Antimitotic and cell growth inhibitory properties of combretastatin A-4-like ethers

Article abstract of DOI:10.1016/S0960-894X(00)00596-5

A series of diarylamines, diaryl and arylbenzyl ethers based on combretastatin A-4 was prepared and evaluated for anticancer activity. 2-Methoxy-5-(3',4',5'-trimethoxyphenoxymethyl)phenol was the most active (IC₅₀, K562 20 nM) and caused significant G2/M cell cycle arrest. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00596-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 51±54
Antimitotic and Cell Growth Inhibitory Properties of
Combretastatin A-4-like Ethers
Nicholas J. Lawrence,a,* David Rennison,a,b Meiki Woo,a Alan T. McGownb and
John A. Had®eldb
aDepartment of Chemistry, UMIST, PO Box 88, Manchester M60 1QD, UK
bCRC Department of Drug Development and Imaging, Paterson Institute for Cancer Research, Christie Hospital NHS Trust,
Wilmslow Road, Manchester M20 4BX, UK
Received 16 August 2000; revised 16 October 2000; accepted 20 October 2000
AbstractÐA series of diarylamines, diaryl and arylbenzyl ethers based on combretastatin A-4 was prepared and evaluated for
anticancer activity. 2-Methoxy-5-(30,40,50-trimethoxyphenoxymethyl)phenol was the most active (IC50, K562 20 nM) and caused
signi®cant G2/M cell cycle arrest. # 2000 Elsevier Science Ltd. All rights reserved.
Combretastatin A-4 (1a), isolated from the African
bush willow, Combretum ca€rum,1 shows exciting
potential as an anticancer agent binding strongly to
tubulin at a site shared with, or close to, the colchicine
binding site.2 It inhibits cell growth at low concentra-
tions (IC50, P388 murine leukaemia cell line 2.6 nM) and
shares many structural features common to other tubu-
lin-binding agents3 such as colchicine and podophylo-
toxin. The phosphate salt 2,4 which has better water
solubility than 1a, is soon to enter phase II clinical
trials.5 It is the ability of 1a and 2 to damage tumour
vasculature, thereby e€ectively starving tumours of
nutrients, which makes them such exciting molecules.6
It is now clear that inhibition and modulation of
angiogenesis and vasculature function will have a sig-
ni®cant impact on the clinical treatment of cancer.7
Evidently lacking from this series were the ether and
amine analogues of 1a where X=O or NH and OCH2.
We now report both the synthesis and biological activity
of such a series of compounds.
The spatial relationship between the two aromatic rings
of combretastatin A-4, colchicine and similar drugs is
an important structural feature that determines their
ability to bind to tubulin.8 We have already reported the
synthesis and biological evaluation of chalcones similar
to 1a. Chalcone 3, the most potent compound of the
series, is highly cytotoxic and binds to tubulin at the
colchicine-binding site.9 It is clear that the X group in
the combretastatin A-4-like compounds 1±j is important
in determining their biological activity (Table 1).10
Table 1. Cell growth inhibitory properties of diaryl containing
compounds 1a±j
1
X
IC50/mM
1
X
IC50/mM
a
b
c
d
e
Z-CHˆCH-11 0.0026a
E-CHˆCH-13 0.16a
e
f
g
h
j
(S,S)-C(OH)HC(OH)H-12
(R,R)-C(OH)HC(OH)H-12
-(RS)-C(OH)HCH2-15
-(RS)-CH2CH(OH)-12
Ðd,17
2a
19a
-CꢀC-14
21b
0.033a
33a
-CH2CH2-13 0.11a
-CO-16
0.01a
>5 0 c
aP388 murine leukemia cell line.
*Corresponding author at present address: Department of Chemistry,
Cardi€ University, PO Box 912, Main Building, Park Place, Cardi€
CF10 3TB, UK. Tel.: +44-29-2087-4023; fax:+44-29-2087-4030;
e-mail: lawrencenj1@cardi€.ac.uk
bK562 human chronic myelogenous leukaemia cell line.
cL1210 murine leukemia cell line.
dThe aryl rings are bonded directly to each other.
