Syntheses and biological evaluation of ring-C modified colchicine analogs

Article abstract of DOI:10.1016/j.bmcl.2010.03.056

Ring-C modified alkaloids were synthesized from colchicine using iminonitroso Diels-Alder reactions in a highly regio- and stereoselective fashion. Several analogs exhibited cytotoxic activity similar to that of colchicine itself against PC-3 and MCF-7 cancer cell lines, by serving as prodrugs of colchicine through retro Diels-Alder reactions under the assayed conditions. In vitro microtubule polymerization assays indicated that these modifications affected their interaction with tubulin.

Full text of DOI:10.1016/j.bmcl.2010.03.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3831–3833
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and biological evaluation of ring-C modified colchicine analogs
Baiyuan Yang, Zhiqing C. Zhu, Holly V. Goodson, Marvin J. Miller *
Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Ring-C modified alkaloids were synthesized from colchicine using iminonitroso Diels–Alder reactions in a
highly regio- and stereoselective fashion. Several analogs exhibited cytotoxic activity similar to that of
colchicine itself against PC-3 and MCF-7 cancer cell lines, by serving as prodrugs of colchicine through
retro Diels–Alder reactions under the assayed conditions. In vitro microtubule polymerization assays
indicated that these modifications affected their interaction with tubulin.
Received 18 January 2010
Revised 9 March 2010
Accepted 11 March 2010
Available online 15 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Colchicine
Nitroso Diels–Alder
2
-Nitrosopyridine
Cytotoxicity
Microtubule
Natural (À)-(aS,7S)-colchicine (1) (Fig. 1), the major alkaloid
MeO
MeO
OMe
4
OMe
isolated from Colchicum autumnale L. (Liliaceae), is a highly potent
MeO
MeO
1
antimitotic agent that derives its activity by binding to protein
12
A
A
1
1
12a
1
tubulin, the basic subunit component of microtubules. Though
MeO1
0
B
C
O
C
B
its use has been limited because of its toxicity, colchicine can still
be used as a lead compound for the generation of potential anti-
cancer drugs. Thus, numerous analogs of colchicine have been syn-
thesized in the hope of developing novel, useful drugs with more
O
8
7
MeO
HN
HN
1
2
O
O
2
favorable pharmacological profiles. Structure–activity studies re-
Figure 1. Colchicine (1) and isocolchicine (2).
veal that the trimethoxy benzene ring (A) and the methoxy tro-
pone ring (C) of colchicine comprise the minimal structural
features of the molecule needed for its high affinity binding to
tubulin. One notable example is that isocolchicine (2), a colchicine
analog differing only in the relative position of the methoxy and
carbonyl groups of ring-C, is virtually inactive and is unable to in-
the ring-C using iminonitroso Diels–Alder (NDA) reactions for
modular enhancement of Nature’s diversity (MEND). We also de-
scribe the results of in vitro anticancer activity of the analogs
against MCF-7 and PC-3 cell lines as well as their tubulin binding
affinity.
3
hibit tubulin assembly. Ring-C is also characterized by two facially
differentiated 1,3-diene moieties, most suitable for cycloaddition
reactions of the alkaloid. While Diels–Alder reactions of colchicine
The use of 2.0 equiv of 6-methyl-2-nitrosopyridine 3a has been
demonstrated to react with colchicine to give adduct 6a as a single
isomer in 82% yield (Table 1, entry 1). To further explore the reac-
4
7
with several symmetrical hetero- and carbo-dienophiles have been
5
reported, the biological consequence of this modification has
tivity of alternate nitroso species, nitrosobenzene 4, as a represen-
tative arylnitroso agent, and ayclnitroso agent 5 derived from
in situ oxidation of benzohydroxamic acid, as a representative
transient acylnitroso agent, were also examined in the cycloaddi-
tion reactions with colchicine (entries 2 and 3). However, none
were as effective as pyridinylnitroso agent 3a. Reaction of colchi-
cine with 4 generated the corresponding cycloadduct 6 only in less
never been disclosed, although these studies should further the
understanding of the role of ring-C in the development of new col-
chicine drugs. Our group also reported one example of hetero
6
Diels–Alder reaction of colchicine with a pyridinylnitroso agent.
