Cytotoxic substituted indolizines as new colchicine site tubulin polymerisation inhibitors

Article abstract of DOI:10.1080/14756366.2020.1801671

A potential microtubule destabilising series of new indolizine derivatives was synthesised and tested for their anticancer activity against a panel of 60 human cancer cell lines. Compounds 11a, 11b, 15a, and 15j showed a broad spectrum of growth inhibitor

Full text of DOI:10.1080/14756366.2020.1801671

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Cytotoxic substituted indolizines as new colchicine
site tubulin polymerisation inhibitors
Monica-Cornelia Sardaru , Anda Mihaela Craciun , Cristina-Maria Al
Matarneh , Isabela Andreea Sandu , Roxana Maria Amarandi , Lacramioara
Popovici , Catalina Ionica Ciobanu , Dragos Peptanariu , Mariana Pinteala ,
Ionel I. Mangalagiu & Ramona Danac
To cite this article: Monica-Cornelia Sardaru , Anda Mihaela Craciun , Cristina-Maria Al
Matarneh , Isabela Andreea Sandu , Roxana Maria Amarandi , Lacramioara Popovici , Catalina
Ionica Ciobanu , Dragos Peptanariu , Mariana Pinteala , Ionel I. Mangalagiu & Ramona
Danac (2020) Cytotoxic substituted indolizines as new colchicine site tubulin polymerisation
inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1581-1595, DOI:
10.1080/14756366.2020.1801671
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1581–1595
https://doi.org/10.1080/14756366.2020.1801671
RESEARCH PAPER
Cytotoxic substituted indolizines as new colchicine site tubulin
polymerisation inhibitors
Monica-Cornelia Sardarua,b, Anda Mihaela Craciuna,b, Cristina-Maria Al Matarneha,b, Isabela Andreea Sandub,
Roxana Maria Amarandia,c , Lacramioara Popovicia, Catalina Ionica Ciobanud, Dragos Peptanariub,
Mariana Pintealab, Ionel I. Mangalagiua
and Ramona Danaca
b
aDepartment of Chemistry, Faculty of Chemistry, “Al. I. Cuza” University of Iasi, Iasi, Romania; “Petru Poni” Institute of Macromolecular
,
c
Chemistry of Romanian Academy, Iasi, Romania; TRANSCEND Research Center, Regional Institute of Oncology, Iasi, Romania;
,
,
dResearch Department, Faculty of Chemistry, “Al. I. Cuza” University of Iasi, Iasi, Romania
,
ABSTRACT
ARTICLE HISTORY
Received 17 May 2020
Revised 22 June 2020
Accepted 19 July 2020
A potential microtubule destabilising series of new indolizine derivatives was synthesised and tested for
their anticancer activity against a panel of 60 human cancer cell lines. Compounds 11a, 11b, 15a, and 15j
showed a broad spectrum of growth inhibitory activity against cancer cell lines representing leukaemia,
melanoma and cancer of lung, colon, central nervous system, ovary, kidney, breast, and prostate. Among
them, compound 11a was distinguishable by its excellent cytostatic activity, showing GI50 values in the
range of 10–100nM on 43 cell lines. The less potent compounds 15a and 15j in terms of GI50 values
showed a high cytotoxic effect against tested colon cancer, CNS cancer, renal cancer and melanoma cell
lines and only on few cell lines from other types of cancer. In vitro assaying revealed tubulin polymerisa-
tion inhibition by all active compounds. Molecular docking showed good complementarity of active com-
pounds with the colchicine binding site of tubulin.
KEYWORDS
Indolizine; anticancer;
tubulin polymerisation
inhibitors;
Phenstatin; pyridyl
Introduction
antimitotic
inconveniences.
agents
to
overcome
the
abovementioned
Recognised as key dynamic structural components in cells, micro-
tubules play an important role in cellular shape organisation,
intracellular movement, cell division, and mitosis1,2. Thus, they
have been considered an attractive target for the development of
Among the large number of microtubule-targeting agents with
diverse scaffolds investigated during last decades1,2,11, Phenstatin
(Figure 1) stands out as one of the simplest molecules that signifi-
cantly inhibit tubulin polymerisation by binding to the colchicine
site of tubulin14,15. Phenstatin is also known for its outstanding
new antiproliferative agents in the past few years3–7
.
Commonly, agents targeting tubulin are divided function of
the site they interact with tubulin. The three major sites of tubulin
are: the paclitaxel binding site (compounds showing microtubule
stabilising effects), vinblastine binding site, and colchicine binding
site (compounds showing inhibition of tubulin polymerisa-
tion)1,3–5,8. The microtubule-targeting strategy in the drug devel-
opment field is validated by the use of microtubule-targeting
agents such as paclitaxel, docetaxel, vinblastine, colchicine, com-
bretastatin A-4 (CA-4), nocodazole, and many others in cancer
antitumor activities on a wide variety of human cancer cells14,15
.
Its biological properties are comparable to CA-4, currently investi-
gated in clinical trials16, but in contrast to CA-4, Phenstatin has a
greater pharmacological potential due to improved metabolic sta-
bility and requires an easier synthesis for large-scale production17.
In the process of drug discovery, this kind of compounds are lead
scaffolds for the development of improved bioactive analogues,
and Phenstatin continues to be a source of inspiration for
researchers in designing new potential anticancer drugs18–25
To increase the structural diversity, the combination of various
types of bioactive moieties and rings has become a practical strat-
egy in the field of medicinal chemistry. Thus, our study was
.
chemotherapy1,8–10
. However, these compounds also present
many drawbacks (high toxicity that induces many side effects, low
oral bioavailability, and development of drug resistance) that limit
their efficiency11–13. Therefore, there is a huge demand of novel inspired by several reported anticancer active Phenstatin
CONTACT Ramona Danac
rdanac@uaic.ro Department of Chemistry, Faculty of Chemistry, “Al. I. Cuza” University of Iasi, 11 Carol I, Iasi 700506, Romania
,
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1582
M.-C. SARDARU ET AL.
Figure 1. Design in the series of the target indolizine derivatives.
analogues having the 30-hydroxy-40-methoxyphenyl ring replaced modified the 3,4,5-trimethoxyphenyl ring of Phenstatin for each
with an indolizine moiety21 (compound A, Figure 1) or a pyr-
series, replacing it with either a 3,5-dimethoxyphenyl, 3,4-dime-
rolo[1,2-b]pyridazine group24 (compound B, Figure 1).
thoxyphenyl, or a 4-bromophenyl ring. The structure–activity rela-
At the same time, the pyridine ring, present in many natural
products and even genetic material, has been noted for its role in
many biological processes as well as in cancer pathogenesis,
which makes it a privileged scaffold in anticancer agents discov-
ery26. Thus, there are reports of a variety of monosubstituted pyri-
dines with cytotoxic activity26–29, and there are also several
pyridyl-containing drugs introduced on the market for their antitu-
mor properties26 (Figure 1, right).
tionships (SARs), effects on tubulin assembly and theoretical
binding interactions were also explored in our study.
Materials and methods
Chemistry
All commercially available reagents and solvents employed were
used without further purification. Melting points were recorded on
In our continuous efforts22,24 to discover more effective micro-
tubule destabilising agents, we have applied a structural combin-
ation strategy to design and synthesise a new series of indolizine-
based Phenstatin analogues and evaluated their anticancer activ-
ity. Thus, we considered unsubstituted indolizines (at the pyridine
ring) (compounds C, Figure 1), as well as several pyridyl-substi-
tuted indolizines (compounds D, E, and F, Figure 1) in order to
investigate a possible beneficial influence of this group to the
binding properties of generated compounds due to the lone pair
electrons in this moiety. In order to increase biological activity, we
also designed an indolizine series that contains two 3,4,5-trime-
€
a A. Kruss Optronic Melting Point Meter KSPI and are uncorrected.
Analytical thin-layer chromatography was performed with com-
mercial silica gel plates 60 F254 (Merck, Darmstadt, Germany) and
visualised with UV light (kmax¼254 or 365 nm). The NMR spectra
were recorded on an Avance III 500 MHz spectrometer (Bruker,
1
Vienna, Austria) operating at 500 MHz for H and 125 MHz for 13C.
Chemical shifts were reported in delta (d) units, part per million
(ppm), and coupling constants (J) in Hz. The following abbrevia-
tions were used to designate chemical shift multiplicities:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, and
thoxybenzoyl groups (compounds G, Figure 1). Furthermore, we bs ¼ broad singlet. Infrared (IR) data were recorded as films on

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1583
potassium bromide (KBr) pellets on an FT-IR (Shimadzu, Kyoto, 106.5 C1, 107.0 C12, C16, 115.5 C6, 119.7 C8, 122.5 C3, 127.9 C7,
Japan) Prestige 8400s spectrophotometer or a Jasco 660 plus FTIR 129.2 C5, 129.4 C2, 140.2 C9, 142.0 C11, 160.8 C13, C15, 164.2 COO,
185.3 C10. Anal. Calcd. for C20H19NO5: C, 67.98; H, 5.42; N, 3.96.
Found: C, 68.00; H, 5.40; N, 3.90.
spectrophotometer. Analyses indicated by the symbols of the ele-
ments or functions were within 0.4% of the theoretical values.
