Tubulin-binding dibenz[c,e]oxepines as colchinol analogues for targeting tumour vasculature

Article abstract of DOI:10.1039/c0ob00500b

Various methoxy- and hydroxy-substituted dibenz[c,e]oxepines were prepared via the copper(i)-induced coupling of ether-tethered arylstannanes or the dehydrative cyclisation of 1,1′-biphenyl-2,2′-dimethanols, assembled using the Ullmann cross-coupling of ortho-bromoaryl carbonyl compounds. The dibenzoxepines were screened for their ability to inhibit tubulin polymerisation and the in vitro growth of K562 human chronic myelogenous leukemia cells. The most active was 5,7-dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol, whose tubulin inhibitory and cytotoxicity (IC₅₀) values were 1 μM and 40 nM, respectively.

Full text of DOI:10.1039/c0ob00500b

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PAPER
Tubulin-binding dibenz[c,e]oxepines as colchinol analogues for targeting
tumour vasculature†
David J. Edwards,a,b John A. Hadfield,b,c Timothy W. Wallace*a and Sylvie Ducki‡c
Received 27th July 2010, Accepted 12th October 2010
DOI: 10.1039/c0ob00500b
Various methoxy- and hydroxy-substituted dibenz[c,e]oxepines were prepared via the copper(I)-induced
coupling of ether-tethered arylstannanes or the dehydrative cyclisation of 1,1¢-biphenyl-2,2¢-
dimethanols, assembled using the Ullmann cross-coupling of ortho-bromoaryl carbonyl compounds.
The dibenzoxepines were screened for their ability to inhibit tubulin polymerisation and the in vitro
growth of K562 human chronic myelogenous leukemia cells. The most active was 5,7-dihydro-
3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol, whose tubulin inhibitory and cytotoxicity (IC50) values
were 1 mM and 40 nM, respectively.
under development as an antitumour agent in the form of the
Introduction
phosphate prodrug 4 (OXi4503).8 Other structures undergoing
Tumour growth requires the support of an associated blood
evaluation as VDAs include the combretastatin analogue 5
supply, making tumour vasculature a potential target for anti-
(AVE8062), which abruptly and irreversibly stops tumour blood
flow in a range of cancer cell lines,9 the analogue 6 of the marine
cancer therapy. This principle has inspired decades of research
into the pathways of angiogenesis (the formation of new blood
vessels), leading to the identification of a family of vascular
endothelial growth factors (VEGFs) that stimulate this process.1
The subsequent search for VEGF inhibitors yielded inter alia the
monoclonal antibody bevacizumab (Avastinꢀ), which in 2004
natural product dolastatin,10 the xanthenylacetic acid 7,11 the
substituted heterocycles 8,12 9,13 10,14 11,15 1216 and 13,17 and the
phosphate prodrug 14 of N-acetylcolchinol.18 With the notable
exception of 7, which appears to act both through the host immune
system, e.g. by stimulating the production of tumour necrosis
factor (TNF),19 and directly by inducing vascular endothelial
cell apoptosis,20 the biological target of these potential VDAs is
tubulin.
R
became the first angiogenesis inhibitor to be approved in the
USA for clinical use in the treatment of cancer.2 While the
underlying principles and clinical practices of anti-angiogenic
therapy continue to evolve,3 an alternative antivascular strategy
is emerging. Tumour vasculature is structurally and functionally
abnormal, being disorganised and prone to inefficiencies caused by
branching, uneven diameter, shunts, etc., rendering it susceptible
to the effects of vascular disrupting agents (VDAs).4 Various small
molecules have come under scrutiny in this context,5 the most
prominent being combretastatin A-4 (CA-4) 1, which was isolated
by Pettit and coworkers from the African bush willow Combretum
caffrum6 and shown to induce shutdown of tumour vasculature
within minutes, while leaving normal vasculature intact.7 Clinical
trials of the water-soluble prodrug CA-4P 2 (Zybrestat) were
initiated in 1998, and the congener combretastatin A-1 3 is also
Tubulin, in the form of various isotypes, is an abundant
component of the cytoplasm of animal cells. Two GTP-binding
monomers, a- and b-tubulin, form a heterodimer whose poly-
merisation generates microtubules, which occupy a pivotal role
in a range of intracellular processes involving structure, shape,
signalling and transport, including chromosome segregation and
positioning during mitosis. The dynamics of microtubule assembly
and disassembly are responsive to the identity of the nucleotide
bound to the b-tubulin of its terminal heterodimer unit, the
microtubule being viable when this is GTP but prone to rapid
shrinkage when it is exchanged for GDP.21 The dynamics of
tubulin–microtubule interconversion are delicately poised and
can be disrupted by coordinating species: some of the tubu-
lin binders in clinical use (taxanes, epothilones) stabilise the
GDP-bound tubulin in the microtubule, thereby inhibiting its
disassembly,22 while others (vincristine, etoposide) bind to the
ab-tubulin heterodimer and inhibit microtubule assembly.23 The
origin and subtleties of the latter type of inhibition have become
more apparent since the acquisition of high-resolution crystal-
lographic structures of tubulin-drug complexes by Knossow and
coworkers.24,25 Tubulin binding agents can exert a direct cytotoxic
effect by perturbing microtubule dynamics, thereby undermining
aSchool of Chemistry, The University of Manchester, Oxford Road, Manch-
ester, UK M13 9PL. E-mail: tim.wallace@manchester.ac.uk; Fax: +44 (0)
161 275 4939
bDrug Development Group, Paterson Institute for Cancer Research, Christie
Hospital NHS Trust, Manchester, UK M20 4BX
cKidscan Laboratories, Centre for Molecular Drug Design, School of
Environment and Life Sciences, University of Salford, Salford, UK M5 4WT
† Electronic supplementary information (ESI) available: Further experi-
mental details. See DOI: 10.1039/c0ob00500b
‡ Present address: Laboratoire de Chimie des He´te´rocycles et des Glucides,
ENSCCF, Clermont Universite´, 63174 Aubie`re, France.
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mitosis (which requires the rapid turnover of microtubules at all
stages) and bringing about cell death. Microtubules also play
a prominent role in maintaining the physical structure of the
endothelial cells lining new tumour vasculature, which lack the
well-defined actin cytoskeleton and other strengthening features
of mature endothelial cells, and it has been established that
some tubulin-binding VDAs induce morphological changes in the
endothelial cells of immature tumour vasculature, e.g. rounding
and detachment, leading to reduced blood flow and tumour
necrosis.4,18b,26 Significantly, vascular shutdown by CA-4 1 is
achieved using substantially less than the maximum tolerated dose
(MTD), illustrating one of the potential advantages of targeting
tumour vasculature rather than the mitotic apparatus, which
generally involves less favourable therapeutic margins.
In common with CA-4 1,27 the structures 8–14 bind to tubulin at
or close to the same site as colchicine 15, the best known tubulin-
binding agent and ‘spindle toxin’.28 The toxicity of colchicine 15
precludes its clinical use as an antimitotic agent, but it is clear
that the colchicine binding site of tubulin can accommodate a
diverse range of structures and so offers considerable scope for the
design of binding agents with minimised pharmacokinetic half-
lives and cardiostimulatory effects,29 the latter being a potentially
generic problem with tubulin-targeting VDAs.18d,30 Our interest in
the axial chirality of colchicine 15 led us to analyse the variation
of the interaryl dihedral angle in a series of heterocyclic variants
of the bridged biaryl core,31 and we observed that the degree
of helicity in dibenz[c,e]oxepines closely matches that found in
colchicine 15. In pursuing this line we synthesised a series of new
dibenz[c,e]oxepines and assessed their ability to inhibit tubulin
polymerisation, which led to the identification of 5,7-dihydro-
3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol 16 as a new lead in
the search for effective VDAs.32 Our results, herein described in
detail, suggest that a dibenz[c,e]oxepine unit may be capable of
providing the helical core of a new series of tubulin-binding small
molecules.33
Synthesis of materials
Initially we assessed the methods available for biaryl synthe-
sis, seeking to identify those suited for use in approaches to
polysubstituted dibenz[c,e]oxepines. Although not ideal from an
environmental viewpoint, the copper(I)-mediated intramolecular
coupling of tethered arylstannane units had been shown by Piers
et al.34 to be an effective source of the target heterocyclic system,
and we therefore chose to apply this method to our first targets. To
prepare the required series of doubly-stannylated dibenzyl ethers,
we first acquired stocks of the stannylated trimethoxybenzyl
bromides 2034 and 21 (Scheme 1) and then used analogous
lithiation–transmetallation sequences to convert benzyl alcohol 22
and veratryl alcohol 24 into the respective tributyltin derivatives
23 and 25 (Scheme 2).
To extend the series, the protected vanillin 26 was reduced to
27 for use in the lithiation–stannylation sequence. However, in
this case the yield of the desired alcohol 28 was poor (28%) and
the by-products included the doubly-stannylated species 29 (11%).
