Tubulin-binding dibenz[c,e]oxepines: Part 2.1Structural variation and biological evaluation as tumour vasculature disrupting agents

Accepted Manuscript

Tubulin-binding dibenz[c,e]oxepines: Part 2.¹Structural variation and biologi-

cal evaluation as tumour vasculature disrupting agents

Steven B. Rossington, John A. Hadfield, Steven D. Shnyder, Timothy W.

Wallace, Kaye J. Williams

PII:

S0968-0896(16)31314-1

DOI:

http://dx.doi.org/10.1016/j.bmc.2017.01.027

BMC 13506

Reference:

Bioorganic & Medicinal Chemistry

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Revised Date:

Accepted Date:

29 November 2016

11 January 2017

17 January 2017

Please cite this article as: Rossington, S.B., Hadfield, J.A., Shnyder, S.D., Wallace, T.W., Williams, K.J., Tubulin-

binding dibenz[c,e]oxepines: Part 2.¹Structural variation and biological evaluation as tumour vasculature disrupting

agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.027

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dibenz[c,e]oxepines: Part 2.

Structural variation and biological

evaluation as tumour vasculature

disrupting agents

Steven B. Rossington, John A. Hadfield, Steven D. Shnyder, Timothy W. Wallace* and Kaye J.

Williams

School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK

School of Environment and Life Sciences, University of Salford, Salford M5 4WT, UK

Institute of Cancer Therapeutics, University of Bradford, Richmond Road, Bradford BD7 1DP, UK

Manchester Pharmacy School, University of Manchester, Oxford Road, Manchester M13 9PT, UK

OMe

MeO

OMe

Br

O

Induces vascular shutdown,

necrosis and growth delay in

tumour xenografts

cat. Pd(OAc)₂aq. HCl

MeO

OMe

O

ligand, base

DMA, heat

MeOH

OMe

OMOM

OMe

OH

OMe

Bioorganic & Medicinal Chemistry

Tubulin-binding dibenz[c,e]oxepines: Part 2.¹Structural variation and biological

evaluation as tumour vasculature disrupting agents

Steven B. Rossington,^aJohn A. Hadfield,^bSteven D. Shnyder,^cTimothy W. Wallace^a, and Kaye J.

Williams^d

^aSchool of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK

^bSchool of Environment and Life Sciences, University of Salford, Salford M5 4WT, UK

^cInstitute of Cancer Therapeutics, University of Bradford, Richmond Road, Bradford BD7 1DP, UK

^dManchester Pharmacy School, University of Manchester, Oxford Road, Manchester M13 9PT, UK

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether

precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in

vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown,

necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological

properties of 1 and related compounds can be attributed to their ability to inhibit

microtubule assembly at the micromolar level, by binding reversibly to the same site of the

tubulin -heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Available online

Keywords:

dibenz[c,e]oxepine

intramolecular direct arylation

tubulin targeting

tumour growth inhibition

vascular shutdown

colchicinoid

———

 Corresponding author. Tel.: +44-161-306-4521; fax: +44-161-275-4939; e-mail: tim.wallace@manchester.ac.uk

1. Introduction

by virtue of its ability to match, in both degree and sense, the

conformational helicity of colchicine 2, which is crucial to the

latter's ability to bind to tubulin,⁷and by analogy with N-

acetylcolchinol methyl ether (NCME) 3, whose binding to

tubulin is strong but rapidly reversible, i.e. compatible with drug-

As the conduit for oxygen, nutrients and waste products, the

vascular system that supports a tumour is a rational target for

anticancer therapy. Two strategies have emerged for the clinical

exploitation of this principle,²the first being the use of

angiogenesis inhibitors, which target growth factors so as to

prevent the formation of new vasculature.³The pioneering

example of this approach is the monoclonal antibody

bevacizumab (Avastin).⁴The second strategy is to employ

vascular disrupting agents (VDAs) to attack newly-formed

tumour vasculature, which is structurally flawed by excessive

branching, uneven diameter, shunts, etc., and more sensitive than

normal host vasculature to small molecules that perturb the

morphology and functionality (migration, adhesion, proliferation)

of the nascent endothelial cells.⁵Microtubules play a prominent

role in maintaining the physical structure of these cells, and most

VDAs are tubulin-binding agents that undermine tubulin-

microtubule dynamics at sub-micromolar concentrations.

like pharmacokinetics.⁸

Indeed the parent phenol N-

acetylcolchinol (NAC) 4 was developed as a VDA in the form of

the phosphate prodrug ZD6126 5, although the project was

curtailed following the observation of adverse cardiac events in

phase I clinical trials.^9,10Various alternative structures have

progressed to clinical trials as VDAs,¹¹the best known being

combretastatin A-4 (CA-4) 6 which, as the phosphate prodrug 7

(CA-4P; fosbretabulin; Zybrestat), has featured in human trials as

a single agent¹²and in combinations with cytotoxic agents

(paclitaxel, carboplatin), radiotherapy, or the antiangiogenic

agent, bevacizumab.¹³Other stilbenes in development include

combretastatin A-1 (CA-1) 8, in the form of the prodrug 9

(OXi4503),¹⁴and AVE8062 10 (ombrabulin).¹⁵The benzofuran

11 (BNC105),¹⁶the diketopiperazine 12 (plinabulin),¹⁷the

pyrimidine 13¹⁸and the chromene 14 (crolibulin)¹⁹have also

progressed to clinical trials. Despite their structural diversity, all

of these candidate VDAs bind to tubulin at, or close to, the same

site as colchicine 2, which is located at the interface of the two

subunits of the ,-tubulin heterodimer.²⁰

In seeking new structures for screening as VDAs, we

identified the dibenz[c,e]oxepinol 1 as a potent inhibitor of

microtubule assembly and a possible lead in this context.¹

A

crystallographic analysis of the dibenz[c,e]oxepine nucleus⁶led

us to conclude that it was well equipped to serve in this capacity

OMe

MeO

A

7

5

B

R

O

S

1

C

B

NHAc

OH

MeO

OR

OMe

OR

8 R = H CA-1

MeO

O

1

2

3 R = Me NCME

4 R = H NAC

5 R = PO₃H₂

6 R = H CA-4

7 R = PO₃Na₂

9 R = PO₃Na₂

OMe

H

N

OMe

MeO

OMe

MeO

Br

N

MeO

O

HN

N

NH

O

CN

NH₃⁺Cl^–

MeO

HN

O

N

NH

HN

OH

Me₂N

OMe

O

NH₂

MeO

OH

N

Bu^t

O

H

10 AVE8062

11 BNC105

12 NPI-2358

13 CYT997

14 EPC2407

OMe

While the colchicine binding site of tubulin can accommodate

a range of structures, it is not an ideal target for therapeutics.

Agents that inhibit microtubule assembly by binding to this site

are associated with dose-limiting cardiac events (ischemia,

infarction, ventricular tachycardia),²¹and this has been attributed

to cell-cycle arrest in the endothelial cells of the myocardium.²²

Zybrestat 7 shows neurotoxic and cardiac effects (prolonged QT

interval), and clinical trial protocols include measures to

counteract hypertension and cardiac ischemia.^13a,23Prolonged

QT interval is also observed with OXi4503 9¹⁴and CYT997

13.^18aCardiotoxicity is thus a generic issue with tubulin-

targeting VDAs, highlighting the need for finely-balanced

therapeutics that are not compromised by this problem.²⁴

MeO

A

7

5

MeO

O

B

O

1

C

OPO₃Na₂

O

OMe

15

16

OMe

17

OMe

MeO

O

NHAc

OSO₂CF₃

OMe

X

OPO₃Na₂

18

19 X = NO₂

20 X = NH₂

21

The dibenz[c,e]oxepine pharmacophore is easily accessible^1,25

and potentially tunable with respect to binding, metabolic and

transport characteristics.²⁶In this paper we describe new routes

to the lead structure 1 and its biological evaluation as a VDA,

along with the analogues 15–20. The results show that in human

tumour xenografts in mice, the prodrugs 15 (derived from 1) and

21 (from the clinically tested VDA 5) are similar in their ability

to induce necrosis, and that 1 inhibits tumour growth in vivo.

the desired heterocycle 34 in moderate yield, confirming that this

is a viable approach to dibenz[c,e]oxepines. Subsequent removal

of the MOM group from 34 using aq. HCl completed the new

route to the target 1.

Br

MeOCH₂Br

Prⁱ₂NEt

CHO

OH

NaBH₄

OH

MeOH

89%

DMF

92%

OMOM

OMe

30

29

28

NaH, THF

ArCH₂Br 31

86%

2. Results and discussion

2.1. Synthesis of materials

Ar = 3,4,5-trimethoxyphenyl

OMe

NMe₂

PCy₂

Ar

MeO

Our original route to the key structure 1, based on a

conventional Ullmann biaryl synthesis,¹was improved by

switching to iodoarene cross-coupling partners (Scheme 1). The

C-ring precursor 23 was conveniently prepared by direct

iodination of o-vanillin mesylate 22 using iodine and periodic

acid,²⁷and the same method provided the A-ring precursor 24

Br

O

aq. HCl

33

O

1

MeOH

80%

0.1 eq. Pd(OAc)₂

0.1 eq. 33

2.0 eq. K₂CO₃

DMA

OMOM

OMe

145 ˚C, 24 h

56%

OMe

from 3,4,5-trimethoxybenzaldehyde.

The outcome of the

32

34

Ullmann coupling of 23 and 24 was strongly dependent on the

reaction conditions, but guided by the painstaking analysis of this

type of reaction by Brown and coworkers,²⁸we obtained

acceptable yields of 25 using a solvent-free 3:1 mixture of the

two components, a 5-fold excess of Cu powder, and careful

control of the reaction time and temperature. Ring-closure of the

diol 26 to 27 was induced with aq. HBr, and the methanesulfonyl

protecting group was cleanly removed from 27, to give 1, using a

modified version of Carreira's method.²⁹

Scheme 2. Intramolecular direct arylation route to 1.

