Discovery of 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[ b ]furan (BNC105), a tubulin polymerization inhibitor with potent antiproliferative and tumor vascular disrupting properties

Article abstract of DOI:10.1021/jm200454y

A structure-activity relationship (SAR) guided design of novel tubulin polymerization inhibitors has resulted in a series of benzo[b]furans with exceptional potency toward cancer cells and activated endothelial cells. The potency of early lead compounds has been substantially improved through the synergistic effect of introducing a conformational bias and additional hydrogen bond donor to the pharmacophore. Screening of a focused library of potent tubulin polymerization inhibitors for selectivity against cancer cells and activated endothelial cells over quiescent endothelial cells has afforded 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105, 8) as a potent and selective antiproliferative. Because of poor solubility, 8 is administered as its disodium phosphate ester prodrug 9 (BNC105P), which is rapidly cleaved in vivo to return the active 8. 9 exhibits both superior vascular disrupting and tumor growth inhibitory properties compared with the benchmark agent combretastatin A-4 disodium phosphate 5 (CA4P).

Full text of DOI:10.1021/jm200454y

ARTICLE
pubs.acs.org/jmc
Discovery of 7-Hydroxy-6-methoxy-2-methyl-3-(3,4,5-
trimethoxybenzoyl)benzo[b]furan (BNC105), a Tubulin
Polymerization Inhibitor with Potent Antiproliferative and Tumor
Vascular Disrupting Properties
Bernard L. Flynn,*,†,‡ Gurmit S. Gill,† Damian W. Grobelny,† Jason H. Chaplin,† Dharam Paul,†
Annabell F. Leske,† Tina C. Lavranos,† David K. Chalmers,‡ Susan A. Charman,§ Edmund Kostewicz,§
David M. Shackleford,§ Julia Morizzi,§ Ernest Hamel,|| M. Katherine Jung,^ and Gabriel Kremmidiotis†
†Bionomics Ltd., 31 Dalgleish Street, Thebarton, South Australia, 5031, Australia
‡Medicinal Chemistry and Drug Action and §Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences,
Monash University, 381 Royal Parade, Parkville, 3052, Victoria, Australia
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
^Division of Metabolism and Health Effects, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health,
Bethesda, Maryland 20892, United States
S Supporting Information
b
ABSTRACT: A structureꢀactivity relationship (SAR) guided design
of novel tubulin polymerization inhibitors has resulted in a series of
benzo[b]furans with exceptional potency toward cancer cells and
activated endothelial cells. The potency of early lead compounds has
been substantially improved through the synergistic effect of introdu-
cing a conformational bias and additional hydrogen bond donor to the
pharmacophore. Screening of a focused library of potent tubulin
polymerization inhibitors for selectivity against cancer cells and
activated endothelial cells over quiescent endothelial cells has afforded
7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo-
[b]furan (BNC105, 8) as a potent and selective antiproliferative.
Because of poor solubility, 8 is administered as its disodium phosphate
ester prodrug 9 (BNC105P), which is rapidly cleaved in vivo to return the active 8. 9 exhibits both superior vascular disrupting and
tumor growth inhibitory properties compared with the benchmark agent combretastatin A-4 disodium phosphate 5 (CA4P).
’ INTRODUCTION
search for safer and more effective agents that are capable of
treating resistant phenotypes.2
Microtubules are protein biopolymers formed through
polymerization of heterodimers of R- and β-tubulin.1 The
polymerization is reversible, and the dynamic assembly and
disassembly of microtubules are involved in a number of
cell functions, including cell division, migration, and shape
change. Microtubules are also involved in a host of cell
signaling pathways, including those related to apoptosis.
Compounds that bind to R,β-tubulin heterodimers and dis-
rupt the dynamics of microtubule assembly and disassembly
have emerged as some of the most effective chemotherapeu-
tics for the treatment of cancer. Notable examples include the
Catharanthus (or vinca) alkaloids vinblastine and vincristine
and the taxoids paclitaxel and docetaxel.2 These drugs are
powerful antimitotic agents that inhibit cancer cell prolifera-
tion and induce apoptosis. While they have proven to be
successful in the treatment of cancer, their narrow therapeutic
index and the emergence of resistance have inspired efforts to
The Catharanthus alkaloids and the taxoids bind at separate
sites on the R,β-tubulin heterodimer, and another natural
product, colchicine 1 (Figure 1), binds to a third distinct site.1
Compounds that bind to the colchicine site have been the subject
of intense investigation by researchers seeking to identify new
agents capable of addressing the limitations of existing tubulin
targeting drugs.3,4 In addition to being potent antimitotics,
compounds that bind to the colchicine site also show potent
vascular disrupting properties, which result from the effects of
these compounds on vascular endothelial cells.5,6 Such vascular
disrupting agents (VDAs) take advantage of the significant
differences that exist between the vasculature of normal, healthy
tissues and that of tumors. The selective shutdown of tumor
Received: April 14, 2011
Published: July 20, 2011
r
2011 American Chemical Society
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Table 1. SAR Studies of Tubulin Polymerization Inhibitors I
tubulina,b
MCF-7b
entry
compd
X
R
IC50 (μM)
IC50 (nM)
1
2
3
4
5
6
4
1.8 ( 0.2
11 ( 4
10
11
14
12
13
S
H
>40c
2000
S
OH
3.4 ( 0.2
1.0 ( 0.1
1.3 ( 0.2
1.6 (both runs)
520 ( 400
300 ( 400
39 ( 18
45 ( 5
O
OH
OH
NH
a Inhibition of extent of microtubule assembly was the parameter
measured. b All data were obtained from the same laboratory: refs
17ꢀ20. c The rate but not the extent of tubulin polymerization was
inhibited by 50%.
Figure 1. Tubulin binding agents with affinity for the colchicine site.
in vivo without exhibiting undue toxicity. In order to further
offset any increase in toxicity that may result from a moderate
increase in half-life, we also sought a selective compound that
would exhibit higher potency toward cancer cells and tumor
vascular endothelial cells relative to normal, healthy tissues.
These efforts have led to the identification of 8 (BNC105), and
we have recently described aspects of this compound’s vascular
disrupting and tumor growth inhibitory properties in animal
models.16 Herein, we describe the SAR-guided discovery process
that led to 8 and the key biological data that led to its selection as a
candidate for further development, including its potency and
selectivity toward various cancer cell lines, effectiveness against
multidrug resistant phenotypes, and favorable pharmacokinetics.17
vasculature starves tumors of oxygen and nutrients, leading to
significant cancer cell death through necrosis and apoptotis.
While the antimitotic and vascular disrupting properties of
colchicine have been known for over 50 years, toxicity issues
have prevented its clinical development as an anticancer agent.