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00596-5

52
N. J. Lawrence et al. / Bioorg. Med. Chem. Lett. 11 (2001) 51±54
The target ether 4 was prepared via the new copper-
catalyzed Ullmann-type coupling recently described by
Buchwald and co-workers (Scheme 1).18 This
involved the treatment of benzyl-protected phenol 7
with 3,4,5-trimethoxybromobenzene. We found that the
yield of the ether 8 was improved if xylene was used as
the reaction solvent. The phenol 7 was prepared by
protection of isovanillin (5 ! 6), followed by sequential
Baeyer±Villiger oxidation (MCPBA) and ester metha-
nolysis. Deprotection of the benzyl ether 8, via palla-
dium-catalyzed hydrogenolysis, proceeded in excellent
yield to reveal the required diaryl ether 4.
In a similar fashion we prepared two other diaryl ethers
14 and 15 and the diarylamine 16, from the commer-
cially available phenols or aniline and aryl halides via
the Buchwald protocols. The yields of these coupling
reactions are reported in Table 2.
Table 2. Synthesis of diaryl ethers and anilines Ar1XAr2
Ar1
Ar2
X
Yield (%)
14
15
16
3,4,5-(MeO)3C6H2
3,4-Me2C6H4
3,4,5-(MeO)3C6H2
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
O
O
NH
84
58
60
We also prepared the two benzyl ethers 17 and 18,
which have the two aryl groups separated by two atoms,
thereby mimicking more closely the arrangement pre-
sent in combretastatin A-4 (Schemes 3 and 4). Firstly
benzyl ether 20 was prepared from the phenol 1920 and
3,4,5-trimethoxybenzyl bromide, itself made from
commercially available 3,4,5-trimethoxybenzyl alcohol.
Scheme 1. Reagents and conditions: (i) PhCH2Cl, K2CO3, DMF, rt
18 h. 82%; (ii) MCPBA, CH2Cl2, rt, 2 h, 84%; (iii) NaOMe, MeOH,
rt, 30 min, 79%; (iv) 3,4,5-(MeO)3C6H2Br, Cu, Ce2CO3, xylene,
140 ꢁC, 4 days, 52%; (v) Pd/C (10%), H2, CH2Cl2, 94%.
The combretastatin A-4 diaryl aniline analogue 13 was
prepared in a similar manner via Pd(II)-catalyzed
coupling of 3,4,5-trimethoxybromobenzene and the
benzyl-protected aniline 11 using another of the
Buchwald protocols (Scheme 2).19
Scheme 3. Reagents and conditions: (i) 3,4,5-(MeO)3C6H2CH2Br,
K2CO3, DMF, rt 18 h, 41%; (ii) H2O2, H2SO4 (cat.), MeOH, rt,
2 days, 59%; (iii) NaBH4, EtOH, rt, 30 min, 94%.
We chose to use a formyl group as a latent hydroxyl
group since a strategy incorporating a benzyl protecting
group, as in the synthesis of 4, would clearly present
selectivity problems. Acid-catalyzed oxidation of the
aldehyde 20 with hydrogen peroxide, a protocol devel-
oped by Matsumoto and co-workers,21 directly gave the
phenol 17. This reaction was superior to the MCPBA
promoted Baeyer±Villiger reaction, as separation from
by-products was much more convenient. Reduction of
the aldehyde 20 provided another ether analogue, the
alcohol 21.
Scheme 2. Reagents and conditions: (i) K2CO3, PhCH2Br, DMF, rt,
ꢁ
.
18 h, 98%; (ii) SnCl2 2H2O, EtOH:EtOAc 1:1, 70 C, 3 h, 88%; (iii)
3,4,5-(MeO)3C6H2Br, NaO-tert-Bu, Pd(OAc)2 (8 mol%), DPEphos
(12 mol%), toluene, 80 ꢁC, 18 h, 56%; (iv) Pd/C (10%, cat.), EtOAc,
H2 (1 atm), rt, 5 days, 80%.
