4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin A-4/lamellarin T hybrids: Synthesis and evaluation as anti-mitotic and cytotoxic agents

Bioorganic & Medicinal Chemistry 14 (2006) 4627–4638

4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin

A-4/lamellarin T hybrids: Synthesis and evaluation as

anti-mitotic and cytotoxic agents

Martin G. Banwell,^a,*Ernest Hamel,^bDavid C. R. Hockless,^aPascal Verdier-Pinard,^b,

Anthony C. Willis^aand David J. Wong^a

^aResearch School of Chemistry, Institute of Advanced Studies, The Australian National University, Canberra, ACT 0200, Australia

^bToxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,

National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA

Received 10 January 2006; revised 8 February 2006; accepted 8 February 2006

Available online 28 February 2006

Abstract—The 4,5-diarylated-1H-pyrrole-2-carboxylates 3–8 have each been prepared as hybrids of the potent anti-mitotic agent

combretastatin A-4 (1) and the similarly active marine alkaloid lamellarin T (2). The key steps involved selective lithium-for-halogen

exchange at C5 within the N-PMB protected 4,5-dibromopyrrole 22 and Negishi cross-coupling of the derived zincated species with

the relevant aryl iodide. The ensuing 5-aryl-4-bromopyrrole then engaged in Suzuki–Miyaura cross-coupling with the appropriate

arylboronic acid to give the 4,5-diarylated pyrroles 4, 6 and 8. TFA-promoted removal of the N-PMB group within these last com-

pounds then gave the N-unsubstituted congeners 3, 5 and 7. Compounds 3–8 have all been evaluated for their anti-mitotic and cyto-

toxic properties and two of them, 3 and 5, display useful activities although they are less potent than combretastatin A-4.

1. Introduction

water and its potential to isomerise to the thermody-

namically more stable (and essentially inactive) trans-

isomer have prompted an extensive eﬀort to ﬁnd

analogues that might display greater stability and/or im-

proved solubilities in therapeutically relevant solvent

systems.^1–6Many structural variations have been inves-

tigated including those wherein there is a one-, two- or

three-atom bridge linking the two multiply oxygenated

and cis-related aromatic rings that appear to be essential

requirements for biological activity.^1–6The many eﬀorts

in this area have been summarised in two recent

reviews.^1,2

Combretastatin A-4 (1) is arguably the most biologically

signiﬁcant member of a group of cis-stilbenes isolated

from a range of sources including the deciduous African

willow tree Combretum caﬀrum Kuntz, extracts of which

have been used in traditional medicines.^1–3As a result of

its capacity to dock in the colchicine binding site of

tubulin, compound 1 displays potent anti-mitotic prop-

erties that have led to its examination as an anti-angio-

genic agent. Indeed, a prodrug form of this natural

product is now undergoing (phase I/II) clinical evalua-

tion in the US and Europe as an agent for the treatment

of various multi-drug resistant solid tumors.^1–3Clearly

then, combretastatin A-4 displays some highly attractive

biological properties. However, its limited solubility in

OH

OMe

OH

MeO

OH

MeO

1

O

MeO

N

O

OMe

Keywords: Anti-mitotic; cis-Stilbene; Combretastatin A-4; Cytotoxic;

Lamellarin T.

OMe

1

2

*

Corresponding author. Tel.: +61 2 6125 8202; fax: +61 2 6125

8114; e-mail: mgb@rsc.anu.edu.au

Another intriguing class of natural product displaying

related structural and biological properties is the

lamellarins, a family of pyrrolic marine alkaloids.⁷

Present address: Departments of Molecular Pharmacology and of

Obstetrics and Gynecology and Women’s Health, Albert Einstein

College of Medicine, Bronx, NY 10461, USA.

doi:10.1016/j.bmc.2006.02.018

4628

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

Compound 2 (lamellarin T) is a representative example

of that subclass incorporating a 6H-[1]-benzopyrano-

[4⁰,3⁰:4,5]pyrrolo[2,1-a]isoquinolinone core and carrying

an aryl substituent at C1 and which display topoisomer-

ase I inhibitory, cytotoxic, reversion of multi-drug resis-

tance, anti-bacterial and/or anti-mitotic properties. As

such they are regarded as important new lead com-

pounds in a number of therapeutic areas.⁷Inspection

of the structure of compound 2 reveals that it incorpo-

rates a combretastatin A4-like substructure and, further-

more, because the two relevant aryl rings are attached at

the C4- and C5-positions (pyrrole numbering) of the

central pyrrole ring they are necessarily rigidly main-

tained in a cis-relationship to one another. On this basis,

and as part of a program underway in our laboratories⁸

to identify natural product-inspired anti-mitotic and

anti-angiogenic agents, we saw merit in preparing a ser-

ies of combretastatin A-4/lamellarin T hybrids for bio-

logical evaluation, particularly as anti-mitotic agents.⁷

Speciﬁcally, we sought to prepare examples of the title

compounds wherein each aryl unit would incorporate

the same or closely related oxygenation patterns to those

seen in compounds 1 and 2. This type of study would al-

low for an assessment of the utility, or otherwise, of the

methyl 4,5-diaryl-1H-pyrrole-2-carboxylate unit as a

scaﬀold for preparing new anti-mitotic agents.^9,10The

presence of the carbomethoxy group within such con-

structs was of particular interest because of the possibil-

ity such a unit, or derivatives thereof, might oﬀer for

addressing the solubility issues associated with the devel-

opment of compound 1 as a drug candidate. A further

intriguing possibility associated with the target scaﬀold

arises because of the presence of the embedded pseu-

do-a-amino acid residue and the capacity, therefore, to

use this as a site for conjugation with speciﬁc peptide

sequences that might deliver constructs likely to be more

cytoselective anti-tumour agents.¹¹

obvious ﬁrst approach to the target compounds 3–8.

The lack of symmetry in the requisite 4,5-dihalogeno-

1H-pyrrole-2-carboxylate coupling partner and the

similar nature of the target hybrids immediately raised

issues about whether a regioselective reaction of such a

substrate with the appropriate arylboronic acid was pos-

sible. Our initial eﬀorts, therefore, were focused on

investigating such matters. To these ends (Scheme 1),

methyl 1H-pyrrole-2-carboxylate (9)¹³was treated with

two equivalents of N-bromosuccinimide (NBS) and the

required coupling partner 10¹⁴obtained, but in only

29% yield. It was hoped that by using carefully con-

trolled conditions and equimolar quantities of phen-

ylboronic acid (11) a regioselective Suzuki–Miyaura

cross-coupling reaction with dibromide 10 might occur.

Unfortunately, when equimolar quantities of the sub-

strates were heated at 90 ꢁC in dimethylacetamide

(DMA), and in the presence of sodium carbonate and

Pd(PPh₃)₄, a complex mixture of reaction products

was produced. Mass spectrometric analysis of this mix-

ture indicated the presence of the diphenylated product

12 but not of any monoarylated precursors. As a result,

this approach to the target compounds was abandoned.

OH

OMe

MeO

HO

CO₂Me

N

R

N

R

MeO

OMe

3 R = H

4 R = PMB

5 R = H

6 R = PMB

O

HO

CO₂Me

N

R

MeO

On the basis of the foregoing, we viewed the 4,5-diaryl-

1H-pyrrole-2-carboxylates 3–8 as representing prototyp-

ical hybrids of the title natural products. In particular,

compounds 3 and 5 seem the best amalgamation of

structures 1 and 2, while congener 7 represents an ana-

logue lacking the 3,4,5-trimethoxyaryl residue often

regarded as essential for the anti-mitotic properties of

combretastatin A-4.^1,2The N-p-methoxybenzyl- or N-

PMB-substituted systems 4, 6 and 8, which were neces-

sary precursors (see below) to their N-unsubstituted

counterparts 3, 5 and 7, were also evaluated for their

anti-mitotic and cytotoxic properties. The following

section details the methodologies developed for the

purposes of preparing the target hybrids 3–8.

7 R = H

8 R = PMB

In earlier work,⁹we had established that methyl 3,4-

dibromo-1-(triisopropylsilyl)-1H-pyrrole-2-carboxylate

engaged in carbomethoxy-group directed lithium-for-

C3-bromine exchange on exposure to phenyllithium.

The ensuing lithio-species could then be transmetallated,

Br

NBS, DMF

CO₂Me

Br

0˚C to 18˚C, 16 h

N

H

N

H

9

10

Pd(PPh₃)₄, Na₂CO₃, DMA,

90˚C, 20 h

+

2. Synthetic studies

In connection with studies directed towards the

synthesis of the simpler lamellarins, we established that

Suzuki–Miyaura cross-coupling reactions between 3,4-

dihalogenated pyrroles and the relevant arylboronic acid

provided a useful method for assembling the 3,4-diaryl-

pyrrole substructures of these natural products.^9,12Con-

sequently, the application of such protocols seemed an

B(OH)₂

CO₂Me

N

H

11

12

Scheme 1.

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

4629

Br1

using ZnCl₂, to give the corresponding zincated species.

This last compound was shown to engage in smooth

Negishi cross-coupling reactions with various aryl ha-

lides to give, in a completely regioselective manner, the

corresponding 3-aryl-4-bromo-1H-pyrrole-2-carboxyl-

ate. By repeating this sort of reaction sequence on the

last compound, the C4-bromine could be replaced by

an aryl unit and so provide various methyl 3,4-diaryl-

1H-pyrrole-2-carboxylates.⁹We sought to apply this

approach as a route to compounds 3–8. This required

working with an N-protected form of the methyl

4,5-dibromo-1H-pyrrole-2-carboxylate because the

organozinc species used as a reaction partner in the ini-

tial Negishi cross-coupling reaction would be basic.

