyl chain (at position 4) through an ether linkage that
terminated with a polar alcohol group (compound 11) and separately a methoxy moiety (compound
8
) resulted in loss of inhibitory activity. Incorporation of a hydrogen bond donor at the allylic
position on the seven-member ring (compounds 37 and 49) and substituted saturation of the double
bond (compounds 3 and 15) both reduced inhibition of tubulin polymerization. The lack of tubulin
activity observed with compound 56 was unanticipated, since semi-rigidity of the seven-membered
ring was maintained through a double bond one carbon removed from the stereogenic center, and
its parent benzosuberene analogue (dimethoxy group on the fused aryl ring) demonstrated a
moderate degree of inhibition of tubulin polymerization (IC50 = 3.1 μM) reported in our previous
2
5
study. The lack of activity of compound 57 in regard to inhibition of tubulin polymerization
suggested that the trimethoxy pendant aryl ring situated at the benzylic position on the fused-ring
system was closely correlated to biological efficacy (at least in regard to inhibition of tubulin
polymerization). While we have previously described similar pendant ring shifts in related
benzosuberene analogues, this β-position substitution with a trimethoxy aryl ring has not been
previously investigated.8
Among the eighteen benzosuberene and dihydronaphthalene analogues investigated in this study
(Fig. 3), the most cytotoxic agents were compounds 6, 13, 18, 19, 28, 68 (GI50 = 0.0314, 0.0221,
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0
.0648, 0.0384, 0.0403 μM, and 0.00690 μM, respectively, against the SK-OV-3 ovarian cancer
cell line, for example). Judiciously selected structural modifications to the 4, 8, and 9-positions in
the parent benzosuberene scaffold accounted for the majority of the highly potent analogues
evaluated in this study. While the strong cytotoxicity of this sub-set of molecules is encouraging,
it is noteworthy that all molecules proved less cytotoxic than the lead benzosuberene KGP18 and
less cytotoxic (with the partial exception of the Maderna compound 68) than the natural product
CA4, despite demonstrating similar inhibition of tubulin polymerization (cell free assay). These
observations provided an important extension to the known SAR considerations regarding
structural modifications to KGP18. As anticipated (and similarly observed for combretastatin A-
4 phosphate),15,71 the benzosuberene phosphate prodrug salt 69 was inactive as an inhibitor of
tubulin polymerization in this cell-free assay, presumably due to the lack of phosphatase enzymes
necessary to cleave the prodrug to its parent phenolic (biologically active) agent. Prodrug 69 was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
an active cytotoxic agent since non-specific phosphatase activity is present in these cancer cell-
based cytotoxicity assays.72,73
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Inhibition of tubulin polymerization, percent inhibition of colchicine binding, and
cytotoxicity of the benzosuberene and dihydronaphthalene analogues.
Inhibition of
% Inhibition of colchicine
tubulin
Compound
GI50 (μM) SRB assaya
binding ± SD
polymerization
IC50 (μM) ± SD
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SK-OV-3
0.00455
0.00119
0.0000543e
0.0029f
0.0789
0.00134
0.394
NCI-H460
DU-145
1.0b
0.00223c
0.00327c
CA4
CA4P
KGP18
KGP03
Doxorubicin
Paclitaxel
3
84 ± 3 (1 μM), 98 ± 0.007 (5 μM)
>40b
0.85 ± 0.02d
0.5f
ND
73 ± 5 (1 μM), 95 ± 0.5 (5 μM)
90 ± 2 (1 μM), 98 ± 0.3 (5 μM)
ND
0.00194c
0.0000418e
0.0032f
0.123
0.00323c
0.0000249e
0.00040f
0.134
ND
NR
NR
0.00176
0.173
0.00147
0.0330
0.141
>20
ND
6
1.2 ± 0.1
>20
72 ± 2 (5 μM)
ND
0.0314
6.52
0.0476
1.90
8
5.66
1
1
3
>20
ND
3.22
0.855
3.76
1
0.39 ± 0.06
4.9 ± 0.1
>20
88 ± 1 (5 μM)
35 ± 5 (1 μM)
0 (5 μM)
0.0221
0.312
0.0353
0.449
0.0362
0.423
14
15
18
19
20
3.03
3.86
4.04
1.1 ± 0.1
0.37 ± 0.08
3.2 ± 0.08
1.0 ± 0.07
0.63 ± 0.03
3.8 ± 0.5
>20
62 ± 0.7 (5 μM)
73 ± 0.6 (5 μM)
61 ± 3 (5 μM)
47 ± 1 (5 μM)
76 ± 2 (5 μM)
28 ± 4 (5 μM)
0.4 ± 0.6 (5 μM)
0 (5 μM)
0.0648
0.0384
0.0572
0.299
0.434
0.860
0.0605
0.0847
0.353
ND
0.0402
0.631
27
28
37
49
0.0403
0.263
0.0628
0.432
ND
0.439
1.20g
ND
0.334
56
57
>20
27.2
70.5
26.0
4.45
>20
0 (5 μM)
5.77
2.00
6
8
9
0.48 ± 0.08
16 ± 0.7
68 ± 1 (0.5 μM), 95 ± 0.8 (5 μM)
45 ± 2 (5 μM)
0.00690
0.0153
0.0581
0.0291
0.0976
0.0239
6
a
Average of n ≥ 3 independent determinations (unless otherwise noted)
