Synthesis of Nonracemic Pyrrolo-allocolchicinoids Exhibiting Potent Cytotoxic Activity

Article abstract of DOI:10.1002/ejoc.201601069

An efficient eight-step semisynthetic approach towards nonracemic pyrrolo-allocolchicinoids starting from natural colchicine was developed by exploiting Pd-catalyzed domino Sonogashira coupling/5-endo-dig cyclization of a 2-iodo-trifluoroacetanilide intermediate to build up the heterocyclic ring system. The N-Me substitution of the pyrrole ring enhanced the antitumor activity of the prepared molecules by 2–3 orders of magnitude. Among the active compounds, the N-methylated colchicinoid exhibited powerful cytotoxic and antiproliferative properties at concentrations < 1 nm.

Full text of DOI:10.1002/ejoc.201601069

A Journal of
Accepted Article
Title: Synthesis of non-racemic pyrrolo-allocolchicinoids exhibiting potent cytotoxic
activity
Authors: Ekaterina Sergeevna Shchegravina; Dmitry Igorevich Knyazev; Irina Pe-
trovna Beletskaya; Elena Viktorovna Svirshchevskaya; Hans-Gu¨nther Schmalz;
Alexey Yu. Fedorov
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601069
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601069
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European Journal of Organic Chemistry
10.1002/ejoc.201601069
COMMUNICATION
Synthesis of non-racemic pyrrolo-allocolchicinoids exhibiting
potent cytotoxic activity.
Ekaterina S. Shchegravina,a Dmitry I. Knyazev,a Irina P. Beletskaya,b Elena V. Svirshchevskaya,c
Hans-Günther Schmalz,d and Alexey Yu. Fedorova*
Abstract: An efficient eight-step semi-synthetic approach towards
non-racemic pyrrolo-allocolchicinoids starting from natural colchicine
was developed exploiting a Pd-catalyzed domino Sonogashira
pronounced cytotoxic effects (Figure 1). In this context, we
recently demonstrated that compounds 3 and rac-4, with an
extra furan or pyrrole unit annealed to ring C of the allocolchicine
skeleton, exhibit unique antitumoral activity.18-20
coupling/5-endo-dig cyclization of
a
2-iodo-trifluoroacetanilide
intermediate to build up the heterocyclic ring system. The N-Me
substitution of the pyrrole ring enhances the antitumoral activity of
the prepared molecules by 2-3 orders of magnitude. Among the
active compounds, the N-methylated colchicinoid 14 exhibits
powerful cytotoxic and anti-proliferative properties at concentrations
below 1 nM.
Notably, the furano-allocolchicinoid 3 inhibits tumor growth in
mice without noticeable negative neurological symptoms,
weights loss or lethality of experimental animals.19 The (racemic)
pyrrolo-allocolchicinoid (rac-4) demonstrated high level of
proliferation inhibition and apoptosis induction against different
lymphoma cells, while its unspecific cytotoxicity is relatively
low.18
Introduction
Colchicine (1), an alkaloid isolated from Colchicum autumnale,
was the first discovered tubulin-destabilizing agent (Figure 1).1
On a molecular level, it blocks the self-assembly of tubulin to
microtubules by interacting at the interface between α- and β-
tubulin heterodimers (colchicine binding site of tubulin).2 This
results in both in an antimitotic activity and the suppression of
cell motility. Clinically, colchicine is approved for the treatment of
Familial Mediterranean fever, Behcet’s disease, as well as acute
gout, chondrocalcinosis and other types of microcrystalline
arthritis.3-6 Other therapeutical indications include primary biliary
cirrhosis, psoriasis, amyloidosis, various forms of dermatitis,
relapsing polychondritis, necrotizing vasculitis, Sweet’s
syndrome and leukocytoclastic vasculitis.7-10 Colchicine can be
useful for the treatment of cardiovascular diseases,11 such as
particular pericarditis, atrial fibrillation caused by inflammation,
and ischemic episodes.12 Besides, it is considered as an
anticancer agent, although its clinical use in sufficient doses is
hampered by relatively high general toxicity, resulting from its
accumulation in the gastro-intestinal tract as well as
neurotoxicity.13
Figure 1. Colchicine (1), allocolchicine (2) and their analogs 3 and 4.