Thus (Scheme 2), compound 9¹³was N-benzylated using

benzyl bromide (13) in the presence of base. Product

14¹⁵(95%) was treated with two equivalents of NBS to

aﬀord compound 15 (93%), which would serve as the

substrate in the pivotal Negishi reaction. Initially phe-

nylzinc chloride (16) was used as the reaction partner

for investigating the selectivity of cross-coupling pro-

cesses involving dibromide 15. The monoarylated and

crystalline pyrrole 17 (74%) was the only reaction prod-

uct and its structure was obtained from a single-crystal

X-ray analysis (see Fig. 1 and Section 5). Selective for-

mation of product 17 is consistent with other observa-

tions¹²that, in the absence of other directing groups,

metallation (including palladation) of pyrroles takes

place preferentially adjacent to the ring nitrogen. When

the monoarylated system 17 was subjected to cross-cou-

pling with p-methoxyphenylzinc chloride (18) the

anticipated product 19 was obtained in 81% yield. Clear-

ly, then, such cross-coupling processes oﬀer considerable

potential for the assembly of the desired methyl 4,5-

diarylated-1H-pyrrole-2-carboxylates. However, we

C2

N1

C3

O2'

C11'

C1

C2'

C4

C12'

C10'

C1'

O1'

C13'

C15'

C3'

C9'

C14'

C8'

C4'

C5'

C7'

C6'

Figure 1. ORTEP derived from a single-crystal X-ray analysis of

compound 17. (Non-hydrogen atoms have been labelled using a

crystallographic numbering scheme).

were unable to eﬀect the cleavage of the N-benzyl group

of 19 with a wide variety of agents. Thus, an alternate

nitrogen-protecting group was required for the synthesis

of compounds 3, 5 and 7. The use of the PMB-group to

protect the pyrrole nitrogen met all synthetic

requirements.

The successful syntheses of compounds 3 and 4 are

shown in Scheme 3. The reaction sequence began with

the N-protection of pyrrole 9 using p-methoxybenzyl

chloride (PMB-Cl, 20). The N-substituted 21 (75%)

was brominated with NBS to give the dibromide 22 in

83% yield. To explore further the utility of the Negi-

shi-coupling reaction, compound 22 was subjected to a

lithium-for-bromine exchange, followed by transmetal-

lation with ZnCl₂. The ensuing zincated species was

cross-coupled with aryliodide 23¹⁶and the C5-arylated

pyrrole 24 was thereby obtained in 82% yield. The struc-

ture of this last compound follows from a single-crystal

X-ray analysis of derivative 3 (see below). Suzuki–Miya-

ura cross-coupling of bromide 24 with the previously

unreported but readily prepared arylboronic acid 25

(see Section 5) gave, after extended reaction times

(110 h), compound 4 in 14% yield. The cause of the inef-

ﬁcient nature of this second cross-coupling step is un-

clear. Such diﬃculties were not encountered while

carrying out the analogous steps associated with the

preparation of the structurally related compounds 6

and 8. The spectral data derived from compound 4 were

in full accord with the assigned structure but ﬁnal con-

ﬁrmation followed removal of the PMB-group by treat-

ment with TFA/DCM in the presence of anisole (as

cation scavenger), as described by Evans et al.¹⁷The

structure of the resulting congener 3 (99%) was deter-

mined from a single-crystal X-ray analysis (see Fig. 2

and Section 5).

CO₂Me

N

Br

NaOH, DCM

+

9

TBAH, 18˚C

16 h

13

14

NBS, DMF,

0˚C to 18˚C,

17 h

Br

CO₂Me

Br

N

ZnCl

+

Pd(PPh₃)₄,THF,

–78˚C to 66˚C,

24 h

16

17

15

MeO

Pd(PPh₃)₄,THF,

+

–78˚C to 66˚C,

56 h

CO₂Me

MeO

N

The reaction sequence leading to hybrids 5 and 6 is

shown in Scheme 4, and used the same strategy as just

described for the preparation of congeners 3 and 4.

Thus, the C5-zincated species derived from dibromide

22 was crossed-coupled with iodide 26¹⁸to give the

ZnCl

18

19

Scheme 2.

4630

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

Br

Cl

CO₂Me

N

CO₂Me

N

NaOH, DCM,

NBS, DMF,

+

9

TBAH, 18˚C, 16 h

0˚C to 18˚C, 16 h

OMe

20

21

22

(i) PhLi, THF, –78˚C, 5 min,

then ZnCl₂, 5 min

Br

+

OTBDMS

MeO

(ii) 23, Pd(PPh₃)₄,

–78˚C to 66˚C, 50 h

CO₂Me

MeO

I

MeO

N

+

B(OH)₂

MeO

OMe

25

24

23

OH

MeO

anisole,

Pd(PPh₃)₄, Na₂CO₃, DMA,

90˚C, 110 h

1:4 v/v TFA/DCM,

MeO

3

CO₂Me

N

37˚C, 48 h

MeO

4

OMe

Scheme 3.

TBDMSO

I

C7'

O3

C6'

C5'

+

22

O4

C8'

MeO

26

C9'

(ii) 26, Pd(PPh₃)₄,

–78˚C to 66˚C, 96 h

(i) PhLi, THF, –78˚C, 5 min,

then ZnCl₂, 5 min

C3'

C4'

Br

OMe

C3

C4

TBDMSO

MeO

C2

C13'

CO₂Me

+

O2

N

C2'

C1

MeO

B(OH)₂

C11'

C1'

N1

O5

C10'

C18'

27

28

OMe

C12'

C16'

O1

C14'

Pd(PPh₃)₄,

Na₂CO₃, DMA,

90˚C, 50 h

C15'

O6

C17'

O7

OMe

MeO

Figure 2. ORTEP derived from a single-crystal X-ray analysis of

compound 3. (Non-hydrogen atoms have been labelled using a

crystallographic numbering scheme.)

anisole

MeO

HO

1:4 v/v TFA/DCM,

5

37˚C, 48 h

CO₂Me

N

anticipated product 27 in 56% yield. Suzuki–Miyaura

cross-coupling of compound 27 with arylboronic acid

28,¹⁹readily prepared from iodide 23, proceeded slowly

(50 h) but aﬀorded the N-benzylated and 4,5-diarylated

pyrrole 6 in 52% yield. Removal of the PMB-group

within pyrrole 6 was again achieved using the Evans’

protocol¹⁷and compound 5 thus obtained in 62% yield.

The spectral data derived from product 5 were closely

related to but not identical with those obtained from iso-

mer 3, the structure of which was obtained by X-ray

analysis. These similarities, together with the synthetic

routes employed, provide strong evidence in support

of the structure assigned to target 5.

MeO

6

OMe

Scheme 4.

detailed above. Speciﬁcs are presented in Scheme 5.

Thus, Suzuki–Miyaura cross-coupling of bromopyrrole

27 with commercially available boronic acid 29 aﬀorded,

after 38 h, product 8 in 87% yield. Removal of the PMB-

group¹⁷gave compound 7 in 60% yield. The spectral

data derived from these compounds were in full accord

with the proposed structures, but ﬁnal conﬁrmation of

the structure of pyrrole 7 was obtained from a single-

crystal X-ray analysis (see Fig. 3 and Section 5). This

The preparation of the ﬁnal pair of target hybrids, com-

pounds 7 and 8, proceeded in much the same way as

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

4631

of [³H]colchicine to bovine brain tubulin. As a conse-

quence, these compounds also proved to be potent

inhibitors of tubulin polymerisation, with IC₅₀s essen-

tially identical with that of combretastatin A-4 (1).

Compound 7 was signiﬁcantly less active while the three

N-PMB-substituted systems, 4, 6 and 8, were essentially

inactive. These biochemical results were mirrored in the

eﬀects of the various compounds on the growth of CA46

Burkitt lymphoma cells. Thus, the IC₅₀values obtained

for compounds 3 and 5 in this assay were 29 and 31 nM,

respectively, while hybrids 4, 6 and 8 displayed values

>1000. These values compare with an IC₅₀of 3.2 nM

observed for combretastatin A-4 (1). The increased

mitotic indices obtained for the same cells, treated with

concentrations of 1, 3 and 5 10-fold higher than the

IC₅₀s, further conﬁrm that tubulin is the probable target

of 3 and 5, as well as of 1.

O

27

Pd(PPh₃)₄, Na₂CO₃,

DMA,

HO

MeO

+

CO₂Me

N

90˚C, 38 h

O

OMe

8

B(OH)₂

anisole,

1:4 v/v TFA/DCM,

29

7

37˚C, 48 h

Scheme 5.

O3'

C6'

C5'

C4'

C7'

O4'

C8'

C9'

4. Conclusions

C3'

C2

C3

Compounds 3 and 5, the hybrids most closely resem-

bling combretastatin A-4 in structure, have potent

anti-mitotic and cytotoxic properties, owing to their

binding to the colchicine site on tubulin. Compound 7,

lacking the 3,4,5,-trimethoxyaryl residue, showed a 4-

fold loss of activity relative to compounds 3 and 5 as

an inhibitor of tubulin assembly. This result is consistent

with other reports.^1,2The lack of activity displayed by

the N-PMB-substituted systems 4, 6 and 8 suggests that

contiguous substitution at four adjacent positions on the

pyrrole backbone disrupts the relevant ligand–protein

binding. Such extensive substitution may prevent the

two vicinally related aryl units from adopting the neces-

sary conformation for binding at the colchicine site on

tubulin. The removal of the N-substituent in compounds

3 and 5 would reduce such restrictions so that appropri-

ate binding conformations can occur. It is particularly

interesting that hybrids 3 and 5 are equally active, thus

showing that either aryl unit could be at positions C4

and C5 on the pyrrole backbone. In such constructs

each unit is 2-atoms removed from the carbon bearing

the carbomethoxy group. In contrast, we previously⁹

found that the isomeric compounds 30 and 31 are essen-

tially inactive as anti-mitotic agents.²⁰This lack of activ-

ity may arise because of conformational impediments

imposed by the C2-carbomethoxy group associated with

O2

C11'

C12'

C1

C4

C2'

C10'

C1'

O1

N1

C13'

C16'

C14'

C15'

O6

O5

Figure 3. ORTEP derived from a single-crystal X-ray analysis of

compound 7. (Non-hydrogen atoms have been labelled using a

crystallographic numbering scheme).

analysis provides additional evidence that the assigned

structures of compounds 27, 5 and 6 are correct.