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b
c
d
e
f
24
74
24
37
Data from ref. 24 and ref. 74
For additional data, see ref. 24
For additional data, see ref. 37
For additional data, see ref. 8
For additional data, see ref. 36 and ref. 75
8
36
75
g
Average of n = 2 independent determinations (of duplicates)
ND = Not Determined
NR = Not Relevant (paclitaxel enhances microtubule assembly)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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Assessment of Vascular Damage
Ultimately, VDAs will be used in vivo, and thus it is crucial to understand both efficacy of vascular
disruption and potential off target toxicity in vivo. As a preliminary investigation, the extent of
vascular damage was assessed in a human prostate tumor line in rats treated with the water-soluble
prodrug salt 69 compared to CA4P as control. Many imaging methods have been developed to
1
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assess vascular disruption non-invasively in vivo. We favor dynamic bioluminescence imaging
BLI) for initial validation of VDA activity since it provides a fast, non-invasive and easy method
(
and allows comparison of repeat or sequential investigations. BLI does require the use of cells
transfected to express luciferase (luc), but these are commonly available and we have used this
approach extensively11,25,39,53,75 as have others. We used the human prostate cancer PC3 line, in
which the tumor suppressor protein DAB2IP had been knocked down and luciferase introduced.77
BLI requires administration of luciferin substrate, which readily crosses membranes and is carried
throughout the vasculature. The measurement of light emission dynamics is related to vascular
76
7
8
delivery of the luciferin substrate, and thus it provides a measure of vascular patency. Disruption
of tumor vasculature blocks delivery of the substrate and consequently results in a quantifiable
decrease in bioluminescent signal. The extent of vascular shutdown was evaluated using IP doses
of 10 and 40 or 80 mg/kg of 69 and compared to a dose of 30 mg/kg of CA4P, which had been
shown previously to cause extensive vascular shutdown in rats at this dose.79–81 It should also be
noted that the lead benzosuberene KGP18 and the dihydronaphthalene KGP03 (both as their
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corresponding water-soluble phosphate prodrug salts (KGP265 and KGP04, respectively), along
with other structurally modified benzosuberene analogues, demonstrated vascular shutdown (as
evidenced by similar BLI imaging studies or color Doppler ultrasound).25,37,75
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Prodrug 69 administered at 10 mg/kg resulted in minimal change in light emission. A subsequent
dose of 40 mg/kg 69 resulted in substantially diminished light emission (signal reduction 95%;
Figs. 4 and 5), but with substantial recovery by 24 h. When CA4P (30 mg/kg) was administered 4
days later it caused a very similar effect in terms of extent and longevity of diminished BLI signal
as a surrogate for vascular shutdown. Similar activity was observed when a treatment naïve rat
was given 40 mg/kg 69 (Fig. 5) with substantial recovery at 24 h. At 48 h the rats appeared healthy
and the signal increased marginally. A dose of 80 mg/kg was also well tolerated, but this higher
dose did not show additional vascular disruption.
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Figure 4. BLI assessment of vascular response to VDA. Left) Heat maps overlaid on photographs
of male Copenhagen rat with subcutaneous PC3-DAB2IP-luc human prostate tumor xenograft
showing light emission about 20 min after administration of D-luciferin (120 mg/kg) at various
times with respect to VDA administration IP. Right) Corresponding dynamic light emission curves
acquired over about 30 min following luciferin administration. Blue shows baseline; red about 4 h
after VDA and green at 24 h. A) 10 mg/kg compound 69 indicating no vascular perturbation, but
increased signal at 24 h consistent with rapid tumor growth. B) 6 h later 40 mg/kg compound 69
was administered to the same rat generating about 95% reduced signal at 4 h, consistent with
substantial vascular shutdown and showing substantial recovery by 24 h. C) Four days later 30
mg/kg CA4P was administered to this rat eliciting BLI response similar to the BLI response shown
in B.