The significant toxicity of colchicine (LD50 < 5 mg/kg in
rodents) is attributed to its very slow dissociation from tubulin
(t1/2 = 35 h), which leads to persistence in sensitive tissues for
several days.7ꢀ9 Compounds that bind more reversibly to the
colchicine site often have shorter half-lives in vivo and are much
better tolerated.10 N-Acetylcolchicinol 2 and combretastatin A-4
(CA4) 4 are examples of reversible binders to the colchicine site
that have short half-lives in vivo and that are much better
tolerated than colchicine.5,6,10 Since vascular disruption in tu-
mors peaks after just a few minutes, safe and effective vascular
shutdown can be achieved by tubulin binders that have reversible
binding kinetics and short half-lives. Both 2 and 4 have entered
clinical trials, where they are administered as the soluble dis-
odium phosphate ester prodrugs 3 (ZD6126) and 5 (CA4P),
which are rapidly cleaved in vivo to the active agents.10ꢀ12
A significant drawback of the short half-lives of 2 and 4 is that
a short period of in vivo exposure is often insufficient to elicit a
significant cell kill via the antimitotic effect of these compounds.13
As a result, significant tumor growth from the outer rim of the
tumor is maintained, since this region is often fed by normal,
healthy vasculature and is not susceptible to the vasculature
disrupting effects of tubulin binders.14 The more potent tumor
growth inhibitory properties of 7 (Oxi4503), a phosphate
prodrug of combretastatin A-1 (CA1) 6, are attributed to its
capacity to act as both a VDA and a cytotoxic agent.15 After
in vivo deesterification of 7, to give the short half-life VDA 6,
further metabolism occurs to produce a cytotoxic o-quinone
(not shown). Thus 7, represents a dual mode agent capable of
targeting the vasculature of the internal regions of the tumor and
proliferative cancer cells of the viable rim.15 Since tubulin binding
compounds are cytotoxic in their own right, we considered that a
moderate half-life agent might act as both a VDA and a cytotoxin
’ RESULTS AND DISCUSSION
Background SAR. Early studies by Pinney and co-workers
identified benzo[b]thiophene 10 as weak inhibitor of tubulin
polymerization that binds to the colchicine binding site (entry 2,
Table 1).18 X-ray crystal structure analysis of 10 revealed the
A-ring to be rotated toward the D-ring in a pseudo-π-stacking
arrangement.18b It was concluded that this conformation is likely
to be important to binding, and early SAR studies within our
group indicated that the A- and D-rings of 10 may coincide with
the A- and B-rings of 4.18ꢀ20 For example, the introduction of a
30-hydroxyl to the D-ring of 10 (compound 11) increased
structural analogy to 4 and increased potency (entry 3, Table 1).
Also, removal of the B-ring in 11 was well tolerated, giving an
even more potent compound 14 (entry 4, Table 3). On the other
hand, we also found that small changes in the substitution pattern
of the B-ring of 10, such as removal or relocation of the C6-
methoxy to C5, completely eliminated activity, indicating that
the B-ring does contribute to activity when present (data not
shown).19 We also identified that the benzo[b]furan 12 and
indole 13 analogues of benzo[b]thiophene 11 were much more
potent (compare entries 3, 5, and 6, Table 1) and that for all
systems I (X = O, S, NH) the carbonyl linker between the A- and
C-rings is essential for activity (data not shown).21,22 Later, Hseih
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Table 2. MCC Reaction
and co-workers reported that indole 15 (BPROL075), an
analogue of 13 in which the D-ring is absent, is also active.23
On the basis of similarities in the SAR of 15 and 4, they
concluded that the A- and B-rings of 15 correlate with the
A- and B-rings of 4. These results presented the possibility that
the D-ring in compounds I (10ꢀ13) may be redundant and that
in fact the B-ring in I plays an equivalent role to the B-ring of 4.
On the other hand, π-stacking interactions between the A- and
D-rings in I may induce a conformational change such that
compounds I present a different pose to the tubulin binding site
than does 15, that is, where the A- and D-rings in I equate to the
A- and B-rings of 4. Therefore, in order to understand the SAR
and generate more potent analogues of I and 15, a closer
evaluation of the relative contributions of the B- and D-rings in
I would be required. Since benzo[b]furans generally gave the
most effective leads, based on potency and stability (metabolic
and thermal), we focused our investigations on I (X = O).
Compound Synthesis. The compounds generated in the
course of this work were prepared either by use of a multi-
component coupling (MCC) reaction or by a modified Larock-
type coupling (Table 2 and Scheme 1).17,24,25 The one-pot MCC
reaction involves deprotonation of an o-iodophenol 16 and a
terminal alkyne 17 with methylmagesium chloride to generate a
magnesium phenolate and magnesium acetylide that then couple
to give 18 in the presence of a catalytic amount of Pd(PPh3)2Cl2
(3 mol %) in THF at reflux (Table 2). The THF is then
exchanged for DMSO. The third coupling partner, aryl iodide
19, is introduced and the reaction mixture heated under an
atmosphere of CO (g) (balloon). This produces a carbonylative,
heteroannulative coupling reaction between 18 and 19, giving the
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Scheme 1. Modified Larock Coupling
3-aroylbenzo[b]furans 20ꢀ24 in moderate yields (30ꢀ55%).17
This step involves initial oxidative insertion of the palladium into
the CꢀI bond of 19, followed by CO insertion to give an
electrophilic ArC(O)PdI complex that coordinates to the alkyne
bond of 18, promoting cyclization to give a 3-[ArC(O)Pd]-
benzo[b]furan complex. Reductive elimination then affords the
3-aroylbenzo[b]furans 20ꢀ24. A number of products 20ꢀ24
required deprotection in order to afford the desired test compounds.
The benzyl protecting groups in 20 and 22 were cleaved by hydro-
genation to give 25 (34%) and 26 (63%), respectively.17 The iso-
propyl ether protecting groups in 23 and 24 were cleaved using AlCl3
in dichloromethane, giving 27 (51%) and 28 (75%), respectively.17
In order to better explore the SAR of the C2 position of the
benzo[b]furan lead series (see below) and to aid in the genera-
tion of a focused library of active compounds, we devised a
concise method for the preparation of 2-substituted benzo[b]-
furans (Scheme 1). This approach involves a modified Larock-
type coupling between o-iodophenol 16a,b and the 3-silyl-1-
arylpropynone 29 to give 2-silylbenzo[b]furans 30a,b.25,26
Silanes 30a,b were subjected to treatment with TBAF in metha-
nol to remove the silyl groups and, in the case of 30b, reaction
with AlCl3, to remove the isopropyl group, giving 31a and 31b in
good yields (83% and 86%, respectively). Bromo-desilylation of
30a was achieved in reasonable yield to give the 2-bromobenzo-
[b]furan 33a (59%).25 In the case of 30b, it was necessary to first
exchange an isopropyl for an acetyl group (32) in order to avoid
competitive C4-bromination of the benzo[b]furan in the bromo-
desilylation step. Subsequent conversion of 32 to bromide 33b
proceeded smoothly (69%).25 The bromo substituent in 33a and
33b proved to be a versatile functionality that could be readily
substituted by way of palladium mediated coupling or by nucleo-
philic displacement to produce a host of analogues 35ꢀ48
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(Methods AꢀE).17,25 The acetyl group was generally cleaved
under the reaction conditions used for C2-substitution or by
methanolysis (MeOH, K2CO3) of the crude product. In the case
of 48, hydrogenation of the benzyl substituent was required to
afford the desired test compound, amine 49.17
Biological Evaluation. The endothelial cells within tumors
are constantly exposed to proangiogenic growth factors and, as a
consequence, are in a constant state of activation and angio-
genesis.27 Since we were primarily interested in identifying a new
compound capable of selective inhibition of tumor over normal
Table 3. Biological Evaluation of Benzo[b]furans II
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Table 3. Continued
a The tubulin concentration was 10 μM. Inhibition of extent of assembly was the parameter measured (n = 2). b Cells were grown for 48 h at 37 ꢀC in a
humidified 5% CO2 atmosphere. Cell protein was the parameter measured. At least two independent experiments were performed with each compound.
c For activated growth conditions HUVEC cells were seeded at 2500 and 500 cells/well, respectively, and cultured in EGM-2 medium (Lonza) or F12K
medium containing 0.03 mg/mL endothelial cell growth supplement. d For quiescent growth conditions HUVEC and HAAE-1 cells were seeded at
15 000 and 5000 cells/well, respectively, in basal medium (EBM-2 or F12K) containing 0.5% fetal calf serum and antibiotics. e Selectivity ratio (IC50
quiescent)/(IC50 activated).
endothelial cells, all new compounds were screened for their
capacity to selectively inhibit human umbilical vein endothelial
cells (HUVECs) grown in the presence (activated) or absence
(quiescent) of growth factors (Table 3). Key compounds were
also evaluated for their capacity to inhibit tubulin polymerization
and the proliferation of MCF-7 cancer cells (Table 3).
indicated that this would be the most appropriate location
(Figure 2). The introduction of a C7-OH to 31a, giving 31b,
was tolerated and afforded a compound of similar potency to 31a,
which was slightly more selective for activated over quiescent
endothelial cells (entry 7, Table 3). This is consistent with the
SAR of 4, where removal of the B-ring hydroxyl has little effect on
potency.30 Interestingly, the introduction of a C7-OH to indole
15 has recently been shown to reduce potency (∼20-fold
increase in IC50 values across multiple cell lines), albeit to a
lesser degree than a C5-OH.31 While 31a and 31b are of similar
potency, the introduction of a C7-OH to 12 afforded a much
more potent compound, 27 (compare entries 2 and 8, Table 3).