Results and Discussion
In our previous studies we prepared pyrrolo-allocolchicines of
types 5a/5b in racemic form by total synthesis (11 steps, 3-18%
overall yield) starting from 3,4,5-trimethoxyphenylpropionic acid
and exploiting the Suzuki coupling and the Friedel-Crafts
cyclization as the key C-C bond forming steps (Scheme 1).18
While colchicine (1) itself cannot be used in cancer
chemotherapy, it still represents an important lead structure in
the search for new anticancer agents. Several structurally
related compounds, such as allocolchicine (2),14 Z-stilbenes,15 4-
arylcoumarins16 and related compounds17 were shown to exhibit
Herein, we report
enantiomerically pure pyrrolo-allocolchicines of type 5’ bearing a
4-bromo and 2’’-oxymethyl substituent. Starting from
a conceptionally different synthesis of
a
[a]
Ekaterina S. Shchegravina, Dmitry I. Knyazev, Alexey Yu. Fedorov
Department of Organic Chemistry, Nizhny Novgorod State
University, Gagarina av. 23, Nizhny Novgorod 603950, Russian
Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
Irina P. Beletskaya
Department of Chemistry, M.V. Lomonosov Moscow State
University, Vorobyevy Gory, 119992 Moscow, Russian Federation
Elena V. Svirshchevskaya
commercially available natural (-)-(aR,7S)-colchicine (1) the
semi-synthetic pathway proceeds in only 8 steps under the
conservation of the chirality center at C-7. This specific
substitution pattern was selected because we had previously
[b]
[c]
observed, that the presence of a hydroxylmethyl substituent
18a,18c
either at C-7 of ring B
or on the heterocyclic ring (like in
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS,
Moscow, Tel/Fax +7 (495)3304011
compound 3)19 significantly increases the anti-proliferative
activity, as does a halogen atom at ring A.21
[d]
Hans-Günther Schmalz
Department of Chemistry, University of Cologne, Greinstrasse 4,
50939 Köln, Germany, Fax: +49 221-470-3064
For the construction of the indolyl moiety in 5’ we devised the
strategy depicted in Scheme 2, which relies on a Sonogashira
cross-coupling with subsequent in situ transition-metal mediated
Supporting information for this article is given via a link at the end of
the document
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European Journal of Organic Chemistry
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COMMUNICATION
cyclization.22 The required ortho-iodoaniline precursor 6 could be
prepared through Curtius degradation and iodination from 4-
bromo-allocolchicinic acid (7), which, in turn, is readily
accessible from colchicine (1) by the known ring contraction and
bromination.19, 23
to a similar reactivity of three aromatic positions. Similarly, we
were not able to achieve a selective C-4 halogenation of
allocolchicinic acid (8).
Therefore, we decided to perform the halogenation at C-4 prior
to the C-ring contraction. The described protocol for this
transformation21 failed in our hands. However, the reaction of 1
with either NCS or NBS works well in the presence of TFA to
give the 4-halogenated colchicines 10a and 10b in 55% and
95% yield, respectively. Interestingly, these two compounds
behave quite differently in the base-induced ring contraction.
While 10a reacted rather sluggishly to give 7a (43% yield) the
corresponding bromide 10b cleanly afforded the allocolchicinic
acid 7b in 89% isolated yield. To our surprise, the C-4
halogenation proved to have a dramatic effect on the tendency
of the allocolchicinic acids (7) to undergo Curtius degradation.
Under the proven conditions, used formerly for the one-pot
conversion of 8 into 9, only trace amounts of the expected
aniline products were obtained. Instead, the stepwise treatment
of bromide 7b with NaN3 in the presence of Boc2O afforded the
acylazide 11 in 87% yield. Interestingly, compound 7a did not
afford the corresponding product at all. Nevertheless, the
rearrangement of the acylazide 11 could be achieved by heating
with an excess of Boc2O in tert-BuOH to afford the Boc-
protected amine 12 in 81% yield. Finally, the Boc-group was
cleaved, and on treatment with NIS/AcOH the resulting aniline
gave the desired ortho-iodinated product 6 in 45% yield (over
two steps).
