Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation

Article abstract of DOI:10.1016/j.bmcl.2012.09.104

Colchicine was modified at the 10-OCH₃ position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62-99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III β-tubulin. Importantly, unlike colchicine, the analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective delivery.

Full text of DOI:10.1016/j.bmcl.2012.09.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7693–7696
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of colchicine C-ring analogues tethered
with aliphatic linkers suitable for prodrug derivatisation
Jérémie Fournier-Dit-Chabert a, Victoria Vinader a, Ana Rita Santos a, Mariano Redondo-Horcajo b,
Aurore Dreneau a, Ramkrishna Basak a, Laura Cosentino a, Gemma Marston a, Hamdy Abdel-Rahman a,
Paul M. Loadman a, Steven D. Shnyder a, José Fernando Díaz b, Isabel Barasoain b, Robert A. Falconer a,
Klaus Pors a,
⇑
a Institute of Cancer Therapeutics, University of Bradford, BD7 1DP, UK
b Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Colchicine was modified at the 10-OCH3 position of the C-ring by reaction with heterocyclic amines or
commercially available amines to afford a library of target colchicinoids in high yields (62–99%).
Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy
and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be
equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in
resistant A2780AD or HeLa cells with mutant Class III b-tubulin. Importantly, unlike colchicine, the
analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective
delivery.
Received 24 August 2012
Revised 25 September 2012
Accepted 27 September 2012
Available online 10 October 2012
Keywords:
Colchicine
MDR (ABCB1, P-glycoprotein)
Class III b-tubulin resistance
Prodrug
Ó 2012 Elsevier Ltd. All rights reserved.
Matrix metalloproteinases (MMPs)
Colchicine, the main alkaloid of the poisonous plant meadow
saffron (Colchicum autumnale L.), is used for the treatment of famil-
ial Mediterranean fever, Behçet’s syndrome (BS), Sweet’s syn-
drome, scleroderma, amyloidosis, liver cirrhosis and gout.1,2
Although colchicine is not used clinically to treat cancer due to
toxicity, it does exert anti-proliferative effects through the inhibi-
tion of microtubule formation leading to mitotic arrest and cell
death by apoptosis.3 Perhaps more significantly, colchicine has also
been shown to produce anti-vascular effects,4 a key feature of
many microtubule disrupting agents. This may be a result of multi-
ple direct actions on endothelial cells resulting in a much greater
reduction in blood flow in tumors than in normal tissues.5,6 A num-
ber of microtubule disrupting agents that possess anti-vascular ef-
fects are currently undergoing clinical trials.7,8 Despite the promise
of these agents in cancer treatment, early results suggest that their
therapeutic value is compromised by intrinsic systemic toxicity.9,10
Our research is aimed at exploring ways to selectively deliver
effective doses of chemotherapeutic agents to solid tumors, there-
by reducing side effects associated with such treatments. The pres-
ence of matrix metalloproteinases (MMPs) within the tumor
microenvironment provides an opportunity as a drug target. The
MMP family comprises 24 zinc-dependent endopeptidases with
structural similarity, but known to play a pivotal role in tumori-
genesis and tumor progression.11–13 The increased activity of
selected membrane-type MMPs (MT-MMPs) within the tumor
microenvironment14 may be targeted by taking advantage of the
capacity of these enzymes to metabolize specific peptide
sequences.14 By conjugating a highly toxic drug to such a peptide,
it is rendered safe and inactive until such time that the peptide is
cleaved selectively in the tumour, releasing the toxic payload.
Although colchicine is not directly amenable to derivatisation,
closely related analogues with similar in vitro potency such as
colchiceinamide15 (Fig. 1) may be used instead. We have recently
demonstrated16 that a non-toxic peptide-conjugated colchiceina-
mide (ICT2588) is selectively metabolized by MT-MMPs to release
the active agent in the tumour. Importantly, a significant growth
delay of human fibrosarcoma HT1080 xenografts without any asso-
ciated systemic toxicity was observed.