Encouraged by these results, we were interested to further explore
the scope of this cycloaddition chemistry of colchicine. Herein, we
report the synthesis of a series of colchicine analogs modified at
1
than 10% yield based on H NMR analysis of the crude mixture;
however, adduct 6 was not able to be isolated in pure form by
chromatography. On the other hand, no NDA reaction occurred
upon the treatment with acylnitroso agent 5. Clearly, 2-pyridinyl-
nitroso 3a, as a stabilized iminonitroso reagent, constitutes an
*
Corresponding author.
E-mail address: mmiller1@nd.edu (M.J. Miller).
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.056

3
832
B. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3831–3833
Table 1
Table 2
Nitroso Diels–Alder reaction of colchicine (1) with different nitroso agents
Nitroso Diels–Alder reaction of colchicine (1) with various iminonitroso agents
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OMe
OMe
1
0
OMe
MeO
1
2
R
O
O
N
O
N
O
N
O
12
(
2.0 equiv)
R
H
MeO10
O
1
2a
H
R
MeOH, rt
8
N
MeO
MeO
8
HN
HN
OMe
HN
6
b-k
Syn-endo
R
O
O
1
6, 6a
O
O
N
N
(
3b-k)
(2.0 equiv)
MeO
MeO
MeO
MeO
+
Entry
Nitroso
Product
Yield (%)
82a
MeOH, rt
OMe
O
HN
O
O
N
1
3a
4
O
6a
1
H
O
N
N
N
R
HN
O
1
O
6
<10b
0
N
7b-k
Syn-endo
O
2c
5
—d
O
Ph
N
Ratioc
3:1
Yieldd (%)
78
a
b
c
Entry
Nitroso
Product
Isolated yield.
Determined by 1H NMR analysis of crude mixture.
In situ oxidation trapping required.
1a
3b
O
6b+7b
N
N
N
ideal combination of reactivity and stability for NDA reactions with
colchicine.
Next we investigated the scope of iminonitroso agents 3, in par-
ticular, pyridinylnitroso derivatives, in the nitroso Diels–Alder
reactions with colchicine. A series of iminonitroso agents 3b–k
was synthesized from the commercially available amine precur-
2
3c
—
—
0
O
N
N
3a
3d
6d+7d
6:1
61
O
N
N
8
sors and each was treated with colchicine (1) separately. The
O
4
3e
6e
—
62
N
results are summarized in Table 2. Cycloaddition with 2-nitroso-
pyridine 3b gave two regio-isomeric adducts 6b and 7b in a 3:1 ra-
tio and 78% yield (entry 1). The major isomer 6b had the same
configuration as 6a by comparison of 1H and C NMR profiles,
which was formed from the cycloaddition at the 8,12-diene moiety
of colchicine and syn to the NH-group, with endo preference. The
structure of the minor adduct, 6b, was assigned as the regioisomer
of 6a, which also shared the syn-endo configuration based on 1D
and 2D NMR analyses of related analogs.9 Interestingly, when 3-
methyl-2-nitrosopyridine 3c was used, no cycloaddition reaction
occurred (entry 2). We assumed that this low reactivity resulted
from the steric effect of the methyl substituent adjacent to the ni-
troso functional group. To further confirm this assumption, reac-
tion of 1 with 4-methyl-2-nitrosopyridine 3d was conducted. As
expected, a mixture of adducts 6d and 7d, with 6d predominate,
was generated in 61% yield (entry 3). A single isomeric adduct 6e
was obtained in 62% yield from the reaction with 6-ethyl-2-nitros-
opyridine 3e (entry 4). It appeared that halide substituents on the
F
5b
6b
3f
6f+7f
7:1
7:1
6:1
75
78
74
O
13
N
N
Cl
3g
3h
6g+7g
6h+7h
O
N
N
Br
7b
O
N
N
2
O N
8b
3i
6i+7i
6j+7j
6k+7k
7:1
8:1
2:1
73
72
80
O
N
N
9b
3j
O
N
N
10
3k
O
O
N
N
a
Cycloadducts 6b and 6d were obtained pure after column chromatography,
along with a mixture of two isomeric adducts (6b and 7b, 6d and 7d), respectively.