The formation of the latter, which was characterised inter alia by
1
the H NMR signals from its diastereotopic SnCH2Si group [dH
(400 MHz, CDCl3) 0.00 (1 H, d, J 19.4 Hz), -0.07 (1 H, d, J
19.4 Hz)], serves as a reminder35 of the increased susceptibility
of a t-butyldimethylsilyloxy group towards deprotonation by an
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for testing purposes and they therefore remain to be optimised. A
sample of the MOM-protected variant 42 was transformed into
the corresponding phenol 44 using a mild procedure.36
Other dibenz[c,e]oxepines were synthesised using the Ullmann
reaction37 to effect the coupling of appropriate substituted
haloarenes. Although conventional Ullmann cross-couplings tend
to give mixtures containing homocoupled products, they offer
rapid access to certain types of target structure and can be
optimised by modifying the reaction conditions and stoichiometry
so as to inhibit homocoupling,38,39 or by the quantitative formation
of the intermediate arylcopper species from one of the reaction
partners prior to its exposure to the other.40 In our first Ullmann
approach (Scheme 3), we found that combining mole equivalents
of the bromoaldehyde 45 and the bromoester 46 gave an acceptable
yield of 47, although chromatography was required to isolate
it from the homocoupled product 48 (12%). Reduction of the
carbonyl functions of 47 provided, successively, the ester-alcohol
49 and the diol 50. The latter was transformed into the oxepine 51
upon treatment with aqueous acid.41
Scheme 1
In a second Ullmann cross-coupling (Scheme 4), reacting 45
with 6-bromopiperonal 52 gave the dialdehyde 53 (22%), the
low isolated yield in this case being partly due to the use
of crystallisations to remove the contaminating dialdehyde 54.
The dialdehyde 5338 and related structures are useful lignan
precursors that can also be prepared via Suzuki–Miyaura coupling
protocols.42 Reduction of 53 to the diol 55, followed by ring closure
as before, gave the dibenzoxepine 56 as a crystalline solid.
Based on our analysis of the structure–activity relationships
in known colchicine, allocolchicine and combretastatin deriva-
tives, we surmised that the substitution pattern present in the
dibenzoxepine 16 was particularly worthy of study. The proposed
route to this compound required a hydroxyl protecting group
that would withstand the conditions of the Ullmann coupling
reaction, and encouraged by a literature precedent43 we elected
to use a methanesulfonyl (mesyl) group in this capacity. The
route to the target therefore began with the mesylation of
the commercially available bromoaldehyde 57 under standard
conditions (Scheme 5). This provided a good yield of the mesylate
58, together with a by-product identified as the sultone 59 (22%)
derived from 58 via the base-induced condensation of the mesylate
and aldehyde groups. We surmise that the formation of 59, whose
heterocyclic core is rare, is assisted by the buttressing effect of
the flanking OMe and Br functions. Ullmann coupling of the
mesylate 58 with the bromoaldehyde 45 (3 equiv.), followed by
chromatography, gave an acceptable yield of the dialdehyde 60,
alkyllithium when a second lithium coordination site, in this case
the methoxy group, is located nearby. Repeating the sequence with
the MOM-protected vanillin 31 also proved troublesome, with
poor conversion to the desired alcohol 32 (23%) and the formation
of a comparable amount of the regioisomeric product 33 (19%).
Using an alternative stannylation procedure based on lithium–
bromine exchange, the bromo alcohol 34 provided a fair yield of
35 in straightforward fashion.
The alcohols 23, 25, 32 and 35 were converted into the respective
dibenzyl ethers 36–39 using the appropriate aryl bromide 20 or 21.
These etherifications were more efficient when using the trimethyl-
stannyl bromide 20, presumably for steric reasons. Subjecting
the ethers 36–39 to the conditions of the cyclisation process34
gave the desired dibenz[c,e]oxepines 40–43 in yields that were,
at best, modest, but allowed the isolation of sufficient material
Scheme 2 Reagents: i, Montmorillonite K10, CH2Cl2, RT, 3 h.
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Scheme 3
Scheme 4
and subsequent reduction followed by acid-induced cyclisation Compound evaluation
gave the desired mesylate 62. Alkaline hydrolysis of 62 provided
the target dibenzoxepine 16 as a white crystalline solid, m.p. 145–
147 ◦C (MeOH). The 1H NMR spectrum of 16 (Fig. 1) illustrates
the line broadening for the diastereotopic methylene signals that
is typical of compounds of this type, in which the biaryl unit is
non-planar and fluctional, undergoing axis inversion slowly on the
NMR time scale.44
New materials were screened for their ability to inhibit microtubule
assembly45 and for growth inhibitory activity (IC50) against the
K562 human chronic myelogenous leukemia cell-line.46 Both
of these assays are routinely used for the evaluation of test
compounds and provide a useful comparison with benchmarks
such as CA-4 1 and colchicine 15. The results (Table 1) show
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Scheme 5
Fig. 1 1H NMR spectrum (400 MHz, CDCl3, 25 ◦C) of the dibenzoxepine 16.
that, as might be expected, biological activity is dependent on the
aromatic substitution pattern, and reveal useful levels of activity in
the dibenzoxepines 16, 51 and 56 (entries 3, 9 and 10). In particular,
the phenol 16 inhibits tubulin assembly to the same extent as CA-4
1, whose substituent array is the same, and returned an IC50 value
of 40 nM against the K562 cell-line. None of the other compounds
inhibited tubulin polymerisation to a significant degree, although
43 and 44 (entries 7 and 8) displayed some in vitro cytotoxicity.
(IC50 > 50 mM)48 and 72 (IC50 1.5 mM),49 and indicates that at least
one oxygen substituent in the C-ring is a prerequisite for tubulin-
binding activity. However, the hexamethoxy series comprising
63 and 66–70, which had been prepared from the diol 64 for
our crystallographic study,31 was almost devoid of activity. The
cytotoxicity of the dibenzazepine 66 (entry 13) is intriguing, given
that its C-ring oxygenation pattern cannot be viewed as optimal,
although it is recognised that the link between cytotoxicity and the
ability to inhibit tubulin polymerisation is potentially complex.
The properties of the dibenzoxepine 16 are consistent with
its structural analogy to the colchicinoids, e.g. 14 and 15. The
interaction of colchicine 15 with tubulin has long been under
intense scrutiny, now informed by the 1SA0 crystal structure,
Discussion
The difference in potency of 40 and 51 as tubulin polymerisation
inhibitors mirrors that of their respective carbocyclic analogues 71
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Table 1 Activities (IC50) of substituted dibenzoxepines, dibenzazepines
and dibenzothiepines in the microtubule assembly and K562 cytotoxicity
assays
Tubulin assembly
K562 MTT assay
Entry
Compound
IC50/mMa
IC50/mMb
1
2
3
4
5
6
7
8
1
1.3
3.9
1.0
0.0010c
0.0022d
0.04
>20
>20
>20
3
15
16
40
41
42
43
44
51
56
62
63
66
67
68
69
70
>10
>10
>10
>10
>10
7.4
>10
>10
>10
>10
>10
>10
>10
>10
19
9
0.13
0.10
>20
>400
73
10
11
12
13
14
15
16
17
360
>400
>400
>400
a Concentration required for 50% inhibition of tubulin assembly.
b Concentration that inhibits the growth of the K562 cell line by 50% after
incubation for 5 days. Each drug concentration was tested in triplicate, and
the standard error of each value is <10%. c All entries in this column are
normalised to this value for 1, which varied over the range 0.0010–0.0022
between batches. d Value taken from ref. 47.
Fig. 2 (a) The tubulin-binding conformation of DAMA-colchicine 73,
extracted from the crystal structure of the DAMA-colchicine/tubulin:RB3
conjugate (ref. 24), together with the nearby b-Lys352 and a-Thr179
residues (numbering system from ref. 24). (b) The energy-minimised
structure of dibenzoxepine 16 (MacroModel 8, MM3 force field).
1 and the colchicinoids 14 and 15, the dibenzoxepine 16 binds to
tubulin in a similar manner.
which gives a detailed picture of the interaction of N-deacetyl-
N-(2-mercaptoacetyl)colchicine (DAMA-colchicine) 73 with the
ab-tubulin heterodimer.24 A comparison of the tubulin-bound 73
with a model of the dibenzoxepine 16 (Fig. 2) reveals some clear
parallels. We speculate that, as has been proposed for CA-4 1,50 the
phenolic hydroxyl of 16 is suitably placed to emulate the side-chain
N–H of the colchicinoids in H-bonding to the carbonyl oxygen of
the residue Thr179 located on the adjacent a-tubulin chain, while
the 3-methoxy group of 16 can interact with the side-chain nitrogen
of the b-tubulin residue Lys352. In the absence of substituents at
C(1), C(5) and C(7), the biaryl unit of 16 has a configurationally
unbiased axis of the tropos type,51 i.e. whose low inversion barrier
renders it free to adopt the (aR) arrangement required for binding.
It is therefore proposed that, while chemically distinct from CA-4
Conclusion
In the course of this study we have synthesised a series of
heterocyclic colchinol analogues and monitored their ability to
inhibit microtubule assembly and the in vitro growth of K562
human chronic myelogenous leukemia cells, leading us to identify
5,7-dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol 16 as a
potent tubulin-binding and cytotoxic agent. Our results suggest
that replacing the central methylene group in the three-atom
bridge of colchinol analogues with an oxygen atom does not
diminish the ability of suitably substituted derivatives of the
system to bind to tubulin and, by inference, interfere with cell
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division processes and the integrity of tumour neovasculature.
We consider the structure 16 to be the leading member of a
new series of potential therapeutic agents based on the hitherto
unexploited dibenz[c,e]oxepine pharmacophore, and are currently
pursuing this principle in the context of a search for clinically
useful antitumour agents and VDAs.
organic extract was washed with brine (20 mL/mmol of alcohol),
dried, evaporated, and the residue purified by chromatography.