This 10-fold increase in potency is not attributed to any particular
pharmacophoric feature of the C2-aryl in 27, as many other C2-
substituted systems bearing a C7-OH exhibit similar levels of
potency (compare entries 8ꢀ23, Table 3). Morevover, the SAR
data accumulated in this study indicate a synergistic (>additive)
effect for the introduction of both a C7-OH and a C2-substituent.
This synergism is apparent for a range of different C2-substitutents
where the antiproliferative activity of lead series II exhibits a SAR as
follows: [R1 = OH, R2 ¼ H] . [R1 =H, R2 =H] ≈[ R1 = OH, R2 =
H] ≈ [R1 = H, R2 ¼ H] (compare entries 2, 6, 7, and 8, Table 3)
Presumably, the C2-substituent in II plays a steric role,
favoring cisoid-II over transoid-II because of increased steric
compression in the latter, forcing the A- and B-rings into the
preferred orientation for binding to tubulin (Figure 3). This is
supported by quantum chemical calculations on 31b (II, R1 =
OH, R2 = H) and 8 (II, R1 = OH, R2 = Me). Calculations using
the B3LYP method with the 6-311g** basis set show that
although the energy differences are not large, in 31b the transoid
conformation is favored by 1.1 kJ/mol while in compound 8 the
cisoid conformation is favored by 0.3 kJ/mol.
In our SAR studies of the benzo[b]furan lead structure II
(Table 3), we first sought to investigate whether the presence of a
C2-aryl group (D-ring) in II influences its mode of binding and
which of the two rings B or D in II equates to the B-ring in 4. To
do this, we generated three new D-ring analogues of 12,
compounds 21, 25, and 26, all of which should be capable of a
similar pseudo-π-stacking interaction with the A-ring as seen in
the crystal structure of 10 (entries 3ꢀ5, Table 3). If the D-ring in
II plays the role of the B-ring in 4, then based on previous SAR
studies of 4,28ꢀ30 compounds 25 and 21 should be of similar
potency to 12, whereas 26 should be inactive. However, all new
analogues 21, 25, 26 exhibited similar potency to 12 (compare
entries 2ꢀ5, Table 3). Furthermore, these compounds were all of
similar potency to the C2-unsubstituted analogue 31a (a benzo-
[b]furan equivalent of 15) (compare entries 2ꢀ6, Table 2).
These data indicate that while the D-ring in II is tolerated, it does
not contribute to the potency of this series. Most likely, all
compounds II present a similar pose to tubulin irrespective of
whether a C2-aryl substituent (D-ring) is present or not (see also
docking studies below). Thus, the B-ring in I/II plays an
equivalent role to the B-ring in 4 and the presence of a C2-aryl
does not influence this conformation. This is also consistent with
our earlier observation that a C6-methoxy is required for the
potency of I (X = S), even when a C2-aryl is present.19 An
alternative explanation for the increase in potency of 11 relative
to 10 (entries 2 and 3, Table 2) may be that the inhibitory effects
of 10 are limited by poor solubility at higher assay concentrations
and that the presence of a hydroxyl in 11 improves solubility.
Satisfied that the B-ring in II corresponds to the B-ring in 4,
irrespective of the presence of a C2-aryl, we next investigated the
effect of introducing a hydroxyl group ortho to the C6-methoxy
in 31a and 12, giving 31b and 27, respectively (entries 7 and 8,
Table 3). We introduced the hydroxyl to the C7-position rather
than the C5-position, since superimposing 4, colchicine, and 31b
Generally speaking, both hydrophobic and hydrophilic sub-
stituents are well tolerated in the C2-position of II, giving potent
antiproliferative compounds in most cases, particularly for C7-
OH analogues (entries 7ꢀ23, Table 3).32 Nonetheless, some C2
substituents had significant effects on potency. The presence of a
strong base at C2, as in compounds 45 and 46, tended to reduce
potency (entries 14 and 21, respectively, Table 3). At this stage, it
is uncertain as to whether this is as a result of reduced affinity of
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biological evaluation. Compound 8 exhibited excellent potency
against a panel of different cancer cell lines (Table 4). The
selectivity observed for 8 against activated over quiescent HU-
VECs was also observed in human aortic arterial endothelial cells
(HAAECs). Again, this selectivity was not seen with 4. Further-
more, 8 generally exhibited greater potency than 4, up to 10-fold
in most cases.
We also evaluated 8 for multidrug resistance. Acquired resis-
tance of human cancer cells to chemotherapeutic agents is one of
the major causes of treatment failure.36 A key mechanism of
acquired resistance is the increased expression of the transporter,
multidrug resistance protein MDR-1 (P-glycoprotein). This
transporter effluxes drug agents from the cytoplasm of the cell,
reducing the drug concentration to ineffective levels. Since more
than 80% of current chemotherapeutics are substrates for MDR-
1, it is important to identify new chemotherapeutic agents that
are not MDR-1 substrates and that are able to treat multidrug
resistant phenotypes. The renal carcinoma cell line 786-0 has
high levels of expression of MDR-1, and the potency of drugs that
are MDR-1 substrates is significantly reduced by this transporter.
The three key tubulin targeting drugs paclitaxel, vincristine, and
vinblastine are all known to be MDR-1 substrates and show
enhanced activity against 786-0 when coadministered with the
MDR-1 inhibitor verapamil (Table 5). By contrast, 8 was
equipotent against 786-0 cells with and without verapamil,
indicating that 8 is not a substrate for the MDR-1 transporter
(Table 5).
Figure 2. Superpositioning of colchicine 1 (yellow), 4 (pink), and 27a
(green). Oxygen atoms are red. Nitrogen atoms are blue.
Figure 3. Cisoid and transoid rotamers of II.
MDR-1 overexpression is also a key element of the multidrug
resistance acquired by the human ovarian cancer cell line A2780
upon continuous exposure to adriamycin, producing the A27-
80ADR phenotype.37 Both doxorubicin and cisplatin, which are
MDR-1 substrates, showed reduced potency toward this cell line,
whereas 8 exhibited similar potency against both A2780 and
A2780ADR (Table 6). Compound 8 also exhibited good potency
toward the cisplatin resistant cell line A2780cis (Table 6).
A2780cis cells exhibit cross-resistance to a number of cytotoxins
and radiation in an MDR-1 independent manner.37
these compounds for the target or due to partial sequestration of
the active into acidic vesicles in the cell, reducing the concentra-
tion in the proximity of the microtubules. Additionally, certain
five-membered ring heterocycles in the C2-position, furan 36,
thiophene 37, and N-methylpyrazole 28, afforded very potent
compounds (entries 10, 15, and 16, respectively, Table 3). Of all
the C2-substitutients evaluated,32 only the C2-methyl 8 showed
any significant selectivity for activated HUVECs over quiescent
HUVECs (81-fold) (entry 9, Table 3). 8 also exhibited good
potency against MCF-7 cancer cells (IC50 = 2.4 nM).
In order to achieve a dual mode agent, capable of acting as a VDA
and an antimitotic, we were interested in producing a compound
that exhibits increased exposure relative to 4 to improve cell kill
levels through the antimitotic effect. Compared with 4, 8 exhibits a
longer half-life and larger AUC0ꢀ∞ in pharmacokinetic studies in
rats (Table 7). It also exhibits a lower level of plasma protein binding
(PPB) than 4, resulting in a significantly higher free drug exposure
of 8 relative to 4. This increased exposure is expected to produce an
increased cell kill through the antimitotic effect of 8, particularly at
the viable rim of the tumor that survives vascular disruption.
