Chimeric microtubule disruptors

Article abstract of DOI:10.1039/c002558e

A chimeric approach is used to discover microtubule disruptors with excellent in vitro activity and oral bioavailability; a ligand-protein interaction with carbonic anhydrase that enhances bioavailability is characterised by protein X-ray crystallography. Dosing of a representative chimera in a tumour xenograft model confirms the excellent therapeutic potential of the class. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c002558e

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Chemical Communications
www.rsc.org/chemcomm
Volume 46 | Number 17 | 7 May 2010 | Pages 2873–3036
ISSN 1359-7345
COMMUNICATION
Barry V. L. Potter et al.
Chimeric microtubule disruptors

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Chimeric microtubule disruptorsw
Mathew P. Leese,a Fabrice Jourdan,a Meriel R. Kimberley,a Gyles E. Cozier,a
Nethaji Thiyagarajan,b Chloe Stengel,c Sandra Regis-Lydi,d Paul A. Foster,c
Simon P. Newman,c K. Ravi Acharya,b Eric Ferrandis,d Atul Purohit,c
c
Michael J. Reedz and Barry V. L. Potter*a
Received (in Cambridge, UK) 8th February 2010, Accepted 4th March 2010
First published as an Advance Article on the web 20th March 2010
DOI: 10.1039/c002558e
A chimeric approach is used to discover microtubule disruptors
with excellent in vitro activity and oral bioavailability; a
ligand–protein interaction with carbonic anhydrase that
enhances bioavailability is characterised by protein X-ray
crystallography. Dosing of a representative chimera in a tumour
xenograft model confirms the excellent therapeutic potential of
the class.
Fig. 1 Structures of 2-methoxyestradiol 1 and colchicine 2. The
pharmacophore elements used to construct the chimera are boxed.
Microtubule disruptors inhibit the normal dynamic assembly
of a,b-tubulin into polymeric microtubules, a process key to
mitosis, triggering cell cycle arrest and ultimately cell death.1
Their cytotoxic effects are thus targeted towards rapidly
dividing cancer cells. Although arguably the most successful
class of anti-cancer drugs, issues with structural complexity,
oral bioavailability, therapeutic window, formulation and
acquired resistance remain.2 Herein, we outline a chimeric
approach to the discovery of simple small molecule inhibitors
of both tubulin polymerization and angiogenesis which
overcome many of the problems associated with existing
clinical agents.
overcome the limitations of parent compounds. Thus, a
bicyclic motif bearing elements of the steroidal AB-rings of
2-MeOE2 was attached to a trimethoxyaryl group. Nitrogen
incorporation in the bicycle provided a logical point through
which the trimethoxyaryl motif could be attached and also
allows for salification and enhanced aqueous solubility.
A tetrahydroisoquinoline (THIQ) mimic was thus targeted
and synthesized from benzyl vanillin 3 (Scheme 1). The desired
THIQ 4 core was accessed via a Pomeranz–Fritsch approach.6
N-Benzylation of 4 followed by hydrogenation delivered the
prototypical chimeras 5a and 5b. The compounds were
evaluated for their ability to inhibit DU-145 (prostate cancer)
and MDA MB-231 (breast cancer) cell proliferation (Table 1).