The aniline 11 was prepared from the commercially
available nitrophenol 9 by sequential benzylation and
reduction with tin(II) chloride. The coupling of the tri-
methoxybromobenzene and aniline 11 proceeded
smoothly in the presence of the bisphosphine DPEphos
and palladium(II) acetate. Deprotection of the benzyl
group within the aniline 12 gave the required
combretastatin analogue 13.
The regioisomeric benzyl ether 18 was prepared, via the
Williamson ether synthesis (Scheme 4), from commer-
cially available 3,4,5-trimethoxyphenol and the sub-
stituted benzyl chloride 24 (prepared from allyl-
protected isovanillin 23 by sequential treatment with
sodium borohydride and thionyl chloride). Deprotec-
tion of the allyl group in 25 with Wilkinson's catalyst

N. J. Lawrence et al. / Bioorg. Med. Chem. Lett. 11 (2001) 51±54
Table 3. Cell growth inhibitiona against the K562 cell line
53
and DABCOTM, using conditions developed by Corey
and Suggs,22 gave the required benzyl ether 18. Dihy-
droxylation of the allyl ether 25 gave the diol 26, which
is e€ectively a glyceryl analogue of Z-1, which we hoped
would possess improved water solubility. Oxidative
cleavage of this diol using sodium periodate gave the
aldehyde-containing analogue 27.
Compound
IC50 (mM)
Compound
IC50 (mM)
18
4
0.02
0.05
0.12
0.17
0.38
2.5
16
12
15
25
8
3
15
24
30
51
60
90
17
20
14
27
13
21
26
2.6
aAs measured by the MTT assay after 5 days incubation of the drug
with the cells cultured at 37 ꢁC.
relative to the untreated control, consistent with the
behaviour of tubulin-binding agents. Strangely, the most
potently antimitotic compound was the aniline 13, even
though it is moderately cytotoxic. Further experiments
to determine the origin of its antimitotic e€ect reveal
that it is probably not binding to tubulin. It neither
prevents the assembly of microtubules nor binds to the
colchicine binding site (Table 5).
Table 4. E€ects upon the cell cyclea
Compound
G0±G1
S-phase
G2/M
Control
25.3
4.3
3.4
3.8
3.5
9.1
47.2
22.3
9.4
17.9
14.0
33.1
27.5
73.4
87.2
78.3
82.5
57.8
4
13
17
18
20
Scheme 4. Reagents and conditions: (i) CH2ˆCHCH2Br, K2CO3,
DMF, rt 18 h, 95%; (ii) NaBH4, EtOH, rt, 30 min, 99%; (iii) SOCl2, rt,
120 min, 91%; (iv) 3,4,5-(MeO)3C6H2OH, NaH, DMF, rt, 18 h, 99%;
(v) (PPh3)3RhCl (5 mol%), DABCOTM (1 eq), EtOH, re¯ux, 18 h,
59%; (vi) potassium osmate (0.2 mol%), NMNO (1.1. eq), aq acetone,
rt, 18 h, 86%; (vii) NaIO4, Et2O:H2O 2:1, rt, 18 h, 84%.
aAs measured by ¯ow cytometry (according to the methods described
in ref 25).
The cell growth inhibitory properties of the com-
bretastatin A-4 analogues (summarized in Table 3) were
determined in the K562 human chronic myelogenous
leukaemia cell line using the MTT assay as detailed by
Edmondson et al.23 We have reported the full details of
this procedure elsewhere.24 The IC50 value represents
the concentration which results in a 50% decrease in cell
growth after 5 days incubation.
Table 5. E€ects upon tubulin binding
Tubulin assemblya
Colchicine displacementb
IC50 (mM)
IC50 (mM)
4
13
17
18
20
16.3
>100
22.5
6.6
36
298
226
36
0.6
4
aAssembly of tubulin was carried out as previously described (ref 26).