3. Biological studies

The evaluation of the title hybrids 3–8 (Table 1) as po-

tential anti-mitotic agents was undertaken using well-es-

tablished assays (see Section 5), with direct comparisons

with authentic samples of combretastatin A-4 (1), gener-

ously provided by G. R. Pettit, Arizona State Universi-

ty. Hybrids 3 and 5 most closely resembled the natural

product 1 in terms of inhibitory eﬀects on the binding

Table 1. Evaluation of 4,5-diaryl-1H-pyrrole-2-carboxylates 3–8 as anti-mitotic and cytotoxic agents

Compound

%Inhibition of

colchicine binding^a

Inhibition of tubulin polymerisation^b

Inhibition of CA46 Burkitt

lymphoma cell growth^cIC₅₀(nM)

Mitotic index^d

(drug concn.)

%

IC₅₀(lM)

1

3

4

5

6

7

8

95

74

—

74

—

19

—

1.1 ( 0.1)

1.4 ( 0.1)

>40

3.2

29

24 (30 nM)

15 (300 nM)

>1000

31

—

19 (300 nM)

1.3 ( 0.2)

>40

>5000

—

>1000

—

5.8 ( 1.0)

>40

^aResults of two experiments—[tubulin] = 1 lM, [³H]colchicine and inhibiting drug at 5 lM.

^bResults of three experiments.

^cResults of two experiments.

^d400 cells counted (one experiment). Cells examined for growth after 24 h. Without drug, 2% of the cells were in mitosis.

4632

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

the latter compounds. Clearly, the position of the carbo-

methoxy group will be of critical importance in the de-

sign, for example, of compounds linked to short

peptide sequences that might facilitate tumour recogni-

tion and provide less toxic, more selective anti-cancer

agents.¹¹Work directed towards examining such possi-

bilities is now underway in our laboratories.

(lit. mp 158–159 ꢁC).¹⁴¹H NMR (500 MHz) d 9.60 (br

s, 1H), 6.89 (d, J 2.7 Hz, 1H), 3.88 (s, 3H). ¹³C NMR

(75 MHz) d 160.4 (C), 123.6 (C), 118.0 (CH), 107.3

(C), 100.6 (C), 52.2 (CH₃); m/z (70 eV) 285, 283 and

281 (42%, 71% and 44%, all M^+Å), 253, 251 and 249

(61, 100 and 61), 226, 224 and 222 (8, 15 and 8).

5.1.4. Methyl 1-benzyl-1H-pyrrole-2-carboxylate (14).

Benzyl bromide (13) (3.14 g, 18.4 mmol) was added

dropwise to a magnetically stirred mixture of pyrrole 9

(2.09 g, 16.7 mmol), sodium hydroxide (150 mL of a

50% w/v aqueous solution) and tetra-n-butylammonium

hydroxide (1 mL of a 40% w/v aqueous solution) in

DCM (150 mL) maintained at 0 ꢁC. The ensuing mix-

ture was allowed to warm to 18 ꢁC over 1 h, stirred at

this temperature for 15 h then acidiﬁed with 3 M HCl

to pH 1. The separated aqueous phase was extracted

with DCM (3· 150 mL) and the combined organic ex-

tracts dried (MgSO₄), ﬁltered and concentrated under

reduced pressure to aﬀord a tan-coloured oil. Subjection

of this material to ﬂash chromatography (silica, 1:5 v/v

ethyl acetate/hexane elution) and concentration of the

appropriate fractions (R_f0.8) furnished the title pyrrole

14¹⁵(3.39 g, 95%) as a pale-green solid, mp 31–32 ꢁC (lit.

mp 31–32 ꢁC).¹⁵¹H NMR (300 MHz) d 7.34–7.25 (com-

plex m, 1H), 7.30 (d, J 7.6 Hz, 2H), 7.11 (d, J 7.6 Hz,

2H), 7.01 (m, 1H), 6.90 (m, 1H), 6.20 (m, 1H), 5.58 (s,

2H), 3.77 (s, 3H); ¹³C NMR (75 MHz) d 161.2 (C),

138.1 (C) 128.9 (CH), 128.4 (2· CH), 127.2 (CH),

126.6 (2· CH), 121.7 (C), 118.2 (CH), 108.3 (CH),

51.8 (CH₃), 50.8 (CH₂); m/z (70 eV) 215 (77%, M^+Å),

183 (32), 156 (19), 91 (100, C₇H₇⁺).

OMe

HO

MeO

OMe

OH

OMe

MeO

CO₂Me

N

H

N

H

30

31

5. Experimental

5.1. Synthetic studies

5.1.1. General procedures. ¹H and ¹³C NMR spectra were

recorded on a Varian Gemini 300 spectrometer using deu-

terochloroform as solvent. Infrared spectra were record-

ed using KBr plates on either a Perkin-Elmer 683 or 1800

FTIR instrument. Unless otherwise speciﬁed, mass spec-

tral analyses were carried out in electron-impact mode

and on a VG Micromass 7070F Double-Focusing Spec-

trometer. Thin layer chromatographic analyses were car-

ried out on aluminium-backed 0.2 mm thick silica gel 60

GF₂₅₄plates supplied by Merck while ﬂash chromato-

graphic puriﬁcations were conducted according to the

method of Still et al.²¹using Merck silica gel 60 (230–

400 mesh) as adsorbent. All solvents and common re-

agents were puriﬁed by established procedures.²²

5.1.5. Methyl 4,5-dibromo-1-benzyl-1H-pyrrole-2-car-

boxylate (15). A solution of NBS (5.39 g, 30.3 mmol)

in DMF (20 mL) was added dropwise to a magnetically

stirred solution of methyl 1-benzyl-1H-pyrrole-2-car-

boxylate (14) (3.26 g, 15.2 mmol) in DMF (50 mL)

maintained at 0 ꢁC. The mixture was allowed to warm

to 18 ꢁC over ca. 1 h, stirred at this temperature for

16 h then partitioned between diethyl ether (150 mL)

and cold water (150 mL). The separated aqueous phase

was extracted with diethyl ether (3· 100 mL) and the

combined organic phases washed with water

(10· 100 mL), dried (MgSO₄), ﬁltered and concentrated

under reduced pressure to aﬀord a pale-yellow solid.

Recrystallisation of this material (diethyl ether/hexane)

aﬀorded methyl 4,5-dibromo-1-benzyl-1H-pyrrole-2-

carboxylate (15) (5.20 g, 93%) as a colourless solid, mp

111–113 ꢁC (Found: C, 42.00; H, 2.98; N, 3.68; Br,

42.52. C₁₃H₁₁Br₂NO₂requires C, 41.86; H, 2.97; N,

3.75; Br, 42.84%). ¹H NMR (500 MHz) d 7.36–7.21

(complex m, 3H), 7.09 (s, 1H), 7.02 (d, J 7.1 Hz, 2H),

5.73 (s, 2H), 3.75 (s, 3H); ¹³C NMR (75 MHz) d 159.7

(C), 136.6 (C) 128.5 (2· CH), 127.4 (CH), 126.3

(2· CH), 123.5 (C), 120.0 (CH), 113.6 (C), 99.6 (C),

51.5 (CH₃), 51.1 (CH₂); m_max(KBr) 3150, 2943, 1701,

1391, 1254 cm^ꢀ1; m/z (70 eV) 375, 373 and 371 (6, 12

and 7%, all M^+Å), 91(100, C₇H₇⁺).

5.1.2. Methyl 1H-pyrrole-2-carboxylate (9). The title

compound was prepared according to literature proce-

dures¹³and obtained in 87% yield as a pale-pink solid,

mp 70–73 ꢁC (lit. mp 73–74 ꢁC).¹³¹H NMR

(300 MHz) d 9.15 (br s, 1H), 6.98 (m, 1H), 6.95 (m,

1H), 6.27 (m, 1H), 3.88 (s, 3H).

5.1.3. Methyl 4,5-dibromo-1H-pyrrole-2-carboxylate

(10). A solution of NBS (1.47 g, 8.3 mmol) in DMF

(15 mL) was added, dropwise, to a magnetically stirred

solution of pyrrole 9 (516 mg, 4.1 mmol) in DMF

(30 mL) maintained at 0 ꢁC under a nitrogen atmo-

sphere. The mixture was allowed to warm to 18 ꢁC over

1 h, and stirring continued at this temperature for 15 h.