3,4,5-Trimethoxy-2-tributylstannylbenzyl alcohol 19 was pre-
pared from 17 (4.00 g, 20.2 mmol). Chromatography (hexane–
ethyl acetate, 6 : 1) gave the title compound 19 (4.20 g, 43%) as a
colourless oil. Elemental analysis, 1H NMR spectroscopy and MS
indicated the presence of residual Bu3SnCl. Other data: nmax/cm-1
3464, 2955, 2928, 2866, 2854, 1584, 1561, 1460, 1379, 1313, 1196,
1157, 1099; dH (400 MHz, CDCl3) 6.81 (1 H, s, 6-H), 4.54 (2 H,
d, J 5.7 Hz, CH2O), 3.85 (3 H, s, OMe), 3.84 (3 H, s, OMe), 3.80
(3 H, s, OMe), 1.71 (1 H, br t, J 5.7 Hz, OH), 1.52 (6 H, m, 3 ¥
CH2CH2Sn), 1.34 (6 H, m, 3 ¥ CH2CH3), 1.09 (6 H, t, J 8.3 Hz, 3 ¥
CH2Sn), 0.90 (9 H, t, J 7.3 Hz, 3 ¥ CH2CH3); dC (100 MHz, CDCl3,
DEPT-135) 12.1 (CH2), 14.1 (CH3), 27.8 (CH2), 29.6 (CH2), 56.3
(CH3), 60.8 (CH3), 61.0 (CH3), 67.4 (CH2), 108.4 (CH); m/z (ES)
489 (MH+, 100%), 332, 291; Rf 0.30 (hexane–ethyl acetate, 6 : 1).
3,4-Dimethoxy-2-(tributylstannyl)benzyl alcohol 25 was pre-
pared from 24 (3.40 g, 20.2 mmol). Chromatography (hexane–
ethyl acetate, 6 : 1) gave the title compound 25 (3.60 g, 39%) as a
colourless viscous oil (Found: C, 55.0; H, 8.5; Sn, 25.9. C21H38O3Sn
requires C, 55.16; H, 8.38; Sn, 25.96%); nmax/cm-1 3367, 2951, 2924,
2870, 2850, 1592, 1464, 1386, 1293, 1269, 1200, 1138, 1037, 870;
Experimental
Melting points were determined using Kofler hot-stage, Buchi 512
or Electrothermal 9100 equipment and are uncorrected. Unless
otherwise indicated, IR spectra were recorded for neat thin films
on NaCl plates, using Perkin–Elmer 1710FT or Nicolet Nexus
670/870 spectrometers. NMR spectra were measured on Bruker
AC300 or DPX400 instruments, and assigned with the aid of
COSY, HMBC, HSQC and DEPT spectra where appropriate.
Coupling constants (J values) are quoted to the nearest 0.1 Hz.
Low-resolution mass spectra were measured on a Micromass LCT
instrument using a Waters 2790 separations module with electro-
spray (ES+) ionisation and TOF fragment detection, or a Kratos
MS-50 spectrometer with FAB ionisation. High-resolution mass
measurements were obtained using ThermoFinnigan MAT95XP
or Kratos Concept S1 instruments. Data for most of the peaks of
intensity <20% of that of the base peak are omitted. Elemental
analyses were carried out by the University of Manchester
microanalytical service.
d
H (400 MHz, CDCl3) 7.14 (1 H, d, J 8.1 Hz, 5-H), 6.89 (1 H, d, J
8.1 Hz, 6-H), 4.55 (2 H, d, J 5.7 Hz, CH2O), 3.88 (3 H, s, OMe),
3.85 (3 H, s, OMe), 1.71 (1 H, br t, J 5.7 Hz, OH), 1.52 (6 H,
m, 3 ¥ CH2CH2Sn), 1.34 (6 H, m, 3 ¥ CH2CH3), 1.09 (6 H, t, J
8.3 Hz, 3 ¥ CH2Sn), 0.90 (9 H, t, J 7.3 Hz, 3 ¥ CH2CH3); Rf 0.40
(hexane–ethyl acetate, 4 : 1).
Starting materials and solvents were routinely purified by
conventional techniques.52 Most reactions were carried out under
nitrogen or, when appropriate, argon dried by passage through an
anhydrous CaCl2 drying tube and freed from traces of oxygen using
an Oxysept cartridge (both Aldrich). Tetrahydrofuran (THF) and
N,N,N¢,N¢-tetramethylethylenediamine (TMEDA) were dried us-
ing sodium benzophenone ketyl under argon. Organic solutions
were usually dried using anhydrous MgSO4 and concentrated by
rotary evaporation under reduced pressure. Analytical thin layer
chromatography (TLC) was carried out on Merck silica gel 60
on aluminium plates containing a 254 nm fluorescent indicator.
The chromatograms were visualised by the use of UV light or
the following developing agents; ethanolic vanillin or potassium
permanganate. Unless otherwise indicated, preparative (column)
chromatography was carried out using the flash technique53 on
60H silica gel (Merck 9385). Compositions of solvent mixtures are
quoted as ratios of volume. ‘Petroleum’ refers to a light petroleum
2-Trimethylstannyl-4-(O-silyldimethyl-t-butyl)-3-methoxyben-
zyl alcohol 28 was prepared from 2755 (2.00 g, 7.45 mmol).
Chromatography (hexane–ethyl acetate, 10 : 1) gave the crude
stannane 28 (0.90 g, 28%) as a yellow viscous oil which was
used without further purification; nmax/cm-1 3309, 2955, 2928,
2854, 1581, 1460, 1383, 1282, 1196, 1134, 1002, 948, 893, 819;
dH (400 MHz, CDCl3) 7.01 (1 H, d, J 8.0 Hz, 5-H), 6.79 (1 H, d,
J 8.0 Hz, 6-H), 4.53 (2 H, d, J 5.4 Hz, CH2), 3.76 (3 H, s, OMe),
1.48 (1 H, br t, J 5.4 Hz, OH), 1.00 (9 H, s, CMe3), 0.38 (9 H,
SnMe3), 0.18 (6 H, s, SiMe2); dC (100 MHz, CDCl3, DEPT-135)
-5.7 (CH3), -4.1 (CH3), 26.2 (CH3), 60.8 (CH3), 66.8 (CH2), 122.1
(CH), 124.8 (CH); m/z (ES) 415 (MH+–OH, 100%) (HRMS could
not be performed due to low ionisation levels); Rf 0.30 (hexane–
ethyl acetate, 8 : 1). Other fractions provided a sample of the ether
◦
29 (0.50 g, 11%) as a colourless solid, m.p. 54–56 C, which was
identified from the following data: nmax/cm-1 3320, 2963, 2928,
2854, 1581, 1464, 1386, 1285, 1196, 1134, 1006, 948, 893, 831, 769;
dH (400 MHz, CDCl3) 6.97 (1 H, d, J 8.0 Hz, 5-H), 6.79 (1 H,
d, J 8.0 Hz, 6-H), 4.53 (2 H, d, J 4.6 Hz, OCH2), 3.77 (3 H, s,
OMe), 1.51 (1 H, br m, OH), 0.97 (9 H, s, CMe3), 0.33 (9 H, s,
ArSnMe3), 0.22 (3 H, s, SiMe), 0.05 (9 H, s, CH2SnMe3), 0.00 (1
H, d, J 19.4 Hz, CHSnMe3), -0.07 (1 H, d, J 19.4 Hz, CHSnMe3);
dC (100 MHz, CDCl3, DEPT-135) -8.4 (CH2), -7.3 (CH3), -5.7
(CH3), -2.5 (CH3), 26.6 (CH3), 60.8 (CH3), 66.8 (OCH2), 121.9
(CH), 124.7 (CH); m/z (ES) 577 (MH+–OH, 100%) (HRMS could
not be performed due to low ionisation levels); Rf 0.35 (hexane–
ethyl acetate, 8 : 1).
◦
fraction, b.p. 60–80 C, unless otherwise stated. ‘Ether’ refers to
diethyl ether. The preparative routes to the dibenzoxepines 16,
51 and 56 have also been described in a patent application.32
Compounds 18,34 20,34 6331 and 67–7031 were prepared using
published procedures.
General procedure for alcohols 19, 25, 28 and 32
To a stirred solution of n-BuLi (2.5 eq.) in dry ether (10 mL mmol-1
◦
of alcohol) at -78 C was added TMEDA (2.5 eq.) and stirring
was continued for 10 min. The neat alcohol was added and the
solution was allowed to warm to RT. After stirring for 3 h, the
reaction was cooled to -78 ◦C and the trialkyltin chloride (1.5 eq.)
was added. The reaction was warmed to RT and stirred for 2 h.