In seeking a water-soluble version of the dibenzoxepinol 1, we

used a conventional reaction sequence³³to prepare the disodium

phosphate 15 via the intermediates 35 and 36. Mixing the

dihydrogen phosphate 36 with two molar equivalents of freshly

prepared sodium methoxide in methanol, followed by

evaporation and drying in vacuo, provided the disodium salt 15

1

I

OMe

as white powder. Although essentially homogeneous by H, ¹³C

CHO

OMs

CHO

OMs

MeO

and ³¹P NMR spectroscopy, this material proved to be

hygroscopic, and microanalytical samples always retained small

amounts of water and methanol.

I₂

HIO₄2H₂O

+

HOAc

H₂O, H₂SO₄

87%

CHO

OMe

22

OMe

I

OMe

23

24

MeO

Cu

185 ˚C

47–58%

CHO

O

0.3 h

O

OMe

O

P

OR

MeO

OMe

OR

NO₂

NaHMDS

THF

aq. HBr

MeCN

35 R = CH₂Ph

36 R = H

37

MeO

CHO

O

1

75%

98%

The dibenzoxepinol 1 was also converted into the derived

triflate 18 by conventional means. The amine 20 was acquired in

three steps from the biaryl dialdehyde 37, which proved

accessible via an Ullmann cross-coupling reaction. Subsequent

reduction and cyclisation gave the nitro compound 19, which was

transformed into the amine 20 by catalytic hydrogenation.

OMs

OMe

25 X = CHO

27

NaBH₄

83%

26 X = CH₂OH

Scheme 1. Improved Ullmann cross-coupling route to 1.

We also developed a potentially more versatile approach to 1

featuring a Pd-catalysed intramolecular direct arylation (IDA)

reaction as the cyclisation step. This strategy called for the

construction of a dibenzyl ether bearing the latent A- and C-ring

substituents of the target compound, together with a single

halogen atom to mediate the metalation step. To prepare such a

precursor for 1, the MOM-protected aldehyde 29 was reduced to

the alcohol 30, which was then alkylated using 3,4,5-

trimethoxybenzyl bromide 31 (Scheme 2). The resulting ether 32

was subjected to the IDA cyclisation conditions first described by

Fagnou and coworkers^30,31and adapted by us for the preparation

of dibenz[c,e]azepines.³²Chromatography of the product gave

2.2. Biological evaluation

2.2.1. Inhibition of microtubule assembly and in

vitro antiproliferative activity

Dibenzoxepines were routinely screened for their ability to

inhibit microtubule assembly^34,35and for growth inhibitory

activity (IC₅₀) against the K562 human chronic myelogenous

leukaemia cell line.³⁶Both of these assays are routinely used for

evaluating test compounds and provide a useful comparison with

SR

0.04

0.19

0.30

0.04

0.28

0.17

0.39

benchmarks such as colchicine 2 and CA-4 6. The results (Table

1) show that the ability of the phenolic compound 1 to inhibit

microtubule assembly is matched by the phosphate 15 (entry 6),

with the latter also showing sub-nanomolar IC₅₀values in the

K562 assay. The activity shown by the triflate 18 (entry 9) is

also noteworthy, and is consistent with prodrug behaviour.

Non-small

cell lung

Colon

NCI-H522

HCT-116

HCT-15

KM12

SW-620

SF-268

SF-295

SF-539

SNB-19

SNB-75

0.32

0.08

0.05

0.09

0.27

0.05

0.14

0.07

0.30

0.08

0.05

ND

0.03

0.26

1.58

0.04

1.00

0.05

0.21

0.07

0.04

0.06

0.26

0.04

0.07

0.22

0.43

0.44

0.30

0.40

0.79

0.32

0.33

0.51

0.54

0.48

0.66

0.27

2.51

0.04

5.37

0.89

0.26

5.50

0.28

0.40

0.21

0.39

2.24

0.24

0.40

1.15

0.05

0.08

0.05

0.46

0.06

0.16

0.98

0.09

0.10

0.21

0.05

>100

0.03

0.10

0.08

0.03

0.08

0.07

0.05

0.13

0.08

0.18

0.20

0.24

0.04

0.03

0.37

0.48

0.36

0.40

1.32

0.33

0.35

0.68

0.46

0.48

0.87

0.23

>100

0.05

0.56

ND

0.21

0.79

0.36

0.25

0.47

0.20

0.37

0.54

0.43

0.40

0.17

CNS

Table 1. Activities (IC₅₀) of compounds in the microtubule

assembly and K562 in vitro cytotoxicity assays.

U251

LOXIMVI

M14

Tubulin assembly

K562 assay

IC₅₀nM^c,d

34

Melanoma

Entry

1^e

2

Compound

IC₅₀M^a,b

MALME-3M

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-62

NCI/ADR-RES

OVCAR-3

SK-OV-3

RXF393

1

2

2.2

2.0

0.7^f

19

g

3

–

4

6

1.3

1.0

0.72

0.23

110

85

5

7

0.9

Ovarian

6

15

16

17

18

19

20

1.4

7

7.4

Renal

Prostate

Breast

DU-145

BT-549

HS578T

MCF7

8

>10

15

9

0.25

16

10

11

>10

MDA-MB-468

10

^aConcentration required for 50% inhibition of tubulin assembly.

^aGI₅₀: concentration required for 50% cell growth inhibition.

^bOnly the cell-lines featuring one GI₅₀value below 100 nM are

^bEntries in this column are corrected for variations in the value for 6, which

was used as a reference for assay batches.

included in this table; see Supplementary Material for the full data

^cConcentration that inhibits the growth of 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%.

set.

^cShading is applied to values below 100 nM; ND = not determined.

^dEntries in this column are normalised to the value for 6, which varied over

the range 1.2–3.0 between batches.

^eValues from ref. 1.

^fValue from ref. 37.

^gNot determined.

The results with the human colorectal tumour cell line HCT-

15, which is known to express multidrug resistance protein (P-

glycoprotein),⁴⁰implies that the inhibitory properties of 1 and 16

are not unduly affected by this efflux pump, which can

undermine the effects of tubulin-targeting anticancer agents.⁴¹

The NCI-60 mean GI₅₀values for 1 and 16 are similar (ca. 0.3

µM), which compares to 0.04 µM for NCME 3.

2.2.2. In vitro screening against NCI-60 cell lines

The dibenzoxepines 1 and 15–17 were evaluated in the US

National Cancer Institute (NCI) anticancer drug screen against

the NCI-60 panel of human tumour cell lines.^38,39The sixty cell

lines of this panel are organised by disease type, and test data

relating to both the levels and the patterns of activity across the

panel can be instructive. Some of the results of the NCI-60 five-

dose assay of the dibenzoxepines are shown in Table 2.

Compounds 1 and 16 reach similar levels in their inhibitory

effects, each with more than twenty instances in which the GI₅₀

value is below 100 nM, whereas 15 and 17 register this level only

with the MDA-MB-435 (melanoma) cell line.

The NCI-60 screening results from the dibenzoxepines 1 and

15–17, together with the public data for the colchicinoids 2–4

and the stilbenes 6 and 7, were analysed using the matrix

COMPARE algorithm.⁴²In this type of analysis, the activity

profile of a 'seed' compound against the NCI-60 cell lines can be

compared to that of any 'target' compound in the same database,

with the COMPARE algorithm being used to generate a series of

correlation coefficients (Table 3). Compounds that exert their

inhibitory effects by similar mechanisms of action can produce

similar patterns of differential antiproliferative data, and the

coefficients within the dibenzoxepine matrix (Table 3a) provide a

reasonable case for the mechanistic correlation of this group of

compounds, the borderline case being 16. However, the extent to

which this correlation is based on interaction with tubulin

remains unclear, as is illustrated by the second section of results

obtained with 2–4, 6 and 7 (Table 3b). Matrix COMPARE

coefficients (r values) less than 0.6 are of questionable

significance,^43,44and there is considerable variation in the

coefficient values across the whole matrix, ranging from notable

homology (2 with 3; 3 with 6; 6 with 7) to almost complete

Table 2. In vitro cell growth inhibition data for various

dibenzoxepines against the NCI-60 panel of human cancer

cell lines.

Cell growth inhibition (GI₅₀, µM)^a,b,c

Panel

Cell line

1

15

16

17

Leukaemia

CCRF-CEM

HL-60(TB)

K-562

0.04

0.03

0.04

0.06

0.29

0.32

0.28

0.40

0.08

0.04

0.09

0.35

0.39

0.21

0.47

MOLT-4

disparity with N-acetylcolchinol 4, which in turn shows a modest

correlation with 16. Taken together, these results provide a

reminder that the relationship between cytotoxicity (or

cytostaticity) and the ability to shutdown vasculature remains

obscure.

NCI standard agents collection of anticancer agents.⁴⁵This

analysis, which can be used to identify the cellular targets of

antitumour agents,^44,46was extended to include compounds 2–4,

6 and 7 as the seeds (Table 3c). The observed correlation of 15

with maytansine and vincristine, which are known to target

microtubule formation,⁴⁷provides further evidence that, as

intended, the biological target of the dibenzoxepines is tubulin.