The poor aqueous solubility of 8 necessitated its conversion to
the disodium phosphate ester 9, providing a soluble prodrug for
in vivo administration (Scheme 2), as has been done for 4 (5)
and 2 (3) (Figure 1).38 Prodrug 9 was prepared from 8 by carbon
tetrabromide mediated esterification with dibenzyl phosphite to
give the dibenzyl phosphate ester 50. Ester 50 was converted to 9
upon sequential treatment with trimethylsilyl bromide and
sodium methoxide. Pharmacokinetic evaluation of 9 in rats and
mice revealed that it is rapidly converted to 8 and that it achieves
similar levels of drug exposure (tumor and other tissues)
compared to direct administration of 8.39
Notwithstanding that there is some variation in activity and
selectivity as a function of C2-substitution in II, the broader
tolerance to structural variation at this position indicates that it is
probably oriented away from the binding pocket, directing its
substituent into an area of relative “free space” (solvent water).32
This and other features of the SAR were supported by docking
studies. Using the X-ray crystal structure of bovine R,β-tubulin
dimer complexed to podophylotoxin (PDB entry 1SA1),33 we
used Glide34 to dock a variety of benzo[b]furan compounds into
the colchicine site (Figure 4), which lies within the β-tubulin
subunit adjacent to its interface with R-tubulin.35 The docked
structures were oriented so that their 3,4,5-trimethoxyphenyl (A)
rings overlapped with that of colchicine and the C6-OMe and
C7-OH substituents overlapped with the methoxy and carbonyl
groups on the tropone ring of colchicine. The benzo[b]furan C7-
OH group makes a hydrogen bond to the side chain of Asn β258,
which also forms a hydrogen bond to the backbone amide
nitrogen of Val 181 in the adjacent R-tubulin subunit. The
C2-position of the benzo[b]furan is oriented toward a gap
between the R- and β-tubulin subunits which can accommodate
large substituents. The D-ring of compound 27 projects into this
gap, lying between residues Thr R179 and Leu β248.
In our previous report on the in vivo efficacy of 9 in murine
tumor models, we demonstrated that 9 was more effective at
tumor vasculature disruption than the disodium phosphate
prodrug of combretastatin A-4 5. Also, in head-to-head studies
In light of the favorable potency and selectivity exhibited by 8,
we subjected this compound to further in vitro and in vivo
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Figure 4. Comparison between the crystal-bound orientation of colchicine (derived from 3E22) (A) and docked orientations of 31b (B), 8 (C), and 27
(D). The tubulin β subunit is shown with light blue carbon atoms. Residue Thr R179 is shown in dark blue. Note the number scheme used is that
described in ref 33.
Table 4. Inhibition of Cell Proliferation by 8 and 4
Table 5. Effect of MDR-1 Inhibition on the Potency of
Antitubulin Drugs toward Renal Carcinoma Cell Line 786-0
8
4
IC50
IC50
(nM)
drug IC50 (nM) against 786-0 cells
entry
cell linea
cell type
(nM)
conditions
paclitaxel
vincristine
vinblastine
8
1
2
3
4
5
6
7
8
9
activated HAAE-1 endothelial cell
quiescent HAAE-1 endothelial cell
0.1 (120)b 2.2 (1.7)b
drug alone
drug + verapamil
31
37
4.5
0.58
0.39
12
1.3
2.6
4
4.0
1.2
0.31
U87-MG
DU145
Calu-6
brain glioblastoma
prostate carcinoma
0.41
0.36
lung anaplastic carcinoma 0.16
0.94
3.2
188
2.1
5.5
2.9
4.4
3.5
Table 6. Potency of Cytotoxic Drugs toward Resistant Phe-
notypes of A2780
MDA-MB-231
A431
breast adenocarcinoma
epidermoid carcinoma
0.63
18.6
A375
malignant skin melanoma 1.5
drug IC50 (nM)
SKOV-3
ovary adenocarcinoma
0.59
cell line
A2780
doxorubicin
cisplatin
8
10 LoVo
colorectal adenocarcinoma 0.24
1.2
194
5.0
329
0.03
0.06
0.01
11 AU565
12 BT549
breast adenocarcinoma
breast carcinoma
5.8
A2780ADR
A2780cis
1103
5021
0.34
a HAAE-1 cells were seeded at 500 cells/well in medium containing 0.03
mg/mL endothelial cell growth supplement (activated) or in basal
medium containing 0.5% fetal calf serum and antibiotics (quiescent).
Cancer cell lines were seeded at an average of 500ꢀ2000 cells/well and
cultured as recommended by the ATCC. b Selectivity ratio (IC50
quiescent)/(IC50 activated) in parentheses.
dosing of 9 at 40 mg/kg (1/2 MTD) completely suppresses
tumor growth and leads to complete eradication of tumors in
14% of the mice after 70 days.16
The superior efficacy and therapeutic index of 9, relative to 5,
can be attributed to a combination of 8’s greater selectivity,
potency, and in vivo exposure. The superior vascular disrupting
properties of 9, relative to 5, may be derived from 8’s greater
potency and higher free drug fraction. The longer half-life
exhibited by 8 (3.5 h), relative to 4 (0.7 h), should enable it to
achieve a higher cell kill at the viable rim, as a higher proportion
of cells will cycle through the most sensitive phase of the cell cycle
(G2-M check point) in the presence of this antimitotic agent.
on tumor growth inhibition, we also observed that 9 is a much
more effective inhibitor of tumor growth than 5 (Figure 5).
When both compounds were dosed at half their maximum
tolerated dose (1/2 MTD) in a murine breast tumor model,40
9 achieved a much greater reduction in tumor growth than did 5
(Figure 5). Furthermore, 9 could achieve a similar reduction in
tumor growth at 10 mg/kg (1/8 MTD) as was achieved by 5 at
150 mg/kg (1/2 MTD). As previously reported, continued
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Table 7. Pharmacokinetic Properties of 4 and 8
significant increase in potency observed (>10 fold). This SAR is
maintained across a broad range of C2-subsitutents, and a
focused library of potent C2-modified analogues of II has been
screened for compounds exhibiting selectivity for activated over
quiescent endothelial cells. Compound 8 exhibited good selec-
tivity toward activated endothelial cells and was also more potent
than 4 against a panel of cancer cell lines. Studies directed toward
the molecular basis of this selectivity are ongoing. Compound 8
also exhibited excellent potency against a range of multidrug
resistant phenotypes and does not appear to be a substrate for the
efflux pump MDR-1. The superior in vivo efficacy of 8, relative to
4, is attributed to a combination of its higher potency and greater
exposure, enabling it to elicit both vascular disrupting and
antimitotic modes of action in vivo. Prodrug 9 is currently
undergoing phase II clinical trials for mesothelioma and renal
cell carcinoma.41
compda
parameter
4
8
dose (μmol/kg)
t1/2 (h)
6
6
0.7
7.3
36
3.5
7.1
48
VSS (L/kg)
AUC0ꢀ∞
PPB (% bound)
98.9
81.4
a Plasma concentrationꢀtime profiles are provided in the Supporting
Information.
Scheme 2. Synthesis of Prodrug 9
’ EXPERIMENTAL SECTION
Chemistry. Melting points were recorded with an Electrothermal
melting point apparatus. Proton (1H) and carbon (13C) NMR spectra
were recorded at 300 and 75 MHz, respectively. Liquid chromatographyꢀ
mass spectrometry (LCMS) was conducted with a C8 column
(4.6 mm ꢁ 150 mm), using an isocratic mobile-phase containing 49%
acetonitrile, 50% and 315 water, and a 1% ammonium formate solution
(this solution was made up of 1 g of acetic acid and 315 mg of
ammonium formate in 1 L of 33% methanol in water) at a constant
flow rate of 0.5 mL/min. UV detection was measured at 214 nm, and MS
analysis was conducted using an atmospheric pressure ionization
(APCI) ion source. High-resolution mass spectrometry (HRMS) was
performed on a time-of-flight mass spectrometer fitted with an electro-
spray (ESI) ion source. Tetrahydrofuran (THF) and diethyl ether were
distilled under nitrogen from sodium benzophenone ketyl. Dichloro-
methane and 1,2-dichloroethane were distilled from calcium hydride
under nitrogen. Analytical thin layer chromatography (TLC) was
conducted on aluminum sheets coated with silica gel 60 GF254. Flash
chromatography was performed on flash grade silica gel. Experimental
methods for the following compounds has been previously reported: 8,
16a, 16b, 30a, 30b, 31a, 32, 33a, 33b, 35, 36, 44, 47.24 All test com-
pounds (Table 3) exhibited >95% purity by LCMS except 26, which was
91% pure by LCMS.