Water (10 mL mmol-1 of alcohol) was added and the mixture was
extracted with ether (3 ¥ 10 mL mmol-1 of alcohol). The combined
2-Trimethylstannyl-3-methoxy-4-methoxymethylbenzyl alcohol
32 was prepared from 3156,57 (3.80 g, 19.2 mmol). Chromatography
(hexane–ethyl acetate, 5 : 1 to 3 : 1) gave the title compound 32
(1.60 g, 23%) as a colourless viscous oil (Found: C, 43.5; H, 6.4;
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Sn, 32.7. C13H22O4Sn requires C, 43.25; H, 6.14; Sn, 32.88%);
max/cm-1 3421, 2971, 2936, 2909, 1464, 1394, 1262, 1192, 1157,
5.7 Hz, OH), 0.28 (9 H, s, SnMe3); m/z (FAB) 332 (M+, 5%), 315
(M+–OH, 100); Rf 0.40 (hexane–ethyl acetate, 1 : 1).
n
1134, 1080, 1002, 773; dH (400 MHz, CDCl3) 7.12 (1 H, d, J 8.2 Hz,
5-H), 7.07 (1 H, d, J 8.2 Hz, 6-H), 5.20 (2 H, s, OCH2O), 4.55 (2
H, s, ArCH2O), 3.88 (3 H, s, ArOMe), 3.52 (3 H, s, CH2OMe), 1.70
(1 H, br s, OH), 0.38 (9 H, SnMe3); dC (100 MHz, CDCl3, DEPT-
135) -5.6 (CH3), 56.6 (CH3), 61.3 (CH3), 66.7 (CH2), 95.3 (CH2),
117.3 (CH), 124.8 (CH); m/z (ES) 345 (MH+–OH, 70%), 315 (55),
206 (100); Rf 0.35 (hexane–ethyl acetate, 4 : 1). Other fractions
provided a sample of the isomeric stannane 33 (1.30 g, 19%) as a
colourless oil, dH (400 MHz, CDCl3) 6.96 (1 H, d, J 1.9 Hz, ArH),
6.94 (1 H, d, J 1.9 Hz, ArH), 5.20 (2 H, s, OCH2O), 4.55 (2 H, s,
ArCH2O), 3.85 (3 H, s, ArOMe), 3.50 (3 H, s, CH2OMe), 1.65 (1
H, br s, OH), 0.35 (9 H, SnMe3); dC (75 MHz, CDCl3, DEPT-135)
-8.1 (CH3), 55.95 (CH3), 58.0 (CH3), 65.6 (CH2), 99.1 (CH2), 112.6
(CH), 126.8 (CH); m/z (ES) 345 (MH+–OH, 70%), 315 (55), 206
(100); Rf 0.18 (hexane–ethyl acetate, 4 : 1).
General procedure for bis(trialkylstannylbenzyl) ethers 36–39
To a suspension of NaH [1.3 eq., pre-washed with pentane
(6 mL mmol-1) and THF (6 mL mmol-1)] in anhydrous DMF
(1 mL mmol-1 of alcohol) at 0 ◦C was added the alcohol (1.1 eq.)
as a solution in anhydrous DMF (1 mL mmol-1) dropwise and
the mixture was stirred for 10 min. The bromide was added as a
so◦lution in DMF (1 mL mmol-1) and the solution was stirred at
0
C for 20 min, then at RT for 16 h. DCM (10 mL mmol-1)
and water (10 mL mmol-1) was added and the mixture was
extracted with DCM (2 ¥ 5 mL mmol-1 of alcohol). The combined
extract was washed with brine (10 mL/mmol of alcohol), dried,
evaporated, and the residue purified by chromatography.
Tributyl(2-((3,4,5-trimethoxy-2-(trimethylstannyl)benzyloxy)-
methyl)phenyl)stannane 36 was prepared from alcohol 2354
(960 mg, 2.42 mmol) and bromide 2034 (930 mg, 2.19 mmol).
Chromatography (hexane–ethyl acetate, 10 : 1) gave the title com-
pound 36 (1.08 g, 67%) as a clear oil (Found: C, 51.9; H, 7.4;
Sn, 31.8. C32H54O4Sn2 requires C, 51.92; H, 7.35; Sn, 32.08%);
3,4,5-Trimethoxy-2-tributylstannylbenzyl bromide 21
A solution of triphenylphosphine (1.62 g, 6.2 mmol) in dry DCM
(50 mL) at 0 ◦C was treated dropwise with bromine (0.32 mL,
1.0 g, 6.2 mmol). The yellow colour that persisted was discharged
by the addition of a few more crystals of triphenylphosphine,
and the solution was stirred for a further 20 min. Imidazole
(450 mg, 6.6 mmol) was then added in one portion and the stirring
continued for a further 20 min. A solution of the alcohol 19
(2.31 g, 4.74 mmol) in DCM (10 mL) was added and the mixture
was stirred at 0 ◦C for 20 min and RT for 1 h. Pentane (7 mL)
was added and the white suspension filtered through a cake of
silica gel (ca. 10 g), rinsing with ether (100 mL). Evaporation
of the filtrate and chromatography of the residue (110 g silica gel,
hexane–ethyl acetate, 6 : 1) gave the title compound 21 (1.80 g, 69%)
as a colourless oil which was used without further purification; dH
(400 MHz, CDCl3) 6.68 (1 H, s, 6-H), 4.37 (2 H, s, CH2Br), 3.81
(3 H, s, OMe), 3.79 (3 H, s, OMe), 3.75 (3 H, s, OMe), 1.45 (6
H, m, 3 ¥ CH2CH2Sn), 1.27 (6 H, m, 3 ¥ CH2CH3), 1.09 (6 H, t,
J 8.3 Hz, 3 ¥ CH2Sn), 0.90 (9 H, t, J 7.3 Hz, 3 ¥ CH2CH3); dC
(100 MHz, CDCl3, DEPT-135) 12.2 (CH2), 14.0 (CH3), 27.8 (CH2),
29.5 (CH2), 37.9 (CH2), 56.4 (CH3), 60.8 (CH3), 61.0 (CH3), 110.3
(CH); Rf 0.50 (hexane–ethyl acetate, 8 : 1).
n
max/cm-1 3048, 2955, 2924, 2847, 1584, 1561, 1480, 1464, 1375,
1351, 1313, 1192, 1161, 1049, 1017; dH (300 MHz, CDCl3) 7.48
(1 H, d, J 6.8 Hz, ArH), 7.30 (1 H, t, J 6.7 Hz, ArH), 7.31–7.25
(2 H, m, ArH), 6.76 (1 H, s, 5-H), 4.46 (2 H, s, CH2O), 4.39 (2
H, s, CH2O), 3.67 (3 H, s, OMe), 3.65 (6 H, s, 2 ¥ OMe), 1.52 (6
H, m, 3 ¥ CH2CH2Sn), 1.31 (6 H, m, 3 ¥ CH2CH3), 1.07 (6 H, t,
J 8.2 Hz, 3 ¥ CH2Sn), 0.90 (9 H, t, J 7.3 Hz, 3 ¥ CH2CH3), 0.26
(9 H, s, SnMe3); dC (100 MHz, CDCl3, DEPT-135) -6.3 (CH3),
10.7 (CH2), 14.1 (CH3), 27.8 (CH2), 29.6 (CH2), 56.4 (CH3), 60.9
(CH3), 61.2 (CH3), 73.2 (CH2), 74.0 (CH2), 109.0 (CH), 127.4 (2
¥ CH), 128.4 (CH), 137.3 (CH); m/z (ES) 741 (MH+, 100%); Rf
0.55 (hexane–ethyl acetate, 6 : 1).
Tributyl(6-((3,4-dimethoxy-2-(tributylstannyl)benzyloxy)me-
thyl)-2,3,4-trimethoxyphenyl)stannane 37 was prepared from alco-
hol 25 (1.60 g, 3.50 mmol) and bromide 21 (1.70 g, 3.09 mmol).
Chromatography (hexane–ethyl acetate, 10 : 1) gave the title com-
pound 37 (1.00 g, 35%) as a clear oil (Found: C, 55.9; H, 8.4;
Sn, 25.9. C43H76O6Sn2 requires C, 55.74; H, 8.27; Sn, 25.63%);
n
max/cm-1 2951, 2928, 2847, 1584, 1565, 1464, 1375, 1317, 1262,
1103, 1041, 1013; dH (400 MHz, CDCl3) 7.10 (1 H, d, J 8.1 Hz,
6¢-H), 6.86 (1 H, t, J 8.1 Hz, 5¢-H), 6.85 (1 H, s, 5-H), 4.40 (2 H, s,
OCH2), 4.33 (2 H, s, OCH2) 3.88 (9 H, br s, 3 ¥ OMe), 3.85 (6 H,
br s, 2 ¥ OMe), 1.52 (12 H, m, 6 ¥ CH2CH2Sn), 1.31 (12 H, m, 6 ¥
CH2CH3), 1.10 (6 H, t, J 8.5 Hz, 3 ¥ CH2Sn), 1.04 (6 H, t, J 8.2 Hz,
3 ¥ CH2Sn), 0.89 (18 H, m, 6 ¥ CH2CH3); dC (100 MHz, CDCl3,
DEPT-135) 12.1 (CH2), 12.15 (CH2), 14.1 (CH3), 14.12 (CH3), 27.8
(CH2), 27.9 (CH2), 29.6 (CH2), 29.7 (CH2), 55.8 (CH3), 56.3 (CH3),
60.8 (CH3), 61.0 (CH3), 61.0 (CH3), 73.1 (CH2), 73.4 (CH2), 108.2
(CH), 112.6 (CH), 125.4 (CH); Rf 0.45 (hexane–ethyl acetate, 6 : 1).