Using the standard COMPARE protocol, the antiproliferative

activity profiles of 1 and 15–17 were compared with those of the

Table 3. Results of COMPARE analyses involving the dibenzoxepines 1 and 15–17. (a) Matrix COMPARE data for the

dibenzoxepines. (b) Standard COMPARE data for colchicinoids 2–4 and stilbenes 6 and 7. (c) Correlations from a standard

COMPARE analysis of dibenzoxepines with the NCI standard agent database.^a,b

Target

vector

1

15

16

Seed vector

1

15

0.81

16

0.84

0.66

17

0.79

0.73

0.86

2

3

4

6

7

0.81

0.84

0.79

a

0.66

0.73

0.50

0.48

0.33

0.43

0.53

0.56

0.62

0.45

0.50

17

0.87

0.53

0.42

0.62

0.31

0.40

0.54

0.53

0.49

0.45

0.47

2 colchicine 0.50

3 NCME

4 NAC

6 CA-4

7 CA-4P

maytansine

vincristine

paclitaxel

rhizoxin

0.52

0.45

0.31

0.38

0.56

0.60

0.52

0.48

0.74

0.22

0.02

0.51

0.71

0.01

0.49

0.58

0.12

0.77

0.43

0.44

0.40

0.58

0.51

0.57

0.51

0.43

0.74

0.22

0.51

0.49

0.76

0.78

0.54

0.74

0.75

b

0.02

0.71

0.58

0.87

0.81

0.36

0.83

0.89

0.01

0.12

0.47

0.41

0.49

0.38

0.29

0.77

0.67

0.58

–

0.64

0.45

0.50

–

0.59

0.44

c

vinblastine

^aMatrix (r) values are Pearson's correlation coefficients,³⁸based on a comparison of the NCI GI₅₀mean graphs for each compound; see Supplementary Material

for full search parameters and results.

^bShading is arbitrarily applied to r values ≥0.55. NCI descriptors: 2, NSC 757; 3, NSC 51046; 4, NSC 51045; 6, 613729; 7, NSC 645646; maytansine, NSC

153858; vincristine, NSC 67574; paclitaxel, NSC 125973; rhizoxin, NSC 332598; vinblastine, NSC 49842.

2.2.3. In vivo antivascular effects of 15

after the administration of the compound. This state persisted

throughout the 24-h study period for 21, whereas for 15 some

recovery of vasculature was evident from 4 h post dose.

Given its close structural analogy and progress to clinical

trials, we selected the erstwhile drug candidate 5,^9,48in the

form of its disodium salt 21, as a benchmark for assessing the

ability of 15 to function as a VDA at levels beneath the

maximum tolerated dose (MTD). In a side-by-side single-dose

study, seven groups of athymic nude mice (n=3 per group)

bearing subcutaneous DLD-1 human colon adenocarcinoma

xenografts were used to assess the effects of treatment with the

test compounds 15 and 21 on the functional vasculature in

tumours. Once an established tumour vascular network was in

place, mice were treated with a single intravenous dose (400

mg/kg) of 15 or 21 (three groups per compound). The results

(Figure 1) provided a clear demonstration of the vascular

shutdown induced by both compounds, peaking ca. one hour

The extent of tumour necrosis (Figure 2) was only seen to

be different to the control at 24 hours post dose for both

compounds, which is consistent with the time delay expected

for necrosis to occur following loss of blood supply to the

tumour cells. The ability of the dibenzoxepine 15 to target

experimental tumours is clearly evident, and parallels that of

the related allocolchinol 21.

Representative images from the functional vasculature and

necrosis studies are provided in Figures 3 and 4 respectively.

200

OMe

MeO

OMe

MeO

150

100

50

MeO

O

NHAc

OPO₃Na₂

21

OMe

15

0

control

1 h

4 h

24 h

1 h

4 h

24 h

Figure 1. Chart showing the time-dependent vascular shutdown of DLD-1 human colon adenocarcinoma xenografts in mice following a single intravenous

dose (400 mg/kg) of 15 or 21 (mean ± SEM).

600

OMe

MeO

500

400

300

200

100

0

O

NHAc

OPO₃Na₂

OMe

21

15

control

1 h

4 h

24 h

1 h

4 h

24 h

Figure 2. Chart showing the quantification of tumour necrosis in DLD-1 human colon adenocarcinoma xenografts in mice following a single intravenous

dose (400 mg/kg) of 15 or 21 (mean ± SD).

Figure 3. Representative images of DLD-1 human colon adenocarcinoma xenografts showing remaining functional vasculature using Hoechst 33342

staining, following a single intravenous dose of 15 or 21 at 400 mg kg^–1at 1, 4 and 24 h post dose.

Figure 4. Representative images of haematoxylin- and eosin-stained DLD-1 human colon adenocarcinoma xenografts following a single intravenous dose of

15 or 21 at 400 mg kg^–1at 1, 4 and 24 h post dose.

2.2.4. In vivo antitumour effects of 1

5a). The antitumour properties of 1 were then monitored in a

10-day study of mice with implanted Calu-6 xenografts. Doses

of one-quarter or one-half of the nominal MTD on days 1 and

5 had approximately the same inhibiting effect on tumour

growth for 24 h, with regrowth then resuming at the original

rate (Figure 5b). The dibenzoxepine 1 was thus demonstrated

The potential of the benz[c,e]oxepine series as a source of

in vivo antitumour agents was investigated using benzoxepinol

1 in a two-dose study with mice bearing subcutaneous Calu-6

lung tumour xenografts. Calu-6 is well vascularised and has

been used previously to study VDAs.⁴⁹Guidelines for the

MTDs of various dibenzoxepines in mice were obtained by

monitoring the effects of a single exposure on body weight

over two weeks, and a limit of 270 mg/kg was set for 1 (Figure

to be carcinostatic, inducing

a growth delay in the

experimental tumours at one-quarter of the nominal MTD, and

was well tolerated in tumour-bearing mice.

Figure 5. The dibenzoxepine 1 is well tolerated in tumour-bearing mice and induces a highly significant growth delay in subcutaneous Calu-6 lung tumour

xenografts. (a) shows average mouse weight, (b) shows average tumour volume (n=5 control, n=6 treated mice per group). Arrows indicate administration of 1

at the noted dosages. The doses 67.5 mg/kg and 135 mg/kg equate to 0.25 and 0.5 MTD respectively. Compound 1 was dosed in 5% DMSO in peanut oil.

2.2.5. Mechanistic considerations

hydroxy group can function as an H-bond donor in the domain

normally occupied by the amide NH of the colchicinoid.

The tubulin-binding capability of the dibenzoxepine 1 is

consistent with its structural analogy with colchicine. The 1SA0

crystal structure provides a detailed picture of the interaction of

N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-colchicine)

38 with the interchain boundary of the -tubulin heterodimer,²⁰

which accounts for the disruptive effect of colchicinoids on the

finely-poised dynamics of this interface. We speculate that,

while chemically distinct from colchicine

2

and the

combretastatins such as 6, the dibenzoxepine 1 binds to the -

tubulin heterodimer at the same location and in a similar

manner.¹Our mechanistic model for this interaction, shown in

Figure 6a, assumes the favourable locations of H-bond donor and

acceptor sites a and b that can anchor the C-ring of the -tubulin-

bound ligand to the nearby -tubulin. It is generally assumed

that β-Cys241 provides colchicinoids with the third anchor c,

although the case for this being solely through H-bonding is less

compelling, as the trimethoxy motif is not indispensible.⁵⁰

(a)

(b)

(c)

Figure 7. Calculated and crystallographic models overlaid on atoms

C(11A), C(8) and C(10) of 1. (a) The dynamic 3D-structure of 1 in aqueous

solution. Carbon atoms are shown in grey and oxygen in red. (b) The bound

conformation of 38 from 1SA0 (residue 701, carbon atoms in grey) and the

closest matching preferred solution conformer of 1 (carbon atoms in green)

aligned in the same pose. (c) The crystal structure of 3 (carbon atoms in

grey) with the closest matching solution conformer of 1 (carbon atoms in

green) and model of 6 (carbon atoms in pink) aligned in the same pose. N

atoms are shown in blue and O in red; H are omitted for clarity.

-CYS241

(a)

SH

OMe

c

The mechanistic model represented in Figure 6 should extend

to the amine 39, which also bears a C(4)-substituent suited to the

role of the H-bond donor in anchor a, capable of binding to the

residue Thr179 located on the -tubulin chain (Figure 6b). On

the same basis, and by analogy with ombrabulin 10, we speculate

that a range of amides 40 derived from 39 may be potent tubulin

binding agents, and hence potential VDAs.

MeO

38

MeO

O

OH

SH

N

H

-tubulin

NH

a

MeO

O

HN

-tubulin

b

O

-SER178

H

NH

-THR179

N

HO

3. Conclusions

O

-ALA180

N

In seeking new vascular disrupting agents, we evaluated the

accessibility and biological properties of a series of substituted

dibenz[c,e]oxepines, identifying candidate compounds by their

ability to inhibit the growth of experimental tumour cell lines in

vitro. The most potent of the compounds studied was the

benzoxepinol 1, which in the NCI-60 anticancer drug screen

manifested broad-spectrum antiproliferative activity whose

profile (COMPARE analysis) indicated tubulin as the biological

target. The results of in vivo studies confirm that the disodium

phosphate prodrug 15, derived from 1, induces vascular

shutdown and necrosis in DLD-1 human colon adenocarcinoma

xenografts to an extent approaching that displayed by N-

acetylcolchinol 4, a structurally related allocolchicine derivative

and recent drug candidate. The phenol 1 has also been shown to

inhibit the growth of Calu-6 lung tumour xenografts in mice at a

dose of one-quarter of the nominal MTD.

O

-VAL181

(b)

OMe

c

9

MeO

8

10

7

MeO

11A

1 X = O

39 X = NH

40 X = NCO.R

O

5

4

XH

3

OMe

a

b

Figure 6. Depictions of (a) the supposed binding interactions within (or

leading to) the DAMA-colchicine:-tubulin:RB3 (1SA0) crystal structure

(ref. 24a) and (b) the proposed analogy of 1 and 39.