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Figure 5. Relative light emission following administration of VDAs. A) Relative signal intensity
is shown about 20 min after administration of D-luciferin subcutaneously in the foreback neck
region of a rat with a subcutaneous PC3-DAB2IP-luc prostate tumor xenograft in the thigh. Top)
baseline (no prior drug), center) 4 h after 40 mg/kg 69, and bottom) 24 h after 69. B) Corresponding
light emission dynamic curve at baseline (blue), 4 h after 69 (red) and 24 h after 69 (green). C)
Normalized BLI signal at various times for the rat in Fig 4 receiving 69 sequentially at 10 mg/kg
(black), 40 mg /kg (red) and 30 mg/kg CA4P (green), together with the treatment naive rat in Fig
5
A, B receiving 40 mg/kg 69 (orange).
Conclusions:
These studies have expanded our SAR knowledge regarding the impact of structural modifications
to lead benzosuberene and dihydronaphthalene analogues on inhibition of tubulin polymerization
and cytotoxicity against human cancer cell lines. Amongst this group of seventeen new molecules
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[
along with the Maderna compound (68), accessed (in this study) through separate synthesis],
emerged several promising analogues (compounds 6, 13, 18, 19, 28, 68) that elicited inhibition
(IC50) of tubulin assembly (cell free assay) greater than or comparable to that of the lead natural
product CA4 and our lead benzosuberene analogues KGP18 and KGP156. These compounds
demonstrated potent cytotoxicity (GI50) against SK-OV-3 (ovarian), NCI-H460 (lung), and DU-
145 (prostate) cells typically in the low to mid nM range. Preliminary investigation of water-
soluble benzosuberene phosphate prodrug salt 69 at 40 mg/kg in vivo revealed vascular disruption
in a PC3-DAB2IP-luc human prostate tumor xenograft based on BLI (Figs. 4 and 5), which was
similar to that obtained with CA4P.
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Experimental Section
Chemistry
General Materials and Methods
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Tetrahydrofuran (THF), carbon tetrachloride, dichloromethane, methanol,
dimethylformamide (DMF), and acetonitrile were used in their anhydrous forms. Reactions were
performed under nitrogen gas. Thin-layer chromatography (TLC) plates (precoated glass plates
with silica gel 60 F254, 0.25 mm thickness) were used to monitor reactions. Purification of
intermediates and products was carried out with a Biotage Isolera flash purification system using
silica gel (200-400 mesh, 60 Å) or RP-18 pre-packed columns or manually in glass columns.
1
Intermediates and products synthesized were characterized on the basis of their H NMR (500 or
13
6
00 MHz), C NMR (125 or 150 MHz) spectroscopic data using a Varian VNMRS 500 MHz or
Bruker DPX 600 MHz instrument. Spectra were recorded in CDCl3,
All chemicals shifts are expressed in ppm (δ), and peak patterns are reported as broad (br), singlet
s), doublet (d), triplet (t), quartet (q), pentet (p), sextet (sext), septet (sept), double doublet (dd),
D
2
O, (CD
3
)
2
CO, or CD
3
OD.
(
double double doublet (ddd), and multiplet (m).
Purity of the final compounds was further analyzed at 25 °C using an Agilent 1200 HPLC
system with a diode-array detector (λ = 190−400 nm), a Zorbax XDB-C18 HPLC column (4.6 mm
Å~ 150 mm, 5 μm), and a Zorbax reliance cartridge guard-column; Method: solvent A, acetonitrile,
solvent B, H O; gradient, 10% A/ 90% B to 100% A/ 0% B over 0 to 40 min; post-time 10 min;
2
flow rate 1.0mL/min; injection volume 20 μL; monitored at wavelengths of 210, 230, 254, 280,
and 320 nm. Purity of target molecules (with reported biological data) was ≥ 95% (as determined by
HPLC at one or more scanned wavelengths) with the exception of compound 27 (94.3% at 254 nm). Mass
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spectrometry was carried out under positive or negative ESI (electrospray ionization) using a
Thermo Scientific LTQ Orbitrap Discovery instrument.