(2-Methoxy-3-(methoxymethoxy)-6-((3,4,5-trimethoxy-2-
(trimethylstannyl)-benzyloxy)methyl)phenyl)trimethylstannane 38
was prepared from alcohol 32 (1.52 g, 4.21 mmol) and bromide 2034
(2.40 g, 5.7 mmol). Chromatography (hexane–ethyl acetate, 6 : 1)
gave the title compound 38 (2.40 g, 81%) as a clear oil (Found: C,
44.3; H, 6.2; Sn, 34.0. C26H42O7Sn2 requires C, 44.36; H, 6.01; Sn,
33.72%); nmax/cm-1 2932, 2850, 1584, 1561, 1468, 1375, 1317, 1262,
1192, 1161, 1103, 1014, 767; dH (300 MHz, CDCl3) 7.11 (1 H, d,
4,5-Dimethoxy-2-trimethylstannylbenzyl alcohol 35
A stirred solution of 2-bromo-4,5-dimethoxybenzyl alcohol 34
(2.90 g, 11.7 mmol) in THF (10 mL) at -78 ◦C was treated dropwise
with n-BuLi (2.5 M in hexanes, 11.3 mL, 28.3 mmol). The solution
was stirred for 1 h at -78 ◦C, treated dropwise with a solution of
trimethyltin chloride (3.50 g, 17.6 mmol) in THF (4 mL), stirred at
-78 ◦C for a further 1 h and then allowed to warm to RT and stirred
overnight. The mixture was poured into 1 M sulfuric acid (50 mL),
extracted with ether (3 ¥ 50 mL), and the combined extracts dried
and evaporated. Chromatography of the residue (ethyl acetate–
hexane, 8 : 1 to 1 : 1) gave the title compound 35 (2.30 g, 59%)
as a colourless oil (Found: C, 43.6; H, 6.3; Sn, 35.8. C12H20O3Sn
requires C, 43.54; H, 6.09; Sn, 35.86%); nmax/cm-1 3499, 2959, 1577,
1495, 1452, 1324, 1289, 1251, 1045, 866; dH (300 MHz, CDCl3)
6.97 (1 H, s, ArH), 6.89 (1 H, s, ArH), 4.97 (2 H, d, J 5.7 Hz,
CH2O), 3.87 (3 H, s, OMe), 3.84 (3 H, s, OMe), 1.91 (1 H, t, J
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J 8.7 Hz, 6-H), 7.03 (1 H, d, J 8.7 Hz, 5-H), 6.75 (1 H, s, 6¢-H), 5.23
(2 H, s, OCH2O), 4.40 (2 H, s, ArCH2O), 4.35 (2 H, s, ArCH2O),
3.85–3.75 (12 H, 4 ¥ s, 4 ¥ OMe), 3.54 (3 H, s, OCH2OMe), 0.32 (9
H, s, SnMe3), 0.26 (9 H, s, SnMe3); dC (100 MHz, CDCl3, DEPT-
135) -7.7 (CH3), -5.7 (CH3), 56.2 (CH3), 56.4 (CH3), 56.6 (CH3),
60.9 (CH3), 61.3 (CH3), 72.7 (CH2), 73.2 (CH2), 95.4 (CH2), 109.0
(CH), 117.0 (CH), 125.7 (CH); m/z (ES) 727 (MNa+, 100%); Rf
0.38 (hexane–ethyl acetate, 5 : 1).
(400 MHz, CDCl3) 7.11 (1 H, d, J 8.2 Hz, 9-H), 6.97 (1 H, d, J
8.2 Hz, 8-H), 6.75 (1 H, s, 4-H), 4.43 (1 H, d, J 11.3 Hz, 5-H),
4.38 (1 H, d, J 11.3 Hz, 5-H), 4.07 (1 H, d, J 11.2 Hz, 7-H), 4.06
(1 H, d, J 11.2 Hz, 7-H), 3.94 (9 H, s, 3 ¥ OMe), 3.78 (3 H, s,
OMe), 3.65 (3 H, s, OMe); dC (100 MHz, CDCl3, DEPT-135)
56.3 (CH3), 56.4 (CH3), 60.8 (CH3), 61.2 (CH3), 61.3 (CH3), 67.4
(CH2), 67.6 (CH2), 108.1 (CH), 112.4 (CH), 124.8 (CH); m/z (ES)
410 [M(MeCN)Na+, 100%], 369 (MNa+, 12), 347 (MH+, 25), 317
(32); Rf 0.24 (hexane–ethyl acetate, 4 : 1).
(4,5-Dimethoxy-2-((3,4,5-trimethoxy-2-(trimethylstannyl)ben-
zyloxy)methyl)phenyl)-trimethylstannane 39 was prepared from
alcohol 35 (1.00 g, 3.02 mmol) and bromide 2034 (1.20 g,
2.83 mmol). Chromatography (hexane–ethyl acetate, 6 : 1) gave the
title compound 39 (1.20 g, 63%) as a clear oil (Found: C, 44.7; H,
6.1; Sn, 35.2. C25H40O6Sn2 requires C, 44.55; H, 5.98; Sn, 35.23%);
5,7-Dihydro-1,2,3,11-tetramethoxy-10-methoxymethoxydibenz-
[c,e]oxepine 42 was prepared from the ether 38 (2.40 g, 3.41 mmol).
Chromatography (ether–petroleum, 1 : 1) followed by crystallisa-
tion (ethyl acetate–hexane) gave the title compound 42 (650 mg,
51%) as colourless crystals, m.p. 76–77 ◦C (Found: C, 63.6; H, 6.6.
C20H24O7 requires C, 63.82; H, 6.43%); nmax/cm-1 2944, 2854, 1596,
1487, 1464, 1406, 1332, 1266, 1161, 1115, 1068; dH (300 MHz,
CDCl3) 7.17 (1 H, d, J 8.2 Hz, 9-H), 7.04 (1 H, d, J 8.2 Hz, 8-H),
6.71 (1 H, s, 4-H), 5.28 (1 H, d, J 6.7 Hz, OCHOMe), 5.25 (1 H,
d, J 6.7 Hz, OCHOMe), 4.40 (1 H, d, J 11.3 Hz, 5-H), 4.35 (1 H,
d, J 11.3 Hz, 5-H), 4.05 (1 H, d, J 11.2 Hz, 7-H), 4.01 (1 H, d, J
11.2 Hz, 7-H), 3.91 (3 H, s, ArOMe), 3.91 (3 H, s, ArOMe), 3.73 (3
H, s, ArOMe), 3.63 (3 H, s, ArOMe), 3.54 (3 H, s, OCH2OMe); dC
(100 MHz, CDCl3, DEPT-135) 56.4 (CH3), 56.7 (CH3), 61.0 (CH3),
61.2 (CH3), 61.3 (CH3), 67.4 (CH2), 67.6 (CH2), 95.8 (CH2), 108.2
(CH), 116.9 (CH), 124.9 (CH); m/z (ES) 440 [M(MeCN)Na+,
100%], 399 (MNa+, 54); Rf 0.25 (hexane–ethyl acetate, 4 : 1).
5,7-Dihydro-1,2,3,9,10-pentamethoxydibenz[c,e]oxepine 43 was
prepared from the ether 39 (1.10 g, 1.63 mmol). Chromatography
(hexane–ethyl acetate, 6 : 1) followed by crystallisation (ethyl
acetate–hexane) gave the title compound 43 (186 mg, 33%) as
n
max/cm-1 2932, 2909, 2839, 1584, 1557, 1499, 1460, 1379, 1313,
1250, 1161, 1045, 1014, 909, 773, 730; dH (300 MHz, CDCl3) 6.97
(1 H, s, ArH), 6.87 (1 H, s, Ar), 6.71 (1 H, s, ArH), 4.40 (2 H, s,
CH2), 4.37 (2 H, s, CH2), 3.90 (3 H, s, OMe), 3.85 (6 H, s, 2 ¥ OMe),
3.83 (3 H, s, OMe), 3.80 (3 H, s, OMe), 0.26 (9 H, s, SnMe3), 0.25 (9
H, s, SnMe3); dC (100 MHz, CDCl3, DEPT-135) -7.7 (CH3), -5.7
(CH3), 56.2 (CH3), 56.4 (CH3), 56.4 (CH3), 60.9 (CH3), 61.3 (CH3),
73.1 (CH2), 73.4 (CH2), 109.3 (CH), 112.4 (CH), 119.1 (CH); m/z
(ES) 697 (MNa+, 100%); Rf 0.38 (hexane–ethyl acetate, 6 : 1).
General procedure for dibenz[c,e]oxepines 40–43
To a suspension of CuCl (5 eq.) in anhydrous DMF (18 mL mmol-1
of ether) under argon was added dropwise the ether (1 eq.)
as a solution in anhydrous DMF (30 mL mmol-1) and the
mixture was stirred for 2 h. Saturated NH4Cl solution (pH 8, ca.
20 mL mmol-1) was added and the mixture was stirred in an open
vessel until deep blue. The mixture was extracted with ether (2 ¥
40 mL mmol-1 of ether). The combined extract was washed with
brine (10 mL/mmol of ether), dried, evaporated, and the residue
purified by chromatography.