Dose-intensification strategies for microtubule disrupting
agents including compounds able to interact with the colchicine
binding domain may be further refined by incorporating agents
(metabolites) that are able to evade multidrug-resistance (MDR)
mechanisms that are present in refractory tumors. Central to
MDR is the membrane-bound P-glycoprotein (P-gp, ABCB1,
MDR1), which is overexpressed in several different tumor types
and known to be associated with poor response to chemotherapy.17
⇑
Corresponding author.
E-mail address: k.pors1@bradford.ac.uk (K. Pors).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.104

7694
J. Fournier-Dit-Chabert et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7693–7696
MMP recognition sequence
Tumour-selective Release
of Colchiceinamide
HN
O
NH2
NH
O
NH2
NH
OH
O
O
AcHN
O
HO
S
O
O
O
O
O
H
N
H
N
H
N
H
N
O
N
H
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
OH
N-terminal EndCap
OH
OH
ICT2588
MT-MMP scissile bond
Figure 1. ICT2588 is selectively cleaved by MT-MMPs to release colchiceinamide.
Table 1
Physicochemical properties, binding affinities and growth inhibition against human DLD-1 colon cancer cell line
d
Compound
MW
Yielda
CLogPb
MM/GBVIc (Kcal/mol)
DLD-1 IC50 (lM)
4
5
6
7
8
9
10
11
511.6
581.7
511.6
511.6
427.5
483.6
441.5
428.5
541.6
399.4
95
62
99
99
95
87
85
98
63
—
1.986
3.546
1.986
1.986
1.488
3.048
1.749
1.522
3.054
2.586
24.410 (R), 101.754 (S)
106.436 (R), 108.622 (S)
À5.608
8.1 0.3f
>10f
>10
>10
0.9 0.2
>10
À6.526
À14.062
66.045
À17.817
130.1 18.9e
69.2 8.1e
>10
À20.827
12
55.476
Colchicine
À27.655
45.5 6.2e
a
b
c
d
e
f
Yields refer to the final amination step.
CLogP was calculated using the Wildman and Crippen model.
The entropy data was generated using MOE software.
IC50 values are the mean SD of at least three independent assays.
IC50 = nM.
IC50 value of the racemic compound.
Many microtubule disrupting agents are substrates for P-gp, includ-
ing colchicine.15 It is well-known that the A- and C-rings of colchi-
cine are key structural features of the molecule in regard to its high
affinity binding to tubulin and biological activity. Any modification
in the A-ring of colchicine causes complete loss of binding, indicat-
ing that the requirement of the A-ring is stringent. On the other
hand, modification to both the B and C-rings is possible, with some
previous reports showing activity against MDR in vitro.15,18,19 As an
extension of our ongoing efforts to develop novel analogues that
evade traditional resistance mechanisms and possess improved tar-
get selectivity, we here report a library of colchicinoids, which have
been modified at the amino group of colchiceinamide to incorpo-
rate aliphatic linkers with hydroxyl or amino groups suitable for
prodrug derivatisation.
This reduction in entropy is due to a decrease in the conforma-
tional freedom of the colchicine analogue and a result of the effi-
cacy of binding. Compounds with longer linkers (5 and 9) or
methylene linkers with more bulky groups (6, 7 and 12) were
shown to not be easily accommodated in the colchicine binding
domain. Our investigation revealed how sensitive the C-ring is to
synthetic modification, which is in accordance with the literature
(see recent review22 for overview). Figure 2 demonstrates opti-
mized binding models of compound 10 and 12. The N-methyleth-
ylene diamine linker of 10 facilitates hydrogen bonding to the
Val315 residue whereas this interaction is lost by incorporation
of the Boc group.
The target compounds were prepared using previously pub-
lished procedures. The 1-(2-aminoethyl)pyrrolidine- and piperi-
dine linkers were afforded after a two-step procedure involving
alkylation of the cyclic secondary amine by bromoalkylnitrile
(Et3N, THF, 45–50 °C, 30 min, 63–92%) and subsequent reduction
of the nitrile with LiAlH4 (THF, reflux, 5 h, 27–76%) as previously
described23 (Scheme 1). Next, colchicine was reacted with the pre-
pared racemic (4, 5) and enantiomeric (6, 7) heterocyclic amines or
commercially obtained amines to afford the target colchicinoids
4–11 in high yields (62–99%, Table 1) after flash column chroma-
tography.24 Using conventional protection chemistry, the Boc-pro-
tected derivative of the secondary amine was afforded in 63% yield.