While 5a showed no significant activity at concentrations up to
Our design strategy integrates pharmacophore elements
from two series of colchicine site binding natural products
that disrupt microtubule dynamics. 2-Methoxyestradiol
(2-MeOE2, 1) is an anti-tumor agent of poor oral bioavailability
with dual anti-proliferative and anti-angiogenic actions.3 Its
apparent lack of toxicity and mechanism of action led to
numerous clinical trials, yet these were hampered by rapid
inactivating metabolism and conjugative clearance.4 Colchicine 2
is one of a number of potent natural product microtubule
disruptors incorporating a trimethoxyaryl motif.5 Its application
as an anti-cancer agent is compromised by a low therapeutic
index. By incorporation of the sub-pharmacophores (Fig. 1)
from 1 and 2 into a single chimera, we envisaged generation of
novel agents with enhanced drug-like properties that might
a Medicinal Chemistry & Sterix Ltd., Department of Pharmacy &
Pharmacology, University of Bath, Claverton Down, Bath,
BA2 7AY, UK. E-mail: B.V.L.Potter@bath.ac.uk;
Fax: +44 (0)1225-386114; Tel: +44 (0)1225-386639
b Department of Biology & Biochemistry, University of Bath,
Claverton Down, Bath, BA2 7AY, UK
c Department of Endocrinology & Metabolic Medicine & Sterix Ltd.,
Imperial College London, St Mary’s Hospital, London W2 1NY, UK
d Institut de Recherche Henri Beaufour, 91966 Les Ulis Cedex, France
w Electronic supplementary information (ESI) available: Synthetic,
spectroscopic and crystallographic details are provided [crystallo-
graphic data pdb accession code 2WD2.pdb]. See DOI: 10.1039/
c002558e
Scheme 1 Synthesis of THIQ derivatives. Reagents and conditions: (i)
(MeO)2CHCH2NH2 (ii) NaBH4, MeOH (iii) 6 M HCl, dioxane (iv)
NaBH4, TFA (v) (MeO)3BnCl, Et3N, EtOH, MW 130 1C, 1 h (vi) H2,
Pd/C (vii) H2NSO2Cl, DMA (viii) AcCl, NaHCO3, DCM (ix) AlCl3,
AcCl, DCM (x) Et3SiH, TFA (xi) BnBr, NaH, DMF (xii) NaOH,
EtOH.
z Michael J. Reed deceased 2009.
ꢀc
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Table 1 GI50s in mM against the proliferation of human prostatea
and breastb cancer cell lines in vitro
Reduction of the C-7 acyl group followed by benzyl
protection delivered the doubly protected THIQ 8 that could
then be converted to the target phenol 5c and sulfamate 6c
under standard conditions (Scheme 1). In the case of 5c
substitution of the C-7-methoxy group for ethyl proved
detrimental to activity, with the 6-fold reduction in activity
illustrating the importance of the methoxy oxygen in the
phenol series. Conversely, sulfamate 6c proved ca. 100-fold
more active than its parent phenol 5c and 7-fold more active
than 6b against the DU-145 line, thus indicating a divergence
in the SAR of the 6-hydroxy and 6-O-sulfamoyl compounds.
The relative activities of 2-MeOE2 1, prototypical chimera 5b
and modified chimera 6c (DU-145 GI50s 1.22 mM, 650 nM and
41 nM) illustrate the power of the chimeric approach and
value of subsequent optimization.
Entry
R
X
R2
R5
DU-145a MDA MB-231b
5a
5b
5c
6a
6b
6c
H
H
H
O
O
CH2
O
O
OMe
H
H
OMe
H
H
H
4100 4100
OMe
OMe
H
OMe
OMe
0.650 0.620
3.85 5.41
17.0 11.9
SO2NH2
SO2NH2
SO2NH2 CH2
0.297
0.041
0.329
0.052
100 mM, 5b showed excellent activity with GI50 values of 650
and 620 nM, respectively, validating the design concept.
Having previously pioneered the sulfamoylation of phenols
to improve oral bioavailability (vide infra), and considering
that the 6-hydroxyl group would be prone to conjugation, we
next synthesized the 6-O-sulfamates 6a and 6c7 from 5a and 5b
using sulfamoyl chloride in dimethyl acetamide. The N-(2,3,4-
trimethoxy)benzyl derivative 6a showed improved, but still
modest, anti-proliferative activity while 6by proved ca. 2-fold
more active than 5b. Exchange of the methoxy for the isosteric
ethyl group at C-7 was next explored. 6-Methoxytetrahydro-
isoquinoline 7 was thus acylated at N-2; then a Friedel–Crafts
acylation effected concomitant 7-acylation and demethylation
of the 6-methoxyl group.
The ability of the active chimeras to inhibit the Taxol
stimulated polymerization of tubulin was established and
confirmed the mechanism of action; the activities of 5b, 6b
and 6c in this assay correlate with their relative anti-
proliferative effects. Inhibition of angiogenesis alongside cell
proliferation by co-administration of a microtubule disruptor
and an angiogenesis inhibitor8 offers a dual, clinically validated
approach against cancer. The activity of 6c against HUVEC
cell proliferation in vitro (GI50 6 nM), a marker of anti-
angiogenic potential, indicates that the chimeras retain
2-MeOE2’s dual mechanism of action.