The IC50 values shown represent the concentrations which cause a
50% decrease in tubulin assembly as measured by an increase in
turbidity at 350 nm.
bCompetition for the colchicine binding site was carried out as
previously described (ref 26). The IC50 values shown represent
the concentrations which cause a 50% decrease in the binding of
3H-colchicine from puri®ed porcine tubulin.
The ether 4 is remarkably active; the poorer activity of
both 14 and 8 shows that the B-ring 30 hydroxyl group is
bene®cial. Indeed it is gratifying to see that the com-
bretastatin A-4 substitution pattern produces the most
active diaryl ether. This is also the case for the diaryl
amines; 13 is more active than 12 and 16. However the
amines are signi®cantly less active than their ether
counterparts (cf. 4 and 13; 14 and 16). The arylbenzyl
ethers 17 and 18 are both signi®cantly active. The
potent activity of 18 is clearly noteworthy. Again most
of the analogues lacking the B-ring 30 hydroxyl group
(25±27) are much less active. The cytotoxicity of the
aldehyde 20 is relatively high and comparable to that
of the phenol 17. Reduction of the aldehyde (20!21)
results in a dramatic loss of activity.
All of the other compounds inhibit the polymerization
of tubulin. The most active compound in the tubulin
assays, the aldehyde 20, binds exceptionally strongly to
the colchicine binding site. Its ability to prevent the
polymerization of tubulin is impressive. Indeed, the
activity is clearly comparable with that of com-
bretastatin A-4 (1a) itself (IC50 0.5, 2 mM).27,28 However
since 20 is much less cytotoxic than CA-4 it may have
improved in vivo properties. These studies are currently
underway. It is nevertheless tempting at this stage to
speculate that the binding of 20 to tubulin involves
The e€ects of the most active compounds upon the cell
cycle were measured by ¯ow cytometry. The results in
Table 4 show that all the compounds assessed caused
signi®cant arrest of the cell cycle at the G2/M point,

54
N. J. Lawrence et al. / Bioorg. Med. Chem. Lett. 11 (2001) 51±54
covalent reaction of the aldehyde group, by the forma-
tion of a Schi€'s base. The poorer cellular activity
compared to that of 4 and 18 may be due to unspeci®c
reaction with non-tubulin amine residues and/or
biodegradation of the aldehyde functionality.
Invest. New Drugs 1997, 15, 49. O'Reilly, M. S. Invest. New
Drugs 1997, 15, 5.
8. McGown, A. T.; Fox, B. W. Anti-Cancer Drug Des. 1989,
3, 249. Aleksandrzak, K.; McGown, A. T.; Had®eld, J. A.
Anti-Cancer Drugs 1998, 9, 545.
9. Ducki, S.; Forrest, R.; Had®eld, J. A.; Kendall, A.; Lawr-
ence, N. J.; McGown, A. T.; Rennison, D. Bioorg. Med.
Chem. Lett. 1998, 8, 1051.
10. For recent examples of the synthesis of combretastatin A-
4-like molecules see Banwell, M. G.; Flynn, B. L.; Willis, A.
C.; Hamel, E. Aus. J. Chem. 1999, 52, 767. Ohsumi, K.;
Hatanaka, T.; Fujita, K.; Kakagawa, R.; Fukuda, Y.; Nihei,
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In conclusion we have shown that the ether analogues
of CA-4 share its impressive activity. The in vivo
assessment of these agents is ongoing. The results of
these studies will be reported in due course.
Acknowledgements
11. Had®eld, J. A.; McGown, A. T. Synth. Commun. 1998, 28,
1421.
We thank the EPSRC for a studentship (to DR) and for
Research Grants (GR/L52246: NMR spectrometer;
GR/L84391: chromatographic equipment) and the
EPSRC Chemical Database Service at Daresbury.29 We
thank the Cancer Research Campaign and the Chemis-
try Department of UMIST for additional support of
this work and Sally Haran and Tim Ward of the PICR
Cell Culture Unit for valuable assistance during the cell
growth inhibition experiments.
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