The colchicine C-ring modified compounds were evaluated for
their ability to inhibit the growth of DLD-1 colorectal cancer cells
(Table 1). Compounds with smaller linkers (8, 10 and 11) exhibited
the highest potency, which is in good accordance with that pre-
dicted from data obtained from computational chemistry. The cal-
culated LogP values ranged from 1.5 to 3.5 and hence the reduced
The X-ray crystal structure of bovine
a,b-tubulin complexed
with N-deacetyl-N-(2-mercaptoacetyl)-colchicine was obtained
from the protein database (PDB code 1SA0, 3.58 Å) as previously
described.20 MOE software was employed to generate binding
models for compounds 4–12. We employed molecular mechanics
generalized Born interaction energy to study the binding interac-
tion, which is the non-bonded interaction energy between the
receptor and the ligand and comprises van der Waals, Coulomb
and generalized Born implicit solvent interaction energies.21 In
our model, we made the assumption that there were no covalent
bonds established between the protein and the ligand. Upon bind-
ing only non-covalent bonding intermolecular interactions were
shown to stabilize the complex. Table 1 reveals that the incorpora-
tion of the linker groups at the colchicine C-ring position led to a
reduction in entropy for all compounds although compounds 8,
10 and 11 generated binding energies similar to that of colchicine.

J. Fournier-Dit-Chabert et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7693–7696
7695
Table 2
Growth inhibition of A2780 parental and A2780AD resistant ovarian carcinoma cells
by compounds 10 and 11
Compound
A2780
A2780AD
RFb
10
11
502.4 64.1a
71.1 7.3
1.1 0.5
784 52
241.4 24
>1000
1.6
3.3
>900
2.5
Paclitaxel
Colchicine
6.7 0.4
16.8 1.1
a
IC50 values are the mean standard error of at least four independent assays.
The resistance factor (RF) is obtained by dividing the IC50 of the A2780AD cell
b
line with the parental A2780 cell line.
cytotoxicity when compared to colchicine (CLogP of colchi-
cine = 2.6) is deemed not related to any significant differences in
lipophilicity. Substitution of the 10-OCH3 group on the C-ring of
colchicine with medium-to-large linkers and bulky heterocyclic
moieties has previously been shown to lead to a reduction in po-
tency19,25 whereas substitution with an NH2 group results in a
more pronounced antitumour effect and less toxicity than colchi-
cine at an optimal dose.26 The ease of synthetic manipulation at
the C-ring and associated drop in biological activity is desirable
in terms prodrug design. For example, computational modeling
demonstrated that the bulkiness of the Boc group as in compound
12 reduced the binding affinity when compared to the N-methyl
precursor analogue 10. Subsequent evaluation of the cellular po-
tency demonstrated that the incorporation of the simple Boc group
resulted in a loss of potency of a magnitude of at least 75-fold. This
is an encouraging result that offers the opportunity to replace the
Boc group with a prodrug moiety that can be selectively cleaved
within the tumor microenvironment.
Compounds 10 and 11, the two most potent colchicinoids pos-
sessing the smallest linker groups (two methylene units), were also
evaluated in the ovarian A2780 carcinoma cell line and its P-gp
overexpressing A2780AD variant. The cytotoxic activity of 10 and
11 was shown to be reduced by 1.6 and 3.3-fold respectively and
hence affected by drug-resistant mechanisms present in the
A2780AD cells, in similar fashion to colchicine (2.5-fold), but not
as dramatic as paclitaxel, which lost at least 900-fold in potency
(Table 2).
A recent study27 disclosed evidence that several agents able to
bind at the colchicine site of tubulin were unaffected by Class III
b-tubulin resistance, indicating that new compounds with affinity
for the colchicine binding site could play a role as an alternative
Figure 2. Binding models of 10 (A) and 12 (B) in the colchicine binding site.