Ethyl
3-(3,4-dimethoxybenzoyl)indolizine-1-carboxylate
(11c).
Beige solid, yield 56%, mp 196–198 ꢀC. IR ꢀ (cmꢁ1): 1703, 1610,
1514, 1483, 1261, 1211, 1138, 1047; 1H NMR (500 MHz, CDCl3): d
1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 3.96 (s, 3H, OMe), 3.98 (s, 3H, OMe),
4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 6.96 (d, J ¼ 8.5 Hz, 1H, H15), 7.07 (t,
J ¼ 7.0 Hz, 1H, H6), 7.41–7.45 (overlapped signals, 2H, H7, H12), 7.48
(d, J ¼ 8.0 Hz, 1H, H16), 7.86 (s, 1H, H2), 8.39 (d, J ¼ 8.5 Hz, 1H, H8),
9.88 (d, J ¼ 7.0 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3,
56.2 OMe, 56.3 OMe, 60.2 CH2, 106.2 C1, 110.3 C15, 111.8 C12,
115.2 C6, 119.7 C8, 122.5 C3, 123.6 C16, 127.5 C7, 128.5 C2, 129.2
General procedure for synthesis of monoquaternary salts 8a–l
and 13a–d
The corresponding heterocycle (pyridine 1, 4,40-bipyridine 2, 2,40-
bipyridine 3, or 2,20-bipyridine 12) (1 mmol,
1 equiv.) was
dissolved in 5–7 ml acetone. Then, reactive halide (2-bromo-1-
(3,4,5-trimethoxyphenyl)ethanone 4, 2-bromo-1-(3,5-dimethoxy-
phenyl) ethanone 5, 2-bromo-1-(3,4-dimethoxyphenyl) ethanone
6, or 2-bromo-1-(4-bromophenyl) ethanone 7) (1.1 mmol,
1.1 equiv.) was added and the resulted mixture was stirred over- C5, 132.6 C11, 139.9 C9, 149.2 C13, 153.4 C14, 164.3 COO, 184.6 C10.
night at room temperature (r.t.). The formed precipitate was fil- Anal. Calcd. for C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C,
tered and washed with diethyl ether to give the desired product 67.64; H, 5.40; N, 3.93.
Ethyl 3-(4-bromobenzoyl)indolizine-1-carboxylate (11d)30. Beige
which was used in the next reaction without any further
purification.
solid, yield 50%, mp 130–132 ꢀC. IR ꢀ (cmꢁ1): 3086, 2978, 1697,
1612, 1522, 1479, 1339, 1219, 1140, 1043. 1H NMR (500 MHz,
CDCl3): d 1.41 (t, J ¼ 7.2 Hz, 3H, CH3), 4.38 (q, J ¼ 7.2 Hz, 2H, CH2),
General procedure for preparation of compounds 11a–l
7.10 (dt, J ¼ 6.8; 1.2 Hz, 1H, H6), 7.47 (m, 1H, H7), 7.65 (d, J ¼ 8.4 Hz,
and 14a–d
2H, H12, H16), 7.70 (d, J ¼ 8.4 Hz, 2H, H13, H15), 7.75 (s, 1H, H2), 8.40
(d, J ¼ 8.8 Hz, 1H, H8), 9.93 (d, J ¼ 6.8 Hz, 1H, H5). 13C NMR
The cycloimmonium salt (8a–l and 13a–d) (1 mmol, 1 equiv.) and
ethyl propiolate (1.1 mmol, 1.1 equiv.) were added to dichlorome-
thane (DCM) and the obtained suspension was stirred at r.t. Then,
a solution of triethylamine (TEA) (3 mmol, 3 equiv.) in DCM (3 ml)
was added drop-wise over 1 h (magnetic stirring) and the resulting
mixture was then stirred overnight at r.t. Methanol (10 ml) was
added and the resulting solid was collected by filtration and
(125 MHz, CDCl3): d 14.5 CH3, 60.2 CH2, 106.5 C1, 115.4 C6, 119.5
C8, 122.1 C3, 126.2 C14, 127.9 C7, 128.8 C2, 129.1 C5, 130.5 C13, C15,
131.6 C12, C16, 140.0 C9, 138.6 C11, 163.9 COO, 184.1 C10. Anal.
Calcd. for C18H14BrNO3: C, 58.08; H, 3.79; N, 3.76. Found: C, 58.04;
H, 3,80; N, 3.80.
Ethyl 7-(pyridin-4-yl)-3-(3,4,5-trimethoxybenzoyl)indolizine-1-carb-
washed with 5 ml methanol. The product was then purified by oxylate (11e). Beige solid, yield 70%, mp 194–196 ꢀC. IR ꢀ (cmꢁ1):
crystallisation from DCM/methanol (1/1, v/v) and/or column chro- 1701, 1622, 1206, 1580, 1528, 1471, 1352, 1206, 1132, 1051. 1H
NMR (500 MHz, CDCl3): d 1.42 (as, 3H, CH3), 3.93 (s, 6H, 2 x OMe),
3.96 (s, 3H, OMe), 4.41 (bs, 2H, CH2), 7.10 (s, 2H, H12, H16), 7.37 (as,
1H, H6), 7.69 (as, 2H, 2 ꢂ Hpy), 7.91 (s, 1H, H2), 8.75 (overlapped
signals, 3H, 2 ꢂ Hpy, H8), 9.96 (as, 1H, H5). 13C NMR (125 MHz,
CDCl3): d 14.7 CH3, 56.52 ꢂ OMe, 60.5 CH2, 61.2 OMe, 107.6 C1,
106.9 C12, C16, 113.7 C6, 117.3 C8, 121.62 ꢂ CHpy, 122.8 C3, 129.0
C2, 129.6 C5, 134.9 C14, 137.0 C7, 139.9 C9, 141.6 C11, 145.3 Cpy,
150.82 ꢂ CHpy, 153.2 C13, C15, 164.1 COO, 184.9 C10. Anal. Calcd.
for C26H24N2O6: C, 67.82; H, 5.25; N, 6.08. Found: C, 67.84; H, 5.20;
N, 6.09.
matography using DCM/methanol (99.5/0.5, v/v).
General procedure for preparation of compounds 15a–j
The monoindolizine 11 (1 mmol, 1 equiv.) and reactive halide
derivative (4, 5, or 6) (2 mmol, 2 equiv.) were suspended in acet-
one (10 ml) and the resulted reaction mixture was refluxed under
magnetical stirring overnight. The resulting precipitate was col-
lected by filtration and then washed with acetone. Compounds
15a–i were further purified by crystallisation from chloroform/
methanol (1:1, v/v).
Ethyl
3-(3,5-dimethoxybenzoyl)-7-(pyridin-4-yl)indolizine-1-carb-
oxylate (11f). Beige solid, yield 55%, mp 208–210 ꢀC. IR ꢀ (cmꢁ1):
1717, 1684, 1595, 1514, 1468, 1362, 1246, 1207, 1159, 1045. 1H
NMR (500 MHz, CDCl3): d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.86 (s, 6H, 2
ꢂ OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2), 6.68 (s, 1H, H14), 6.95 (s, 2H,
H12, H16), 7.36 (ad, J ¼ 7.0 Hz, 1H, H6), 7.67 (as, 2H, 2 ꢂ Hpy), 7.90
(s, 1H, H2), 8.74 (overlapped signals, 3H, 2 ꢂ Hpy, H8), 9.99 (ad,
J ¼ 7.0 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3, 55.8 2 ꢂ
OMe, 60.4 CH2, 104.0 C14, 107.1 C12, C16, 107.6 C1, 113.8 C6, 117.3
C8, 121.4 2 ꢂ CHpy, 122.8 C3, 129.4 C2, 129.7 C5, 137.0 C7, 139.9
C9, 141.6 C11, 145.3 Cpy, 150.82 ꢂ CHpy, 160.8 C13, C15, 164.0 COO,
184.4 C10. Anal. Calcd. for C25H22N2O5: C, 69.76; H, 5.15; N, 6.51.
Found: C, 69.74; H, 5.10; N, 6.50.
Ethyl 3-(3,4,5-trimethoxybenzoyl)indolizine-1-carboxylate (11a).
Beige solid, yield 51%, mp 197–198 ꢀC. IR ꢀ (cmꢁ1): 1701, 1622,
1
1584, 1526, 1485, 1350, 1223, 1202, 1130, 1055. H NMR (500 MHz,
CDCl3): d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 3.92 (s, 6H, 2 ꢂ OMe), 3.95
(s, 3H, OMe), 4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 7.08 (s, 2H, H12, H16),
7.10 (t, J ¼ 7.0 Hz, 1H, H6), 7.46 (t, J ¼ 7.5 Hz, 1H, H7), 7.88 (s, 1H,
H2), 8.40 (d, J ¼ 8.5 Hz, 1H, H8), 9.91 (d, J ¼ 6.5 Hz, 1H, H5). 13C NMR
(125 MHz, CDCl3): d 14.7 CH3, 56.52 ꢂ OMe, 60.3 CH2, 61.2 OMe,
106.5 C1, 107.8 C12, C16, 115.4 C6, 119.7 C8, 122.5 C3, 127.8 C7,
128.9 C2, 129.3 C5, 135.2 C11, 140.1 C9, 141.4 C14, 153.2 C13, C15,
164.2 COO, 184.8 C10. Anal. Calcd. for C21H21NO6: C, 65.79; H, 5.52;
N, 3.65. Found: C, 65.74; H, 5.52; N, 3.70.