5
-position of the pyridine ring of the nitroso agents did not affect
reactivity and selectivity. Thus, adducts 6f–h and 7f–h were
formed in good yields with 6f–h as the major products (entries
b
Cycloadducts were obtained as a mixture of two regioisomers after column
chromatography.
c
Determined by 1H NMR of the crude reaction mixture.
5
2
–7). Reaction of a more electron deficient nitroso agent, 5-nitro-
-nitrosopyridine 3i, gave results similar to those previously ob-
tained upon reactions with 3f–h (entry 8). When quinoline-based
nitroso agent 3j was used, a mixture of adducts 6j and 7j was ob-
tained in 72% yield with an 8:1 ratio (entry 9). These results indi-
cated that having a 6-substitutent close to nitrogen is important
to induce high regioselectivity in the NDA reactions with colchi-
cine. An alternate iminonitroso agent, 5-methyl-3-nitrosoisoxazole
are very effective dienophiles with exquisite sensitivity to elec-
tronic and steric influences of the diene-containing colchicine
alkaloid.
With these ring-C modified colchicine analogs at hand, in vitro
cytotoxicity assays against both PC-3 (prostate cancer) and MCF-7
(breast cancer) tumor cell lines were conducted (Table 3). In gen-
eral, most analogs showed similar activity to colchicine itself.
However, relatively low activity was observed with compounds
6i, 6k and 7k. Since in the most cases, the activity was essentially
identical, we decided to determine the stability of those adducts to
see if they were eventually serving as prodrugs of colchicine itself,
3
k, was also reacted with colchicine. Compared to the reactions
with pyridinylnitroso agents, cycloaddition between 3k and 1
afforded a mixture of two isomeric adducts 6k and 7k in 80% yield
with a compromised regioselectivity. The isomers were separated
by prep HPLC. These studies indicated that 2-nitrosopyridines 3

B. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3831–3833
3833
Table 3
MeO
MeO
MeO
MeO
MeO
OMe
+
Cytotoxic activity against PC-3 and MCF-7 cell line for colchicine nitroso adducts
OMe
Compd
IC50, PC-3 (nM)
IC50, MCF-7 (nM)
O
N
O
DMSO
7ºC
Colchicine (1)
20
25
28
14
23
15.6
10
24
250
250
245
12
20
17
10
22
15
15.6
20
230
190
250
H
MeO
O
N
N
N
6
6
6
6
6
6
6
6
6
7
a
b
d
e
3
O
HN
HN
6a
1
3a
O
O
a
f
g
a
a
Equation 1. Retro Diels–Alder reaction of colchicine nitroso adduct 6a at 37 °C.
h
a
i
k
k
In conclusion, a series of ring-C modified colchicine analogs
using iminonitroso Diels–Alder reactions was synthesized and
evaluated for cytotoxic and anti-microtubule activity. The cycload-
dition reactions occurred exclusively with the 8,12-diene moiety
and often in a highly regio- and stereoselective fashion. Most ana-
logs showed cytotoxic activity against PC-3 and MCF-7 cancer cell
lines, by serving as prodrugs of colchicine through retro Diels–Al-
der reactions under the assayed conditions. In vitro microtubule
polymerization assays indicated that these analogs changed the
interactive properties of colchicine with tubulin.
a
Adducts 6f–i were tested with a small amount of regio-isomeric adducts 7f–i
present (for ratios, see Table 2).
Acknowledgments
We gratefully acknowledge Mrs. Patricia Miller for performing
MCF-7 and PC-3 cellular assays. We thank the National Institutes
of Health (GM 075855) and The University of Notre Dame for sup-
port of this research. We also thank the Lizzadro Magnetic Reso-
nance Research Center at Notre Dame for NMR facilities and
Nonka Sevova for mass spectroscopic analyses.
Figure 2. In vitro microtubule polymerization assay.
perhaps by retro Diels–Alder reaction under the assayed condi-
tions. The experiments were carried out by treating two represen-
tative adducts, 6a and 6k, in deuterated DMSO at 37 °C, since they
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.056.
1
exhibited different cytotoxicity. H NMR monitoring indicated that
indeed 13% of cycloadduct 6a underwent the retro Diels–Alder
reaction to release colchicine within 5 h. After 24 h, half of the
starting cycloadduct 6a was converted to colchicine (Eq. 1). The
resultant pyridinylnitroso 3a dimerized and lost oxygen to gener-
References and notes
1. (a) Capraro, H. G.; Brossi, A.. In The Alkaloids; Brossi, A., Ed.; Academic: New
York, 1984; Vol. 23, pp 1–70; (b) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.;
Jourdain, I.; Iachkar, S.; Sobel, A.; Knossow, M. Nature 2004, 428, 198.