R2
n
R2
R1
NC
R2
R1
R1
MeO
4
5
6
7
(amine 1)
(i)
NH
(ii)
(iii)
(amine 2)
N
m
n
n
MeO
N
N
H
H2N
(amine 3-S)
(amine 3-R)
O
O
MeO
m
1
2
(R1 = H, R2 =OH, n = 2, m = 1)
(R1 = H, R2 =OH, n = 2, m = 5)
R
3-S (R1 = CH2OH, R2 =H, n = 1, m = 1)
3-R (R1 = CH2OH, R2 =H, n = 1, m = 1)
MeO
MeO
MeO
MeO
NH
MeO
NH
8
9
(R = HN(CH2)2NH2)
(R = HN(CH2)6NH2)
(iv) Commercial
amine
NH
O
MeO
MeO
10 (R = HN(CH2)2NHCH3)
11 (R = HN(CH2)2OH)
O
O
O
MeO
O
O
MeO
12
H3C
NH
(v)
OCH3
Colchicine
R
10
N
Boc
Scheme 1. Synthesis of amino sidechains and target colchicinoids. (i) 1.1 equiv bromoalkylnitrile, 1.5 equiv Et3N, anhyd THF, 50 °C, 1 h, 63–92%; (ii) 3 equiv LiAlH4, anhyd
THF, 6 h, rt, 27–76%; (iii) colchicine, 10–25 equiv amines (1, 2, 3-S or 3-R), CH2Cl2, 70 °C, 4 h, for yields, see Table 1; (iv) as under iii, amines used: H2N(CH2)2NH2,
H2N(CH2)6NH2, H2N(CH2)2NHCH3, H2N(CH2)2OH, for yields, see Table 1; (v) 10, 1.2 equiv Boc2O, 1,5 equiv Et3N, THF, 45 °C, 16 h, 63%.

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J. Fournier-Dit-Chabert et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7693–7696
Table 3
Department, The University of Texas Health Science Center, San
Antonio, Texas, USA).
Growth inhibition of HeLa parental and HeLa b-III transfected cervix carcinoma cells
by compounds 10 and 11
Compound
HeLa
HeLa bIII
RFb
References and notes
10
11
585.9 49.9a
192.3 16.3
1.0 0.1
554.3 50
164.1 16.3
11.2 0.1
9.7 0.3
0.95
0.85
11.2
1.2
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over MgSO4, filtered and evaporated to afford a yellow powder 10 (418 mg,
98%). dH (600 MHz, CDCl3): 1.86 (m, 1H), 2.00 (s, 3H), 2.19 (m, 1H), 2.28 (m,
1H), 2.39 (dd, 1H, J 5.9; 12.9 Hz), 3.50 (m, 2H), 3.57 (s, 3H), 3.85 (s, 3H), 3.91 (s,
3H), 3.95 (m, 2H), 4.64 (ddd, 1H, J 6.4; 6.8; 11.8 Hz), 5.56 (br s, 1H, OH), 6.49 (s,
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37.1 (CH2), 45.7 (CH2), 52.3 (CH), 56.0 (CH3), 59.8 (CH2), 61.2 (CH3), 61.3 (CH3),
107.2 (CH), 109.1 (CH), 122.6 (CH), 126.6 (C), 130.6 (C), 134.4 (C), 139.5 (CH),
141.4 (C), 150.9 (C), 151.2 (C), 152.8 (C), 154.8 (C), 169.9 (C@O), 174.9 (C@O)
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Acknowledgments
This work was supported by YCR Program Grant (R.A.F., K.P.),
FCT (A.R.S., SFRH/BD/46871/2008), BIO2010-16351 from Ministe-
rio de Economia y Competitividad (J.F.D.) and grant S2010/BMD-
2457 BIPEDD2 from Comunidad Autónoma de Madrid (J.F.D.).
Parental HeLa and transfected b-III cervix carcinoma cell lines were
a kindly gift from Professor Richard F. Ludueña (Biochemical
34. Pors, K.; Shnyder, S. D.; Teesdale-Spittle, P. H.; Hartley, J. A.; Zloh, M.; Searcey,
M.; Patterson, L. H. J. Med. Chem. 2006, 49, 7013.