The mixture was poured into diethyl ether (150 mL) and

water (150 mL) and the separated aqueous phase

extracted with diethyl ether (3· 150 mL). The combined

organic extracts were washed with water (3· 500 mL)

and brine (1· 500 mL) before being dried (MgSO₄), ﬁl-

tered and concentrated under reduced pressure to aﬀord

a pale-yellow oil. Subjection of this material to ﬂash

chromatography (1:5 v/v ethyl acetate/hexane elution)

and concentration of the appropriate fractions (R_f0.3)

aﬀorded a pale-yellow oil that crystallised upon tritur-

ation (ether/hexane) to give the title methyl ester 10¹⁴

(333 mg, 29%) as colourless needles, mp 158–160 ꢁC

5.1.6. Methyl 4-bromo-1-benzyl-5-phenyl-1H-pyrrole-2-

carboxylate (17). tert-Butyllithium (9.60 mL of a 1.7 M

solution in pentane, 16.4 mmol) was added dropwise

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

4633

to a magnetically stirred solution of bromobenzene

(1.29 g, 8.2 mmol) in dry THF (10 mL) maintained at

ꢀ78 ꢁC. Stirring was continued for a further 0.2 h, and

anhydrous zinc chloride (1.12 g, 8.2 mmol) was added

and the cold bath removed. Whilst warming, methyl 4,

5-dibromo-1-benzyl-2-pyrrolecarboxylate (15) (509 mg,

1.4 mmol) and Pd(PPh₃)₄(473 mg, 0.4 mmol) were

added to the reaction mixture, which was then stirred

at reﬂux for 22 h. After this time, the reaction mixture

was cooled, treated with ﬂash chromatography-grade

silica (2.31 g) and concentrated under reduced pressure.

The resulting free-ﬂowing solid was added to the top of

a ﬂash chromatography column ﬁlled with silica, and

this was eluted with 2:3 v/v DCM/hexane. Concentra-

tion of the appropriate fractions (R_f0.5) aﬀorded a

pale-yellow oil trituration (diethyl ether/hexane) of

which aﬀorded methyl 4-bromo-1-benzyl-5-phenylpyr-

role (17) (370 mg, 74%) as colourless needles, mp 78–

80 ꢁC (Found: C, 61.45; H, 4.39; N, 3.61; Br, 21.34.

C₁₉H₁₆BrNO₂requires C, 61.64; H, 4.36; N, 3.78; Br,

21.58%). ¹H NMR (500 MHz) d 7.35 (m, 3H), 7.26

(m, 2H), 7.21–7.16 (complex m, 4H), 6.80 (d, J 7.0 Hz,

2H), 5.52 (s, 2H), 3.73 (s, 3H); ¹³C NMR (75 MHz) d

160.5 (C), 139.1 (C), 138.4 (C), 130.6 (2· CH), 129.6

(C), 129.0 (CH), 128.4 (4· CH), 127.0 (CH), 125.7

(2· CH), 122.2 (C), 120.1 (CH), 97.1 (C), 51.3 (CH₃),

49.8 (CH₂); m_max(KBr) 3565, 3319, 1710, 1453, 1258,

1190 cm^ꢀ1; m/z (70 eV) 371 and 369 (both 47%, M^+Å),

340 and 338 (both 7), 91 (100, C₇H₇⁺).

(KBr) 3313, 1736, 1700, 1455, 1247, 1175 cm^ꢀ1; m/z

(70 eV) 397(100%, M^+Å), 382(5), 366(7), 306(14), 91(82,

C₇H₇⁺).

5.1.8. Methyl 1-(4-methoxybenzyl)-1H-pyrrole-2-carbox-

ylate (21). A magnetically stirred mixture of aqueous

sodium hydroxide (150 mL of a 50% w/v solution) and

methyl 1H-pyrrole-2-carboxylate (9) (4.41 g, 35.2 mmol)

in DCM (50 mL) maintained at 18 ꢁC was treated with

PMB-Cl (20) (6.07 g, 38.8 mmol). The mixture was stir-

red for 16 h then neutralised with 3 M HCl. The result-

ing mixture was extracted with DCM (3· 150 mL) then

dried (MgSO₄), ﬁltered and concentrated under reduced

pressure to aﬀord a brown oil. Subjection of this mate-

rial to ﬂash chromatography (silica, 3:2 v/v DCM/hex-

ane elution) and concentration of the appropriate

fractions (R_f0.3) aﬀorded methyl 1-(4-methoxybenzyl)-

1H-pyrrole-2-carboxylate (21) (6.50 g, 75%) as a pale-

yellow oil (Found: M^+Å, 245.1049. C₁₄H₁₅NO₃requires

M^+Å, 245.1052). ¹H NMR (300 MHz) d7.08 (d, J

8.5 Hz, 2H), 6.98(m, 1H), 6.86 (m, 1H), 6.83 (d, J

8.5 Hz, 2H), 6.15 (m, 1H), 5.48 (s,2H), 3.77 (s, 3H),

3.76 (s, 3H); ¹³C NMR (75 MHz) d 161.3 (C), 158.8

(C), 130.1 (C), 128.6 (CH), 128.3 (2· CH), 121.6 (C),

118.2 (CH), 113.8 (2· CH), 108.2 (CH), 55.0 (CH₃),

51.3 (CH₂), 50.8 (CH₃); m_max(KBr) 3000, 2950, 1704,

1612, 1514, 1437, 1107 cm^ꢀ1; m/z (70 eV) 245 (30%,

M^+Å), 230 (12), 213 (7), 121 [100, (C₇H₆OCH₃)⁺].

5.1.9. Methyl 4,5-dibromo-1-(4-methoxybenzyl)-1H-pyr-

role-2-carboxylate (22). A solution of NBS (4.61 g,

25.9 mmol) in DMF (15 mL) was added dropwise to

a magnetically stirred solution of methyl 1-(4-meth-

oxybenzyl)-1H-pyrrole-2-carboxylate (21) (3.68 g, 13.0

mmol) in DMF (50 mL) maintained at 0 ꢁC. The mix-

ture was allowed to warm to 18 ꢁC over ca. 1 h, stirred

for 16 h then partitioned between diethyl ether

(100 mL) and cold water (150 mL). The aqueous phase

was extracted with diethyl ether (3· 100 mL), and the

combined organic phases were washed with water

(10· 100 mL) before being dried (MgSO₄), ﬁltered and

concentrated under reduced pressure to aﬀord a pale-

yellow oil. Crystallisation of this material (diethyl

ether/hexane) then aﬀorded methyl 4,5-dibromo-1-(4-

methoxybenzyl)-1H-pyrrole-2-carboxylate (22) (4.33 g,

83%) as a clear, colourless solid, mp 66–67 ꢁC (Found:

C, 41.42; H, 3.03; N, 3.56; Br, 39.64. C₁₄H₁₃Br₂NO₃

requires C, 41.72; H, 3.25; N, 3.48; Br, 39.65%). ¹H

NMR (300 MHz) d 7.08 (s, 1H), 7.06 (d, J 8.7 Hz,

2H), 6.83 (d, J 8.7 Hz, 2H), 5.66 (s, 2H), 3.78 (s, 3H),

3.77 (s, 3H); ¹³C NMR (75 MHz) d 159.8 (C), 158.8

(C), 128.8 (C), 128.0 (2· CH), 123.4(C), 120.0 (CH),

113.9 (2· CH), 113.4 (C), 99.6 (C), 55.1 (CH₃), 51.5

(CH₃), 50.6 (CH₂); m_max(KBr) 2952, 1712, 1613, 1514,

1436, 1250 cm^ꢀ1; m/z (70 eV) 405, 403 and 401 (15, 24

and 16%, all M^+Å), 121 [100, (C₇H₆OCH₃)⁺].

5.1.7. Methyl 1-benzyl-4-(4-methoxyphenyl)-5-phenyl-

1H-pyrrole-2-carboxylate

(19).

tert-Butyllithium

(5.50 mL of a 1.7 M solution in pentane, 9.4 mmol)

was added dropwise to a magnetically stirred solution

of p-methoxybromobenzene (880 mg, 4.7 mmol) in dry

THF (10 mL) maintained at ꢀ78 ꢁC. Stirring was con-

tinued for 0.2 h at this temperature, and anhydrous zinc

chloride (641 mg, 4.7 mmol) was added to the reaction

mixture and the cold bath removed. Whilst warming,

compound 17 (290 mg, 0.8 mmol) and Pd(PPh₃)₄

(91 mg, 0.1 mmol) were added to the reaction mixture,

which was then stirred at reﬂux for 56 h. After this time,

the reaction mixture was cooled, treated with ﬂash chro-

matography-grade silica (480 mg) and concentrated un-

der reduced pressure. The resulting free-ﬂowing solid

was added to the top of a ﬂash chromatography column

ﬁlled with silica, and this was eluted with 2:3 v/v DCM/

hexane. Concentration of the appropriate fractions (R_f

0.2) aﬀorded a pale-yellow oil. Crystallisation (diethyl

ether/hexane) of this material aﬀorded methyl 1-

benzyl-4-(4-methoxyphenyl)-5-phenyl-1H-pyrrole-2-car-

boxylate (19) (225 mg, 81%) as colourless needles, mp

131–132 ꢁC (Found: C, 78.47; H, 5.76; N, 3.30.