On the basis of the tubulin binding model shown in Figure 6,

the large body of SAR data available for colchicinoids, and the

characteristics of the compounds that we have prepared to date,

we propose that the dibenz[c,e]oxepine pharmacophore offers a

series of colchicinoid analogues with tunable tubulin binding

characteristics. Exploitation of the IDA strategy (Scheme 2)

should allow rapid access to new VDAs with design features that

would prove difficult to achieve by the modification of natural

colchicinoids. Amides of the form 40 are proposed as a focus for

further development in this context.

Conformational analysis of 1 using the methodology of

Blundell et al.⁵¹gave a detailed view of its dynamic 3D-structure

in dilute aqueous solution (Figure 7a). Overlaying the resulting

conformers of 1, in which the biaryl axis is free to assume the

tubulin-binding (aR) configuration, and the bound colchicinoid

38 in 1SA0 (Figure 7b) indicates that the C(3) methoxy group of

1 coincides with the carbonyl oxygen of the (distorted) tropolone

ring. In an alternative comparison (Figure 7c), conformers of 1

and a model of combretastatin A-4 6 were overlaid with the X-

ray structure of colchicine 3.⁵²This provides further support for

the idea of a distinctive binding motif of 1, in which the C(4)

3.36 (3 H, s, SO₂Me), 3.95 (3 H, s, OMe), 7.26 (1 H, dd, J 8.2,

1.6 Hz, 5-H), 7.38 (1 H, apparent dt, J ca. 8, 0.7 Hz, 4-H), 7.53

(1 H, dd, J 7.8, 1.6 Hz, 3-H) and 10.33 (1 H, d, J 0.7 Hz, CHO);

_C(100 MHz, CDCl₃) 39.5, 56.6, 118.3, 120.5, 128.2, 131.3,

140.3, 152.2 and 188.4; R_f0.35 (EtOAc - hexane, 1:1); R_f0.57

(EtOAc - toluene, 1:9, three elutions).

4. Experimental section

4.1. Chemistry

Melting points were determined using Kofler hot-stage, Buchi

512 or Stuart Scientific SMP10 equipment and are uncorrected.

Unless otherwise indicated, IR spectra were recorded for neat

thin films using Perkin-Elmer 1710FT or Nicolet Nexus 670/870

spectrometers. NMR spectra were measured using a Bruker

Avance III 400 spectrometer and are calibrated by reference to

signals from the solvent (CDCl₃at 77.16 ppm and CD₃OD at

49.00 ppm for ¹³C spectra; residual protium in CDCl₃at 7.26

ppm, CD₃OD at 3.31 ppm and D₂O at 4.79 ppm for ¹H spectra).⁵³

Chemical shifts for ¹⁹F and ³¹P spectra are quoted relative to

CFCl₃and 85% H₃PO₄at 0 ppm respectively. Coupling

constants (J values) are given in Hz; multiplicities are given as

singlet (s), doublet (d), triplet (t), quartet (C), quintet (qn) or

multiplet (m). NMR spectra were assigned with the aid of

COSY, HMBC, HMQC and DEPT spectra where appropriate.

Low-resolution mass spectra were recorded on a Micromass Trio

2000 instrument using the electrospray ionisation method; data

for peaks of intensity <20% of that of the base peak are omitted.

High-resolution (accurate mass) data were recorded using a

Thermo Finnigan MAT95XP instrument. Elemental analyses

were carried out by the University of Manchester microanalytical

service.

4.1.1.2. 3-Iodo-2-formyl-6-methoxyphenyl

methanesulfonate (23)

To apply the iodination procedure described,²⁷the aldehyde

22 (2.763 g, 12.0 mol) was dissolved in a warm solvent mixture

(60 mL; from acetic acid - water - conc. sulfuric acid 100 : 10 : 3

v/v/v). Iodine (1.28 g, 5.04 mmol) was added, followed by

periodic acid dihydrate (602 mg, 2.64 mmol). The flask was

closed with a balloon to retain vapours and the mixture was

stirred at 60–65 ˚C for 24 h, after which time the dark mixture

had become a clear red solution. With continued stirring, the

mixture was diluted with water (50 mL), decolourised by the

portionwise addition of solid Na₂S₂O₅(total 500 mg), and poured

into water (100 mL). The resulting yellow precipitate was

collected, washed with water and dried at the pump, giving the

crude title compound 23 (3.71 g, 87%) which was crystallised

from ethanol (10 mL) and dried in vacuo, giving pale yellow

prismatic needles (3.19 g, 75%), m.p. 102–104 ˚C (Found: C,

30.56; H, 2.48; I, 35.81; S, 8.87. C₉H₉IO₅S requires C, 30.35; H,

Starting materials and solvents were routinely purified by

conventional techniques.⁵⁴Reactions were routinely carried out

in a nitrogen atmosphere. Tetrahydrofuran (THF) was dried

immediately prior to use, by distillation from sodium-

benzophenone ketyl under nitrogen. Organic solutions were

dried using anhydrous MgSO₄and concentrated by rotary

evaporation under reduced pressure. Analytical thin layer

chromatography (TLC) was carried out using Macherey-Nagel

Polygram SIL G/UV₂₅₄plates and the chromatograms were

routinely visualised using UV light (254 nm). Preparative

column (flash) chromatography was carried out on 60H silica gel

(Merck 9385) using the flash technique.⁵⁵Compositions of

solvent mixtures are quoted as ratios of volume. 'Petroleum'

refers to a light petroleum fraction, b.p. 60–80 ˚C, unless

otherwise stated. 'Ether' refers to diethyl ether. Details of the

preparations of 19 and 20 are provided in Supplementary

Material.

–1

2.55; I, 35.63; S, 9.00%); 

/cm 3094, 3045, 3020, 3010,

max

2941, 2912, 2887, 2841, 1702, 1588, 1563, 1467, 1437, 1390,

1361, 1325, 1293, 1277, 1218, 1181, 1162, 1120, 1059, 971, 877,

822, 791, 707; _H(400 MHz, CDCl₃) 10.05 (1 H, s, CHO), 7.86

(1 H, d, J 8.8 Hz, 4-H), 6.94 (1 H, d, J 8.8 Hz, 5-H), 3.93 (3 H, s,

OMe), 3.38 (3 H, s, SMe); _C(100 MHz, CDCl₃) 40.0 (CH₃),

56.8 (CH₃), 85.3 (C), 118.6 (CH), 130.4 (C), 138.9 (C), 139.7

(CH), 153.4 (C), 192.0 (CH); m/z (ES⁺) 411 (67%), 379 (MNa⁺,

100), 374 (MH₂O⁺, 73), 357 (MH⁺, 18); R_f0.26 (EtOAc - toluene,

1:9) [22 has R_f0.20 under the same conditions].

4.1.1.3. 2-Iodo-3,4,5-trimethoxybenzaldehyde (24)

To apply the iodination procedure described,²⁷3,4,5-

trimethoxybenzaldehyde (58.86 g, 0.30 mol) was dissolved by

warming in a mixture of acetic acid (700 mL) and 2 M sulfuric

acid (70 mL). Iodine (32.00 g, 126 mmol) was added, followed

by periodic acid dihydrate (15.04 g, 66 mmol). The flask was

closed with a septum cap to retain vapours and the mixture was

stirred for 4 h at 60–65 ˚C, during which the colour changed from

opaque purple to clear orange-brown. The stirred mixture was

treated dropwise with a solution made by dissolving Na₂S₂O₅

(14.5 g, 76 mmol) in water (50 mL) and then poured into water

(2.0 L). The precipitated solid was collected on a Büchner

funnel, rinsed with water, dried on the filter and then in vacuo,

giving the aldehyde 24 (76.69 g, 79%) as a cream solid, m.p. 67–

68 ˚C [lit.^25d67 ˚C (cyclohexane)]; _H(400 MHz, CDCl₃) 10.04

(1 H, s, CHO), 7.34 (1 H, s, 6-H), 3.96 (3 H, s, OMe), 3.91 (3 H,

s, OMe), 3.90 (3 H, s, OMe); _C(100 MHz, CDCl₃) 56.4 (CH₃),

61.2 (CH₃), 61.3 (CH₃), 91.7 (C), 108.7 (CH), 130.7 (C), 147.9

(C), 153.1 (C), 154.1 (C), 195.4 (CH); R_f0.20 (EtOAc - hexane,

1:4).

4.1.1. Improved Ullmann route to 5,7-dihydro-

3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1

4.1.1.1. 2-Formyl-6-methoxyphenyl

methanesulfonate (22)^{5 6}

Methanesulfonyl chloride (17.76 g, 155 mmol, 12.0 mL) was

added to a solution of o-vanillin (21.60 g, 142 mmol) in

dichloromethane (200 mL) at 0 °C. The mixture was left to stir

for 5 min, then triethylamine (17.42 g, 172.2 mmol, 24.0 mL)

was added dropwise, keeping the internal temperature at 0–5 °C.

The mixture was stirred for a further 1 h at 0–5 ˚C and then at

room temperature for a further 0.5 h. The precipitate was

collected on a Buchner funnel and rinsed with dichloromethane

(80 mL). The filtrate was washed with water (120 mL), aq. HCl

(1 M; 2 x 70 mL), saturated sodium hydrogen carbonate (120

mL) and brine (120 mL). Drying and evaporation under reduced

pressure gave the crude mesylate 22 (32.0 g, 98%) as a pale

yellow oil which rapidly solidified. Crystallisation from ethanol

(ca. 1 mL/g) gave colourless prisms (two crops, total 26.11 g,

80%), m.p. 78–80 ˚C (lit.⁵⁶79–80 ˚C); _H(400 MHz, CDCl₃)

4.1.1.4. 2,6'-Diformyl-4,2',3',4'-

tetramethoxybiphenyl-3-yl methanesulfonate (25)

The mesylate 23 (3.56 g, 10.0 mmol) and aldehyde 24 (9.66 g,

30.0 mmol) were placed in a 100 mL round-bottomed flask and

the mixture was melted by gentle heating with a hot-air gun.