5,7-Dihydro-1,2,3-trimethoxydibenz[c,e]oxepine 40 was pre-
pared from the ether 36 (1.00 g, 1.35 mmol). Chromatography
(hexane–ethyl acetate, 6 : 1) gave a sample of 40 (320 mg, 83%) as a
clear oil which crystallised on standing and was shown by 1H NMR
spectroscopy to contain a co-eluting by-product. Crystallisation
(ether–hexane) gave the pure title compound 40 (100 mg, 26%),
m.p. 79–80 ◦C (Found: C, 71.4; H, 6.3. C17H18O4 requires C, 71.31;
H, 6.34%); nmax/cm-1 2955, 2932, 2854, 1596, 1487, 1460, 1452,
1402, 1332, 1254, 1231, 1192, 1146, 1118, 1091, 1052, 1005; dH
(300 MHz, CDCl3) 7.64 (1 H, d, J 7.8 Hz, 11-H), 7.40–7.27 (3 H,
m, 8,9,10-H), 6.70 (1 H, s, 4-H), 4.42 (1 H, br m, 5-H), 4.31 (1 H,
br m, 5-H), 4.10 (1 H, br m, 7-H), 3.96 (1 H, br m, 7-H), 3.88 (3
H, s, OMe), 3.86 (3 H, s, OMe), 3.60 (3 H, s, OMe); dC (100 MHz,
CDCl3, DEPT-135) 56.5 (CH3), 61.3 (CH3), 61.5 (CH3), 67.91
(CH2), 67.93 (CH2), 109.1 (CH), 128.1 (CH), 128.4 (CH), 129.9
(CH), 130.0 (CH); m/z (ES) 287 (MH+, 23%), 257 (100), 224 (30);
Rf 0.18 (hexane–ethyl acetate, 6 : 1).
◦
colourless crystals, m.p. 135–136 C (lit.58 124–125 ◦C) (Found:
C, 66.1; H, 6.6. C19H22O6 requires C, 65.88; H, 6.40%); nmax/cm-1
2936, 2854, 1608, 1515, 1491, 1460, 1410, 1375, 1328, 1247, 1122,
1091, 1049, 1014, 990, 854, 734; dH (300 MHz, CDCl3) 7.30 (1
H, s, 11-H), 6.93 (1 H, s, 8-H), 6.78 (1 H, s, 4-H), 4.42 (2 H,
br m, 5-H2), 4.17–4.07 (1 H, br m, 7-H), 4.05–3.95 (1 H, br m,
7-H), 3.99–3.93 (12 H, 4 ¥ s, 4 ¥ OMe), 3.68 (3 H, s, ArOMe);
dC (100 MHz, CDCl3, DEPT-135) 56.3 (CH3), 56.4 (CH3), 61.2
(CH3), 61.6 (CH3), 67.7 (CH2), 68.0 (CH2), 109.2 (CH), 112.5
(CH), 112.9 (CH) (in accord with published data58); m/z (ES) 410
[M(MeCN)Na+, 100%], 371 (45), 347 (MH+, 5), 317 (94), 302 (20);
Rf 0.20 (hexane–ethyl acetate, 4 : 1).
5,7-Dihydro-10-hydroxy-1,2,3,11-tetramethoxydibenz[c,e]oxepine
44
To a solution of the oxepine 42 (100 mg, 0.27 mmol) in dry DCM
(2 mL) was added Montmorillonite clay K10 (100 mg, washed with
dry DCM, dried in vacuo).36 The mixture was stirred at RT for 3 h
and then concentrated in vacuo. Chromatography of the residue
(10 g silica gel, hexane–ethyl acetate, 3 : 2) gave the title compound
◦
44 (55 mg, 62%) as a colourless solid, m.p. 166–167 C (Found:
5,7-Dihydro-1,2,3,10,11-pentamethoxydibenz[c,e]oxepine 41 was
prepared from the ether 37 (1.00 g, 1.08 mmol). Chromatography
(hexane–ethyl acetate, 6 : 1) followed by crystallisation (ethyl
acetate) gave the title compound 41 (130 mg, 35%) as colourless
prisms, m.p. 159–160 ◦C (Found: C, 66.0; H, 6.6. C19H22O6 requires
C, 65.88; H, 6.40%); nmax/cm-1 2994, 2963, 2940, 2858, 1600, 1577,
1487, 1460, 1410, 1324, 1274, 1196, 1157, 1111, 1060, 1010; dH
C, 65.2; H, 6.0. C18H20O6 requires C, 65.05; H, 6.07%); nmax/cm-1
3379, 2941, 2862, 1598, 1489, 1465, 1406, 1328, 1259, 1194, 1150,
1112, 1064; dH (300 MHz, CDCl3) 7.09 (1 H, d, J 8.1 Hz, 9-H),
7.00 (1 H, d, J 8.1 Hz, 8-H), 6.78 (1 H, s, 4-H), 6.00 (1 H, s, OH),
4.43 (1 H, d, J 11.4 Hz, 5-H), 4.39 (1 H, d, J 11.3 Hz, 7-H), 4.09
(1 H, d, J 11.3 Hz, 7-H), 4.02 (1 H, d, J 11.4 Hz, 5-H), 3.95 (3
H, s, OMe), 3.95 (3 H, s, OMe), 3.70 (3 H, s, OMe), 3.43 (3 H, s,
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OMe); dC (75 MHz, CDCl3) 56.4 (CH3), 60.8 (CH3), 61.4 (CH3),
61.5 (CH3), 67.4 (CH2), 67.6 (CH2), 108.5 (CH), 115.1 (CH), 123.1
(CH), 125.6 (C), 128.5 (C), 129.1 (C), 131.3 (C), 142.8 (C), 144.7
(C), 149.4 (C), 151.5 (C), 154.1 (C); m/z (ES) 396 [M(MeCN)Na+,
100%], 303 (25), 286 (23); Rf 0.23 (hexane–ethyl acetate, 7 : 1).
m/z (ES) 301 (MH+–CH2O, 100%); Rf 0.28 (hexane–ethyl acetate,
4 : 1).
3-Bromo-2-formyl-6-methoxyphenyl methanesulfonate 5832
To
a
stirred solution of 6-bromo-2-hydroxy-3-methoxy-
benzaldehyde 57 (0.33 g, 1.43 mmol) and triethylamine (0.17 g,
1.71 mmol) in DCM (5 mL) was added methanesulfonyl chloride
(0.19 g, 2.3 mmol) at 0 ◦C. The reaction was stirred at 0 ◦C
for 10 min and then at RT for 30 min, by which time a brown
colour had developed. Water (20 mL) was added, the mixture
was extracted with ethyl acetate (2 ¥ 20 mL). The combined
extract was washed with brine, dried and evaporated to give a
crude solid (0.42 g). Chromatography (60 g silica gel, hexane–ethyl
acetate, 3 : 1) gave the mesylate 58 (320 mg, 72%) and 5-bromo-
8-methoxybenzo[e][1,2]oxathiine 2,2-dioxide 59 (90 mg, 22%) as
white solids. The title compound 58 had m.p. 95–97 ◦C; nmax/cm-1
1705, 1565, 1468, 1399, 1363, 1293, 1219, 1165, 1130, 971, 878,
800; dH (400 MHz, CDCl3) 10.28 (1 H, s, CHO), 7.55 (1 H,
d, J 8.9 Hz, 4-H), 7.09 (1 H, d, J 8.9 Hz, 5-H), 3.93 (3 H, s,
OMe), 3.38 (3 H, s, SMe); dC (100 MHz, CDCl3) 40.1 (CH3), 56.9
(CH3), 115.2 (C), 118.1 (CH), 128.9 (C), 133.9 (CH), 138.6 (C),
152.6 (C), 189.8 (CH); m/z (ES) 374/372 [M(MeCN)Na+, 100%],
333/331 (MNa+, 80); Rf 0.35 (hexane–ethyl acetate, 1 : 1). 5-
Bromo-8-methoxybenzo[e][1,2]oxathiine 2,2-dioxide 59 had m.p.
186–188 ◦C (Found: C, 37.3; H, 2.2; S, 10.9; Br, 27.2. C9H7BrO4S
requires C, 37.13; H, 2.42; S, 11.01; Br, 27.45%); nmax/cm-1 (nujol
mull) 3091, 2959, 2924, 2854, 1608, 1569, 1468, 1367, 1309, 1270,
1169, 1080, 909; dH (300 MHz, CDCl3) 7.62 (1 H, d, J 10.5 Hz,
4-H), 7.47 (1 H, d, J 10.5 Hz, 6-H), 6.96 (1 H, d, J 10.5 Hz, 7-H),
6.88 (1 H, d, J 10.5 Hz, 3-H), 3.88 (3 H, s, OMe); dC (75 MHz,
CDCl3) 57.0 (CH3), 113.7 (C), 116.2 (CH), 120.3 (C), 123.9 (CH),
129.9 (CH), 135.8 (CH), 142.0 (C), 149.1 (C); Rf 0.60 (hexane–ethyl
acetate, 1 : 1).
Dibenz[c,e]oxepines 16, 51 and 56 via Ullmann cross-coupling
reactions
The sequences leading to 16, 51 and 56 proceeded along conven-
tional lines, as illustrated by the route to 16 (Scheme 5) below. Full
details of Schemes 3 and 4 are provided in the ESI.†
5,7-Dihydro-1,2,3,9-tetramethoxydibenz[c,e]oxepine 51
A solution of 50 (170 mg, 0.51 mmol) in THF (2 mL), 2 M
hydrochloric acid (2 mL) and conc. hydrochloric acid (1 mL)
was stirred under reflux for 3 h. Water (15 mL) and ethyl acetate
(15 mL) were added to the reaction, the layers were separated and
the aqueous layer was extracted with ethyl acetate (2 ¥ 10 mL). The
combined organic extract was dried over Na2SO4 and concentrated
in vacuo. Chromatography of the residue (20 g silica gel, hexane–
ethyl acetate, 4 : 1) gave the title compound 51 (150 mg, 93%)
as a colourless solid, m.p. 151–153 ◦C (Found: C, 68.5; H, 6.5.