1

C₂₆H₂₃NO₃requires C, 78.57; H, 5.83; N, 3.52%). H

NMR (500 MHz) d 7.32–7.27 (complex m, 4H), 7.22–

7.15 (complex m, 5H), 7.07–7.05 (complex m, 2H),

6.84 (d, J 7.5 Hz, 2H), 6.72–6.70 (complex m, 2H),

5.50 (s, 2H), 3.76 (s, 3H), 3.72 (s, 3H); ¹³C NMR

(125 MHz) d 161.4 (C), 157.7 (C), 139.1 (C), 137.7 (C),

131.6 (C), 131.0 (2· CH), 128.8 (2· CH), 128.6

(2· CH), 128.5 (CH), 128.3 (2· CH), 127.5 (C), 126.7

(CH), 125.8 (2· CH), 123.2 (C), 121.8 (C), 117.5 (CH),

113.6 (2· CH), 55.1 (CH₃), 51.1 (CH₃), 48.9 (CH₂); m_max

5.1.10. 5-Iodo-1,2,3-trimethoxybenzene (23). Sodium ni-

trite (6.78 g, 98.3 mmol) was added in portions to a mag-

netically stirred solution of 3,4,5-trimethoxyaniline

(15.0 g, 81.9 mmol, ex. Aldrich Chemical Co.) in

H₂SO₄(150 mL of a 6% w/v aqueous solution,

91.8 mmol) maintained at 0 ꢁC. After 1 h, the yellow

4634

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

solution was added dropwise over 1 h to a magnetically

stirred solution of potassium iodide (16.3 g, 98.2 mmol)

in water (50 mL) maintained at 0 ꢁC. The mixture was

stirred for a further 2 h at 0 ꢁC then poured into ethyl

acetate (200 mL). The separated aqueous phase was

extracted with ethyl acetate (3· 200 mL), and the com-

bined organic extracts were dried (MgSO₄), ﬁltered

and concentrated under reduced pressure to aﬀord a

dark-yellow oil. Subjection of this material to ﬂash chro-

matography (1:10 v/v ethyl acetate/hexane elution) and

concentration of the appropriate fractions (R_f0.2) fur-

nished 5-iodo-1,2,3-trimethoxybenzene (23)¹⁶(11.5 g,

48%) as a pale-yellow solid, mp 83–85 ꢁC (lit. mp 85–

87 ꢁC).¹⁶¹H NMR (300 MHz) d 6.89 (s, 2H), 3.84 (s,

6H), 3.82 (s, 3H); ¹³C NMR (75 MHz) d 153.7 (2· C),

138.0 (C), 114.7 (2· CH), 86.0 (C), 60.6 (CH₃), 56.1

(2· CH₃); m/z (70 eV) 294 (100%, M^+Å), 279 (56).

1 M aqueous solution) and the separated aqueous phase

extracted with DCM (3· 200 mL). The combined organ-

ic extracts were washed with brine (1· 500 mL) before

being dried (MgSO₄), ﬁltered and concentrated under

reduced pressure to furnish 2-methoxyphenyl acetate¹⁸

(13.4 g, 99%) as a pale-yellow oil.

Step (ii): A solution of 2-methoxyphenyl acetate (13.4 g,

80.6 mmol) in chloroform (200 mL) maintained at 0 ꢁC

under a nitrogen atmosphere was treated, in one por-

tion, with silver triﬂuoroacetate (23.1 g, 105 mmol). To

this mixture was added, dropwise over 1 h, a solution

of molecular iodine (20.5 g, 80.8 mmol) in chloroform

(500 mL). The mixture was allowed to warm to 18 ꢁC

over 1 h, stirred for 4 h then washed with sodium thio-

sulfate (1· 500 mL of a 20% w/v aqueous solution),

dried (MgSO₄), ﬁltered and concentrated under reduced

pressure to furnish 5-iodo-2-methoxyphenyl acetate¹⁸

(17.2 g, 73%) as a light-brown and photosensitive solid.

5.1.11. Methyl 1-(4-methoxybenzyl)-4-bromo-5-(3,4,5-tri-

methoxyphenyl)-1H-pyrrole-2-carboxylate (24). Phenylli-

thium (1.7 mL of a 1.8 M solution in cyclohexane/

diethyl ether, 3.02 mmol) was added dropwise to a mag-

netically stirred solution of dibromopyrrole 22 (1.11 g,

2.7 mmol) in dry THF (20 mL) maintained at ꢀ78 ꢁC

under a nitrogen atmosphere. Stirring was continued

for a further 0.2 h then anhydrous zinc chloride

(411 mg, 3.0 mmol) was added in one portion and the

cold bath removed. Whilst the reaction mixture was

warming up, 3,4,5-trimethoxyiodobenzene (23) (1.29 g,

4.4 mmol) and Pd(PPh₃)₄(317 mg, 0.3 mmol) were add-

ed, and the solution was then stirred at reﬂux for 50 h.

After this time, the reaction mixture was cooled, treated

with ﬂash chromatography-grade silica (2.60 g) and

concentrated under reduced pressure. The resulting

free-ﬂowing solid was added to the top of a ﬂash chro-

matography column ﬁlled with silica, and the column

was eluted with 1:4 v/v ethyl acetate/hexane. Concentra-

tion of the appropriate fractions (R_f0.3) aﬀorded methyl

1-(4-methoxybenzyl)-4-bromo-5-(3,4,5-trimethoxyphenyl)-

1H-pyrrole-2-carboxylate (24) (1.10 g, 82%) as a viscous

yellow gum (Found: M^+Å, 489.0787. C₂₃H₂₄⁷⁹BrNO₆re-

Step (iii): A solution of 5-iodo-2-methoxyphenyl acetate

(17.2 g, 58.9 mmol) in methanol (500 mL) maintained at

18 ꢁC under an atmosphere of nitrogen was treated in

one portion with potassium carbonate (40.7 g,

294 mmol). The mixture was stirred at 18 ꢁC for 16 h then

ﬁltered through a sintered-glass funnel. The ﬁltrate was

concentrated under reduced pressure to furnish 5-iodo-

2-methoxyphenol¹⁸(14.7 g, 99%) as a tan-coloured solid,

mp 85–87 ꢁC (lit. mp 87–88 ꢁC).¹⁸¹H NMR (300 MHz) d

7.23 (d, J 2.2 Hz, 1H), 7.17 (dd, J 8.5 and 2.2 Hz, 1H), 6.60

(d, J 8.5 Hz, 1H), 5.55 (br s, 1H), 3.87 (s, 3H).

Step (iv): TBDMSCl (143 mg, 0.95 mmol) was added

dropwise to a magnetically stirred solution of imidazole

(186 mg, 2.7 mmol) and 5-iodo-2-methoxyphenol

(215 mg, 0.9 mmol) in DMF (5 mL) maintained at

0 ꢁC under a nitrogen atmosphere. The mixture was al-

lowed to warm to 18 ꢁC over 1 h, stirred for 15 h then

partitioned between ice-cold H₃PO₄(10 mL of a 1 M

aqueous solution) and diethyl ether (10 mL). The sepa-

rated aqueous phase was extracted with diethyl ether

(3· 10 mL) and the combined organic extracts washed

with water (3· 50 mL) and brine (1· 50 mL) then dried

(MgSO₄), ﬁltered and concentrated under reduced pres-

sure to aﬀord a pale-yellow oil. Subjection of this mate-

rial to ﬂash chromatography (1:10 v/v ethyl acetate/

hexane elution) and concentration of the appropriate

fractions (R_f0.7) furnished (5-iodo-2-methoxyphen-

oxy)(tert-butyl)dimethylsilane (26)¹⁸(264 mg, 84%) as

a clear, colourless solid, mp 32–34 ꢁC (lit. mp 36 ꢁC).¹⁸

¹H NMR (300 MHz) d 7.20 (dd, J 8.8 and 2.1 Hz,

1H), 7.14 (d, J 2.1 Hz, 1H), 6.59 (d, J 8.8 Hz, 1 H),

3.77 (s, 3H, OCH₃), 0.99 (s, 9H, 3· CH₃), 0.15 (s, 6H,

2· CH₃); m/z (70 eV) 364 (9%, M^+Å), 307 (91), 292 (100).

1

quires M^+Å, 489.0787). H NMR (300 MHz) d 7.14 (s,

1H), 6.83–6.76 (complex m, 4H), 6.43 (s, 2H), 5.46

(s, 2H), 3.88 (s, 3H), 3.79 (s, 3H), 3.75 (s, 3H), 3.66 (s,

6H); ¹³C NMR (75 MHz) d 160.6 (C), 158.5 (C), 152.9

(2· C), 138.9 (C), 130.9 (2· C), 126.9 (2· CH), 124.7

(C), 122.2 (C), 120.0 (CH), 113.8 (2· CH), 107.7 (2·

CH), 96.8 (C), 60.8 (CH₃), 55.9 (2· CH₃), 55.2 (CH₃),

51.4 (CH₃), 49.5 (CH₂) (one signal obscured or overlap-

ping); m_max(KBr) 1709, 1584, 1513, 1473, 1444, 1252,

1127, 1090 cm^ꢀ1; m/z (70 eV) 491 and 489 (both 20%,

M^+Å), 121 [100, (C₇H₆OCH₃)⁺].