General Procedure A: Multicomponent Coupling of 16,
17, and 19. To a solution of 2-iodophenol 16 (1 mmol) and alkyne
(1.2 mmol) in dry THF (5 mL) under nitrogen at 0 ꢀC was added
MeMgCl (3.0 M solution in THF, 2.5 mmol), and the mixture was
allowed to warm to room temperature. Pd(Ph3P)2Cl (3 mol %) was
added and the mixture heated to 65 ꢀC for 4ꢀ8 h (monitoring by TLC).
The reaction mixture was then cooled to room temperature and the
THF removed under vacuum. Aryl iodide 19 (1.1 mmol) was added, and
the mixture was dissolved in DMSO (12 mL/mmol). The nitrogen
atmosphere was replaced with carbon monoxide (balloon). The aryl
iodide (1.05 mmol) was added, and the mixture was heated to 80 ꢀC
overnight, then quenched with 10% NH4Cl (aq) and extracted with
ethyl acetate. The organic layer was washed with brine and the solvent
removed under vacuum. The residue was concentrated onto silica gel
and purified by flash column chromatography.
Figure 5. Tumor growth inhibition by 9 and 5 in a MDA-MB-231
breast cancer model: 5 at 150 mg/kg (1/2 MTD), dosed on days 1 and 5
(0); 9 at 10 mg/kg (1/8 MTD), dosed days 1 and 5 (b); 9 at 40 mg/kg
(1/2 MTD), dosed days 1 and 8 (1).
Unlike colchicine, though, 8 is completely cleared from healthy
tissues over 24 h, which, in combination with its higher selectivity
for activated over quiescent phenotypes, minimizes its toxicity.16
As previously reported, 8 is trapped in tumor tissue because of
the vascular shutdown effect, such that 64% of drug remains in
the tumor after 24 h.16
’ CONCLUSION
The potency of early leads I has been improved by the
introduction of a C7-OH. The presence of both a C7-OH
and a C2-substituent are required for optimal potency. The
C2-substituent sterically interacts with the trimethoxybenzoyl
unit in II, favoring the required cisoid-conformer (cisoid-II,
Figure 3). The C7-OH group enables an additional hydrogen
bonding interaction with Asn β258 to be achieved. Only when
both effects are engendered (C7-OH + C2-substituent) is a
2-(3-N,N-Dibenzyl-4-methoxy-phenyl)-6-methoxy-3-(3,4,
5-trimethoxybenzoyl)benzo[b]furan 20. 20 was prepared from
16a, 17a, and 19 in accordance with general procedure A. Flash
chromatography: silica gel, eluent = hexane/diethyl ether 8:2, yield
31%. 1H NMR (300 MHz, CDCl3) δ 7.50 (d, J = 8.71 Hz, 1H), 7.27ꢀ
7.05 (m, 16H), 6.88 (dd, J = 8.68, 2.23 Hz, 1H), 4.03 (br s, 4H), 3.87
(s, 6H), 3.80 (s, 3H), 3.58 (s, 6H).
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6-Methoxy-2-(N-methyl-5-indolyl)-3-(3,4,5-trimethoxy-
benzoyl)benzo[b]furan 21. 21 was prepared from 16a, 17b, and 19
in accordance with general procedure A. Flash chromatography: silica
gel, eluent = hexane/diethyl ether 8:2, yield 32%. 1H NMR (300 MHz,
CDCl3) δ 7.91 (d, J = 1.19 Hz, 1H), 7.60 (d, J = 8.65 Hz, 1H), 7.32ꢀ7.36
(m, 2H), 7.14 (d, J = 8.63 Hz, 1H), 7.11 (s, 2H), 7.01 (d, J = 3.11 Hz,
1H), 6.92 (dd, J = 9.95, 2.27 Hz, 1H), 6.41 (d, J = 3.08 Hz, 1H) 3.89
(s, 6H), 3.76 (s, 3H), 3.74 (s, 3H), 3.61 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 191.0, 159.1, 158.0, 154.5, 152.4, 141.7, 136.7, 132.6, 129.6,
127.9, 122.0, 121.9, 121.27, 120.8, 113.9, 112.1, 108.7, 107.05, 101.6,
95.4, 60.4, 55.6, 55.4, 32.5. LCMS: tR = 2.71 min, >99%. MS m/z = 472
(M + H)+, 100%. HRMS: calcd for C28H26NO6+ = 472.1760, found =
472.1761.
dichloromethane (2 ꢁ 5 mL), and the solvent was removed under
vacuum. Purification by flash chromatography (silica gel, eluent =
hexane/ethyl acetate, 6:4) gave a yellow paste that was recrystallized
from hexane and dichloromethane (vapor diffusion) to afford 26 as a
yellow solid (23.2 mg, 63%). 1H NMR (300 MHz, CDCl3) δ 7.49 (dd,
2 ꢁ J = 8.36 Hz, 3H), 7.11 (s, 2H), 7.07 (d, J = 2.06 Hz, 1H), 6.89 (dd, J =
8.62, 2.18 Hz, 1H), 6.74 (d, J = 8.69 Hz, 2H), 3.87 (s, 3H), 3.86 (s, 3H),
3.69 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 191.0, 158.1, 157.4, 157.2,
154.3, 152.5, 142.2, 132.3, 129.7, 127.1, 121.7, 121.3, 115.2, 114.1, 112.3,
107.3, 95.4, 60.6, 55.8, 55.4. LCMS: tR = 2.38 min, >90%. MS m/z = 435
(M + H)+, 38%; 157, 100%. HRMS: calcd for C25H23NO7+ = 435.1444,
found = 435.1442.
7-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-6-methoxy-
3-(3,4,5-trimethoxybenzoyl)benzo[b]furan 27. To a solution of
23 (62 mg, 0.11 mmol) in dry dichloromethane (3 mL) was added AlCl3
(37 mg, 0.28 mmol). The reaction mixture was stirred vigorously at
room temperature for 5 h. Another portion of AlCl3 (18 mg, 0.14 mmol)
was added, and stirring continued for 20 min (TLC). The reaction was
quenched with saturated NH4Cl (aq), and the aqueous layer was
extracted with ethyl acetate (20 mL). The organic layer was washed
with water (15 mL), dried over MgSO4, and concentrated under vacuum.
The crude product was purified by flash chromatography (silica gel, gradient
elution = hexane/diethyl ether 2:8, then neat diethyl ether) to afford 27 as a
crystalline yellow solid (27 mg, 51%). 1H NMR (300 MHz, CDCl3) δ 7.25
(d, J = 2.34 Hz, 1H), 7.12 (d, J = 8.92 Hz, 1H), 7.10 (dd, J = 8.36, 2.14 Hz,
1H), 7.10 (s, 2H), 6.91 (d, J = 8.59 Hz, 1H), 6.70 (d, J = 8.45 Hz, 1H), 5.71
(br s, 1H), 5.52 (br s, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.69
(s, 6H). LCMS: tR = 1.62 min, >99%. MS m/z = 481 (M + H)+, 100%.
HRMS: calcd for C26H24O9+ = 481.1499, found = 481.1501.