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-((Tert-butyldimethylsilyl)oxy)-2-methoxy-5-(3,4,5-trimethoxyphenyl)-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-5-ol (2).25 To an oven dried flask, THF (10 mL) and 3, 4, 5-
trimethoxyphenyl bromide (0.89 g, 3.6 mmol) were added, and the solution was cooled to -78 °C.
n-BuLi (1.44 mL, 3.60 mmol) was added dropwise to the reaction mixture, which was then stirred
at -78 °C for 1 h. TBS-protected ketone (1) (0.77 g, 2.4 mmol) in THF (5 mL) was then added
slowly to the flask, and the reaction mixture was stirred while warming from -78 °C to room
temperature over 12 h. The reaction mixture was quenched with water and extracted with EtOAc
(3 x 30 mL). The combined organic phase was dried over sodium sulfate and evaporated under
reduced pressure. The crude reaction product was purified by flash chromatography using a pre-
packed 50 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 2%A / 98%B (1 CV),
2
%A / 98%B → 20%A / 80%B (10 CV), 20%A / 80%B (2 CV); flow rate: 50 mL/min; monitored
1
at 254 and 280 nm] to afford tertiary alcohol 2 (1.05 g, 2.15 mmol, 89%) as a clear oil. H NMR
(
(
(
500 MHz, CDCl
3
) δ 7.15 (1H, d, J = 9 Hz), 6.69 (1H, d, J = 9 Hz), 6.50 (2H, s), 3.84 (3H, s), 3.80
3H, s), 3.75 (6H, s), 3.29 (1H, m), 2.56 (1H, m), 2.26 (1H, m), 2.12 (2H, m), 1.90 (1H, m), 1.75
1
3
2H, m), 0.99 (9H, s), 0.17 (3H, s), 0.15 (3H, s). C NMR (125 MHz, CDCl ) δ 153.1, 149.4,
3
142.0, 141.9, 138.7, 137.3, 132.9, 119.8, 108.0, 104.4, 80.2, 61.0, 56.2, 54.8, 41.4, 27.1, 26.4, 26.2,
5.5, 19.1, -3.8, -4.0.
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2
-Methoxy-5-(3,4,5-trimethoxyphenyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,5-diol
2
5
(3). TBS-protected tertiary alcohol 2 (0.41 g, 0.84 mmol) was dissolved in THF (6 mL), and
TBAF (1.01 mL, 1 M in THF, 1.01 mmol) was added, and the reaction mixture was stirred at room
temperature for 4 h. The solution was washed with water and extracted with EtOAc (3 x 20 mL).
The combined organic phase was dried over sodium sulfate and evaporated under reduced
pressure. The crude reaction product was purified by flash chromatography using a pre-packed 10
g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 3%A / 97%B (1 CV), 3%A /
0
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7%B → 30%A / 70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 12 mL/min; monitored at 254
1
and 280 nm] to afford phenol (0.11 g, 0.29 mmol, 35%) as a colorless oil. H NMR (500 MHz,
CDCl
3
) δ 7.04 (1H, d, J = 9 Hz), 6.70 (1H, d, J = 9 Hz), 6.52 (2H, s), 5.79 (1H, s), 3.91 (3H, s),
3
1
.84 (3H, s), 3.76 (6H, s), 3.23 (1H, m), 2.56 (1H, m), 2.35 (1H, m), 2.11 (1H, m), 1.92 (1H, m),
1
3
,75 (2H, m), 1.47 (1H, m). C NMR (125 MHz, CDCl ) δ 153.1, 145.6, 142.7, 141.9, 139.4,
3
137.3, 127.2, 118.2, 107.3, 104.4, 80.2, 61.0, 56.3, 56.0, 41.5, 26.8, 26.3, 24.7. HRMS: Obsvd
+
3
97.1623 [M + Na ], Calcd for C21
H
26
6
O Na: 397.1622. HPLC: 16.33 min.