Ethyl 3-(3,5-dimethoxybenzoyl)indolizine-1-carboxylate (11b).
Beige solid, yield 50%, mp 178–180 ꢀC. IR ꢀ (cmꢁ1): 1699, 1628,
1593, 1528, 1458, 1356, 1200, 1155, 1045. 1H NMR (500 MHz,
CDCl3): d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 3.86 (s, 6H, 2 ꢂ OMe), 4.38
(q, J ¼ 7.0 Hz, 2H, CH2), 6.66 (s, 1H, H14), 6.93 (s, 2H, H12, H16), 7.09
Ethyl
3-(3,4-dimethoxybenzoyl)-7-(pyridin-4-yl)indolizine-1-carb-
oxylate (11g). Beige solid, yield 56%, mp 192–195 ꢀC. IR ꢀ (cmꢁ1):
3079, 2979, 1691, 1596, 1514, 1347, 1266, 1219, 1141, 1020. 1H
NMR (500 MHz, CDCl3): d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.97 (s, 3H,
OMe), 3.99 (s, 3H, OMe), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 6.98 (d,
(t, J ¼ 7.0 Hz, 1H, H6), 7.46 (t, J ¼ 7.5 Hz, 1H, H7), 7.87 (s, 1H, H2), J ¼ 8.5 Hz, 1H, H15), 7.35 (d, J ¼ 7.0 Hz, 2H, H6), 7.45 (s, 1H, H12),
8.40 (d, J ¼ 8.0 Hz, 1H, H8), 9.95 (d, J ¼ 7.0 Hz, 1H, H5). 13C NMR 7.51 (d, J ¼ 7.0 Hz, 1H, H16), 7.66 (d, J ¼ 5.0 Hz, 2H, 2 ꢂ Hpy), 7.89
(125 MHz, CDCl3): d 14.7 CH3, 55.82 ꢂ OMe, 60.3 CH2, 103.8 C14, (s, 1H, H2), 8.75 (overlapped signals, 3H, 2 ꢂ Hpy, H8), 9.93 (d,

1584
M.-C. SARDARU ET AL.
J ¼ 7.0 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3, 56.2 9.98 (d, J ¼ 7.5 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3,
OMe, 56.3 OMe, 60.4 CH2, 107.3 C1, 110.3 C15, 111.7 C12, 113.5 C6, 60.6 CH2, 108.5 C1, 114.0 C6, 118.3 C8, 122.7 CHpy, 123.0 C3, 124.4
117.3 C8, 121.4 2 ꢂ CHpy, 123.0 C3, 123.7 C16, 128.7 C5, 129.5 C2, CHpy, 126.8 C14, 129.1 C2, 129.7 C5, 130.7 C13, C15, 132.0 C12, C16,
132.2 C11, 136.6 C7, 139.6 C9, 145.5 Cpy, 149.3 C13, 150.8 2 ꢂ CHpy, 138.5 C11, C7, 139.5 C9, 140.2 CHpy, 147.6 CHpy, 153.0 Cpy, 164.0
152.6 C14, 164.2 COO, 184.7 C10; Anal. Calcd. for C25H22N2O5: C, COO, 184.5 C10. Anal. Calcd. for C23H17BrN2O3: C, 61.48; H, 3.81; N,
69.76; H, 5.15; N, 6.51. Found: C, 69.72; H, 5.11; N, 6.55.
6.23. Found: C, 61.49; H, 3.78; N, 6.26.
Ethyl 3-(4-bromobenzoyl)-7-(pyridin-4-yl)indolizine-1-carboxylate
Ethyl 5-(pyridin-2-yl)-3-(3,4,5-trimethoxybenzoyl)indolizine-1-carb-
(11h). Yield 65%. All spectral data are in agreement with the oxylate (14a). Cream solid, yield 35%, mp 161–163 ꢀC. IR ꢀ (cmꢁ1):
literature31.
Ethyl 7-(pyridin-2-yl)-3-(3,4,5-trimethoxybenzoyl)indolizine-1-carb-
2984, 1686, 1639, 1585, 1520, 1419, 1350, 1234, 1130, 1059, 752.
1H NMR (500 MHz, CDCl3): d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 3.87 (s,
oxylate (11i). Yellow solid, yield 41%, mp 202–203 ꢀC. IR ꢀ (cmꢁ1): 6H, 2 ꢂ OMe), 3.95 (s, 3H, OMe), 4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 7.08
2994, 2926, 1700, 1620, 1580, 1480, 1352, 1204, 1136, 780. 1H (d, J ¼ 6.0 Hz, 1H, H6), 7.13 (s, 2H, H12, H16), 7.20 (dd, J ¼ 6.5; 4.5 Hz,
NMR (500 MHz, CDCl3): d 1.43 (t, J ¼ 7.0 Hz, 3H, CH3), 3.94 (s, 6H, 2 1H, H20), 7.46 (dd, J ¼ 9.0; 7.0 Hz, 1H, H7), 7.64 (s, 1H, H2), 7.72 (d,
ꢂ OMe), 3.97 (s, 3H, OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 7.11 (s, 2H, J ¼ 8.0 Hz, 1H, H22), 7.91 (t, J ¼ 7.5 Hz, 1H, H21), 8.27 (d, J ¼ 4.0 Hz,
H12, H16), 7.35 (dd, J ¼ 7.5; 5.0 Hz, 1H, Hpy), 7.85 (dt, J ¼ 8.0; 2.0 Hz, 1H, H19), 8.47 (d, J ¼ 8.5 Hz, 1H, H8). 13C NMR (125 MHz, CDCl3): d
1H, Hpy), 7.90 (s, 1H, H2), 7.92 (dd, J ¼ 7.5; 2.0 Hz, 1H, H6), 7.97 (d, 14.7 CH3, 56.5 2 x OMe, 60.2 CH2, 61.1 OMe, 105.9 C1, 107.1 C12,
J ¼ 8.0 Hz, 1H, Hpy), 8.77 (d, J ¼ 4.5 Hz, 1H, Hpy), 9.01 (bs, 1H, H8), C16, 118.1 C6, 120.2 C8, 122.2 C22, 124.0 C20, 126.5 C7, C3, 127.3 C2,
9.95 (d, J ¼ 7.0 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3, 133.3 C11, 138.6 C5, 138.7 C21, 141.4 C9, 141.8 C14, 148.5 C19, 153.0
55.62 ꢂ OMe, 60.5 CH2, 61.2 OMe, 107.0 C12, C16, 108.0 C1, 113.9 C13, C15, 155.2 C17, 164.4 COO, 183.4 C10. Anal. Calcd. for
C6, 118.1 C8, 122.5 CHpy, 123.2 C3, 124.3 CHpy, 128.9 C2, 129.6 C5, C26H24N2O6: C, 67.82; H, 5.25; N, 6.08. Found: C, 67.81; H, 5.23;
134.9 C11, 138.9 C7, 139.5 C9, 140.2 CHpy, 141.7 C14, 147.9 CHpy, N, 6.11.
153.2 Cpy, 153.3 C13, C15, 164.1 COO, 184.9 C10. Anal. Calcd. for
Ethyl
3-(3,5-dimethoxybenzoyl)-5-(pyridin-2-yl)indolizine-1-carb-
C26H24N2O6: C, 67.82; H, 5.25; N, 6.08. Found: C, 67.85; H, 5.17; oxylate (14b). Yellow solid, yield 77%, mp 149–150 ꢀC. IR ꢀ (cmꢁ1):
N, 6.13.