Scheme 1. Total- and semi-synthetic approaches towards pyrrolo-
allocolchicines.
Our attempts to perform the Pd/Cu-catalyzed Sonogashira
coupling of unprotected ortho-iodoaniline 6 with several terminal
alkynes failed. Instead, trifluoroacetamide 13, prepared from 6
(Scheme 4) readily underwent the Larock annulation to afford
the desired pyrrolo-allocolchicines 5’’ in a good yield. Notably,
the N-trifluoroacetamide group is cleaved under the reaction
conditions. The choice of alkynes was based on our previous
studies,19 which indicated that the presence of a CH2OH,
CH(CH3)OH or CH2OAc group in the side chain of the
heterocyclic ring leads to higher in vitro antitumor activity. In
order to determine the influence of substituents at the nitrogen
atom on the biological activity, N-methylated pyrrolo-allocolchine
14 was prepared in 3 steps from amide 13. In the first step, the
indole 5f’’ was obtained through tandem Sonogashira/cyclization
sequence, using TBDMS-protected propargyl alcohol. Then,
after the N-methylation (NaH/MeI), and the selective cleavage of
the silyl group, compound 14 was isolated in 28% overall yield
(over 3 steps).
Scheme 2. Strategy for the semi-synthesis of pyrrolo-allocolchicinoids of type
5’ starting from colchicine (1).
The in vitro cytotoxicity of the synthesized compounds toward
Colo (human pancreatic adenocarcinoma), HEK (human em
bryonic kidney) and Mia (human pancreatic carcinoma) cell lines
was investigated. A tetrazolium-based assay was used to
determine the drug concentration required to inhibit cell growth
by 50% after incubation in the culture medium for 72 h. The
calculated IC50 values are summarized in Table 1. Compound
5a’’ was found to be completely inactive, while a modest
cytotoxic effect was observed for compounds 5b-e’’.
The synthesis starts with the base-induced tropolone ring
contraction23 of 1 (in wet MeOH) to give allocolchicinic acid 8 in
excellent yield (Scheme 3). After a careful optimization, the
subsequent Curtius degradation proceeded smoothly using
NaN3 in combination with Boc2O as an activating agent to afford
the amine 9 in 64% yield. However, all attempts to achieve a
selective iodination of
9 failed, and only complex and
inseparable mixtures of iodinated products were obtained, due
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European Journal of Organic Chemistry
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COMMUNICATION
Reaction conditions: a: MeONa (solid, 17 eq.), MeOH/H2O, 65 °C; b: NaN3 (3 eq.), Boc2O (6 eq.), DME, 75 °C, then CF3COOH (3 M
in DCM), rt; c: NCS or NBS, TFA-AcOH (3:1), rt; d: NaOMe, MeOH, 65 °C; e: NaN3 (6 eq.), Boc2O (8 eq.), THF, rt; f: Boc2O (6 eq.), t-
BuOH, 90 °C; g: 3 M HCl-H2O/ DCM, rt; h: NIS (1,1 eq.), AcOH, rt.
Scheme 3. Conversion of colchicines (1) into iodo-aniline (6)
Reaction conditions: a:
HC≡CCH2OTBDMS (1,2 eq.),
Pd(dppf)Cl2 (0,05 eq.), CuI (0,1 eq.), DIPEA (3 eq.), MeCN,
90 °C, 6 h; b: NaH (60% in mineral oil, 2,3 eq.), MeI (1,5 eq.),
THF, 0→65 °C; c: 1,0 M TBAF in THF.
Reaction conditions: a: alkyne (1,2 eq.), [Pd]-source (0,05 eq.),
CuI (0,1 eq.), base, solvent; b: (CF3CO2)2O (1,8 eq.), pyridine
Scheme 5. Synthetic route to N-methylated pyrrolo-allocolchicinoids.
o
(2,6 eq.), DCM, 0 C; c: alkyne (1,2 eq.), Pd(OAc)2 (0,05 eq.),
CuI (0,1 eq.), PPh3 (0,15 eq.), AcOK, (3 eq.), MeCN, 80 oC.