An important challenge for new agents is the circumvention of
acquired resistance that often stems from upregulation of drug
efflux pumps. The MCF-7DOX cell line, by upregulation of
P-glycoprotein (PgP), is resistant to the DNA intercalator
doxorubicin and the taxanes.9 6c similarly inhibited proliferation
of both wild type human breast cancer MCF-7 cells (GI50 20 nM)
and their resistant MCF-7DOX counterparts (GI50 10 nM),
indicating that the chimera is not a substrate for the PgP pump
and has potential for use in the therapy of taxane resistant tumors.
Having established a desirable in vitro profile 6c was
progressed to in vivo evaluation. The effects of 6c on the
Fig. 2 Effects of 6c on the growth of MDA MB-231 xenografts.
Fig. 3 Protein X-ray crystal structure of hCAII-9 complex; left panel: active site funnel region of hCAII-9 complex; right panel: ligand 9 shown
viewing into active site, showing potential for further elaboration of pendant aryl group, with the protein shown as a molecular surface; acidic
residues are coloured red, basic residues in blue and the zinc ions are shown in brown.
ꢀc
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growth of MDA MB-231 xenografts in athymic female nude
mice were investigated. Oral dosing at 20 mg kgꢁ1 was carried
out over 28 days using a simple aqueous medium. This initial
experiment (Fig. 2) revealed that, even at a non-optimized
dose or formulation, 6c greatly inhibits the growth of the
breast cancer xenograft and with no apparent signs of toxicity.
The high oral bioavailability of selected aryl sulfamates is
believed to arise from sequestration by red blood cell (rbc)
carbonic anhydrase (hCAII)10 which allows avoidance of first
pass liver metabolism. We defined the interaction of the
sulfamoylated chimeras and hCAII using protein crystallo-
graphy. Of a panel of inhibitors complexed with hCAII,
crystals obtained with THIQ derivative 9 (the N-[3-methoxy-
benzyl] analogue of 6a) were of suitable quality for X-ray
analysis. The protein crystal structure (Fig. 3) shows the
co-ordination of the catalytic Zn by the sulfamate nitrogen
and one of its oxygens, whilst the flanking methoxy group
H-bonds to Thr-200. The THIQ nitrogen also appears to pick-up
a bonding interaction (to Gln-92), while the N-benzyl group
projects into a hydrophobic region that tantalizingly could be
further utilized to enhance potency for this carrier.
stirred at rt under nitrogen for 24 hours. After addition of water
(5 mL) and KHCO3 (150 mg, 1.5 mmol) the reaction mixture was
extracted into ethyl acetate (2 ꢂ 50 mL), the organic layers washed
with water and brine, then dried (MgSO4) and evaporated. The crude
product was purified by flash chromatography (DCM/ethyl acetate
gradient) and precipitated from diethyl ether to give the desired
product as a yellow powder (125 mg, 57%), mp 143–144 1C.
1H NMR (270 MHz, CDCl3) d 2.70–2.84 (4H, m), 3.58 (2H, s), 3.61
(2H, s), 3.81 (3H, s), 3.83 (3H, s), 3.84 (6H, s), 5.10 (2H, br s), 6.61 (1H,
s), 6.63 (1H, s) and 7.07 (1H, s), 13C NMR (67.5 MHz, CDCl3) d 28.1,
50.4, 55.7, 56.2, 56.4, 61.0, 62.8, 105.7, 111.2, 124.2, 127.6, 133.7,
134.6, 137.0, 137.4, 149.4 and 153.3. HRMS (ESI) calcd for
C20H27N2O7S [M + H]+, 439.1533; found 439.1525. Microanalysis:
C: 54.3% (expected 54.78%); H: 5.99% (expected 5.98%); N: 6.30%
(expected 6.39%).
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Since this new class of orally available agent overcomes
numerous issues limiting the optimal clinical application of
microtubule disruptors it will be attractive to optimise the best
representatives towards early clinical entry in oncology.
We acknowledge the support of scientists (station IO3) at
Diamond Light source, UK and Ipsen Ltd. for funding.
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Notes and references
y Preparation of 7-methoxy-2-(3,4,5-trimethoxybenzyl)-6-O-sulfamoyl-
1,2,3,4-tetrahydroisoquinoline 6b: a solution of 6-hydroxy-7-methoxy-
2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (180 mg,
0.5 mmol) and sulfamoyl chloride (1 mmol) in DMA (1 mL) was
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2907–2909 | 2909