2974, 1715, 1630, 1595, 1421, 1188, 1155, 754. 1H NMR (500 MHz,
Ethyl
3-(3,5-dimethoxybenzoyl)-7-(pyridin-2-yl)indolizine-1-carb- CDCl3): d 1.39 (t, J ¼ 7.0 Hz, 3H, CH3), 3.81 (s, 6H, 2 ꢂ OMe), 4.37
oxylate (11j). Yellow solid, yield 40%, mp 165–166 ꢀC. IR ꢀ (cmꢁ1): (q, J ¼ 7.0 Hz, 2H, CH2), 6.66 (bs, 1H, H14), 7.00 (d, J ¼ 2.0 Hz, 2H,
2934, 1696, 1587, 1448, 1352, 1209, 1152, 1044, 774. 1H NMR H12, H16), 7.09 (d, J ¼ 7.0 Hz, 1H, H6), 7.18 (dd, J ¼ 6.5; 5.5 Hz, 1H,
(500 MHz, CDCl3): d 1.43 (t, 3H, J ¼ 7.0 Hz, CH3), 3.87 (s, 6H, 2 ꢂ H20), 7.47 (dd, J ¼ 8.5; 7.5 Hz, 1H, H7), 7.65 (s, 1H, H2), 7.71 (d,
OMe), 4.41 (q, 2H, J ¼ 7.0 Hz, CH2), 6.68 (t, J ¼ 2.5 Hz, 1H, H14), 6.96 J ¼ 8.0 Hz, 1H, H22), 7.88 (t, J ¼ 7.5 Hz, 1H, H21), 8.30 (d, J ¼ 4.5 Hz,
(d, J ¼ 2.5 Hz, 2H, H12, H16), 7.34 (dd, J ¼ 7.0; 5.0 Hz, 1H, Hpy), 7.84 1H, H19), 8.47 (d, J ¼ 8.5 Hz, 1H, H8). 13C NMR (125 MHz, CDCl3):
(dt, J ¼ 7.5; 2.0 Hz, 1H, Hpy), 7.89 (s, 1H, H2), 7.92 (dd, J ¼ 7.5; 14.7 CH3, 55.82 ꢂ OMe, 60.2 CH2, 105.1 C14, 105.9 C1, 107.3 C12,
2.0 Hz, 1H, H6), 7.97 (d, J ¼ 8.0 Hz, 1H, Hpy), 8.77 (d, J ¼ 4.5 Hz, 1H, C16, 118.2 C6, 120.2 C8, 122.0 C22, 123.9 C20, 126.5 C3, 126.7 C7,
Hpy), 9.00 (d, J ¼ 1.0 Hz, 1H, H8), 9.99 (d, J ¼ 7.5 Hz, 1H, H5). 13C 127.8 C2, 138.5 C21, 138.7 C5, 140.2 C11, 141.6 C9, 148.5 C19, 155.2
NMR (125 MHz, CDCl3): d 14.7 CH3, 55.82 ꢂ OMe, 60.4 CH2, 103.9 C17, 160.7 C13, C15, 164.4 COO, 183.6 C10. Anal. Calcd. for
C14, 107.0 C12, C16, 107.5 C1, 114.0 C6, 116.8 C8, 121.0 CHpy, 122.8 C25H22N2O5: C, 69.76; H, 5.15; N, 6.51. Found: C, 69.78; H, 5.13;
C3, 123.6 CHpy, 129.4 C2, 129.5 C5, 137.3 CHpy, 138.6 C7, 140.3 C9, N, 6.54.
141.9 C11, 150.1 CHpy, 154.4 Cpy, 160.8 C13, C15, 164.2 COO, 185.6
C10. Anal. Calcd. for C25H22N2O5: C, 69.76; H, 5.15; N, 6.51. Found: oxylate (14c). Yellow solid, yield 35%. IR ꢀ (cmꢁ1): 2933, 1719,
C, 69.79; H, 5.10; N, 6.53.
1676, 1624, 1593, 1514, 1417, 1269, 1224, 1024, 766. 1H NMR
Ethyl
Ethyl
3-(3,4-dimethoxybenzoyl)-5-(pyridin-2-yl)indolizine-1-carb-
3-(3,4-dimethoxybenzoyl)-7-(pyridin-2-yl)indolizine-1-carb- (500 MHz, CDCl3): d 1.39 (t, J ¼ 7.0 Hz, 3H, CH3), 3.98 (s, 3H, OMe),
oxylate (11k). Yellow solid, yield 44%, mp 199–200 ꢀC. IR ꢀ (cmꢁ1): 4.00 (s, 3H, OMe), 4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 6.95 (d, J ¼ 8.5 Hz,
2974, 2931, 1699, 1608, 1517, 1472, 1426, 1347, 1267, 1199, 773. 1H, H15), 7.08 (d, J ¼ 6.0 Hz, 1H, H6), 7.19 (dd, J ¼ 6.5; 4.5 Hz, 1H,
1H NMR (500 MHz, CDCl3): d 1.44 (t, J ¼ 7.0 Hz, 3H, CH3), 3.98 (s, H20), 7.42 (bs, 1H, H12), 7.47 (dd, J ¼ 8.5; 7.0 Hz, 1H, H7), 7.48 (d,
3H, OMe), 4.00 (s, 3H, OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 6.99 (d, J ¼ 8.0 Hz, 1H, H16), 7.64 (s, 1H, H2), 7.71 (d, J ¼ 8.0 Hz, 1H, H22),
J ¼ 8.5 Hz, 1H, H15), 7.46 (bs, 1H, H12), 7.33 (dd, J ¼ 7.0; 5.0 Hz, 1H, 7.90 (t, J ¼ 7.5 Hz, 1H, H21), 8.28 (d, J ¼ 4.0 Hz, 1H, H19), 8.47 (d,
Hpy), 7.85 (t, J ¼ 7.5 Hz, 1H, Hpy), 7.88 (s, 1H, H2), 7.90 (dd, J ¼ 7.5; J ¼ 8.5 Hz, 1H, H8). 13C NMR (125 MHz, CDCl3): d 14.6 CH3, 56.2
1.5 Hz, 1H, H6), 7.97 (d, J ¼ 8.0 Hz, 1H, Hpy), 8.77 (d, J ¼ 4.5 Hz,1H, OMe, 56.3 OMe, 60.2 CH2, 106.0 C1, 110.3 C15, 111.9 C12, 118.2 C6,
Hpy), 7.52 (dd, J ¼ 8.0; 1.5 Hz, 1H, H16), 9.00 (bs, 1H, H8), 9.93 (d, 120.2 C8, 122.1 C22, 123.5 C16, 124.0 C20, 126.5 C3, 126.6 C7, 127.6
J ¼ 7.5 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 CH3, 56.2 C2, 132.5 C11, 138.7 C5, 138.6 C21, 141.5 C9, 148.5 C19, 149.1 C13,
OMe, 56.3 OMe, 60.3 CH2, 107.2 C1, 110.3 C15, 111.8 C12, 113.8 C6, 153.3 C14, 155.2 C17, 164.3 COO, 184.5 C10. Anal. Calcd. for
116.8 C8, 121.0 CHpy, 123.0 C3, 123.5 CHpy, 123.7 C16, 128.8 C2, C25H22N2O5: C, 69.76; H, 5.15; N, 6.51. Found: C, 69.79; H, 5.12;
129.2 C5, 132.5 C11, 137.2 CHpy, 138.2 C7, 140.0 C9, 149.2 C13, 150.1 N, 6.55.
CHpy, 152.5 C14, 154.5 Cpy, 164.3 COO, 184.6 C10. Anal. Calcd. for
C25H22N2O5: C, 69.76; H, 5.15; N, 6.51. Found: C, 69.78; H, 5.09;
N, 6.53.
Ethyl 3-(4-bromobenzoyl)-5-(pyridin-2-yl)indolizine-1-carboxylate
(14d)30. Yellow crystals, yield 40%, mp 229–231 ꢀC. IR ꢀ (cmꢁ1):
2925, 1725, 1694, 1650, 1586, 1419, 1342, 1070, 750. 1H NMR
Ethyl 3-(4-bromobenzoyl)-7-(pyridin-2-yl)indolizine-1-carboxylate (500 MHz, DMSO-d6): d 1.31 (t, J ¼ 7.0 Hz, 3H, CH3), 4.29 (q,
(11l). Yellow solid, yield 40%, mp 197–198 ꢀC. IR ꢀ (cmꢁ1): 2928, J ¼ 7.0 Hz, 2H, CH2), 7.24 (dd, J ¼ 7.0; 4.5 Hz, 1H, H20), 7.35 (dd,
1694, 1620, 1526, 1479, 1342, 1200, 1079. 1H NMR (500 MHz, J ¼ 7.5; 1.0 Hz, 1H, H6), 7.42 (s, 1H, H2), 7.67–7.70 (overlapped sig-
CDCl3): d 1.43 (t, J ¼ 7.0 Hz, 3H, CH3), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), nals, 3H, H12, H16, H7), 7.74 (d, J ¼ 8.5 Hz, 2H, H13, H15), 7.81 (d,
7.34 (dd, J ¼ 7.5; 4.5 Hz, 1H, Hpy), 7.68 (d, J ¼ 8.0 Hz, 2H, H12, H16), J ¼ 8.0 Hz, 1H, H22), 7.93 (dt, J ¼ 7.5; 1.5 Hz, 1H, H21), 8.13 (d,
7.73 (d, J ¼ 8.0 Hz, 2H, H13, H15), 7.80 (s, 1H, H2), 7.84 (dt, J ¼ 7.5; J ¼ 4.0 Hz, 1H, H19), 8.38 (dd, J ¼ 9.0; 1.0 Hz, 1H, H8). 13C NMR
2.0 Hz, 1H, Hpy), 7.94 (dd, J ¼ 7.5; 2.0 Hz, 1H, H6), 7.97 (d, J ¼ 8.0 Hz, (125 MHz, DMSO-d6): d 14.5 CH3, 59.8 CH2, 104.4 C1, 118.3 C6,
1H, Hpy), 8.77 (d, J ¼ 4.5 Hz, 1H, Hpy), 9.01 (d, J ¼ 1.0 Hz, 1H, H8), 119.0 C8, 122.2 C22, 124.3 C20, 125.1 C2, 126.2 C3, 126.8 C14, 127.7

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1585
C7, 131.2 C12, C16, 131.8 C13, C15, 136.2 C11, 138.4 C21, 138.8 C5, J ¼ 7.5 Hz, 1H, H5). 13C NMR (125 MHz, DMSO-d6): d 14.4 CH3, 55.6
140.4 C9, 148.3 C19, 154.2 C17, 163.3 COO, 182.2 C10. Anal. Calcd. 2 ꢂ OMe, 56.4 2 ꢂ OMe, 60.3 CH2, 60.4 OMe, 65.7 C23, 103.5 C14,
for C23H17BrN2O3: C, 61.48; H, 3.81; N, 6.23. Found: C, 61.51; H, 106.0 C26, C30, 106.8 C12, C16, 108.2 C1, 113.9 C6, 119.0 C8, 123.1
3.77; N, 6.25.