5.1.12. 3-{[(tert-Butyl)dimethylsilyl]oxy}-4-methoxyphenyl-

boronic acid (25) and (5-iodo-2-methoxyphenoxy)(tert-

butyl)dimethylsilane (26). Step (i): Acetyl chloride

(7.33 g, 93.4 mmol) was added dropwise to a magneti-

cally stirred solution of 2-methoxyphenol (guaicol)

(10.0 g, 80.6 mmol) and pyridine (14.4 mL, 178 mmol)

in DCM (200 mL) maintained at 0 ꢁC under a nitrogen

atmosphere. The mixture was allowed to warm to 18 ꢁC

over 1 h, and stirring continued for 23 h. The resulting

solution was poured into ice-cold H₃PO₄(200 mL of a

Step (v): tert-Butyllithium (711 lL of a 1.7 M solution

in pentane, 1.21 mmol) was added dropwise to a mag-

netically stirred solution of (5-iodo-2-methoxyphen-

oxy)(tert-butyl)dimethylsilane (26) (200 mg, 0.6 mmol)

in toluene (5 mL) maintained at ꢀ78 ꢁC under a nitro-

gen atmosphere. After 30 min, trimethylborate

(152 mg, 1.5 mmol) was added to the reaction mixture

and the resulting solution allowed to warm to 18 ꢁC over

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

4635

1 h and, after 17 h, it was poured into brine-water

(30 mL of a 1:1 v/v mixture). The mixture was acidiﬁed

to pH 3 with 3 M HCl. The separated aqueous phase

was extracted with DCM (3· 100 mL), and the com-

bined organic extracts were dried (Na₂SO₄), ﬁltered

and concentrated under reduced pressure to aﬀord 3-

{[(tert-butyl)dimethylsilyl]oxy}-4-methoxyphenylboron-

ic acid (25) (123 mg, 80%) as a tan-coloured oil, and

containing of a variable mixture of anhydrides. Due to

the presence of these anhydrides, this material gave rise

to highly variable and complex NMR spectra. Acid 25

was used without puriﬁcation in the next step of the

reaction sequence.

nyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2-carboxyl-

ate (3) (39 mg, 99%) as colourless crystals, mp 168–

170 ꢁC (Found: M^+Å, 413.1472. C₂₂H₂₃NO₇requires

1

M^+Å, 413.1475). H NMR (300 MHz) d 9.02 (br s, 1H),

6.99 (d, J 2.8 Hz, 1H), 6.95 (m, 1H), 6.80 (m, 2H), 6.57

(s, 2H), 5.56 (s, 1H), 3.88 (s, 6H), 3.87 (s, 3H), 3.72

(s, 6H); ¹³C NMR (75 MHz) d 161.8 (C), 153.2 (2· C),

145.3 (2· C), 137.8 (C), 133.0 (C), 128.7 (C), 127.2 (C),

123.7 (C), 121.5 (C), 120.4 (CH), 116.8 (CH), 115.0

(CH), 110.5 (CH), 105.1 (2· CH), 60.9 (CH₃), 56.1

(3· CH₃), 51.6 (CH₃) (one signal obscured or overlap-

ping); m_max(KBr) 2926, 1736, 1699, 1244, 1126 cm^ꢀ1

m/z (70 eV) 413 (100%, M^+Å), 398(25), 381(42), 366(50).

;

5.1.13. Methyl 1-(4-methoxybenzyl)-4-(3-hydroxy-4-

methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrole-

2-carboxylate (4). Na₂CO₃(8.4 mL of a 2.0 M aqueous

solution, 16.8 mmol) was added to a solution of bromo-

pyrrole 24 (330 mg, 0.7 mmol), arylboronic acid (25)

(800 mg, 2.8 mmol) and Pd(PPh₃)₄(80 mg, 0.1 mmol)

in DMA (20 mL). The solution was deoxygenated (by

subjecting it to six cycles of exposure to 16 mm Hg pres-

sure then back ﬁlling with nitrogen) and stirred rapidly

while heating at 90 ꢁC for 110 h. The reaction mixture

was cooled to 18 ꢁC, acidiﬁed with 3 M HCl and extract-

ed with diethyl ether (4· 50 mL). The combined organic

extracts were washed with H₂O (12· 60 mL) before

being dried (MgSO₄), ﬁltered and concentrated under

reduced pressure to aﬀord a dark-coloured oil. This

material was subjected to ﬂash chromatography (silica,

2:5 v/v ethyl acetate/hexane elution), and concentration

of the appropriate fractions (R_f0.4) aﬀorded methyl

1-(4-methoxybenzyl)-4-(3-hydroxy-4-methoxyphenyl)-5-

3,4,5-trimethoxyphen-yl)-1H-pyrrole-2-carboxylate (4)

(14%) as a pale-yellow oil (Found: M^+Å, 533.2047.

C₃₀H₃₁NO₈requires M^+Å, 533.2050). ¹H NMR

(300 MHz) d 7.25 (s, 1H), 6.85 (d, J 2.1 Hz, 1H), 6.80–

6.77 (complex m, 4H), 6.67 (d, J, 8.4 Hz, 1H), 6.62

(dd, J 8.4 and 2.1 Hz, 1H), 6.32 (s, 2H), 5.48 (br s,

1H), 5.42 (br s, 2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.81 (s,

3H), 3.75 (s, 3H), 3.54 (s, 6H); ¹³C NMR (75 MHz) d

161.4 (C), 158.4 (C), 153.0 (2· C), 145.2 (C), 144.9

(C), 138.1 (C), 137.5 (C), 131.6 (C), 128.4 (C), 127.0

(2· CH), 126.7 (C), 122.8 (C), 121.8 (C), 119.3 (CH),

117.3 (CH), 113.9 (CH), 113.7 (2· CH), 110.4 (CH),

108.0 (2· CH), 60.9 (CH₃), 55.9 (3· CH₃), 55.2 (CH₃),

51.2 (CH₃), 48.6 (CH₂); m_max(KBr) 2925, 1703, 1583,

1513, 1248, 1095 cm^ꢀ1; m/z (70 eV) 533 (43%, M^+Å),

412 (8), 121 [100, (C₇H₆OCH₃)⁺].

5.1.15. Methyl 4-bromo-5-{[(tert-butyl)dimethylsilyloxy]-

4-methoxyphenyl}-1-(4-methoxybenzyl)-1H-pyrrole-2-

carboxylate (27). Phenyllithium (1.8 mL of a 1.8 M solu-

tion in cyclohexane/diethyl ether, 3.3 mmol) was added

dropwise to a magnetically stirred solution of compound

22 (1.32 g, 3.3 mmol) in dry THF (10 mL) maintained at

ꢀ78 ꢁC. Stirring continued for a further 0.2 h then anhy-

drous zinc chloride (491 mg, 3.6 mmol) was added in one

portion and the cold bath removed. Whilst warming,

compound 26 (1.20 g, 3.6 mmol) and Pd(PPh₃)₄

(378 mg, 0.3 mmol) were added to the reaction mixture

which was then heated and stirred at reﬂux for 96 h. After

this time, the reaction mixture was cooled, treated with

ﬂash chromatography-grade silica (3.6 g) and

concentrated under reduced pressure. The resulting free-

ﬂowing solid was added to the top of a ﬂash chromatogra-

phy column ﬁlled with silica, and the column was eluted

with 1:9 v/v ethyl acetate/hexane. Concentration of the

appropriate fractions (R_f0.2) aﬀorded methyl 4-bromo-

5-{[(tert-butyl)dimethylsilyloxy]-4-methoxyphenyl}-1-

(4-methoxybenzyl)-1H-pyrrole-2-carboxylate

(1.03 g, 56%) as a colourless and viscous gum (Found:

(27)

M^+Å, 559.1382. C₂₇H₃₄⁷⁹BrNO₅Si requires M^+Å,

1

559.1390). H NMR (300 MHz) d 7.10 (s, 1H), 6.85–

6.84 (complex m, 2H), 6.76–6.75 (complex m, 5H),

5.46 (s, 2H), 3.83 (s, 3H), 3.76 (s, 3H), 3.74 (s, 3H),

0.95 (s, 9H), 0.10 (s, 6H); ¹³C NMR (75 MHz) d 160.7

(C), 158.5 (C), 151.7 (C), 144.7 (C), 138.9 (C), 130.7

(C), 127.1 (2· CH), 124.3 (CH), 123.2 (CH), 122.1

(C), 121.8 (C), 120.1 (CH), 113.8 (2· CH), 111.6

(CH), 97.1 (C), 55.4 (CH₃), 55.1 (CH₃), 51.3 (CH₃),

49.2 (CH₂), 25.6 (3· CH₃), 18.4 (C), ꢀ4.7 (2· CH₃); m_max

(KBr) 2953, 2930, 2856, 1711, 1612, 1576, 1470, 1252,

1173 cm^ꢀ1; m/z (70 eV) 561 and 559 (6 and 5%, M^+Å),

504 and 502 (21 and 19), 121[100, (C₇H₆OCH₃)⁺].

5.1.14. Methyl 4-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-

trimethoxyphenyl)-1H-pyrrole-2-carboxylate (3). A solu-

tion of pyrrole 4 (50 mg, 0.1 mmol), anisole (101 mg,

0.9 mmol) and triﬂuoroacetic acid (1.0 mL) in DCM

(4.0 mL) was stirred at 37 ꢁC for 48 h, at which time

the excess triﬂuoroacetic acid was removed under a

stream of nitrogen. The residue was then concentrated

further under reduced pressure. The resulting dark-

brown oil was subjected to ﬂash chromatography (silica,

3:2 v/v ethyl acetate/hexane elution), and concentration

of the appropriate fractions (R_f0.4) aﬀorded a colourless

solid. Recrystallisation (diethyl ether/hexane) of this

material aﬀorded methyl 4-(3-hydroxy-4-methoxyphe-

5.1.16. 3,4,5-Trimethoxyphenylboronic acid (28). The title

compound was prepared, in 84% yield, from iodide 23

using the same protocol as employed for the conversion

26 ! 25. Like boronic acid 25, congener 28¹⁹was ob-

tained as a tan-coloured oil and as a variable admixture

with various anhydride forms. Due to the presence of

these anhydrides, this material gave rise to highly vari-

able and complex NMR spectra. Acid 28 was used with-

out puriﬁcation in the next step of the reaction sequence.