7-Hydroxy-6-methoxy-2-(4-N-methylpyrazolyl)-3-(3,4,5-
trimethoxybenzoyl)benzo[b]furan 28. To a solution of 24
(85 mg, 0.18 mmol) in dry dichloromethane (3 mL) was added AlCl3
(48 mg, 0.36 mmol). The reaction mixture was stirred vigorously at
room temperature for 10 min. Another portion of AlCl3 (10 mg, 0.08
mmol) was added, and stirring continued for 20 min (TLC). The
reaction was quenched with saturated NH4Cl (aq), and the aqueous
layer was extracted with ethyl acetate (20 mL). The organic layer was
washed with water (15 mL), dried over MgSO4, and concentrated under
vacuum. The crude product was purified by flash chromatography (silica
gel, gradient elution = hexane/diethyl ether 2:8, then neat diethyl ether)
to afford the title compound as a crystalline yellow solid (58 mg, 75%).
1H NMR (300 MHz, CDCl3) δ 8.15 (s, 1H), 8.03 (s, 1H), 7.13 (s, 2H),
6.79 (d, J = 8.62 Hz, 1H), 6.69 (d, J = 8.58 Hz, 1H), 3.92 (br s, 6H), 3.91 (s,
3H), 3.77 (s, 6H). LCMS: tR = 1.63 min, >99%. MS m/z = 439 (M + H)+,
100%. HRMS: calcd for C23H22NO7+ = 439.1505, found = 439.1510.
7-Hydroxy-6-methoxy-3-(3,4,5-trimethoxybenzoyl)benzo-
[b]furan 31b. Step 1. Tetrabutylammonium fluoride (76.5 μL, 0.076
mmol, 1 M solution in THF) was added to a stirred solution of 30b24
(0.066 mmol) in THF (1 mL). The reaction mixture was stirred at room
temperature for 20 min (TLC), diluted with ethyl acetate (10 mL), and
washed with 1 M HCl (5 mL). The organic layer was dried over MgSO4,
and the solvent was removed under vacuum. The crude product was
purified by flash chromatography (silica gel, eluent = hexane/diethyl
ether, 7:3) to afford the 3-(3,4,5-trimethoxybenzoyl)-6-methoxy-7-
isopropoxybenzo[b]furan as a light yellow paste (23 mg) that was used
directlyinthe nextstep. 1H NMR (300 MHz, CDCl3) δ 8.00(s, 1H), 7.78
(d, J = 8.60 Hz, 1H), 7.15 (s, 2H), 7.04 (d, J = 8.61 Hz, 1H), 4.73 (m, 1H),
3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 1.37 (d, J = 6.14 Hz, 6H).
Step 2. A solution of the product from step 1 (23 mg, 0.058 mmol) in
dry dichloromethane (1 mL) was treated with AlCl3 (16 mg, 0.116
mmol). The reaction mixture was stirred for 0.5 h at room temperature,
then quenched with saturated NH4Cl (aq) and extracted with ethyl
acetate (10 mL). The organic layer was washed with water (5 mL), dried
over MgSO4 and concentrated under vacuum. The crude product was
2-(4-Benzyloxyphenyl)-6-methoxy-3-(3,4,5-trimethoxyben-
zoyl)benzo[b]furan 22. 22 was prepared from 16a, 17c, and 19 in
accordance with general procedure A. Flash chromatography: silica gel,
1
eluent = hexane/diethyl ether, 8:2 and then 7:3, yield 29%. H NMR
(300 MHz, CDCl3) δ 7.54 (d, J = 8.96 Hz, 2H), 7.49 (d, J = 8.67 Hz,
1H), 7.36ꢀ7.38 (m, 5H), 7.11 (s, 2H), 7.07 (d, J = 2.19 Hz, 1H), 6.90
(dd, J = 8.60, 2.20 Hz, 1H), 6.86 (d, J = 8.92 Hz, 2H), 5.04 (br s, 2H) 3.87
(s, 3H), 3.86 (s, 3H), 3.67 (s, 6H).
7-Isopropoxy-2-(3-isopropoxy-4-methoxyphenyl)-6-me-
thoxy-3-(3,4,5-trimethoxybenzoyl)benzo[b]furanbenzo[b]furan
23. 23 was prepared from 16b, 17d, and 19 in accordance with
general procedure A. Flash chromatography: silica gel, eluent =
hexane/diethyl ether, 8:2 and then 7:3, yield 46%. 1H NMR
(300 MHz, CDCl3) δ 7.26 (d, J = 8.58 Hz, 1H), 7.18 (dd, J = 8.44,
2.14 Hz, 1H), 7.13 (s, 2H), 7.07 (d, J = 2.02 Hz, 1H), 6.93 (d, J = 8.65
Hz, 1H), 6.81 (d, J = 8.52 Hz, 1H), 4.77 (quintet, 1H), 4.22 (quintet,
1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.68 (s, 6H), 1.42 (d, J =
6.17 Hz, 6H), 1.23 (d, J = 6.17 Hz, 6H).
7-Isopropoxy-6-methoxy-2-(4-N-methylpyrazolyl)-3-(3,4,
5-trimethoxybenzoyl)benzo[b]furan 24. 24 was prepared from
16b, 17f, and 19 in accordance with general procedure A. Flash chro-
matography: silica gel, eluent = hexane/diethyl ether, 8:2 and then 7:3,
yield 55%. 1H NMR (300 MHz, CDCl3) δ 8.12 (s, 1H), 7.98 (s, 1H),
7.13 (s, 2H), 6.84 (d, J = 8.63 Hz, 1H), 6.79 (d, J = 8.67 Hz, 1H), 4.73
(quintet, 1H), 3.93 (br s, 6H), 3.88 (s, 3H), 3.77 (s, 6H), 1.41 (d, J = 6.16
Hz, 6H). LCMS: tR = 2.27 min, >99%. MS m/z = 481 (M + H)+, 100%.
HRMS: calcd for C24H26NO7+ = 481.1975, found = 481.1974.
2-(3-Amino-4-methoxyphenyl)-6-methoxy-3-(3,4,5-trimeth-
oxybenzoyl)benzo[b]furan 25. Catalyst 10% Pd/C (60 mg) was
added to a solution of 20 (60 mg, 0.093 mmol) in ethyl acetate (12 mL),
methanol (5 mL), and water (2.5 mL), followed by 1 drop of HCl (aq)
(6 M), and the reaction mixture was stirred under an atmosphere of
hydrogen (balloon) for 1 h. The mixture was filtered through Celite and
washed with dichloromethane (2 ꢁ 5 mL) and the solvent removed
under vacuum. The crude product was purified by preparative layer
chromatography (eluent = hexane/ethyl acetate/triethylamine 6:4:1) to
give a yellow paste that was recrystallized from hexane and dichloro-
methane (vapor diffusion) to afford 25 as a yellow solid (14.7 mg, 34%).
1H NMR (300 MHz, CDCl3) δ 7.47 (d, J = 8.64 Hz, 1H), 7.12
(s, 2H), 7.09ꢀ7.05 (m, 2H), 6.99 (dd, J = 8.39, 1.57 Hz, 1H), 6.88 (dd,
J = 8.41, 2.18 Hz, 1H), 6.65 (d, J = 8.38 Hz, 1H), 6.31 (br s, 2H), 3.86 (br
s, 6H), 3.81 (s, 3H), 3.70 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 190.7,
158.1, 157.0, 154.2, 152.5, 148.4, 142.0, 134.7, 132.4, 122.3, 121.7, 121.3,
119.9, 114.4, 114.3, 112.2, 109.6, 107.0, 95.3, 60.6, 55.8, 55.4, 55.2.
LCMS: tR = 2.14 min, >99%. MS m/z = 464 (M + H)+, 100%. HRMS:
calcd for C26H26NO7+ = 464.1709, found = 464.1707.