C3, 124.8 C18, C22, 128.1 C2, 128.8 C25, 129.3 C5, 132.2 C7, 138.0 C9,
140.8 C11, 143.2 C28, 146.6 C19, C21, 152.4 C17, 153.1 C27, C29, 160.4
C13, C15, 162.8 COO, 184.6 C10, 189.7 C24. Anal. Calcd. for
4-(1-(Ethoxycarbonyl)-3-(3,4,5-trimethoxybenzoyl)indolizin-7-yl)-1-
(2-oxo-2-(3,4,5-trimethoxy phenylethyl)pyridin-1-ium bromide (15a).
Orange solid, yield 70%, mp 188 ꢀC. IR ꢀ (cmꢁ1): 2986, 2941, 1713, C36H35BrN2O9: C, 60.09; H, 4.90; N, 3.88. Found: C, 60.03; H, 4.81;
1640, 1583, 1530, 1508, 1465, 1412, 1346, 1323, 1207, 1130. 1H
N, 4.84.
NMR (500 MHz, CDCl3): d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.92 (s, 6H, 2
4-(3-(3,5-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
bromide (15e).
ꢂ OMe), 3.93 (s, 3H, OMe), 3.96 (s, 6H, 2 ꢂ OMe), 3.97 (s, 3H, (3,5-dimethoxyphenyl)-2-oxoethyl)pyridin-1-ium
OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 7.09 (s, 2H, H12, H16), 7.26 (s, Orange solid, yield 75%, mp 149–150 ꢀC. IR ꢀ (cmꢁ1): 2940, 1709,
2H, H23), 7.47–7.49 (3H, overlapped signals, H6, H26, H30), 7.89 (s, 1640, 1603, 1465, 1425, 1348, 1207, 1159, 1062, 758. 1H NMR
1H, H2), 8.35 (bs, 2H, H18, H22), 8.89 (bs, 1H, H8), 9.35 (bs, 2H, H19, (500 MHz, DMSO-d6): d 1.36 (t, J ¼ 7.0 Hz, 3H, CH3), 3.84 (s, 6H, 2 ꢂ
H21,), 9.92 (d, J ¼ 7.0 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d 14.7 OMe), 3.87 (s, 6H, 2 ꢂ OMe), 4.37 (q, J ¼ 7.0 Hz, 2H, CH2), 6.48 (s,
CH3, 56.62 ꢂ OMe, 57.22 ꢂ OMe, 60.9 CH2, 61.1 OMe, 61.2 OMe, 2H, H23), 6.83 (t, J ¼ 2.0 Hz, 1H, H14), 6.93 (d, J ¼ 2.0 Hz, 2H, H12,
66.4 C23, 106.9 C26, C30, 107.0 C12, C16, 109.8 C1, 112.4 C6, 119.9 H16), 6.95 (t, J ¼ 2.0 Hz, 1H, H28), 7.21 (d, J ¼ 2.0 Hz, 2H, H26, H30),
C8, 123.9 C3, 124.1 C18, C22, 128.4 C2, 128.6 C25, 130.0 C5, 130.9 C7, 7.75 (s, 1H, H2), 7.97 (dd, J ¼ 7.5; 1.5 Hz, H6), 8.80 (d, J ¼ 6.5 Hz, 2H,
134.2 C11, 138.2 C9, 142.1 C14, 144.4 C28, 147.0 C19, C21, 153.3 C13, H18, H22), 8.96 (bs, 1H, H8), 9.10 (d, J ¼ 6.5 Hz, 2H, H19, H21), 9.91 (d,
C15, 153.6 C27, C29, 153.9 C17, 163.5 COO, 185.0 C10, 189.3 C24. J ¼ 7.5 Hz, 1H, H5). 13C NMR (DMSO-d6, 125 MHz): d 14.4 CH3, 55.62
Anal. Calcd. for C37H37BrN2O10: C, 59.28; H, 4.98; N, 3.74. Found: C, ꢂ OMe, 55.82 ꢂ OMe, 60.2 CH2, 65.9 C23, 103.5 C14, 106.1 C26, C30,
59.32; H, 4.87; N, 3.79.
106.2 C28, 106.8 C12, C16, 108.2 C1, 113.8 C6, 119.0 C8, 123.1 C3,
124.8 C18, C22, 128.1 C2, 129.3 C5, 132.2 C7, 135.4 C25, 138.0 C9,
1-(2-(3,5-Dimethoxyphenyl)-2-oxoethyl)-4-(1-(ethoxycarbonyl)-3-
(3,4,5-trimethoxybenzoyl) indolizin-7-yl)pyridin-1-ium bromide (15b). 140.8 C11, 146.6 C19, C21, 152.5 C17, 160.4 C13, C15, 160.9 C27, C29,
Orange solid, yield 65%, mp 175 ꢀC. IR ꢀ (cmꢁ1): 2928, 1700, 1638,
1529, 1457, 1351, 1207, 1016. H NMR (400 MHz, DMSO-d6): d 1.37 60.96; H, 4.82; N, 4.06. Found: C, 60.93; H, 4.77; N, 4.13.
162.8 COO, 184.6 C10, 190.5 C24. Anal. Calcd. for C35H33BrN2O8: C,
1
(t, J ¼ 6.8 Hz, 3H, CH3), 3.80 (s, 3H, OMe), 3.86 (s, 12H, 4 ꢂ OMe),
4-(3-(3,5-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
bromide (15f).
4.37 (q, J ¼ 6.8 Hz, 2H, CH2), 6.50 (s, 2H, H23), 6.95 (s, 1H, H28), 7.15 (3,4-dimethoxyphenyl)-2-oxoethyl)pyridin-1-ium
(s, 2H, H12, H16), 7.21 (s, 2H, H26, H30), 7.84 (s, 1H, H2), 7.96 (bs, H6), Orange solid, yield 60%, mp 220–223 ꢀC. IR ꢀ (cmꢁ1): 2924, 2851,
8.80 (bs, 2H, H18, H22), 8.95 (bs, 1H, H8), 9.11 (bs, 2H, H19, H21), 1696, 1640, 1596, 1524, 1463, 1401, 1349, 1200, 1157. 1H NMR
9.87 (bs, 1H, H5). 13C NMR (100 MHz, DMSO-d6): d 14.3 CH3, 55.82 (500 MHz, DMSO-d6): d 1.36 (t, J ¼ 7.0 Hz, 3H, CH3), 3.84 (s, 6H, 2 ꢂ
ꢂ OMe, 56.23 ꢂ OMe, 60.3 CH2, 65.9 C23, 106.1 C26, C28, C30, 106.7 OMe), 3.86 (s, 3H, OMe), 3.92 (s, 3H, OMe), 4.37 (q, J ¼ 7.0 Hz, 2H,
C12, C16, 108.2 C1, 113.7 C6, 119.0 C8, 123.1 C3, 124.8 C18, C22, CH2), 6.45 (s, 2H, H23), 6.83 (bs, 1H, H14), 6.93 (d, J ¼ 2.0 Hz, 2H,
127.9 C2, 129.2 C5, 132.0 C7, 133.9 C11, 135.4 C25, 137.8 C9, 141.0 H12, H16), 7.24 (d, J ¼ 8.5 Hz, 1H, H29), 7.54 (bs, 1H, H26), 7.76 (s, 1H,
C14, 146.6 C19, C21, 152.5 C17, 152.7 C13, C15, 160.9 C27, C29, 162.8 H2), 7.79 (dd, J ¼ 8.0; 1.0 Hz, 1H, H30), 7.97 (dd, J ¼ 7.5; 1.5 Hz, H6),
COO, 184.1 C10, 190.6 C24. Anal. Calcd. for C36H35BrN2O9: C, 60.09; 8.79 (d, J ¼ 6.5 Hz, 2H, H18, H22), 8.97 (bs, 1H, H8), 9.09 (d,
H, 4.90; N, 3.89. Found: C, 60.12; H, 4.87; N, 3.94.