5.1.17. Methyl 1-(4-methoxybenzyl)-5-(3-hydroxy-4-

methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-

2-carboxylate (6). The title compound was prepared by

4636

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

cross-coupling precursors 27 and 28 using essentially the

same protocol as employed for the conversion

24 + 25 ! 4, but with a reaction time of 50 h. In this

manner methyl 1-(4-methyoxybenzyl)-5-(3-hydroxy-4-

methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyr-

role-2-carboxylate (6) (52%) was obtained as pale-yel-

low crystals, mp 47–49 ꢁC (Found: C, 67.59; H, 5.89; N,

2.44. C₃₀H₃₁NO₈requires C, 67.53; H, 5.86; N, 2.63%)

(R_f0.5 in 3:2 v/v ethyl acetate/hexane). ¹H NMR

(300 MHz) d 7.27 (s, 1H), 6.84–6.73 (complex m, 6H),

6.69 (dd, J 2.1 and 7.1 Hz, 1H), 6.40 (s, 2H), 5.62 (br

s, 1H), 5.44 (br s, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 3.79

(s, 3H), 3.75 (s, 3H), 3.63 (s, 6H); ¹³C NMR (75 MHz)

d 161.3 (C), 158.3 (C), 152.7 (2· C), 146.9 (C), 145.6

(C), 137.7 (C), 135.9 (C), 131.0 (C), 130.6 (C), 127.1

(2· CH), 124.5 (C), 123.2 (CH), 123.0 (C), 121.4 (C),

117.2 (CH), 117.0 (CH), 113.6 (2· CH), 110.6 (CH),

104.5 (2· CH), 60.7 (CH₃), 55.9 (CH₃), 55.6 (2· CH₃),

55.1 (CH₃), 51.1 (CH₃), 48.2 (CH₂); m_max(KBr) 3318,

2923, 1699, 1385, 1251, 1033 cm^ꢀ1; m/z(70 eV) 533

(72%, M^+Å), 121 [100, (C₇H₆OCH₃)⁺].

(KBr) 1703, 1513, 1459, 1444, 1249, 1219, 1176 cm^ꢀ1

;

m/z (70 eV) 487 (M^+Å, 61%), 121[100, (C₇H₆OCH₃)⁺].

5.1.20. Methyl 4-(benzo[d][1,3]dioxol-6-yl)-5-(3-hydroxy-

4-methoxyphenyl)-1H-pyrrole-2-carboxylate (7). The ti-

tle compound was prepared from precursor 8 using the

same protocol as employed in the conversion 4 ! 3. In

this manner methyl 4-(benzo[d][1,3]dioxol-6-yl)-5-(3-hy-

droxy-4-methoxyphenyl)-1H-pyrrole-2-carboxylate (7)

(60%) was obtained as colourless crystals, mp 145–

147 ꢁC (Found: C, 65.55; H, 4.98; N, 4.14. C₂₀H₁₇NO₆

requires C, 65.39; H, 4.66; N, 3.81%), (R_f0.4 in 2:5

1

v/v ethyl acetate/hexane). H NMR (500 MHz) d 9.27

(br s, 1H), 7.00 (d, J 2.0 Hz, 1H), 6.96 (d,J 3.0 Hz,

1H), 6.85 (m, 1H), 6.81–6.75 (complex m, 4H), 5.94 (s,

2H), 3.90 (s, 3H), 3.88 (s, 3H) (signal due to OH not ob-

served); ¹³C NMR (75 MHz) d 161.7 (C), 147.5 (C),

146.5 (C), 146.1 (C), 145.7 (C), 132.9 (C), 129.4 (C),

125.1 (C), 123.3 (C), 121.9 (CH), 121.3 (C), 120.0

(CH), 116.9 (CH), 113.9 (CH), 110.8 (CH), 109.1

(CH), 108.3 (CH), 100.8 (CH₂), 55.9 (CH₃), 51.6

(CH₃); m_max(KBr) 1697, 1684, 1464, 1259, 1085,

1038 cm^ꢀ1; m/z367 (70 eV) (100%, M^+Å), 335 (77).

5.1.18. Methyl 5-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole-2-carboxylate (5). The ti-

tle compound was prepared from precursor 6 using the

same protocol as employed in the conversion 4 ! 3. In

this manner methyl 5-(3-hydroxy-4-methoxyphenyl)-4-

(3,4,5-trimethoxyphenyl)-1H-pyrrole-2-carboxylate (5)

(62%) was obtained as colourless needles, mp 172–

174 ꢁC (Found: C, 63.73; H, 5.74; N, 3.65. C₂₂H₂₃NO₇

requires C, 63.92; H, 5.61; N, 3.39%) (R_f0.5 in 3:2 v/v

5.2. X-ray crystallographic studies

5.2.1. Data collection. Intensity data were collected on a

Rigaku AFC6R diﬀractometer using the x ꢀ 2h scan

technique to a maximum 2h value of 120ꢁ. The weak

reﬂections [I < 10.0r(I)] were rescanned (maximum of

four scans), and the counts were accumulated to ensure

good counting statistics. Stationary background counts

were recorded on each side of the reﬂection. The ratio

of peak to background counting time was 2:1.

1

ethyl acetate/hexane). H NMR (300 MHz) d 9.04 (br

s, 1H), 7.04 (d, J 2.8 Hz, 1H), 7.02 (d, J 2.2 Hz, 1H),

6.90 (dd, J 2.2 and 8.4 Hz, 1H), 6.81 (d, J 8.4 Hz, 1H),

6.54 (s, 2H), 5.72 (s, 1H), 3.91 (s, 3H), 3.90 (s, 3H),

3.86 (s, 3H), 3.72 (s, 6H); ¹³C NMR (75 MHz) d 161.8

(C), 153.0 (2· C), 146.6 (C), 145.6 (C), 136.5 (C),

133.2 (C), 131.0 (C), 125.0 (C), 123.4 (C), 121.4 (C),

120.5 (CH), 116.6 (CH), 113.9 (CH), 110.7 (CH), 105.5

(2· CH), 60.9 (CH₃), 56.0 (CH₃), 55.9 (2· CH₃), 51.7

(CH₃); m/z (70 eV) 413(100%, M^+Å), 398(77), 381(42),

366(57).

5.2.2. Crystal data. Compound 3: C₂₂H₂₃NO₇,

ꢀ

M = 413.43, T = ꢀ80 ꢁC, triclinic, space group P1,

˚

Z = 2, a = 8.767(1), b = 9.197(2), c = 13.617(1) A,

a = 98.23(2)ꢁ,

b = 106.90(1)ꢁ,

c = 99.86(1)ꢁ,

3

V = 1013.2(4) A , D_x= 1.355 Mg m^ꢀ3, 3011 unique data

(2h_max= 120.1ꢁ), 2665 with I > 2.0r(I); R = 0.037,

R_w= 0.053, S = 2.352.

˚

5.1.19. Methyl 1-(4-methoxybenzyl)-4-(benzo[d][1,3]diox-

ol-6-yl)-5-(3-hydroxy-4-methoxy-phenyl)-1H-pyrrole-2-

carboxylate (8). The title compound was prepared by

cross-coupling precursors 27 and 29 using the same pro-

tocol as employed for the conversion 24 + 25 ! 4, but

with a reaction time of 38 h. In this manner methyl

1-(4-methoxybenzyl)-4-(benzo-[d][1,3]dioxol-6-yl)-5-(3-

hydroxy-4-methoxyphenyl)-1H-pyrrole-2-carboxylate (8)

(87%) was obtained as a pale-yellow oil (Found: M^+Å,

487.1632. C₂₈H₂₅NO₇requires M^+Å, 487.1631), (R_f0.3

Compound 7: C₂₀H₁₇NO₆, M = 367.36, T = ꢀ80 ꢁC, tri-

ꢀ

clinic, space group P1, Z = 2, a = 8.8204(6),

˚

b = 9.433(1), c = 11.803(1) A, a = 111.838(8)ꢁ,

3

˚

b = 91.606(8)ꢁ, c = 100.445(8)ꢁ, V = 891.6(2) A , D_x=

1.368 Mg m^ꢀ3, 2654 unique data (2h_max= 120.1ꢁ), 2259

with I > 2.0r(I); R = 0.035, R_w= 0.041, S = 2.677.

Compound 17: C₁₉H₁₆BrNO₂, M = 370.24, T = ꢀ80 ꢁC,

monoclinic, space group P2₁/c, Z = 4, a = 9.382(2),

˚

b = 19.034(3),

c = 10.135(2)

A,

b = 116.37(1)ꢁ,

1

3

V = 1621.5(5) A , D_x= 1.517 Mg m^ꢀ3, 2425 unique data

(2h_max= 120.1ꢁ), 2098 with I > 3.0r(I); R = 0.027,

R_w= 0.028, S = 2.424.

˚

in 1:5 v/v ethyl acetate/hexane). H NMR (300 MHz) d

7.19 (s, 1H), 6.80–6.63 (complex m, 10H), 5.88 (s, 2H),

5.61 (br s, 1H), 5.42 (s, 2H), 3.90 (s, 3H), 3.77 (s, 3H),

3.75 (s, 3H); ¹³C NMR (75 MHz) d 161.4 (C), 158.3

(C), 147.2 (C), 146.8 (C), 145.5 (2· C), 137.4 (C), 131.1

(C), 129.3 (C), 127.1 (2· CH), 124.3 (C), 123.1 (CH),

123.0 (C), 121.4 (C), 121.0 (CH), 117.6 (CH), 117.0

(CH), 113.6 (2· CH), 110.5 (CH), 108.3 (CH), 108.1

(CH), 100.6 (CH₂), 55.7 (CH₃), 55.1 (CH₃), 51.1 (CH₃),

48.2 (CH₂) (one signal obscured or overlapping); m_max

5.2.3. Data reduction. Absorption corrections were ap-

plied. The data were corrected for Lorentz and polarisa-

tion eﬀects.