Tert-butyl((3-methoxy-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-
3
7
yl)oxy)dimethylsilane (4). TBS-protected tertiary alcohol 2 (0.64 g, 1.3 mmol) was dissolved in
acetic acid (10 mL), and the reaction mixture was stirred at room temperature for 6 h. The
unreacted acetic acid was removed under reduced pressure. The resulting reaction mixture was
washed with water and extracted with EtOAc (3 x 30 mL). The combined organic extracts were
washed with brine, dried over sodium sulfate, evaporated under reduced pressure and purified by
flash chromatography using a pre-packed 25 g silica column [solvent A: EtOAc, solvent B:
hexanes; gradient: 5%A / 95%B (1 CV), 5%A / 95%B → 50%A / 50%B (10 CV), 50%A / 50%B
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(
2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford a clear oil that solidified as
1
a colorless solid of TBS-protected benzosuberene analogue 4 (0.41 g, 0.87 mmol, 66%). H NMR
(CDCl
3
, 500 MHz) δ 6.68 (1H, d, J = 8.5 Hz), 6.61 (1H, d, J = 8.5 Hz), 6.48 (2H, s), 6.32 (1H, t,
1
1
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1
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5
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0
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2
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J = 7 Hz), 3.85 (3H, s), 3.81 (3H, s), 3.79 (6H, s), 2.76 (2H, t, J = 7 Hz), 2.10 (2H, m), 1.95 (2H,
1
3
m), 1.04 (9H, s), 0.23 (6H, s). C NMR (CDCl
3
, 125 MHz) δ 152.9, 148.8, 143.2, 141.6, 138.8,
137.3, 133.9, 133.4, 127.0, 122.5, 108.5, 105.3, 61.0, 56.2, 54.8, 34.1, 26.3, 25.7, 24.4, 19.2, -3.7.
1
-(Tert-butyldimethylsilyl)oxy)-5-hydroxy-2-methoxy-5-(3,4,5-trimethoxyphenyl)-5,7,8,9-
tetrahydro-6H-benzo[7]annulen-6-one (5). To a solution of TBS-protected benzosuberene 4
0.51 g, 1.1 mmol) dissolved in CH Cl (20 mL) was added m-CPBA (0.36 g, 2.1 mmol) at -5 °C,
and the reaction mixture was stirred for 2 h and then at room temperature for 12 h. The solution
was washed with saturated Na and saturated NaHCO and extracted with CH Cl (3 x 20
(
2
2
2
S
2
O
3
3
2
2
mL). The combined organic layers were dried over sodium sulfate and evaporated under reduced
pressure. The crude product was purified by flash chromatography using a pre-packed 50 g silica
column [solvent A: EtOAc; solvent B: hexanes; gradient: 5%A / 95%B (1 CV), 5%A / 95%B →
6
0%A / 40%B (10 CV), 60%A / 40%B (2 CV); flow rate: 100 mL/min; monitored at 254 and 280
1
nm] to afford tertiary alcohol 5 (0.256 g, 0.51 mmol, 47%) as a yellow oil. H NMR (600 MHz,
CDCl ) δ 7.26 (1H, d, J = 9 Hz), 6.76 (1H, d, J = 9 Hz), 6.42 (2H, s), 5.01 (1H, s), 3.83 (3H, s),
3
3.81 (3H, s), 3.75 (6H, s), 3.10 (2H, m), 2.82 (1H, m), 2.70 (1H, m), 1.98 (1H, m), 1.76 (1H, m).
13C NMR (150 MHz, CDCl
3
) δ 211.1, 153.4, 150.2, 142.1, 138.0, 137.5, 131.4, 131.1, 127.8,
1
09.1, 105.2, 85.6, 61.0, 56.3, 54.8, 39.7, 26.2, 25.8, 24.2, 19.1, -3.7, -3.9.
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,5-Dihydroxy-2-methoxy-5-(3,4,5-trimethoxyphenyl)-5,7,8,9-tetrahydro-6H-
benzo[7]annulen-6-one (6). TBS-protected benzosuberane 5 (0.17 g, 0.33 mmol) was dissolved
in THF (10 mL). TBAF (0.33 mL, 1 M, 0.33 mmol) was added, and the reaction mixture was
stirred at room temperature for 1 h at 0 °C. A brine (30 mL) solution was added, and the reaction
mixture was extracted with EtOAc (3 x 30 mL). The combined organic phase was dried over
sodium sulfate, filtered, evaporated under reduced pressure, and purified by flash chromatography
using a pre-packed 50 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 5%A /
0
1
2
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4
5
6
7
8
9
0
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9
5%B (1 CV), 5%A / 95%B → 60%A / 40%B (10 CV), 60%A / 40%B (2 CV); flow rate: 100
mL/min; monitored at 254 and 280 nm] to afford phenol 6 (123 mg, 0.320 mmol, 96%) as a white
1
solid. H NMR (600 MHz, CDCl
3
) δ 7.17 (1H, d, J = 8.4 Hz), 6.77 (1H, d, J = 9 Hz), 6.43 (2H, s),
5
.88 (1H, s), 5.00 (1H, s), 3.90 (3H, s), 3.82 (3H, s), 3.74 (6H, s), 3.08 (2H, m), 2.84 (1H, m), 2.68
1
3
(1H, m), 1.99 (1H, m), 1.83 (1H, m). C NMR (150 MHz, CDCl ) δ 211.0, 153.3, 146.4, 142.8,
3
138.0, 137.2, 131.6, 125.7, 120.2, 108.4, 105.2, 85.5, 60.9, 56.3, 56.0, 39.4, 25.4, 23.3. HRMS:
+
Obsvd 411.1414 [M + Na ], Calcd for C21
H
24
7
O Na: 411.1414. HPLC: 15.75 min.