Dendritic copper powder (Aldrich 357456; 20.33 g, 0.32 mol)

was mixed into the melt using a spatula, and the flask was then

heated in a Woods metal bath at 185 ˚C for 20 min. The reaction

mixture was then allowed to cool and extracted with EtOAc (100

mL). The resulting suspension was filtered through a pad of

Celite, rinsing with EtOAc, and the filtrate concentrated.

Chromatography of the residue over silica gel (elution with

EtOAc - hexane, gradient 1:2 to 1:1) yielded 4,4',5,5',6,6'-

hexamethoxy-1,1'-biphenyl-2,2'-dicarboxaldehyde¹followed by

the title compound 25 (2.00 g, 47%), which formed colourless

prisms, m.p. 132 ˚C (EtOH) [lit.¹131–132 ˚C (EtOAc)]; _H(400

MHz, CDCl₃) 10.18 (1 H, s, 2-CHO), 9.63 (1 H, s, 6'-CHO), 7.36

(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), 4.03 (3 H, s, OMe), 3.98 (3 H, s, OMe), 3.57 (3 H, s,

OMe), 3.43 (3 H, s, SMe); _C(100 MHz, CDCl₃) 40.0 (CH₃),

56.3 (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 (CH), 190.2 (CH); R_f0.20 (EtOAc - hexane, 2:1), 0.30

(ether). These data are in full accord with those published

earlier.¹In a small-scale version of this procedure, the mesylate

23 (356 mg, 1.0 mmol) gave the dialdehyde 25 (246 mg, 58%) as

a colourless solid.

A solution of 27 (2.265 g, 5.52 mmol) in THF (35 mL) at 0 ˚C

under N₂was treated dropwise with sodium

hexamethyldisilazide in THF (5.0 mL, 10.0 mmol).²⁹After 5 min

the solution was cautiously diluted with aqueous

hydrochloric acid (12 mL) at 0 ˚C, further diluted with water (20

mL) and extracted with EtOAc (3 x 30 mL). The combined

organic extracts were washed with brine (40 mL), dried and

2

M

2

M

evaporated.

The resulting beige solid was dissolved in

dichloromethane and the solution filtered through a short column

of flash silica (h 4 cm, d 3 cm), eluting with dichloromethane.

The eluate gave the title compound 1 (1.37 g, 75%) as a

colourless solid, identical (TLC, ¹H-NMR, ¹³C-NMR) to an

authentic sample.¹

4.1.2. Intramolecular direct arylation route to 1

4.1.2.1. 6-Bromo-3-methoxy-2-

(methoxymethoxy)benzaldehyde (29)

To

a

stirred

solution

of

6-bromo-2-hydroxy-3-

methoxybenzaldehyde 28⁵⁷(4.67 g, 20.2 mmol) and

diisopropylethylamine (4.90 mL, 3.64 g, 28.1 mmol) in DMF (40

mL) at 0 ˚C was added dropwise bromomethyl methyl ether (2.00

mL, 3.062 g, 24.5 mmol). After stirring at 0 ˚C for 10 min and

then at room temperature for 1 h, the mixture was poured into

rapidly stirring water (200 mL). The precipitate was collected on

a filter, washed with water (3 x 40 mL) and dried in vacuo. To

remove polar impurities, a solution of the crude product (5.28 g)

in dichloromethane was filtered through a plug of silica gel (3 cm

4.1.1.5. 2,6'-Bis(hydroxymethyl)-4,2',3',4'-

tetramethoxybiphenyl-3-yl methanesulfonate (26)

Sodium borohydride (1.25 g, 33.0 mmol) was added

portionwise to a stirred suspension of the dialdehyde 25 (6.37 g,

15.0 mmol) in MeOH (90 mL) at room temperature. The

mixture, which became clear and warm, was stirred for a further

1 h and then diluted with water (120 mL). The bulk of the

MeOH was removed by rotary evaporation and the residue was

extracted with EtOAc (3 x 90 mL). The combined extracts were

washed with brine (90 mL), dried and evaporated.

Crystallisation of the residue from EtOAc (20 mL) gave a semi-

solid mass that was broken up using ether (10 mL), collected on a

filter, rinsed with EtOAc - ether (1:1) and dried in vacuo, giving

the title compound 26 (4.54 g, 71%) as a white solid, m.p. 124–

125 ˚C (EtOAc); R_f(EtOAc - hexane 2:1) 0.18. Concentration

and trituration of the residue with EtOAc - ether (1:3) gave a

further 0.80 g (12%) of the product, which was identical (¹H-

NMR, ¹³C-NMR) to material prepared previously.¹

diameter,

6

cm depth), eluting with dichloromethane.

Evaporation of the eluate provided the title compound 29 (5.10 g,

92%) as a cream solid, m.p. 69–71 ˚C (EtOH) (Found: C, 43.29;

H, 3.92; Br, 29.13. C₁₀H₁₁BrO₄requires C, 43.66; H, 4.03; Br,

–1

29.05%); 

/cm 2944, 2835, 1701, 1573, 1464, 1433, 1301,

max

1258, 1157, 1064, 951; _H(400 MHz, CDCl₃) 10.37 (1 H, d, J 0.5

Hz, CHO), 7.36 (1 H, dd, J 8.8, 0.5 Hz, 5-H), 6.96 (1 H, d, J 8.8

Hz, 5-H), 5.17 (2 H, s, OCH₂O), 3.87 (3 H, s, ArOMe), 3.55 (3

H, s, CH₂OMe); _C(100 MHz, CDCl₃) 56.4 (CH₃), 58.1 (CH₃),

100.1 (CH₂), 113.2 (C), 117.4 (CH), 129.2 (C), 129.8 (CH),

148.9 (C), 152.5 (C), 190.8 (CH); m/z (ES⁺) 340/338

(MNa₂H₂O⁺, 100%), 299/297 (MNa⁺, 80); R_f0.27 (EtOAc -

hexane, 1:3).

4.1.2.2. (6-Bromo-3-methoxy-2-

(methoxymethoxy)phenyl)methanol (30)

4.1.1.6. 5,7-Dihydro-3,9,10,11-

tetramethoxybenz[c,e]oxepin-4-yl methanesulfonate

(27)

A stirred solution of the aldehyde 29 (4.125 g, 15.0 mmol) in

MeOH (75 mL) at room temperature was treated portionwise

with sodium borohydride (0.76 g, 20 mmol). The solution was

stirred for a further 2 h and then poured into water (200 mL).

The mixture was extracted with dichloromethane (3 x 30 mL)

and the extract dried and concentrated. Removal of the final

traces of solvent in vacuo gave the title compound 30 (3.714 g,

89%) as a colourless oil which slowly solidified. The analytical

sample had m.p. 60–62 ˚C (EtOAc - hexane, 1:4) (Found: C,

To a solution of 26 (4.285 g, 10.0 mmol) in acetonitrile (50

mL) was added 48% aq. hydrobromic acid (2.5 mL, 22 mmol)

and the solution was stirred at 50–55 ˚C for 1 h. Water (50 mL)

and dichloromethane (40 mL) were added, the layers were

separated and the aqueous layer was extracted with

dichloromethane (40 mL). The extracts were combined, washed

with brine (40 mL), dried and concentrated, giving the title

compound 27 (4.035 g, 98%) as a colourless crystalline solid,

43.56; H, 4.67; Br, 28.73. C₁₀H₁₃BrO₄requires C, 43.34; H,

–1

1

m.p. 158–161 ˚C (MeOH), identical (TLC, H-NMR, ¹³C-NMR)

4.73; Br, 28.83%); 

/cm 3457, 2941, 2837, 1576, 1465,

max

to an authentic sample.¹

1437, 1399, 1298, 1269, 1231, 1197, 1159, 1071, 1011, 960, 924,

799; _H(400 MHz, CDCl₃) 7.31 (1 H, d, J 8.8 Hz, 4-H), 6.77 (1

H, d, J 8.8 Hz, 5-H), 5.09 (2 H, s, OCH₂O), 4.81 (2 H, s,

ArCH₂O), 3.83 (3 H, s, ArOMe), 3.59 (3 H, s, CH₂OMe); _C(100

MHz, CDCl₃) 56.1 (CH₃), 57.8 (CH₃), 60.0 (CH₂), 99.6 (CH₂),

113.2 (CH), 115.4 (C), 128.8 (CH), 134.9 (C), 146.2 (C), 151.8

4.1.1.7. 5,7-Dihydro-3,9,10,11-

tetramethoxybenz[c,e]oxepin-4-ol (1)

(C); m/z (ES⁺) 301/299 (MNa⁺, 100%); R_f0.18 (EtOAc - hexane,

1:2).

h, after which the DMA was evaporated in vacuo. The residue

was diluted with EtOAc and the solution filtered through a plug

of silica gel, eluting with more EtOAc. Evaporation of the eluate

and chromatography of the residue, eluting with acetone - hexane

(1:4), provided the title compound 34 (127 mg, 56%) as

4.1.2.3. 5-Bromomethyl-1,2,3-trimethoxybenzene

(31)^{5 8}

colourless crystals, m.p. 112–114 ˚C (MeOH) (Found: C, 64.04;

–1

H, 6.47. C₂₀H₂₄O₇requires C, 63.82; H, 6.43%); 

/cm

max

To a stirred solution of 3,4,5-trimethoxybenzyl alcohol (10.0

g, 50.5 mmol) in dry dichloromethane (150 mL) under N₂at –5

˚C was added dropwise a solution of phosphorus tribromide (3.50

mL, 10.1 g, 37.2 mmol) in dichloromethane (10 mL). After

stirring at –5 ˚C for 45 min, the mixture was poured on to ice

(200 g), neutralised with sat. aq. NaHCO₃(120 mL), and the

organic layer was separated. The aqueous phase was extracted

with dichloromethane (2 x 20 mL) and the combined organic

phases were washed with brine (30 mL), dried and concentrated

to obtain the title compound 31 (13.0 g, 99%) as a colourless

solid, m.p. 76–78 ˚C (hexane) [lit.^58b86–87 ˚C (petroleum)]; _H

(400 MHz, CDCl₃) 6.61 (2 H, s, 4-H and 6-H), 4.54 (2 H, s,

CH₂Br), 3.86 (6 H, s, 1-OMe and 3-OMe), 3.84 (3 H, s, 2-OMe)

(in accord with published data⁴³); _C(100 MHz, CDCl₃) 34.4,

56.2, 61.0, 106.2, 133.3, 138.2, 153.4.