10
ate the corresponding azo-oxy compound. In contrast, adduct
k, derived from 5-methyl-3-nitrosoisoxazole 3k, was intact even
6
2.
For selected reviews, see: (a) Bbattacbaryya, B.; Panda, D.; Gupta, S.; Banerjee,
M. Med. Res. Rev. 2008, 28, 155; (b) Lee, K. H. Med. Res. Rev. 1999, 19, 569; (c)
Quinn, F. R.; Neiman, Z.; Beisler, J. A. J. Med. Chem. 1981, 24, 636.
after 3 days. Obviously, the different thermal stability of colchicine
analogs might account for their varied cytotoxic activity.
3.
(a) Hastie, S. B.; Williams, R. C., Jr.; Puett, D.; McDonald, T. L. J. Biol. Chem. 1989,
We also noticed that within the 30 min time frame for the stan-
dard microtubule (MT) polymerization assay, colchicine nitroso
adducts were stable and no retro Diels–Alder reactions were de-
tected. Therefore, it was feasible to examine the tubulin binding
affinities of the adducts. In this regard, several representative spec-
troscopically pure cycloadducts, 6a–b, 6k and 7k, were subjected
to MT polymerization assays. In these experiments, the anti-micro-
tubule activities of selected colchicine analogs were evaluated and
compared with reference compounds including colchicine, noco-
dazole, a microtubule destabilizer, and paclitaxel, a microtubule
stabilizer (Fig. 2). Interestingly, unlike colchicine, which nearly
2
64, 6682; (b) Dumortier, C.; Yan, Q.; Bane, S.; Engelborghs, Y. Biochem. J. 1997,
327, 685.
4
.
.
For a review of cycloaddition reactions of 2-methoxytropone, see: Pietra, F.
Chem. Rev. 1973, 73, 293.
Brecht, R.; Haenel, F.; Seitz, G.; Frenzen, G.; Pilz, A.; Massa, W.; Wocadlo, S.
Liebigs Ann./Recueil 1997, 851.
5
6. Li, F. Z.; Yang, B. Y.; Miller, M. J.; Zajicek, J.; Noll, B. C.; Mollmann, U.; Dahse, H.
M.; Miller, P. Org. Lett. 2007, 9, 2923.
The stereochemistry of 6a was conclusively determined by X-ray
crystallographic analysis. See Ref. 6.
7
.
8. 2-Nitrosopyridine derivatives were synthesized in a two-step sequence (N,N-
dimethyl sulfilimine intermediate formation, followed by oxidation using m-
CPBA), see: Taylor, E. C.; Tseng, C. P.; Rampal, J. B. J. Org. Chem. 1982, 47, 552.
The regioselectivity of the reaction was determined by
13
9
.
C
NMR
inhibited the entire MT polymerization at 10
l
M, all assayed col-
measurements; for example, in the case of cycloaddition with nitroso 3k
Table 2, entry 10), the chemical shifts of C-8 of adduct 6k, and C-12 of adduct
k (at 82.8 and 78.7 ppm, respectively) correspond to carbons next to oxygen.
(
7
chicine analogs showed decreased inhibitory activity towards MT
polymerization. Moreover, except for 7k, colchicine adducts 6a–b
and 6k appeared to enhance the MT polymerization. Clearly, intro-
duction of a N–O heterocycle at the C8,12 position of ring-C of col-
chicine changed the structural conformation, and thereby affected
the colchicine-tubulin interaction.
The structure of 7k was further confirmed by 2D NMR studies (gCOSY, gHSQC,
gHMBC and gROESY). In general, all the isomers with same configuration share
similar 1H and C NMR patterns. For example, all the chemical shifts of the C-
13
12 of the major isomers 6a–k are near 82.0 ppm.
1
0. For a detailed discussion of retro iminonitroso Diels–Alder reactions, see: Yang,
B. Y.; Lin, W. M.; Krchnak, V.; Miller, M. J. Tetrahedron Lett. 2009, 50, 5879.