C18H20O5 requires C, 68.34; H, 6.37%); nmax/cm-1 2963, 2936, 2858,
2839, 1612, 1491, 1456, 1332, 1243, 1150, 1104, 1052, 1006; dH
(300 MHz, CDCl3) 7.63 (1 H, d, J 8.4 Hz, 11-H), 6.98 (1 H, dd,
J 2.6, 8.4 Hz, 10-H), 6.96 (1 H, d, J 2.6 Hz, 8-H), 6.75 (1 H, s,
4-H), 4.42 (2 H, m), 4.08 (2 H, m), 3.94 (3 H, s, ArOMe), 3.91
(3 H, s, ArOMe), 3.86 (3 H, s, ArOMe), 3.65 (3 H, s, ArOMe);
dC (75 MHz, CDCl3) 55.7 (CH3), 56.4 (CH3), 61.2 (CH3), 61.5
(CH3) [one CH2 signal obscured], 68.1 (CH2), 109.1 (CH), 114.2
(CH), 114.8 (CH), 126.7 (C), 129.7 (C), 131.1 (CH), 131.4 (C),
136.8 (C), 143.1 (C), 150.9 (C), 153.1 (C), 159.4 (C); m/z (ES) 380
[M(MeCN)Na+, 100%], 287 (MH+–CH2O, 100); Rf 0.39 (hexane–
ethyl acetate, 3 : 1).
2,6¢-Diformyl-4,2¢,3¢,4¢-tetramethoxybiphenyl-3-yl
methanesulfonate 60
5,7-Dihydro-1,2,3-trimethoxybenzo[d][1,3]dioxolo[4,5-h]-
[2]benzoxepine 56
To a suspension of copper bronze (0.646 g, 10 mmol) in an-
hydrous DMF (2 mL) was added a solution of 2-bromo-3,4,5-
trimethoxybenzaldehyde 4538 (0.80 g, 2.91 mmol) and 58 (0.30 g,
0.97 mmol) in anhydrous DMF (1 mL) and the suspension was
stirred at 165 ◦C for 3 h. TLC showed the presence of unreacted 58
and more 45 (100 mg) was added. After a further 1 h at 165 ◦C the
reaction, now complete, was cooled and diluted with ethyl acetate
A solution of 55 (170 mg, 0.48 mmol) in THF (2 mL), 2 M
hydrochloric acid (2 mL) and conc. hydrochloric acid (1 mL)
was stirred under reflux for 3 h. Water (15 mL) and ethyl acetate
(15 mL) were added, the layers were separated and the aqueous
layer was extracted with ethyl acetate (2 ¥ 10 mL). The combined
organic extract was dried over Na2SO4 and concentrated in vacuo.
Chromatography of the residue (20 g silica gel, hexane–ethyl
acetate, 4 : 1) followed by crystallisation (ethyl acetate) gave the
title compound 56 (103 mg, 64%) as large clear crystals, m.p. 154–
156 ◦C (Found: C, 65.3; H, 5.5. C18H18O6 requires C, 65.45; H,
5.49%); nmax/cm-1 2967, 2932, 2866, 1600, 1484, 1460, 1414, 1324,
1239, 1146, 1107, 1045; dH (300 MHz, CDCl3) 7.21 (1 H, s, 12-H),
6.98 (1 H, s, 8-H), 6.75 (1 H, s, 4-H), 6.04 (2 H, d, J 4.8 Hz, 10-
H2), 4.40 (2 H, d, J 11.2 Hz, 5-H2), 4.04 (1 H, d, J 10.8 Hz, 7-HA),
4.01 (1 H, d, J 10.8 Hz, 7-HB), 3.96 (3 H, s, OMe), 3.91 (3 H, s,
OMe), 3.71 (3 H, s, OMe); dC (75 MHz, CDCl3) 56.3 (CH3), 61.2
(CH3), 61.5 (CH3), 67.6 (CH2), 67.8 (CH2), 101.6 (CH2), 109.0
(CH), 109.9 (CH), 110.2 (CH), 126.8 (C), 129.5 (C), 131.3 (C),
131.7 (C), 143.0 (C), 147.3 (C), 147.7 (C), 150.8 (C), 153.3 (C);
R
(20 mL). The resulting suspension was filtered through Celiteꢀ
(3 g) and the filtrate concentrated in vacuo. Chromatography (70 g
silica gel, hexane–ethyl acetate, 2 : 1 to 1 : 1) and crystallisation
from ethyl acetate yielded the title compound 60 (180 mg, 44%),
m.p. 131–132 ◦C (Found: C, 53.7; H, 4.8; S, 7.8. C19H20O9S
requires C, 53.77; H, 4.75; S, 7.56%); nmax/cm-1 2934, 2843, 1701,
1682, 1588, 1561, 1480, 1371, 1336, 1285, 1169, 1111, 1072; dH
(400 MHz, CDCl3) 10.15 (1 H, s, 2-CHO), 9.61 (1 H, s, 6¢-CHO),
7.34 (1 H, s, 5¢-H), 7.26 (1 H, d, J 8.5 Hz, 6-H), 7.15 (1 H, d, J
8.5 Hz, 5-H), 4.00 (3 H, s, OMe), 3.958 (3 H, s, OMe), 3.955 (3 H, s,
OMe), 3.55 (3 H, s, OMe), 3.41 (3 H, s, SMe);dC (100 MHz, CDCl3)
40.0 (CH3), 56.2 (CH3), 56.6 (CH3), 60.9 (CH3), 61.2 (CH3), 105.9
(CH), 116.4 (CH), 127.3 (C), 129.7 (C), 130.3 (C), 130.7 (C), 132.1
(CH), 139.5 (C), 147.3 (C), 150.8 (C), 152.3 (C), 153.8 (C), 189.0
228 | Org. Biomol. Chem., 2011, 9, 219–231
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◦
(CH), 190.2 (CH); m/z (ES+) 447 (MNa+, 100%); Rf 0.20 (ethyl
50 C. The solution was diluted with 2 M aqueous hydrochloric
acid (2 mL) at 0 ◦C, extracted with DCM (3 ¥ 5 mL), washed
with saturated aqueous sodium hydrogen carbonate (5 mL), dried
and evaporated. The residual white solid was chromatographed
(30 g silica gel, ethyl acetate–hexane, 1 : 2) to obtain the title
compound 16 (62 mg, 90%), m.p. 145–147 ◦C (Found: C, 64.8;
H, 6.1. C18H20O6 requires C, 65.05; H, 6.07%); nmax/cm-1 3394,
2932, 2858, 1600, 1480, 1344, 1270, 1250, 1150, 1115, 1087, 1056;
dH (400 MHz, CDCl3) 7.21 (1 H, d, J 8.4 Hz, 1-H), 6.94 (1 H, d,
J 8.4 Hz, 2-H), 6.75 (1 H, s, 8-H), 5.91 (1 H, s, 4-OH), 5.15 (1 H,
d, J 11.1 Hz, 5-CH), 4.40 (1 H, d, J 11.0 Hz, 7-CH), 4.05 (1 H, d,
J 11.0 Hz, 7-CH), 3.95 (3 H, s, OMe), 3.94 (3 H, s, OMe), 3.91 (3
H, s, OMe), 3.85 (1 H, d, J 11.1 Hz, 5-CH), 3.66 (3 H, s, OMe);
dC (100 MHz, CDCl3) 56.1 (CH3), 56.2 (CH3), 59.4 (5-CH2), 61.0
(CH3), 61.2 (CH3), 67.9 (7-CH2), 108.9 (CH), 110.1 (CH), 120.9
(CH), 121.2 (C), 126.5 (C), 130.7 (C), 131.3 (C), 142.7 (C), 143.5
(C), 145.7 (C), 150.8 (C), 152.8 (C); m/z (ES) 396 [M(MeCN)Na+,
55%], 355 (MNa+, 4), 315 (MH+–H2O, 100), 303 (MH+–CH2O,
17); Rf 0.16 (acetone–hexane, 1 : 4); Rf 0.41 (ethyl acetate–hexane,
1 : 1).
acetate–hexane, 2 : 1), 0.30 (ether).