2-(4-Hydroxyphenyl)-6-methoxy-3-(3,4,5-trimethoxyben
zoyl)benzo[b]furan 26. A mixture of 30 (45 mg, 0.09 mmol) and
Pd/C (10%, 40 mg) in a mixture of ethyl acetate (7 mL) and triethyl-
amine (3 drops) was stirred under an atmosphere of hydrogen
for 1 h. The mixture was filtered through Celite and washed with
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Journal of Medicinal Chemistry
ARTICLE
purified by flash chromatography (silica gel, eluent = hexane/diethyl
ether/ethyl acetate, 80:20:1) to afford 31b as a cream crystalline solid
(18 mg, 86%). 1H NMR (300 MHz, CDCl3) δ 8.04 (s, 1H), 7.63 (d, J =
8.53 Hz, 1H), 7.14 (s, 2H), 7.02 (d, J = 8.38 Hz, 1H), 5.69 (s, 1H), 3.97
(s, 3H), 3.93 (s, 3H), 3.89 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 188.7,
152.8, 151.2, 144.5, 143.3, 141.8, 134.0, 130.9, 120.92, 120.6, 112.4,
109.2, 106.1, 60.6, 56.9, 56.0. LCMS: tR = 1.73 min, >99%. MS m/z =
359 (M + H)+, 100%. HRMS: calcd for C19H19O7+ = 359.1131, found =
359.1126.
1% triethylamine), affording the title compound as light yellow solid
(25 mg, 54%). H NMR (300 MHz, CDCl3) δ 9.10 (br s, 1H), 6.95
1
(s, 2H), 6.60 (d, J = 8.44 Hz, 1H), 6.48 (d, J = 8.48 Hz, 1H), 3.90
(s, 3H), 3.84 (s, 3H), 3.83 (s, 6H), 3.82 ꢀ 3.83 (m, 2H), 2.65
(t, J = 5.63 Hz, 2H), 2.34 (s, 6H). LCMS: tR = 1.65 min, >99%. MS
+
m/z = 445 (M + H)+, 100%. HRMS: calcd for C23H29N2O7
=
445.1975, found = 445.1978.
2-Benzylamino-7-hydroxy-6-methoxy-3-(3,4,5-trimethoxy-
benzoyl)benzo[b]furan 48. When the reaction above was per-
formed using benzylamine in the place of N,N-dimethylethylenedia-
mine, product 48 was obtained as a light yellow solid (112 mg, 83%). 1H
NMR (300 MHz, CDCl3) δ 9.27 (bs, NH, 1H), 7.39ꢀ7.32 (m, 5H),
6.96 (s, 2H), 6.61 (d, J = 8.47 Hz, 1H), 6.50 (d, J = 8.10 Hz, 1H,), 4.83
(d, J = 4.95 Hz, 2H), 3.91 (s, 3H), 3.85 (s, 3H), 3.84 (s, 6H).
(E)-3-(7-Hydroxy-6-methoxy-3-(3,4,5-trimethoxybenzoyl)-
benzofuran-2-yl)acrylamide 40. Pd(OAc)2 (5.0 mg, 0.02 mmol)
was added to a solution of 33b (48 mg, 0.10 mmol) and acrylamide 38b
(70 mg, 1 mmol) in a mixture of acetonitrile and triethylamine (2:1,
1.5 mL) and the resulting solution degassed (stirring solution exposed to
a partial vacuum and backfilled with N2 gas 3 times). The reaction
mixture was stirred at reflux for 1 h. More palladium acetate (5.0 mg, 0.02
mmol) was added, and refluxing continued for 7 h (monitored by TLC).
The solvent was removed under vacuum and the residue subjected to
flash chromatography (silica gel, eluent = hexane/diethyl ether 1:1)
affording the title compound 40 as a yellow crystalline solid (7.3 mg,
17%). 1H NMR (300 MHz, CD3OD) δ: 7.48 (d, J = 15.5 Hz, 1H), 7.18
(s, 2H), 7.00 (d, J = 8.65 Hz, 1H), 6.94 (d, J = 15.5 Hz, 1H), 6.88 (d, J =
8.62 Hz, 1H), 5.46 (s, 1H), 4.55 (br s, 2H), 3.91 (s, 3H), 3.87 (s, 3H),
3.79 (s, 6H). LCMS: tR = 1.49 min, >99%. MS m/z = 424 (M + H)+,
100%. HRMS: calcd for C22H22NO8+ = 428.1345, found = 428.1345.
2-(2,3-Dihydro-1H-pyrrol-1-yl)-7-hydroxy-6-methoxy-3-
(3,4,5-trimethoxybenzoyl)benzo[b]furan 42. To a solution of
pyrrole (22 mg, 0.32 mmol) in dry THF (2 mL) was added sodium
hydride (60%, 24 mg, 0.60 mmol), and the resulting suspension was
stirred at room temperature for 24 h. To this solution 33b (50 mg, 0.10
mmol) was added, and the reaction mixture was stirred at room
temperature for 18 h (monitored by TLC), quenched with saturated
NH4Cl (aq) (10% w/v, 3 mL), extracted with dichloromethane
(10 mL), dried over MgSO4, and concentrated under vacuum. The
resultant residue was purified by planar chromatography on silica gel
(eluent = hexane/ethyl acetate 3:1) to give the title compound 42 as a
yellow solid (25 mg, 60%). 1H NMR (300 MHz, CDCl3) δ 8.47 (d, J =
1.21 Hz, 1H), 7.75 (d, J = 1.20 Hz, 1H), 7.27 (d, J = 8.60 Hz, 1H), 7.18
(d, J = 8.66 Hz, 1H), 7.05 (s, 2H), 6.17 (t, J = 2.3 Hz, 2H), 5.27 (s, 1H),
3.94 (s, 3H), 3.74 (s, 6H), 3.73 (s, 3H). LCMS: tR = 1.77 min, >99%. MS
m/z = 424 (M + H)+, 100%. HRMS: calcd for C23H22NO7+ = 424.1396,
found = 424.1396.
2-Amino-7-hydroxy-6-methoxy-3-(3,4,5-trimethoxyben-
zoyl)benzo[b]furan 49. 10% Pd/C (100 mg) was added to a solution
of 48 (105 mg, 0.23 mmol) in a mixture of ethyl acetate/THF/water
3:2:1 (6 mL), and 2 drops of HCl (aq) (6 M) were added. The resultant
mixture was stirred at room temperature for 2.5 h under H2 (g) (balloon).
After this time, the mixture was filtered through Celite, the filtrate
diluted with water (10 mL) and extracted with dichloromethane
(5 mL ꢁ 3). The organic phase was dried over MgSO4 and concentrated
under vacuum. The resultant residue was purified by flash chromatog-
raphy (silica gel, eluent hexane/ethylacetate 4:1) to give 49 as a
1
crystalline yellow solid (79 mg, 93%). H NMR (300 MHz, CDCl3)
δ 6.98 (s, 2H), 6.93 (br s, 2H), 6.63 (d, J = 8.18 Hz, 1H), 6.52 (d, J = 8.41
Hz, 1H), 5.65 (br s, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.84 (s, 6H). LCMS:
tR = 1.628 min, >99%. MS m/z = 374 (M + H)+, 100%. HRMS: calcd
for C19H20NO7+ = 374.1240, found = 374.1237.
Dibenzyl (6-Methoxy-2-methyl-3-(3,4,5-trimethoxyben-
zoyl)benzofuran-7-yl)phosphate 50. Triethylamine (0.295 mL,
2.12 mmol) was added dropwise with stirring to a suspension of 8 (0.36 g,
0.97 mmol), CBr4 (0.383 g, 1.15 mmol), and dibenzyl phosphite
(0.28 mL, 1.27 mmol) in anhydrous acetonitrile (5 mL) at 0 ꢀC under a
N2 (g) atmosphere. The resulting homogeneous mixture was stirred for
2 h at room temperature and evaporated to dryness under reduced
pressure. The residue was diluted to 40 mL with ethyl acetate, washed
with 0.1 M HCl (10 mL), water (10 mL), brine (10 mL), and dried over
anhydrous MgSO4. Filtration and evaporation of the filtrate under
reduced pressure gave 0.68 g of crude product, which was purified
by flash column chromatography (silica gel, hexane/ethyl acetate 7:3)
1
to give of 50 as a cream solid (0.455 g, 74%). H NMR (300 MHz,
CDCl3) δ 7.3ꢀ7.43 (m, 10H), 7.21 (d, J = 8.7 Hz, 1H), 7.08 (s, 2H,
CH), 6.89 (d, J = 8.7 Hz, 1H), 5.32 (m, 4H,), 3.93 (s, 3H), 3.83 (s, 9H),
2.42 (s, 3H).