2937, 2855, 1599, 1579, 1482, 1462, 1436, 1401, 1370, 1333,

1304, 1274, 1242, 1225, 1196, 1154, 1116, 1089, 1060, 991, 968,

798, 734; _H(400 MHz, CDCl₃) 7.43 (1 H, d, J 8.6 Hz, 1-H),

7.00 (1 H, d, J 8.6 Hz, 2-H), 6.75 (1 H, s, 8-H), 5.22 (1 H, br d, J

5.6 Hz, OCHO), 5.18 (1 H, br d, J 5.6 Hz, OCHO), 5.10 (1 H, d,

J 11.2 Hz, 5-H), 4.39 (1 H, d, J 11.3 Hz, 7-H), 4.04 (1 H, d, J

11.2 Hz, 7-H), 3.94 (3 H, s, OMe), 3.92 (3 H, s, OMe), 3.91 (3 H,

s, OMe), 3.83 (1 H, d, J 11.2 Hz, 5-H), 3.66 (3 H, s, OMe), 3.64

(3 H, s, OMe); _C(100 MHz, CDCl₃) 56.0 (CH₃), 56.2 (CH₃),

57.8 (CH₃), 60.3 (CH₂), 61.0 (CH₃), 61.2 (CH₃), 67.9 (CH₂), 99.6

(CH₂), 108.8 (CH), 111.9 (CH), 125.8 (CH), 126.4 (C), 129.4

(C), 130.5 (C), 131.1 (C), 142.7 (C), 144.0 (C), 150.7 (C), 151.6

(C), 152.9 (C); m/z (ES⁺) 399 (MNa⁺, 100); R_f0.21 (acetone -

hexane, 1:4) [1 has R_f0.16 (acetone - hexane, 1:4)].

4.1.2.6. 5,7-Dihydro-3,9,10,11-

tetramethoxybenz[c,e]oxepin-4-ol (1)

4.1.2.4. 1-Bromo-4-methoxy-3-(methoxymethoxy)-2-

(((3,4,5-trimethoxyphenyl)methoxy)methyl)benzene

(32)

A solution of 34 (127 mg, 0.34 mmol) in MeOH (3 mL) and 2

M hydrochloric acid (1 mL, 2 mmol) was heated to 50–55 ˚C for

1.5 h, after which TLC (acetone - hexane 1:3) indicated that no

34 remained. The solution was diluted with water (5 mL) and

extracted with dichloromethane (3 x 5 mL). The combined

organic extract was washed with brine (10 mL), dried and

evaporated. Chromatography of the residue, eluting with hexane

- EtOAc (2:1), gave the title compound 1 (90 mg, 80%), identical

(TLC, ¹H NMR) to that obtained from 27 as described above.

To a stirred suspension of sodium hydride (60% dispersion in

mineral oil; 750 mg, 18.75 mmol) in THF (45 mL) under N₂at

room temperature was added dropwise a solution of the alcohol

30 (2.08 g, 7.5 mmol) in THF (9 mL). After stirring at room

temperature for 1.5 h, the mixture was treated dropwise with a

solution of the bromide 31 (2.35 g, 9.0 mmol) in THF (9 mL) and

the stirred mixture was heated at 45 ˚C for 2.5 h. It was then

poured into water (180 mL) and extracted with dichloromethane

(3 x 60 mL). The combined organic phases were washed with

water (90 mL) and brine (90 mL), dried and evaporated.

Chromatography of the residue over silica gel (elution with

EtOAc - hexane, 1:3) gave the title compound 32 (2.95 g, 86%)

4.1.3. Preparative route to 15

as

a

colourless viscous oil (Found: M+Na⁺, 479.0678;

–1

4.1.3.1. 5,7-Dihydro-3,9,10,11-

tetramethoxydibenz[c,e]oxepin-4-yl

bis(phenylmethyl) phosphate (35)

C₂₀H₂₅BrO₇Na requires 479.0681); 

/cm

max

2939, 2838,

1592, 1505, 1463, 1422, 1397, 1355, 1330, 1273, 1234, 1152,

1128, 1101, 1072, 1008, 957, 829, 805, 780, 691; _H(400 MHz,

CDCl₃) 7.30 (1 H, d, J 8.8 Hz, 5-H), 6.77 (1 H, d, J 8.8 Hz, 6-H),

6.65 (2 H, s, 2'-H, 6'-H), 5.10 (2 H, s, OCH₂O), 4.72 (2 H, s,

OCH₂), 4.55 (2 H, s, CH₂O), 3.86 (6 H, s, ArOMe), 3.822 (3 H, s,

ArOMe), 3.818 (3 H, s, ArOMe), 3.52 (3 H, s, CH₂OMe); _C(100

MHz, CDCl₃) 56.0 (CH₃), 56.1 (2 x CH₃), 57.7 (CH₃), 60.8

(CH₃), 66.7 (CH₂), 72.9 (CH₂), 99.6 (CH₂), 104.8 (2 x CH), 113.5

(CH), 116.7 (CBr), 128.4 (CH), 131.6 (C), 134.2 (C), 137.3 (C),

146.3 (C), 151.9 (C), 153.2 (2 x C); m/z (ES⁺) 481/479 (MNa⁺,

100%); R_f0.18 (EtOAc - hexane, 1:2).

The method of Silverberg et al.³³was adapted thus: A three-

necked flask fitted with a thermometer, nitrogen inlet and

magnetic stirrer was charged with 1 (1.460 g, 4.39 mmol, 1.0

eq.), N,N-dimethylaminopyridine (60 mg, 0.49 mmol, 0.11 eq.)

and anhydrous acetonitrile (25 mL). The resulting solution was

cooled to an internal temperature of –10 ˚C (cooling bath of

acetone, water and solid CO₂pellets) and treated with anhydrous

tetrachloromethane (2.1 mL, 3.35 g, 22 mmol, 5 eq.) followed by

diisopropylethylamine (1.6 mL, 1.19 g, 9.2 mmol, 2.1 eq.). The

mixture was stirred for 1 min and then treated dropwise with

dibenzyl phosphite (1.4 mL, 1.66 g, 6.3 mmol, 1.44 eq.), keeping

the mixture at or below –10 ˚C. The mixture was then stirred at –

10 ˚C for a further 40 min, at which point TLC indicated the

consumption of the organic starting materials. The mixture was

quenched at –10 ˚C by the dropwise addition of a solution of

potassium dihydrogen phosphate (0.68 g) in water (10 mL),

allowed to warm to room temperature, and then extracted with

EtOAc (3 x 30 mL). The combined organic phase was washed

with water (40 mL) and brine (40 mL), dried and concentrated.

The residue was purified by chromatography (silica gel

preconditioned with EtOAc - hexane - Et₃N 50:50:1), eluting

with EtOAc - hexane (1:1 to 3:2), which gave the title compound

35 (2.06 g, 79%) as a white crystalline solid, m.p. 132–133 ˚C

4.1.2.5. 5,7-Dihydro-1,2,3,9-tetramethoxy-8-

(methoxymethoxy)dibenz[c,e]oxepine (34)

In an adaptation of the procedure described by Fagnou et al.,³⁰

the ether 32 (276.2 mg, 0.604 mmol), anhydrous potassium

carbonate (powdered, 167 mg, 1.21 mmol), DavePhos 33 (23.6

mg, 0.06 mmol) and palladium acetate (13.5 mg, 0.06 mmol)

were placed in a round-bottomed flask. The flask was purged

with nitrogen for 10 min and DMA (12 mL) was added. The

solution was then heated, darkening above 130 ˚C to black at 145

˚C. TLC (EtOAc - hexane, 1:2) after 21 h at 145 ˚C suggested

that the reaction was incomplete. Heating was continued for 67

(EtOH) (Found: C, 64.88; H, 5.38; P, 5.18. C₃₂H₃₃O₉P requires C,

64.86; H, 5.61; P, 5.23%) (M+H⁺, 593.1943. C₃₂H₃₄O₉P requires

593.1940); _max/cm^–12943, 2858, 1482, 1461, 1278; _H(400

MHz, CDCl₃) 7.52 (1 H, dd, J 8.6, 1.2 Hz, 1-H), 7.40–7.29 (10

H, m, ArH), 7.04 (1 H, d, J 8.6 Hz, 2-H), 6.75 (1 H, s, 8-H),

5.28–5.20 (4 H, m, OCH₂Ph), 5.12 (1 H, d, J 11.4 Hz, 5-H), 4.39

(1 H, d, J 11.3 Hz, 7-H), 4.04 (1 H, d, J 11.3 Hz, 7-H), 3.95 (3 H,

s, OMe), 3.92 (3 H, s, OMe), 3.86 (3 H, s, OMe), 3.84 (1 H, d, J

11.4 Hz, 5-H), 3.66 (3 H, s, OMe); _C(100 MHz, CDCl₃) 56.1

(CH₃), 56.2 (CH₃), 60.1 (5-CH₂), 61.0 (CH₃), 61.2 (CH₃), 67.9

(7-CH₂), 69.80 (PhCH₂), 69.86 (PhCH₂), 108.8 (8-CH), 112.2 (2-

CH), 125.9 (C), 127.1 (two peaks, 1-CH), 127.9 (Ph 2,6- or 3,5-

CH), 128.2 (C), 128.2 (C), 128.5 (Ph 4-H), 128.6 (Ph 2,6- or 3,5-

CH), 130.5 (C), 131.1 (C), 138.3 (C), 138.4 (C), 142.7 (C), 150.3

(C), 150.3 (C), 150.7 (C), 153.1 (C); _P(162 MHz, CDCl₃) –5.77

(quin, J 7.5 Hz); m/z (CI, NH₃) 593 (MH⁺, 10%); R_f0.22 (EtOAc

- petroleum ether 60–80˚ 1:1).