J ¼ 6.5 Hz, 2H, H19, H21), 9.92 (d, J ¼ 7.5 Hz, 1H, H5). 13C NMR
(125 MHz DMSO-d6): d 14.4 CH3, 55.72 ꢂ OMe, 55.8 OMe, 56.1
1-(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)-4-(1-(ethoxycarbonyl)-3-
(3,4,5-trimethoxybenzoyl) indolizin-7-yl)pyridin-1-ium bromide (15c). OMe, 60.3 CH2, 65.6 C23, 103.6 C14, 106.9 C12, C16, 108.0 C1, 110.1
Orange solid, yield 50%, mp 236–239 ꢀC. IR ꢀ (cmꢁ1): 2925, 1699,
C26, 111.6 C29, 113.7 C6, 119.0 C8, 123.2 C3, 123.5 C30, 124.8 C18,
1638, 1582, 1523, 1460, 1345, 1272, 1205. 1H NMR (400 MHz, C22, 126.3 C25, 128.2 C2, 129.3 C5, 132.3 C7, 138.0 C9, 140.8 C11,
DMSO-d6): d 1.37 (t, J ¼ 6.8 Hz, 3H, CH3), 3.80 (s, 3H, OMe), 3.86 (s, 146.7 C19, C21, 149.0 C27, 152.4 C28, 154.3 C17, 160.5 C13, C15, 162.9
9H, 3 ꢂ OMe), 3.92 (s, 3H, OMe), 4.37 (q, J ¼ 6.8 Hz, 2H, CH2), 6.47
COO, 184.9 C10, 189.1 C24. Anal. Calcd. for C35H33BrN2O8: C, 60.96;
(s, 2H, H23), 7.16 (s, 2H, H12, H16), 7.24 (bs, 1H, H29), 7.55 (bs, 1H, H, 4.82; N, 4.06; Found: C, 60.93; H, 4.80; N, 4.13.
H26), 7.81 (bs, 1H, H30), 7.85 (s, 1H, H2), 7.96 (bs, 1H, H6), 8.80 (bs,
4-(3-(3,4-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
2H, H18, H22), 8.97 (bs, 1H, H8), 9.11 (bs, 2H, H19, H21), 9.89 (bs, 1H, oxo-2-(3,4,5-trimethoxyphenyl)ethyl)pyridin-1-ium bromide (15g).
H5). 13C NMR (100 MHz, DMSO-d6): d 14.3 CH3, 55.8 OMe, 56.0 2 ꢂ Orange solid, yield 70%, mp 132–137 ꢀC. IR ꢀ (cmꢁ1): 2924, 1698,
1
OMe, 56.22 ꢂ OMe, 60.2 CH2, 65.4 C23, 110.3 C26, 106.7 C12, C16, 1640, 1619, 1456, 1410, 1344, 1265, 1206, 1124. H NMR (500 MHz,
108.2 C1, 111.3 C29, 113.7 C6, 118.9 C8, 123.2 C3, 123.4 C30, 124.7 CDCl3): d 1.44 (t, J ¼ 7.0 Hz, 3H, CH3), 3.93 (s, 3H, OMe), 3.97 (s, 6H,
C18, C22, 126.2 C25, 127.9 C2, 129.3 C5, 132.0 C7, 133.9 C11, 137.8 2 ꢂ OMe), 4.00 (s, 6H, 2 ꢂ OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 7.23
C9, 141.0 C14, 146.6 C19, C21, 148.9 C27, 152.4 C28, 152.7 C13, C15, (s, 2H, H23), 6.95 (d, J ¼ 8.5 Hz, 1H, H15), 7.42–7.47 (3H, overlapped
C17, 162.8 COO, 184.1 C10, 189.0 C24. Anal. Calcd. for signals, H12, H16, H6), 7.49 (bs, 2H, H26, H30), 7.84 (s, 1H, H2), 8.32
C36H35BrN2O9: C, 60.09; H, 4.90; N, 3.89. Found: C, 60.10; H, 4.85; (d, J ¼ 5.5 Hz, 2H, H18, H22), 8.84 (bs, 1H, H8), 9.32 (d, J ¼ 5.0 Hz, 2H,
N, 3.93.
4-(3-(3,5-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
H19, H21), 9.87 (d, J ¼ 7.5 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d
14.7 CH3, 56.1 OMe, 56.22 ꢂ OMe, 56.92 ꢂ OMe, 60.8 CH2, 66.4
C23, 106.7 C26, C30, 109.3 C1, 110.1 C15, 111.7 C12, C6, 119.4 C8,
oxo-2-(3,4,5-trimethoxyphenyl)ethyl)pyridin-1-ium bromide (15d).
Orange solid, yield 84%, mp 176–178 ꢀC. IR ꢀ (cmꢁ1): 2913, 1696, 123.7 C16, C3, 123.8 C18, C22, 128.1 C25, 128.4 C2, 129.7 C5, 131.5
1683, 1639, 1590, 1528, 1454, 1402, 1352, 1207, 1159, 1130. 1H
C11, 130.4 C7, 137.8 C9, 144.1 C28, 146.9 C19, C21, 149.3 C13, 152.9
NMR (500 MHz, DMSO-d6): d 1.36 (t, J ¼ 7.0 Hz, 3H, CH3), 3.81 (s, C1, 153.3 C17, 153.4 C27, C29, 163.5 COO, 184.3 C10, 189.4 C24. Anal.
3H, OMe), 3.84 (s, 6H, 2 ꢂ OMe), 3.92 (s, 6H, 2 ꢂ OMe), 4.37 (q,
Calcd. for C36H35BrN2O9: C, 60.09; H, 4.90; N, 3.89. Found: C, 60.13;
H, 4.89; N, 3.93.
J ¼ 7.0 Hz, 2H, CH2), 6.52 (s, 2H, H23), 6.83 (t, J ¼ 2.0 Hz, 1H, H14),
6.93 (d, J ¼ 2.0 Hz, 2H, H12, H16), 7.39 (s, 2H, H26, H30), 7.76 (s, 1H,
4-(3-(3,4-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
H2), 7.97 (dd, J ¼ 7.5; 2.0 Hz, H6), 8.81 (d, J ¼ 7.0 Hz, 2H, H18, H22), (3,5-dimethoxyphenyl)-2-oxoethyl)pyridin-1-ium
bromide
(15h).
8.97 (bs, 1H, H8), 9.10 (d, J ¼ 6.5 Hz, 2H, H19, H21), 9.92 (d, Orange solid, yield 62%, mp 138–139 ꢀC. IR ꢀ (cmꢁ1): 2925, 1698,

1586
M.-C. SARDARU ET AL.
Figure 2. Effects of compounds 11a, 11b, 11k, 14a, 15a, and 15j (10ꢁ5 M) on microtubule dynamics using Paclitaxel (10ꢁ5 M) as microtubule stabilising agent and
Phenstatin (10ꢁ5 M) as microtubule destabilising agent.
1
1641, 1596, 1519, 1458, 1342, 1264, 1204, 1017. H NMR (500 MHz, Tubulin polymerisation assay
CDCl3): d 1.45 (t, J ¼ 7.0 Hz, 3H, CH3), 3.86 (s, 6H, 2 ꢂ OMe), 3.98
Microtubule assembly was studied using the tubulin polymerisa-
(s, 3H, OMe), 4.00 (s, 3H, OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 6.71 (s,
tion assay kit (Cytoskeleton Inc., Denver, CO, Cat. # BK006P),
2H, H23), 6.63 (bs, 1H, H28), 6.94 (d, J ¼ 8.5 Hz, 1H, H15), 7.28 (bs,
2H, H26, H30), 7.43–7.45 (3H, overlapped signals, H12, H16, H6), 7.81
(s, 1H, H2), 8.32 (bs, 2H, H18, H22), 8.82 (bs, 1H, H8), 9.35 (bs, 2H,
H19, H21), 9.91 (d, J ¼ 7.5 Hz, 1H, H5). 13C NMR (125 MHz, CDCl3): d
14.7 CH3, 56.23 OMe, 56.24 2 x OMe, 56.3 OMe, 60.9 CH2, 66.8 C23,
106.6 C26, C30, 107.9 C28, 109.5 C1, 110.2 C15, 111.8 C12, 112.3 C6,
119.7 C8, 123.9 C16, C3, 124.0 C18, C22, 128.3 C2, 129.8 C5, 130.6 C7,
131.6 C11, 135.3 C25, 137.9 C9, 147.1 C19, C21, 149.4 C13, 153.0 C14,
153.6 C17, 161.3 C27, C29, 163.5 COO, 184.5 C10, 190.4 C24. Anal.
Calcd. for C35H33BrN2O8: C, 60.96; H, 4.82; N, 4.06. Found: C, 60.95;
H, 4.79; N, 4.09.
according to the manufacturer’s instructions36,37
.
The polymerisation was monitored using FLUOstar Omega
multi-mode microplate reader (BMG LABTECH, Ortenberg,
Germany). The first step before the analysis is the pre-warming of
the plate to 37 ꢀC for 30 min. Plate temperature is essential for
high polymerisation activity and reproducible results. The tubulin
polymerisation buffer will be composed of general tubulin buffer,
tubulin glycerol buffer, and GTP stock, at 4 ꢀC. Another volume of
500 ml of general tubulin buffer is necessary for dilutions, at r.t.
Ten microlitres of GTB will be pipetted into each well. Two of the
wells will remain with GTB only, as controls. Into the rest, 10 ml of
compounds of ꢂ10 strength or Phenstatin, also of ꢂ10 strength
will be added. The final concentration of the compounds and
Phenstatin will be 10 mM. The plate will be incubated at 37 ꢀC for
2 min. Meanwhile, proceed to the dilution of 10 ml of the Paclitaxel
Stock solution with 190 ml of r.t. general tubulin buffer, to be used
in quantities of 10 ml of this per well. The tubulin will be defrosted
until r.t., placed on ice and diluted with 420 TP cold buffer, reach-
ing a final concentration of 3 mg/ml in 80 mM PIPES (piperazine-
N,N0-bis(2-ethanesulfonic acid) sesquisodium salt), pH ¼ 6.9, 2 mM
MgCl2, 0.5 mM EGTA (ethylene glycol-bis (b-amino-ethyl ether)
N,N,N0,N0-tetra-acetic acid, 1 mM GTP and 10.2% glycerol. The
diluted tubulin will be used immediately by adding 120 ml into
each of the wells with a multichannel pipette. The absorbance
was measured at 340 nm for 1 h at 1 min intervals using a plate
reader at 37 ꢀC. Representative experiment (n ¼ 3) is shown in
Figure 2.