3
-Methoxy-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (7). TBS-
protected benzosuberene 4 (0.41 g, 0.87 mmol) was dissolved in THF (10 mL). TBAF (1.13 mL,
.13 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The
1
solution was washed with water and extracted with EtOAc (3 x 20 mL). The combined organic
phase was dried over sodium sulfate and evaporated under reduced pressure. The crude reaction
product was purified by flash chromatography using a pre-packed 10 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: 12 mL/min; monitored at 254 and 280 nm] to afford phenol
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Page 29 of 84
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
7
(0.25 g, 0.70 mmol, 81%) as a white solid. H NMR (CDCl
3
, 500 MHz) δ 6.71 (1H, d, J = 9
Hz), 6.56 (1H, d, J = 9 Hz), 6.50 (2H, s), 6.34 (1H, t, J = 7.5 Hz), 5.74 (1H, s), 3.91 (3H, s), 3.86
1
3
(3H, s), 3.80 (6H, s), 2.76 (2H, t, J = 7 Hz), 2.14 (2H, m), 1.97 (2H, m). C NMR (CDCl
3
, 125
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz) δ 152.9, 145.2, 142.9, 142.4, 138.6, 134.4, 127.9, 127.4, 121.0, 110.1, 107.8, 105.4, 61.1,
5
6.3, 56.1, 33.7, 25.9, 23.7.
3
-Methoxy-4-(2-(2-methoxyethoxy)ethoxy)-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H-
benzo[7]annulene (8). Phenol 7 (0.11 g, 0.31 mmol) was dissolved in DMF (6 mL), and K
2
CO
3
(
0.12 g, 0.86 mmol) was added. The solution was stirred at room temperature for 20 min. 1-Bromo-
2
-(2-methoxyethoxy) ethane in 90% purity (0.07 mL, 0.5 mmol) was added, the reaction mixture
was stirred at room temperature for 15 h. The solution was washed with water and extracted with
EtOAc (3 x 40 mL). The combined organic phase was dried over sodium sulfate and evaporated
under reduced pressure. The crude reaction product was purified by flash chromatography using a
pre-packed 10 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 3%A / 97%B (1
CV), 3%A / 97%B → 30%A / 70%B (20 CV), 30%A / 70%B (2 CV); flow rate: 12 mL/min;
1
monitored at 254 and 280 nm] to afford ether 8 (25 mg, 0.06 mmol, 18%) as a colorless oil. H
NMR (500 MHz, CDCl ) δ 6.74 (2H, m), 6.48 (2H, s), 6.32 (1H, t, J = 7.5 Hz), 4.18 (2H, t, J = 5
3
Hz), 3.87 (2H, t, J = 5.5 Hz), 3.85 (6H, s), 3.79 (6H, s), 3.76 (2H, m), 3.60 (2H, m), 3.40 (3H, s),
13
2
.78 (2H, t, J = 6.5 Hz), 2.13 (2H, m), 1.93 (2H, m). C NMR (125 MHz, CDCl ) δ 152.9, 151.5,
3
1
45.1, 142.9, 138.5, 137.4, 136.1, 133.8, 127.3, 125.3, 109.3, 105.3, 72.8, 72.2, 70.78, 70.77, 61.0,
+
59.2, 56.2, 55.7, 34.5, 25.7, 24.2. HRMS: Obsvd 481.2198 [M + Na ], Calcd for C26
H
34
7
O Na:
4
81.2197. HPLC: 21.65 min.
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