(CH₃), 56.0 (CH₃), 59.9 (5-CH₂), 61.1 (CH₃), 61.2 (CH₃), 66.5

(7-CH₂), 109.3 (CH), 112.4 (CH), 124.5 (CH), 126.3 (C), 127.8

(C), 129.1 (C), 130.6 (C), 135.9 (C), 140.9 (C), 141.6 (C), 149.3

(C), 151.5 (C), 152.1 (C); _P(162 MHz, D₂O) –0.45 (s).

* The elemental analysis result corresponds to a solvate with a

w/w distribution of 15 (93.0%), water (3.7%), methanol (3.3%);

compound 15 is hygroscopic.

4.1.4. Preparative route to 18

4.1.4.1. 5,7-Dihydro-3,9,10,11-

tetramethoxydibenz[c,e]oxepin-4-yl

trifluoromethanesulfonate (18)

To a 5 mL flask containing the dibenzoxepinol 1 (83.2 mg,

0.25 mmol, 1.0 eq.) and anhydrous pyridine (0.28 mL, 274 mg,

3.46 mmol, 13.8 eq.) in dry dichloromethane (0.3 mL) under

argon was added triflic anhydride (0.053 mL, 89 mg, 0.315

mmol, 1.26 eq.) dropwise at 0 ˚C over a period of 2–3 min. After

stirring the mixture at this temperature for 30 min, TLC indicated

4.1.3.2. 5,7-Dihydro-3,9,10,11-

tetramethoxydibenz[c,e]oxepin -4-ol 4-(dihydrogen

phosphate) (36)

the consumption of starting material.

The mixture was

A solution of 35 (1.481 g, 2.5 mmol) in MeOH (65 mL) and

EtOAc (10 mL) containing palladium on charcoal (10% w/w; 70

mg) at room temperature was stirred under an atmosphere of

hydrogen for 1 h, at which point TLC (EtOAc) indicated that the

starting material had been consumed. The solution was filtered

through a pad of Celite (depth 8 mm), rinsing with MeOH, and

evaporated. The residue was triturated with EtOAc and the solid

collected on filter, washed with EtOAc and dried in vacuo, giving

the title compound 36 (984 mg, 95%) which crystallised from

EtOH as colourless rosettes, m.p. >200 ˚C (dec.) (Found: C,

52.36; H, 5.20; P, 7.52. C₁₈H₂₁O₉P requires C, 52.43; H, 5.13; P,

7.51%) (M+Na⁺, 435.0826. C₁₈H₂₁O₉PNa requires 435.0821);

transferred to a separating funnel containing dichloromethane (2

mL) and 1 M hydrochloric acid (2 mL). The organic layer was

collected and washed with more 1 M hydrochloric acid (2 x 1

mL), saturated aqueous sodium hydrogen carbonate (1 mL) and

brine (0.5 mL), dried and concentrated in vacuo to afford a dark

yellow oil. Chromatography (EtOAc - petroleum, 1:2) afforded

the title compound 18 (90.6 mg, 78%) as a white solid, m.p. 164

˚C (M, 464.0748. C₁₉H₁₉F₃O₈S requires 464.0753); _max/cm^–1

2943, 2866, 1596, 1486, 1412, 1329; _H(400 MHz, CDCl₃) 3.69

(3 H, s, OMe), 3.92 (1 H, d, J 11.5 Hz, 5-H), 3.93 (3 H, s, OMe),

3.95 (3 H, s, OMe), 3.98 (3 H, s, OMe), 4.00 (1 H, d, J 11.5 Hz,

7-H), 4.43 (1 H, d, J 11.5 Hz, 7-H), 4.90 (1 H, d, J 11.5 Hz, 5-H),

6.76 (1 H, s, 8-H), 7.12 (1 H, d, J 8.7 Hz, 2-H), 7.67 (1 H, d, J

8.7 Hz, 1-H); _C(100 MHz, CDCl₃) 56.2 (CH₃), 56.4 (CH₃), 59.9

(CH₂), 61.1 (CH₃), 61.3 (CH₃), 68.0 (CH₂), 98.5 (C), 99.7 (C),

109.0 (CH), 112.5 (CH), 125.0 (C), 129.1 (C), 130.1 (CH), 131.0

(C), 136.9 (C), 142.9 (C), 150.4 (C), 150.7 (C), 153.6 (C); _F

(375 MHz, CDCl₃) –77.98; m/z (EI) 464 (M, 10%); R_f0.35

(EtOAc - petroleum, 1:2).



_max/cm^–12930, 2858, 1590, 1107; _H(400 MHz, CD₃OD) 7.46

(1 H, dd, J 8.6, 1.2 Hz, 1-H), 7.16 (1 H, d, J 8.6 Hz, 2-H), 6.89 (1

H, s, 8-H), 5.15 (1 H, d, J 11.1 Hz, 5-H), 4.41 (1 H, d, J 11.1 Hz,

7-H), 3.96 (1 H, d, J 11.1 Hz, 7-H), 3.93 (3 H, s, OMe), 3.91 (3

H, s, OMe), 3.89 (3 H, s, OMe), 3.81 (1 H, d, J 11.1 Hz, 5-H),

3.61 (3 H, s, OMe); _C(100 MHz, CD₃OD) 56.5 (CH₃), 56.6

(CH₃), 61.0 (5-CH₂), 61.3 (CH₃), 61.5 (CH₃), 68.4 (7-CH₂), 110.1

(CH), 113.3 (CH), 127.3 (C), 127.8 (CH), 129.2 (C), 131.4 (C),

132.2 (C), 140.0 (C), 140.1 (C), 144.1 (C), 151.8 (C), 152.3 (C),

154.5 (C); _P(162 MHz, CD₃OD) –4.81 (s); m/z (ES) 435

(MNa⁺, 80%).

4.2. Biological Evaluation

4.2.1. Inhibition of tubulin assembly and K526

growth inhibition assays

4.1.3.3. 5,7-Dihydro-3,9,10,11-

tetramethoxydibenz[c,e]oxepin-4-ol 4-(disodium

phosphate) (15)

Details of the procedures are described in detail elsewhere.¹

A stirred solution of the dihydrogen phosphate 36 (731 mg,

1.773 mmol) in MeOH (12 mL) was treated with methanolic

sodium methoxide (0.3984 M, 8.90 mL, 3.546 mmol) [prepared

by dissolving freshly cut sodium (2.29 g, 99.6 mmol) in

anhydrous MeOH (250 mL)]. The mixture was stirred for 2 min,

then concentrated by rotary evaporation. The residue was

triturated with ethanol - hexane (1:1) and dried in vacuo, giving

the title compound 15 (802 mg, 99%) as a white powder, m.p.

208–215 ˚C (darkens above 180 ˚C) (Found: C, 45.50; H, 4.81;

Na, 8.35; P, 6.39. C₁₈H₁₉Na₂O₉P requires C, 47.38; H, 4.20; Na,

10.08; P, 6.79%);* (400 MHz, D₂O) 7.13 (1 H, dd, J 8.6, 1.0 Hz,

1-H), 7.05 (1 H, d, J 8.6 Hz, 2-H), 6.87 (1 H, s, 8-H), 5.29 (1 H,

d, J 11.1 Hz, 5-H), 4.31 (1 H, d, J 11.1 Hz, 7-H), 3.84 (3 H, s,

OMe), 3.83 (3 H, s, OMe), 3.81 (3 H, s, OMe), 3.76–3.69 (2 H,

m, 5-H and 7-H), 3.58 (3 H, s, OMe); _C(100 MHz, D₂O) 55.7

4.2.2. In vitro screening against NCI cell lines

Compounds 1 and 15–17 were submitted to the US National

Cancer Institute (NIH, Bethesda, Maryland) for screening against

the NCI-60 panel of human cancer cell lines, a service offered

through the Developmental Therapeutics Program (DTP). Full

details of the methodology for compound testing, data analysis

and use of the COMPARE algorithm are available on the NCI

website.⁴⁵Compounds 1 and 15–17 were tested more than once

in NCI-60 5-dose assays, and the average data were used in

COMPARE analyses. All of these were run with the GI₅₀values

as the target-set endpoints and the default settings: Minimum

correlation 0.2; count results to return 50; minimum count

common cell lines, 40; minimum standard deviation, 0.05.