4-(3-(3,4-Dimethoxybenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-
(3,4-dimethoxyphenyl)-2-oxoethyl)pyridin-1-ium
bromide
(15i).
Orange solid, yield 80%, mp 247–250 ꢀC. IR ꢀ (cmꢁ1): 2972, 1702,
1
1684, 1595, 1518, 1413, 1337, 1268, 1213, 1019. H NMR (500 MHz,
DMSO-d6): d 1.37 (t, J ¼ 7.0 Hz, 3H, CH3), 3.85 (s, 3H, OMe), 3.87 (s,
3H, OMe), 3.90 (s, 3H, OMe), 3.92 (s, 3H, OMe), 4.38 (q, J ¼ 7.0 Hz,
2H, CH2), 6.47 (s, 2H, H23), 7.18 (d, J ¼ 8.5 Hz, 1H, H15), 7.24 (d,
J ¼ 8.5 Hz, 1H, H29), 7.43 (bs, 1H, H12), 7.52 (d, J ¼ 8.5 Hz, 1H, H16),
7.54 (bs, 1H, H26), 7.79 (s, 1H, H2), 7.80 (d, J ¼ 8.0 Hz, 1H, H30), 7.93
(J ¼ 7.0 Hz, d, H6), 8.79 (d, J ¼ 6.5 Hz, 2H, H18, H22), 8.96 (bs, 1H,
H8), 9.10 (d, J ¼ 6.5 Hz, 2H, H19, H21), 9.84 (d, J ¼ 7.5 Hz, 1H, H5). 13C
NMR (125 MHz, DMSO-d6): d 14.4 CH3, 55.6 OMe, 55.7 OMe, 55.8
OMe, 56.0 OMe, 60.2 CH2, 65.4 C23, 107.9 C1, 110.3 C26, 111.0 C15,
111.3 C29, 111.7 C12, 113.4 C6, 119.0 C8, 123.4 C3, C30, 123.6 C16,
124.6 C18, C22, 126.3 C25, 127.3 C2, 129.2 C5, 131.0 C11, 131.8 C7,
137.7 C9, 146.6 C19, C21, 148.7 C13, 148.9 C27, 152.4 C28, 152.6 C14,
154.4 C17, 162.9 COO, 183.8 C10, 189.1 C24. Anal. Calcd. for
C35H33BrN2O8: C, 60.96; H, 4.82; N, 4.06. Found: C, 60.90; H, 4.78;
N, 4.11.
Molecular modelling
4-(3-(4-Chlorobenzoyl)-1-(ethoxycarbonyl)indolizin-7-yl)-1-(2-(4-
methoxy phenyl)-2-oxoethyl) pyridin-1-ium bromide (15j). Yield 93%.
All spectral data are in agreement with the literature32.
Flexible-ligand docking experiments were performed as previously
reported24, with slight modifications. Briefly, the 3D structures of
the ligands were constructed in Avogadro v1.2.038 and were ener-
getically optimised in the MMFF94 force field until a local energy
minimum was achieved. Autodock Vina39 was used for all docking
experiments, using a 22 ꢂ 22 ꢂ 22 Å3 gridbox centred on the col-
chicine binding site of the a,b-tubulin heterodimer (PDB: 4O2B)40.
The co-crystallised colchicine ligand and water molecules were
removed during protein preparation for docking, and the target
protein was kept rigid. Twenty poses were generated for each
Anticancer activity
The compounds were tested against a panel of 60 human cancer
cell lines at the National Cancer Institute (NCI) (Rockville, MD). The
cytotoxicity experiments were performed using a 48 h exposure
protocol which consisted of a sulphorhodamine B assay33–35
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1587
Scheme 1. Synthesis pathway for indolizines 11a–l.
Scheme 2. Synthesis of indolizines 14a–d.
ligand, which were then ranked based on theoretical binding basic medium from the salts 8a–l and 13a–d, to ethyl propiolate
energy. The best ranked models were visually inspected in order (Schemes 1 and 2)21,30,31,41
.
Indolizines 15a–i were obtained in good yields using the sub-
stitution of halides 4–6 generated by the indolizines 11e–g
(Scheme 3)32,42. We also synthesised the previously reported com-
pound 15j32 using a similar procedure.
to assess the consistency of the generated docking solutions rela-
tive to the docking poses of the known inhibitors colchicine and
Phenstatin. In order to evaluate the quality of the docking proto-
col, colchicine was extracted from the crystal structure and re-
docked into the binding site. RMSD between re-docked ligand
and co-crystallised conformation was computed in PyMOL. Visual
inspection, molecular graphics and analyses were made in the
The structures of all new target compounds were fully con-
1
firmed by H and 13C NMR, IR and elemental analyses.
€
PyMOL Molecular Graphics System, Version 1.8.2 (Schrodinger,
Biological activity
LLC, New York, NY) and Discovery Studio Visualiser Version
20.1.0.19295 (Dassault Systemes, BIOVIA Corp., San Diego, CA).
Anticancer activity
All synthesised compounds were submitted to the NCI, and 13
compounds (11a, b, d, e, f, i, j, k, l, 14a, 14d, 15a, and 15j)
were selected for single dose (10ꢁ5 M) screening against a panel
of 60 human tumour cell lines, representing leukaemia, melanoma
and cancer of lung, colon, central nervous system, ovary, kidney,
prostate, and breast33. Representative results for 11 of the com-
pounds are summarised in Table 1.
Results and discussion
Chemistry
The pyridinium salts 8a–l and 13a–d were prepared through the
direct reaction of pyridine 1, 4,40-bipyridine 2, 2,40-bipyridine 3, or
2,20-bipyridine 12, respectively, with 2-bromo-acetophenones 4–7
Indolizines 11a, b showed a very good inhibition effect on almost
in acetone, at r.t. (Schemes 1 and 2) (for spectral data of pyridi- all 60 lines, the best results being registered on leukaemia HL-60 (TB)
nium salts 8a–l and 13a–d see Supplementary data). In the next cells, colon cancer COLO 205 cells, SNC cancer SF-539 cells, melan-
step, for the synthesis of the indolizine ring, we used the 1,3- oma M14 and MDA-MB-435 cells, ovarian cancer cell OVCAR-3, renal
dipolar cycloaddition of the pyridinium ylides generated in situ in cancer A498 and RXF393 and breast cancer MDA-MB-468 cells.

1588
M.-C. SARDARU ET AL.
Scheme 3. Synthesis of indolizines 15a–i.
Table 1. Results of the in vitro growth inhibition (GI %) of tested compounds against human cancer cell lines in the single-dose assaya.
GI (%) (10–5 M)a
Cell line
Compound
CCRF-CEM
K-562
SR
HL-60(TB)
MOLT-4
RPMI-8226
A549/ATCC
HOP-62
NCI-H460
NCI-H522
COLO205
HCT-116
HCT-15
Cell type
11a
11b
11d
11e
11f
11i
11j
11k
14a
15a
15j
0
0
Leukaemia
83
89
65
86
24
23
32
30
34
10
11
10
0
25
0
19
17
23
7
10
1
30
35
0
43
20
10
3
21
0
24
1
8
0
0
20
11
30
28
0
13
11
11
0
19
0
3
0
12
0
0
0
0
0
0
0
14
0
0
2
5
2
3
15
0
4
0
13
0
3
3
0
4
0
0
0
38
0
11
6
0
11
6
4
0
5
0
8
0
0
0
8
–
0
6
100b,p
100b,q
100b,r
67
100b,i
100b,j
0
76
84
0
0
100b,k
75
100b,c
76
100b,l
69
0
0
0
0
0
0
0
100b,s
100b,t
68
89
77
68
12
96
29
57
95
77
74
72
86
72
64
70
85
59
86
88
74
27
53
47
82
100b,o
35
0
17
23
41
23
31
70
76
78
75
15
0
29
16
63
21
34
94
63
–
58
36
70
0
Non-small cell lung cancer
Colon cancer
0
13
0
14
4
12
5
100b,u
100b,v
56
74
34
0
7
0
100b,d
81
11
0
0
0
0
0
100b,w
17
71
99
70
75
25
0
HT-29
SW-620
KM12
90
78
76
100b,x
100b,z
55
100b,l
0
0
0
100b,m
36
0
2
2
0
0
CNS cancer
SF-295
SF-539
SNB-75
U251
SF-268
LOX IMVI
M14
MDA-MB-435
UACC-62
SK-MEL-5
OVCAR-3
NCI/ADR-RES
SK-OV-3
OVCAR-8
OVCAR-4
A498
RXF393
ACHN
786-0
TK10
79
73
55
79
61
53
61
3
3
2
10
11
8
0
0
11
7
0
8
18
12
8
2
3
0
6
11
7
4
1
0
0
1
17
0
1
13
9
4
14
5
0
0
9
5
0
3
0
0
2
3
0
0
19
6
0
0
0
0
0
0
0
9
0
11
0
9
0
6
70
100b,e
67
12
17
26
3
0
5
0
8
0
0
0
6
0
–
0
9
19
9
0
11
15
14
7
0
0
0
1
7
0
0
0
9
–
6
6
10
0
0
10
7
24
0
78
44
53
100b,a
87
21
87
43
24
0
0
100b,b
0
Melanoma
100b,c
45
31
13
18
18
6
100b,f
100b,g
50
96
100b,m
69
62
73
2
Ovarian cancer
Renal cancer
Breast cancer
Prostate cancer
100b,h
89
100b,n
88
63
11
9
23
70
26
27
4
78
67
64
58
28
71
50
51
64
44
78
70
51
44
64
60
46
0
93
0
32
100b,d
20
100b,i
100b,j
41
34
0
0
0
15
0
9
0
65
51
10
10
0
30
16
30
9
100b,e
91
0
45
17
25
77
42
4
87
0
MCF7
MDA-MB-468
T-47D
MDA-MB-231/ATCC
BT-549
PC-3
76
5
10
9
10
13
2
10
6
20
6
0
9
0
100b,f
99
29
17
29
18
49
70
100b,k
53
3
0
0
12
0
2
57
16
18
0
17
0
50
0
62
60
88
75
100b,g
0
100b,h
46
15
0
12
0
13
35
DU-145
0
0
55
aData obtained from NCI’s in vitro 60 cell one dose screening at 10ꢁ5 M concentration; compounds 11l and 14d were also tested, but no GI was exhibited on the
tested cell lines (results are not shown).