6-Methoxy-2-(4-N-methylpiperazino)-3-(3,4,5-trimethoxy-
benzoyl)benzo[b]furan 45. N-Methylpiperazine (50 μL, 0.45 mmol)
was added to a stirred solution of 33a (17 mg, 0.041 mmol) in acetonitrile/
dichloromethane 1:1 (2 mL), and the reaction mixture was stirred at room
temperature for 1 h. After this time the solvent was removed under vacuum,
and the resultant residue was purified by planar chromatography on silica gel
(eluent, hexane/ethyl acetate 4:6 + 1% triethylamine) to give the title
compound as a yellow paste (8 mg, 43%). 1H NMR (300 MHz, CDCl3)
δ 7.07 (s, 2H), 6.93 (d, J = 8.60 Hz, 1H), 6.84 (d, J = 2.25 Hz, 1H), 6.65
(dd, J = 8.64, 2.30 Hz, 1H), 3.92 (s, 3H), 3.83 (s, 6H), 3.78 (s, 3H),
3.66ꢀ3.64 (m, 4H), 2.65ꢀ2.63 (m, 4H), 2.40 (s, 3H). 13CNMR(75MHz,
CDCl3) δ 188.8, 162.2, 155.9, 152.7, 149.1, 141.1, 135.4, 121.7, 119.64,
110.4, 106.1, 155.9, 95.6, 60.6, 55.9, 55.4, 53.9, 47.1, 45.3. LCMS: tR =
1.78 min, >99%. MS m/z = 441 (M + H)+, 100%. HRMS: calcd for
C24H29N2O6+ = 441.2026, found = 441.2030.
2-(2-Dimethylaminoethylamino)-7-hydroxy-6-methoxy-
3-(3,4,5-trimethoxybenzoyl)benzo[b]furan 46. N,N-Dimethyl-
ethylenediamine (500 μL) was added to a stirred solution of 33b
(50 mg, 0.10 mmol) in dry pyridine (2 mL) and the reaction mixture
stirred at 80 ꢀC for 1 h. The reaction mixture was cooled to room
temperature, concentrated under vacuum and the residue purified by
flash chromatography (silica gel, eluent hexane/ethyl acetate 4:1 with
Disodium (6-Methoxy-2-methyl-3-(3,4,5-trimethoxyben-
zoyl)benzofuran-7-yl)phosphate 9. Bromotrimethylsilane (0.3 mL)
was added dropwise to a stirred solution of 50 (0.455 g, 0.719 mmol)
in anhydrous acetonitrile (5 mL) at ꢀ5 ꢀC under a N2 (g) atmosphere.
The reaction mixture was allowed to warm to 0 ꢀC over 1 h, then
evaporated to dryness under reduced pressure. The residue was
dissolved in 90% aqueous acetonitrile (15 mL) and evaporated to
dryness. The crystalline residue was washed with a 50% hexane/
dichloromethane mixture (20 mL) to give the free acid of 9 (0.302 g,
93%). This was suspended in anhydrous methanol (15 mL), and a
solution of MeONa (0.073 g, 1.34 mmol) in methanol (1 mL) was
added, with stirring, at room temperature. The pH of the resulting
mixture was further adjusted to 10 by addition of a small amount of
additional MeONa, and the mixture was evaporated to dryness. The
residue was dissolved in water (2 mL), and acetonitrile (10 mL) was
added. The product crystallized, was removed by filtration, rinsed with
cold (0 ꢀC) acetonitrile (5 mL) and diethyl ether (10 mL), and air-
dried for 24 h to a constant mass to give pure 9 (tetrahydrate) as a
creamy solid (0.33 g, 92%). 1H NMR (300 MHz, D2O) δ 7.10 (s, 2H),
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ARTICLE
7.04 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 2.37 (s, 3H),
3.76 (s, 6H), 3.79 (s, 3H), 3.81 (s, 3H), 4.66 (s, hydrate, 8H). LCMS,
tR = 1.16, >99%. MS m/z = 452.8 (M ꢀ 2Na + 3H), 100%. HRMS:
calcd for C20H20O10Pꢀ = 451.0794, found = 451.0769.
Docking. Molecular docking was performed with Glide 5.6
(Schr€odinger LLC) using XP mode with default settings. Ligands were
prepared using LigPrep, version 2.4. Default settings were used, unless
stated otherwise. Compounds were docked into the crystal structure of
the colchicine binding site of the bovine tubulin/stathmin complex
(PDB entry 1SA1) which contains a podophylotoxin ligand.33
Biology. The following assays were run according to previously
described methods: tubulin polymerization assay42 and MCF-7 prolif-
eration assay.43
Cell Culture and Cell Lines. All in vitro assays were carried out using
endothelial cells derived from human umbilical vein (HUVEC) (Clonetics
Lonza, Walkersville, MD, U.S.) or human abdominal aorta (HAAE-1)
(ATCC, Manassas, VA, U.S.). HUVEC were routinely cultured in EGM-
2 medium (Clonetics), and HAAE-1 cells were cultured in F12K
medium (Gibco, Invitrogen, Auckland, NZ) containing 10% fetal calf
serum, 0.1 mg/mL heparin (Sigma-Aldrich, St. Louis, MO, U.S.),
0.03 mg/mL endothelial cell growth supplement (Sigma-Aldrich,
St. Louis, MO, U.S.), 2 mM penicillinꢀstreptomycinꢀglutamine
(Gibco, Invitrogen, Auckland, NZ), and 10 mM Hepes buffered solution
(Gibco, Invitrogen, Auckland, NZ). Endothelial cell cultures between
passages 2 and 6 were used for all assays. Cancer cell lines were obtained
from ATCC (Manassas, VA, U.S.) and cultured as recommended by the
supplier. All cells were cultured in a humidified incubator at 37 ꢀC with
5% CO2.
’ ABBREVIATIONS USED
CA4, combretastatin A-4; CA4P, combretastatin A-4 disodium
phosphate; HUVEC, human umbilical vein endothelial cell;
HAEEC, human aortic arterial endothelial cell; IC50, inhibitory
concentration 50%; LD50, lethal dose 50%; MCC, multicom-
ponent coupling; MDR, multidrug resistance; MTD, maxi-
mum tolerated dose; SAR, structureꢀactivity relationship;
VDA, vascular disrupting agent
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’ ASSOCIATED CONTENT
S
Supporting Information. Complete listing of benzo-
b
[b]furan compounds tested; synthesis of 17a, 17b, and 17f;
results of pharmacokinetic studies of 4, 5, 8, and 9. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +61 399039650. Fax: +61 99039143. E-mail: bernard.
flynn@monash.edu.
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ARTICLE
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(31) Subsequent to our published patents on C7-OH benzo-
[b]furan, benzo[b]thiophene, and indole analogues of 11 (ref 17),
Cachet and Hseih reported that C7-OH analogues of 15 exhibit reduced
potency, albeit to a lesser extent than does a C5-OH analogue (refs 4l
and 4m). Additionally, in their earlier report (see ref 23), Hseih and co-
workers also observed a modest increase in potency for a C2-methyl
analogue of indole 15 (∼2-fold decrease in IC50 across multiple cell
lines). However, consistent with our findings on benzo[b]furans, we
have shown that the introduction of both C7-OH and C2-Me to 15
affords a much more potent analogue (ref 17b). A more detailed
description of the SAR of indoles and other heterocyclic analogues of
8 will be reported elsewhere.
(32) See Supporting Information for a complete listing of benzofur-
ans evaluated.
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