The test results for 1 and 15–17 were subjected to a matrix

COMPARE analysis⁴²together with the colchicinoids 2–4 and

the combretastatins 6 and 7. In this type of analysis, the profile

of antiproliferative activity of the 'seed' compound against the

panel of cancer cell lines in the NCI-60 in vitro assays is

compared with that of the 'target' compound in the same assays.

The NSC numbers used to retrieve the data for this analysis were:

1, NSC 756015; 15, NSC 756016; 16, NSC 756013; 17, NSC

756014. Data for 2–4, 6 and 7 are available in the NCI

collection. The results provided the source data for Tables 3a

and 3b.

other tumour was immersion fixed in 10% neutral buffered

formalin for 24 h and processed for paraffin embedding. The

control group were processed at the same time as the 24 h time-

point. Frozen sections of 10 m thickness were taken at

approximately 100 m intervals through the tumour, with each

tumour being attributed a random number so that examination

was done blind. Up to five fields from each of 10 random

sections were examined for each tumour under UV illumination

using a Leica DMRB microscope, with images captured digitally

through a JVC 3-CCD camera and processed using AcQuis

(Synoptics, Cambridge, UK) software. Functional vasculature

was assessed by placing a cm²grid over the captured digital

image and counting the number of points on the grid which

overlay fluorescently stained cells. The percentage functional

vasculature was then calculated by taking the total number of

fluorescence-positive points for each field and dividing by the

total number of points. An average percentage for each animal

was calculated. Comparisons were made between percentage

vasculature in control and treated tumours. Statistical analysis of

shutdown was carried out using a two-tailed Student’s t-Test.

The results are shown in Figure 1.

Using the standard COMPARE protocol, the antiproliferative

activity profiles of the 1 and 15–17 were compared with those of

the NCI standard agents collection of anticancer agents. For

perspective, the analysis was also carried out with 2–4, 6 and 7 as

the seeds. The results provided the source data for Table 3c.

All of the relevant data, including the complete Table 2, are

provided in the Supplementary Material.

4.2.3. In vivo antivascular effects of 15

Tumour necrosis: For each animal, 5 m thick paraffin

sections were taken and stained with haematoxylin and eosin to

assess for necrosis. Each tumour was attributed a random

number so that examination was done blind. Digital images were

captured using the same system as above but with bright-field

illumination. Percentage necrosis was assessed by placing a cm²

grid over the captured digital image and counting the number of

points on the grid which overlay necrotic cells. The percentage

necrosis was calculated by taking the total number of necrosis-

positive points for each field and dividing by the total number of

points. An average percentage for each animal was then

calculated. Statistical analysis of shutdown was carried out using

a two-tailed Student’s t-Test. The results are shown in Figure 2.

Female Balb/C immunodeficient nude mice (Harlan UK Ltd.,

Blackthorn, UK) aged 6-12 weeks were kept in cages housed in

isolation cabinets in an air-conditioned room with regular

alternating cycles of light and darkness. They received Teklad

2018 diet (Harlan) and water ad libitum. Accurately weighed

amounts of 15 or 21 were dissolved in sterile water and

administered within 15 minutes of addition of solvent. To

determine the MTD, 15 was administered intravenously on days

0, 2 and 4 at 200 or 400 mg kg^–1to groups of 2 mice, with a

control group remaining untreated. Mice were frequently

weighed and monitored for any visible deleterious effects for 14

d following administration. Percentage bodyweight compared to

bodyweight on day 0 was determined. No major loss in

bodyweight or other deleterious effects were observed at either of

the concentrations of 15 administered. A persistent reduction in

percentage bodyweight to 85% of starting weight would be

considered toxic. Tumours were excised from a donor animal,

placed in sterile physiological saline containing penicillin and

streptomycin and cut into small fragments of approximately

2 mm³. Under brief general isoflurane inhalation anaesthesia,

DLD-1 fragments were implanted in both the left and right flanks

of each mouse using a trocar. Once tumours had reached a

volume of approximately 150 mm³(as measured by callipers), to

ensure that an established tumour vascular network was in place,

the mice were allocated into groups of three by restricted

randomisation to keep group mean tumour size variation to a

minimum.

See Figures 3 and 4 for sample images and Supplementary

Material for further data.

4.2.4. In vivo antitumour effects of 1

Calu-6 human lung carcinoma cells (American Type Culture

Collection, Manassas, VA, USA) were cultured in RPMI

supplemented with 10% FCS and 2 mmol/L glutamine. Cells

were harvested in exponential phase growth and prepared at a

concentration of 2 × 10⁷cells/mL in a 1:1 mix of serum-free

RPMI and Matrigel (phenol red-free; BD Biosciences,

Erembodegem, Belgium).

To initiate tumour xenografts, 0.1 mL of cell suspension was

implanted at an approximate depth of 1 mm under the skin 1 cm

from the tail base of female nu/nu CBA mice aged 10 to 12 wk.

Palpable tumours were evident 5–7 days after cell implantation.

Tumour volume was measured daily using calipers. Once a

tumour volume of approximately 250 mm³was attained, tumours

were randomised into 3 treatment groups (n=6/group): vehicle

(5% DMSO in peanut oil), compound 1 at 0.5 MTD in 5%

DMSO/peanut oil and compound 1 at 0.25 MTD in 5%

DMSO/peanut oil. Compound/vehicle was administered IP at 0.1

mL per 10 g body weight on days 1 and 5. Mouse condition and

body weight were monitored daily and the animals maintained

throughout using the highest welfare standards.

Vascular shutdown: Seven groups of tumour-bearing mice

(n=3 per group) were used to assess the effects of treatment with

the compounds on the functional vasculature in DLD-1 tumours.

Once tumours had reached a volume of approximately 150 mm³,

to ensure that an established tumour vascular network was in

place, mice were treated with a single 400 mg kg^–1intravenous

dose of 15 or 21 (three groups per compound). An untreated

group was maintained as a control (one group). At 1, 4 or 24 h

following treatment (n=3 per group), vascular shutdown was

assessed as follows: Hoechst 33342 dye was dissolved in sterile

saline and injected intravenously by the tail vein at 40 mg kg^–1.

One minute after injection the mice in the relevant treatment

group were sacrificed by cervical dislocation and the tumours

carefully and rapidly excised. One tumour from each mouse was

then wrapped in aluminium foil, immediately immersed in liquid

nitrogen and stored at 80 ˚C until ready for ultracryotomy. The

All procedures had local ethics and Home Office approval and

were conducted under PPL 40/2328.

12. Mooney, C. J.; Nagaiah, G.; Fu, P.; Wasman, J. K.; Cooney, M.

M.; Savvides, P. S.; Bokar, J. A.; Dowlati, A.; Wang, D.;

Agarwala, S. S.; Flick, S. M.; Hartman, P. H.; Ortiz, J. D.;

Lavertu, P. N.; Remick, S. C. Thyroid 2009, 19, 233.

4.2.5. Dynamic 3D-solution structure of 1

Details of this analysis, which utilised the methodology of

Blundell et al.,⁵¹are provided in the Supplementary Material.

13. (a) Rustin, G. J.; Shreeves, G.; Nathan, P. D.; Gaya, A.; Ganesan,

T. S.; Wang, D.; Boxall, J.; Poupard, L.; Chaplin, D. J.; Stratford,

M. R. L.; Balkissoon, J.; Zweifel, M. Br. J. Cancer 2010, 102,

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J.; Carnell, D.; Meer, K.; Padhani, A. R.; Saunders, M. I.; Hoskin,

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Padhani, A. R.; Koh, D.-M.; Ng, M.; Collins, D. J.; Harris, A.;

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S.-P.; Leach, M. O.; Rustin G. J. S.; Judson, I. Clin. Cancer Res.

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14. Patterson, D. M.; Zweifel, M.; Middleton, M. R.; Price, P. M.;

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15. (a) Eskens, F. A. L. M.; Tresca, P.; Tosi, D.; Van Doorn, L.;

Fontaine, H.; Van der Gaast, A.; Veyrat-Follet, C.; Oprea, C.;

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Acknowledgments

We are grateful to The University of Manchester Intellectual

Property (UMIP) for funding this work, the TINHAT project,

through a Proof-of-Principle award, and we warmly thank Dr

Richard Price and Dr Sunita Jones of UMIP for their constant

support, guidance and encouragement. We also thank Prof.

Ludwig Neyses and Dr Elizabeth Cartwright (Institute of

Cardiovascular Sciences, University of Manchester), Dr Ian

Crocker (Institute of Human Development, University of

Manchester), Prof. Alan McGown (University of Salford) and Dr

Charles Blundell (C4X Discovery Ltd.) for their significant

contributions to this project. We are also grateful to Darren Cook

(University of Salford) and Patricia Cooper (University of

Bradford) for assistance with compound testing, and to the staff

of the analytical services of School of Chemistry (University of

Manchester), especially Gareth Smith (MS), Steve Kelly (NMR)

16. (a) Rischin, D.; Bibby, D. C.; Chong, G.; Kremmidiotis, G.;

Leske, A. F.; Matthews, C. A.; Wong, S. S.; Rosen, M. A.; Desai,

J. Clin. Cancer Res. 2011, 17, 5152; (b) Nowak, A. K.; Brown, C.;

Millward, M. J.; Creaney, J.; Byrne, M. J.; Hughes, B.;

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422.

and Martin Jennings (Microanalysis).

We also wish to

acknowledge the use of the EPSRC-funded Chemical Database

Service at Daresbury.⁵⁹

17. (a) Mita, M. M.; Spear, M. A.; Yee, L. K.; Mita, A. C.; Heath, E.

I.; Papadopoulos, K. P.; Federico, K. C.; Reich, S. D.; Romero,

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Tubulin-binding dibenz[c,e]oxepines: Part 2.1Structural variation and biological evaluation as tumour vasculature disrupting agents

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