2,6¢-Bis(hydroxymethyl)-4,2¢,3¢,4¢-tetramethoxybiphenyl-3-yl
methanesulfonate 61
To a solution of 60 (170 mg, 0.40 mmol) in methanol (4 mL)
was added sodium borohydride (45 mg, 1.20 mmol) and the
solution was stirred at RT for 1 h. Water (20 mL) and ethyl acetate
(20 mL) were added to the reaction, the layers were separated
and the aqueous layer was extracted with ethyl acetate (2 ¥
20 mL). The combined organic extract was dried over Na2SO4
and filtered. The solvent was removed in vacuo and the residue
purified by chromatography (25 g silica gel, hexane–ethyl acetate,
1 : 2), followed by crystallisation (ethyl acetate), which yielded the
title compound 61 (164 mg, 96%) as a white crystals, m.p. 124–
126 ◦C (Found: C, 53.30; H, 5.67; S, 7.26. C19H24O9S requires C,
53.26; H, 5.65; S, 7.48%); nmax/cm-1 3219, 2947, 1607, 1484, 1410,
1363, 1328, 1278, 1161, 1111, 1006, 889; dH (400 MHz, CDCl3)
7.07 (1 H, d, J 8.5 Hz, 6-H), 7.02 (1 H, d, J 8.5 Hz, 5-H), 6.89 (1
H, s, 5¢-H), 4.62 (1 H, dd, J 4.2, 12.0 Hz, CHOH), 4.26–4.15 (3 H,
m, CHOH and CH2OH), 3.94 (3 H, s, OMe), 3.91 (3 H, s, OMe),
3.88 (3 H, s, OMe), 3.55 (3 H, s, OMe), 3.51–3.46 (2 H, m, 2 ¥ OH),
3.40 (3 H, s, SMe); dC (100 MHz, CDCl3) 39.3 (CH3), 56.1 (CH3),
56.2 (CH3), 57.5 (CH2), 61.0 (CH3), 61.1 (CH3), 62.5 (CH2), 108.7
(CH), 112.2 (CH), 125.1 (C), 130.1 (CH), 130.2 (C), 135.0 (C),
136.0 (C), 137.8 (C), 141.5 (C), 150.9 (C), 151.3 (C), 153.6 (C);
m/z (ES) 451 (MNa+, 100%); Rf 0.18 (hexane–ethyl acetate, 1 : 2).
6-Benzyl-6,7-dihydro-1,2,3,9,10,11-hexamethoxy-5H-
dibenz[c,e]azepine ( )-66
To
a
solution of the 6,6¢-bis(bromomethyl)-2,2¢,3,3¢,4,4¢-
hexamethoxybiphenyl 65, prepared from the diol 64 (134 mg,
0.34 mmol) as described,31 in dry DMF (1 mL) at 0 ◦C under argon
was added benzylamine (146 mg, 1.36 mmol) and triethylamine
(103 mg, 1.02 mmol) and the mixture was stirred overnight at
RT. Water (10 mL) was added and the mixture extracted with
ethyl acetate (3 ¥ 10 mL). The combined organic extract was
washed with brine (10 mL), dried and evaporated in vacuo.
Chromatography of the residue (25 g silica gel, hexane–ethyl
acetate, 3 : 2) gave the title compound 66 (155 mg, 98%) as a
5,7-Dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-yl
methanesulfonate 62
A solution of 61 (155 mg, 0.362 mmol) in THF (2 mL), 2 M
hydrochloric acid (2 mL) and conc. hydrochloric acid (1 mL)
was stirred under reflux for 3 h. Water (15 mL) and ethyl acetate
(15 mL) were added to the reaction, the layers were separated and
the aqueous layer was extracted with ethyl acetate (2 ¥ 10 mL).
The extracts were combined, dried over Na2SO4, filtered and
concentrated. Chromatography of the residue (20 g silica gel,
hexane–ethyl acetate, gradient 3 : 1 to 1 : 1) gave the title compound
62 (110 mg, 74%) as a colourless solid, m.p. 158–161 ◦C (MeOH)
(Found: C, 55.61; H, 5.34; S, 7.74. C19H22O8S requires C, 55.60;
H, 5.40; S, 7.81%); nmax/cm-1 2940, 1604, 1573, 1484, 1460, 1367,
1282, 1161, 1118, 1072, 1060, 909, 831; dH (400 MHz, CDCl3) 7.61
(1 H, d, J 8.7 Hz, 1-H), 7.09 (1 H, d, J 8.7 Hz, 2-H), 6.75 (1 H, s,
8-H), 5.01 (1 H, br d, J 11.3 Hz, 5-H), 4.42 (1 H, br d, J 11.3 Hz,
7-H), 4.01 (1 H, br d, J 11.3 Hz, 7-H), 3.96 (3 H, s, OMe), 3.94 (3
H, s, OMe), 3.92 (3 H, s, OMe), 3.90 (1 H, br d, J 11.3 Hz, 5-H),
3.68 (3 H, s, OMe), 3.39 (3 H, s, SMe); dC (100 MHz, CDCl3) 39.5
(CH3), 56.2 (CH3), 56.3 (CH3), 60.4 (5-CH2), 61.1 (CH3), 61.2
(CH3), 67.9 (7-CH2), 108.9 (CH), 112.3 (CH), 125.4 (C), 129.0
(CH), 130.2 (C), 130.8 (C), 131.0 (C), 137.1 (C), 142.8 (C), 150.6
(C), 151.2 (C), 153.3 (C); m/z (ES) 433 (MNa+, 100%), 411 (MH+,
58); Rf 0.31 (hexane–ethyl acetate, 1 : 1).
◦
colourless crystalline solid, m.p. 117–118 C, which darkened in
air (Found: M + H+, 466.2216; C27H32O6N requires 466.2225);
n
max/cm-1 2940, 2835, 1600, 1577, 1495, 1456, 1406, 1317, 1231,
1130, 1099, 734, 699; dH (400 MHz, CDCl3) 7.43 (2 H, d, J 7.1 Hz,
2¢,6¢-H), 7.38–7.27 (3 H, m, 3¢,4¢,5¢-H), 6.62 (2 H, s, 4,8-H), 3.91
(6 H, s, 2 ¥ OMe), 3.90 (6 H, s, 2 ¥ OMe), 3.73 (6 H, s, 2 ¥ OMe),
3.75–3.65 (2 H, br m, 12-H2), 3.45 (2 H, d, J 12.0 Hz, 5,7-HA), 3.08
(2 H, d, J 12.0 Hz, 5,7-HB); dC (100 MHz, CDCl3) 55.4 (CH2),
56.4 (CH3), 60.0 (CH2), 61.1 (CH3), 61.4 (CH3), 108.5 (CH), 123.2
(C), 127.7 (CH), 128.9 (CH), 129.7 (CH), 130.6 (C), 142.0 (C),
151.8 (C), 153.1 (C) (1 C coincident); m/z (ES) 466 (MH+, 100%);
Rf 0.22 (hexane–ethyl acetate, 3 : 2).
Biological evaluation
Inhibition of tubulin assembly.45 The assembly of microtubules
from porcine tubulin was monitored spectrophotometrically by
measuring the associated increase in solution turbidity. For each
of the test compounds, six samples were prepared directly in quartz
◦
cuvettes at 0 C and contained 2-(N-morpholino)ethanesulfonic
acid (MES) buffer [740 mL (0.1 M MES, 1 mM EGTA, 0.5 mM
MgCl2, distilled water, pH 6.6)], GTP (100 mL, 10 mM in MES
buffer), tubulin45b (1 mg, 100 mL at 10 mg mL-1 in MES buffer)
and the test compound (10 mL, decreasing concentration starting
at 10 mM in DMSO). The cells were immediately placed in a Varian
5,7-Dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol 16
To a solution of 62 (85 mg, 0.231 mmol) in 1,4-dioxane (0.25 mL)
and methanol (0.25 mL) was added aqueous 3 M potassium
hydroxide (0.5 mL) and the mixture was stirred overnight at
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Cary 300 Bio UV/visible spectrophotometer, preheated at 37 ◦C,
alongside six blank samples each containing MES buffer (1 mL)
and the test compound. The absorbance at l 350 nm was then
recorded during a period of 30 min. The results were compared to
the untreated control cells to evaluate the relative degree of change
in optical density. From the data, an IC50 value (50% inhibition of
tubulin assembly) was determined graphically.
for valuable discussions, Edward Piers (University of British
Columbia) for his kind provision of experimental details, Darren
Cook (University of Salford) for help with compound testing,
and Val Boote and Steve Kelly (University of Manchester) for
assistance with MS and NMR measurements. We also wish to
acknowledge the use of the EPSRC’s Chemical Database Service
at Daresbury.59
K562 growth inhibition by MTT assay.46 K562 cells were
cultured in RPMI medium, free of antibiotics and containing 2-
mercaptoethanol (2 mM) and L-glutamine (2 mM), supplemented
with 10% v/v foetal calf serum (FCS). The cells were adjusted to a
concentration of 4000 cells mL-1 in RPMI medium supplemented
with 10% v/v FCS, and the test compound was made up as
a 10 mM stock solution in DMSO. Starting with eight sterile
centrifuge tubes, a portion of cell solution (2 mL) was added
to the first tube followed by 4 mL of the test compound stock
solution. A portion (1 mL) of the resulting solution was added to
1 mL of cell solution in the adjacent tube, giving a test compound
concentration of one-half of that of the first tube. This series
of dilutions was continued to afford seven samples at different
concentrations. As a control, the eighth centrifuge tube contained
1 mL of cell solution only. Aliquots of 100 mL of the treated cells
were then pipetted in triplicate into a 96-well microtitre testplate
and incubated (37 ◦C, 5% CO2 in air) for 5 days. After this
time the plate was removed from the incubator and 50 mL of a
solution of MTT (3 mg mL-1 in phosphate-buffered saline) was
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minimisation); n = 10, 79.1◦ (79.1◦ after minimisation); n = 11,
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Acknowledgements
We thank the Association for International Cancer Research
(AICR) for financial support of this work (grant ref. 00-090).
We are also grateful to Alan McGown (University of Salford)
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