Table 1. Anti-proliferative properties (IC50, μM)a of the prepared compounds.
Scheme 4. Final steps of the synthesis of pyrrolo-allocolchicines of type 5”
Cell line
Colo
HEK
Mia
Colchicine
5a’’
0.02
>4
0.1
0.32
0.09
0.5
0.007
20
0.8
0.1
0.8
0.008
4
0.04
0.2
0.01
0.16
<0.001
In contrast, compound 14 inhibited cell proliferation at
nanomolar or even subnanomolar concentrations. We presume
that the presence of an alkyl on the N-1 of the pyrrolo-
allocolchicine skeleton is essential for the antitumor activity of
the prepared compounds .2
5b’’
5c’’
5d’’
5e’’
0.8
0.001
14
0.004
[a] Drug concentration that inhibits the growth of the represented cells by
50% after incubation in a liquid medium for 72 h. Each drug concentration
was tested in triplicate, and the SE of each point is <10%.
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European Journal of Organic Chemistry
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COMMUNICATION
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of an allo-colchicinic acid followed by a Larock annulation to
build-up the indole ring system. The developed synthetic
methodology may find future applications in the synthesis of
various pyrrolo-allocolchicines. The N-methylated compound 14
may serve as a leading structure for the development of
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Experimental Section
Synthesis of compound 14. Into a Schlenk flask with a stirring bar,
compound 13 (80 mg, 0,12 mmol), Pd(dppf)2Cl2 (0,05 eq., 4,4 mg, 0,006
mmol), and CuI (0,1 eq., 2,3 mg, 0,012 mmol) were placed. Acetonitrile
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45 mcl of 1N TBAF in THF was added, and the mixture was stirred for 3
hours. The product was purified by column on silica (petroleum ether –
ethyl acetate – ethanol 15:1:1). Compound 14 was obtained as a white
solid in 55% yield (over two steps). M.p. = 175-177 °С. 1H NMR (400
MHz, dmso-d6): δ 8.51 (d, J = 8.3 Hz, 1H, NHAc), 7.46 (s, 1H, C(8)-H),
7.37 (s, 1H, C(12)-H), 6.38 (s, 1H, C(11)-H), 5.22 (t, J = 5.0 Hz, 1H, OH),
4.64 (d, J = 5.1 Hz, 2H, CH2OH), 4.60 – 4.56 (m, 1H, C(7)-H), 3.91 (s,
3H, OMe), 3.86 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.34 (s, 3H, NMe), 3.04
– 2.99 (m, 1H, C(5)-H), 2.10 – 2.05 (m, 1H, C(5)-H), 2.02 – 1.97 (m, 1H,
C(6)-H), 1.93 (s, 3H, C(O)CH3), 1.84 – 1.79 (m, 1H, C(6)-H). 13C NMR
(101 MHz, dmso-d6): δ 168.5, 150.1, 149.1, 145.8, 140.7, 137.2, 133.8,
133.7, 130.2, 125.2, 124.3, 121.2, 112.5, 103.6, 100.1, 61.2, 60.7, 60.2,
55.5, 48.3, 37.0, 29.8, 29.7, 22.80. For the experimental details see
Supporting Information.
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Acknowledgements
This work was supported by the Russian Science Foundation
(Project 16-13-10248), E.S.S. thanks RFBR (Project 16-33-
00942) for the financial support during the work on the
preparation of compound 14
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Keywords: alkaloids • cross-coupling • antitubulin agents •
cytotoxicity • rearrangements
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European Journal of Organic Chemistry
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COMMUNICATION
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Text for Table of Contents
Alkaloids, anti-tubulin agents*
Ekaterina S. Shchegravina, Dmitry I.
Knyazev, Irina P. Beletskaya, Elena V.
Svirshchevskaya, Hans-Günther
Schmalz, Alexey Yu. Fedorov*
Page No. – Page No.
Synthesis of non-racemic pyrrolo-
allocolchicinoids exhibiting potent
cytotoxic activity
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