Structure solution and reﬁnement: The structures were

solved by direct methods²³and expanded using Fourier

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

4637

techniques. The non-hydrogen atoms were reﬁned aniso-

tropically. Hydrogen atom positions were reﬁned for

compounds 7 and 17. All calculations were performed

using the teXsan²⁴crystallographic software package

of Molecular Structure Corporation.

examined under a light microscope, with mitotic cells

deﬁned as those with condensed chromosomes and no

nuclear membrane. Four hundred cells were counted

for each condition examined.

Data deposition: Atomic coordinates, bond lengths and

angles, and displacement parameters have been deposit-

ed at the Cambridge Crystallographic Data Centre

(CCDC reference numbers 289493, 289494 and 289495

for compounds 3, 7 and 17, respectively). These data

can be obtained free-of-charge via the Internet at

Acknowledgments

We thank the Australian Research Council for ﬁnancial

support including the provision of a PhD Scholarship to

D.J.W., Mr. Tony Herlt (RSC, ANU) for expert assis-

tance with the operation of HPLC instrumentation

and Mr. Chris Blake (RSC, ANU) for help with the

acquisition of certain NMR data.

www.ccdc.cam.ac.uk/data_request/cif,

by

emailing

data_request@ccdc.cam.ac.uk, or by contacting The

Cambridge Crystallographic Data Centre, 12 Union

Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

References and notes

5.3. Biological studies

1. Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Curr. Pharm.

Des. 2005, 11, 1655.

2. Cirla, A.; Mann, J. Nat. Prod. Rep. 2003, 20, 558, and

references cited therein.

3. Griggs, J.; Metclafe, J. C.; Hesketh, R. Lancet Oncol.

2001, 2, 82.

5.3.1. Tubulin assays. Bovine brain tubulin was puriﬁed

as described previously.²⁵Assays for the evaluation of

tubulin assembly²⁶and for the binding of [³H]colchicine

to tubulin²⁷were performed as described previously.

The tubulin assembly assay included a preincubation

of the potential inhibitor, at varying concentrations,

with 10 lM tubulin prior to addition of the GTP re-

quired for assembly to occur. After GTP addition, reac-

tions were followed turbidimetrically for 20 min at

30 ꢁC, and the IC₅₀value was deﬁned as the compound

concentration, obtained by interpolation between the

data points, that inhibited the extent of assembly by

50%. In the colchicine binding assay, reaction mixtures

contained 1.0 lM tubulin, 5.0 lM [³H]colchicine, and

the potential inhibitor at 5 lM. Reaction mixtures were

incubated for 10 min at 37 ꢁC, a time point at which the

colchicine binding reaction is about 50% complete.

4. See, for example. (a) Pettit, G. R.; Rhodes, M. R.; Herald,

D. L.; Hamel, E.; Schmidt, J. M.; Pettit, R. K. J. Med.

Chem. 2005, 48, 4087; (b) Pettit, G. R.; Anderson, C. R.;

Gapud, E. J.; Jung, M. K.; Knight, J. C.; Hamel, E.;

Pettit, R. K. J. Nat. Prod. 2005, 68, 1191.

5. Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.;

Rondanin, R.; Piccagli, L.; Baruchello, R.; Rossi, M.;

Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.; Jung, M.

K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Di

Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M.

J. Med. Chem. 2005, 48, 723, and references cited therein.

6. Hadimani, M. B.; Hua, J.; Jonklass, M. D.; Kessler, R. J.;

Sheng, Y.; Olivares, A.; Tanpure, R. P.; Weiser, A.;

Zhang, J.; Edvardsen, K.; Kane, R. R.; Pinney, K. G.

Bioorg. Med. Chem. Lett. 2003, 13, 1505.

5.3.2. Cytotoxicity and mitotic index assays. The Burkitt

lymphoma CA46 cells were grown in 5-mL ﬂasks, and

IC₅₀values were determined by counting the cells in a

Coulter counter (Beckman Coulter, Fullerton, CA),

with the IC₅₀value deﬁned as the drug concentration

that reduced increase in cell number by 50% at 24 h after

drug addition. The cells were grown in RPMI 1640

medium supplemented with 17% fetal bovine serum

and 2 mM ^L-glutamine at 37 ꢁC in a 5% CO₂atmo-

sphere. The dimethyl sulfoxide concentration was 0.1%

(v/v) in all culture ﬂasks.

7. (a) For a very recent and useful review of this area see: (a)

Current Medicinal Chemistry—Anti-Cancer Agents; Bailly,

C., Ed.; Bentham Science Publishers Ltd: Sharjah, United

Arab Emirates, 2004; Vol. 4, p 363; See, also (b) Marﬁl,

´

M.; Albericio, F.; Alvarez, M. Tetrahedron 2004, 60, 8659,

for an up-to-date commentary on the biological proﬁles of

this group of natural products.

8. (a) Banwell, M. G.; Herbert, K. A.; Buckleton, J. R.;

Clark, G. R.; Rickard, C. E. F.; Lin, C. M.; Hamel, E.

J. Org. Chem. 1988, 53, 4945; (b) Banwell, M. G.; Cameron,

J. M.; Collis, M. P.; Crisp, G. T.; Gable, R. W.; Hamel, E.;

Lambert, J. N.; Mackay, M. F.; Reum, M. E.; Scoble, J. A.

Aust. J. Chem. 1991, 44, 705; (c) Banwell, M. G.; Cameron,

J. M.; Corbett, M.; Dupuche, J. R.; Hamel, E.; Lambert, J.

N.; Lin, C. M.; Mackay, M. F. Aust. J. Chem. 1992, 45,

1967; (d) Banwell, M. G.; Fam, M.-A.; Gable, R. W.;

Hamel, E. J. Chem. Soc., Chem. Commun. 1994, 2647; (e)

Banwell, M. G.; Hamel, E.; Ireland, N. K.; Mackay, M. F.

Heterocycles 1994, 39, 205; (f) Banwell, M. G.; Flynn, B. L.;

Willis, A. C.; Hamel, E. Aust. J. Chem. 1999, 52, 767; (g)

Banwell, M. G.; Edwards, A. J.; Jolliﬀe, K. A.; Smith, J. A.;

Hamel, E.; Verdier-Pinard, P. Org. Biomol. Chem. 2003, 1,

296; (h) Banwell, M. G.; Bezos, A.; Burns, C.; Kruszelnicki,

I.; Parish, C. R.; Su, S.; Sydnes, M. O. Bioorg. Med. Chem.

Lett. 2006, 16, 181.

The mitotic index in the Burkitt cell cultures was also

determined at 24 h. About 4.5 mL of cell culture medi-

um was centrifuged at 1000 rpm for 1 min. The pelleted

cells were resuspended in 5 mL of phosphate-buﬀered

saline at room temperature, and the cells were harvested

by centrifuging the suspension as before. The cell pellet

was suspended in 0.5 mL of half-strength phosphate-

buﬀered saline, and the cells were allowed to swell for

10 min. The cells were then ﬁxed by adding 6 mL of

0.5% acetic acid/1.5% ethanol. After 30 min, the cells

were harvested by centrifuging as before. The cells were

resuspended in 1:3 v/v acetic acid/ethanol, and a droplet

of the cell suspension was spread on the slide. The slide

was air-dried and stained with Giemsa. The slide was

9. We have previously reported on the preparation of the

isomeric methyl 3,4-diaryl-1H-pyrrole-2-carboxylates as

lamellarin/colchicine hybrids and, therefore, potential

4638

M. G. Banwell et al. / Bioorg. Med. Chem. 14 (2006) 4627–4638

anti-mitotic agents: Banwell, M. G.; Flynn, B. L.; Hamel,

E.; Hockless, D. C. R. Chem. Commun. 1997, 207.

15. Anderson, H. J.; Griﬃths, S. J. Can. J. Chem. 1967, 45,

2227.

10. Certain 4,5-diarylated pyrroles incorporating electron-

withdrawing substituents have been investigated as anti-

inﬂammatory agents or as drugs for the treatment of

complications arising from diabetes: (a) Wilkerson, W. W.;

Galbraith, W.; Gans-Brangs, K.; Grubb, M.; Hewes, W.

E.; Jaﬀee, B.; Kenney, J. P.; Kerr, J.; Wong, N. J. Med.

Chem. 1994, 37, 988, and references cited therein; (b)

Wilkerson, W. W.; Copeland, R. A.; Covington, M.;

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strategy see: Dharap, S. S.; Wang, Y.; Chandna, P.;

Khandare, J. J.; Qiu, B.; Gunaseelan, S.; Sinko, P. J.;

Stein, S.; Farmanfarmaian, A.; Minko, T. Proc. Natl.

Acad. Sci. U.S.A. 2005, 102, 12962, Also see Ref. 4a.

12. For an up-to-date ‘mini-review’ on palladium-catalysed

cross-coupling reactions involving pyrroles, see: Banwell,

M. G.; Goodwin, T. E.; Ng, S.; Smith, J. A.; Wong, D. J.

Eur. J. Org. Chem. accepted for publication.

1978.

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Edmunds, J. J. PCT Int. Appl. 1996, WO9626195.

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observations.

21. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,

2923.

22. Armarego, W. L. F.; Perrin, D. D. Puriﬁcation of

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quez, B.; Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J.

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4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin A-4/lamellarin T hybrids: Synthesis and evaluation as anti-mitotic and cytotoxic agents

DOI: 10.1016/j.bmc.2006.02.018

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Full text of DOI:10.1016/j.bmc.2006.02.018

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