0
0
c
f
i
j
k
l
r
s
t
v
x
z
bCytotoxic effect; cell growth percent: 0 -25; d0 -32; e0 -7; -22; g-42; h-6; -1; -0.4; -7; -14; m-5; n-5; o-4; p-15; q-45; -16; -9; -22; u-10; -21; w-61; -10; -65; a -74, b -14,
0
0
0
0
0
0
0
0
c -44; d -33; e -82; f -29; g -23; h -6; i -3; j -53; k -27; l -10; m -30.
The best values in terms of growth inhibition are highlighted in bold.
Compound 11a also exhibited a cytotoxic effect on all these lines, selectively inhibited the growth of NCI-H522 and NCI-H460 non-
the best one being registered on melanoma MDA-MB-435 cells.
Interestingly, the substitution of indolizine heterocycle at pos-
ition 7 with a pyrid-4-yl or pyrid-2-yl ring resulted in the loss of
small cell lung cancer, SK-OV-3 ovarian cancer cells and T-47D
breast cancer cells.
Substitution of the indolizine heterocycle at position 5 with a
the activity, compounds 11e–f and 11i, 11j, and 11l (data not pyrid-2-yl ring also led to a loss of growth inhibition effect. Thus,
shown for compound 11l) presenting almost no inhibition effect compounds 14a (Table 1) and 14d showed no inhibition on
on the tested cell lines. As an exception, compound 11k tested cancer cells (data not shown for compound 14d).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1589
Table 2. Results of the 5-dose in vitro human cancer cell growth inhibitiona for compounds 11a, 15a, and 15j and positive control Phenstatin.
Compound
Cell line
11a
GI50 (lM)
11a
LC50 (lM)
15a
GI50 (lM)
15a
LC50 (lM)
15j
GI50 (lM)
15j
LC50 (lM)
Phenstatin
GI50 (lM)
Phenstatin
LC50 (lM)
Cell type
Leukaemia
K-562
HL-60(TB)
SR
0.036
0.032
0.023
0.055
0.077
0.044
0.042
0.041
0.074
0.051
0.035
0.035
0.037
0.036
0.038
0.053
0.032
0.053
0.039
0.053
0.088
0.098
0.133
0.038
<0.010
0.031
0.089
0.067
0.041
0.033
0.039
0.060
0.047
0.027
0.066
0.070
0.044
0.046
0.060
0.051
0.094
0.038
0.090
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
20.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
2.58
2.90
3.28
2.58
n.d.
2.00
2.13
3.29
1.83
n.d.
2.62
1.83
1.76
1.81
1.72
1.86
1.61
1.46
2.17
1.90
2.12
1.87
1.91
1.90
2.32
1.73
2.60
1.81
1.83
>100
19.6
1.75
1.96
n.d.
n.d.
>100
>100
>100
>100
n.d.
7.16
>100
>100
n.d.
n.d.
2.05
0.33
1.75
2.04
n.d.
1.63
1.93
1.88
1.78
n.d.
1.64
1.63
1.38
1.93
1.61
1.70
1.68
1.47
1.70
1.86
1.83
1.82
2.02
1.86
1.82
2.01
2.04
1.77
1.88
3.16
2.01
1.80
1.60
n.d.
n.d.
>100
>100
>100
>100
n.d.
n.d.
7.81
n.d.
n.d.
n.d.
7.87
n.d.
7.70
n.d.
n.d.
6.46
6.35
6.50
7.39
n.d.
n.d.
n.d.
n.d.
7.22
6.79
7.88
9.32
6.60
7.08
>100
6.51
n.d.
6.29
n.d.
6.57
8.11
>100
n.d.
<0.010
0.011
<0.010
0.034
0.040
0.037
0.033
0.027
0.057
0.073
3.05
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CCRF-CEM
MOLT-4
RPMI-8226
NCI-H460
NCI-H522
A549/ATCC
HOP-62
COLO205
HCT-15
HT29
SW-620
KM12
HCT-116
SF-295
SF-539
SNB-75
U251
SF268
SNB-19
LOX IMVI
M14
MDA-MB-435
UACC-62
MALME-3M
SK-MEL-2
SK-MEL-5
OVCAR-3
NCI/ADR-RES
SK-OV-3
786-0
Non-small cell lung cancer
Colon cancer
n.d.
>100
7.38
6.83
6.46
7.10
n.d.
6.77
7.77
n.d.
n.d.
n.d.
n.d.
n.d.
7.82
39.9
7.82
>100
6.93
7.11
>100
>100
6.58
7.55
n.d.
<0.010
2.95
<0.010
<0.010
0.038
0.367
0.011
<0.010
0.043
0.053
0.031
0.013
<0.010
<0.010
0.448
n.d.
0.520
0.040
0.021
0.012
0.623
0.905
2.28
0.296
0.016
0.033
0.031
0.034
30.4
2.71
0.045
0.039
CNS cancer
Melanoma
Ovarian cancer
Renal cancer
A498
CAKI-1
RXF 393
MCF7
HS 578T
BT-549
T-47D
MDA-MB-468
PC-3
DU-145
1.66
1.79
1.74
1.98
1.94
1.66
2.61
1.80
7.27
n.d.
1.40
1.40
1.81
1.87
1.83
1.84
1.93
1.69
Breast cancer
25.0
>100
>100
7.49
>100
6.40
n.d.
7.62
n.d.
Prostate cancer
6.05
GI50: the molar concentration of tested compound causing 50% growth inhibition of tumour cells; LC50: the molar concentration of tested compound causing 50%
death of tumour cells; n.d.: not determined.
The most significant values are highlighted in bold.
aData obtained from NCI’s in vitro 60 cell 5-dose screening35–37
.
The presence of two 3,4,5-trimethoxybenzoyl groups in com- lines at five concentrations33–35. Results from the NCI-60 5-dose
pound 15a led to the best growth inhibition effect on the tested screen are shown in Table 2.
All three compounds displayed good antiproliferative proper-
ties. The best candidate in terms of growth inhibition properties
was compound 11a, with GI50 values < 100 nM against 47 cell
lines, most notably on melanoma MDA-MB-435 (GI50<10 nM) and
UACC-62 (GI50¼31 nM) cells, leukaemia SR cells (GI50¼23 nM), and
renal cancer A498 cells (GI50¼27 nM). Compound 11a displayed
selective cytotoxic activity on the melanoma MDA-MB-435 cell line
cancer cells. Compound 15a is also distinguished by the high
cytotoxic activity displayed on 18 cell lines, including cell lines
from each panel except prostate cancer. Of the same serious,
compound 15j showed similar behaviour to 15a on most line
cells, but with much lower GI % values on NCI-H522 lung cancer
cells, SF-268 CNS cancer cells, MDA-MB-231/ATCC and BT-549
breast cancer cells.
As shown in Table 1, the substitution of the 3,4,5-trimethoxy- (LC50¼20.4 mM). Even if the overall antiproliferative activity of com-
phenyl ring produced different effects in series of compounds C pound 11a is lower in comparison with control Phenstatin, there
and D (Figure 1). Thus, in series C (11a–d), replacing the 3,4,5-tri- are 11 cancer cell lines against which compound 11a showed bet-
methoxyphenyl ring with 3,5-dimethoxyphenyl does not alter the ter GI50 values and several other lines against which compound
potency substantially, while substitution with 4-bromophenyl ring 11a had comparable GI50 values.
Although displaying excellent growth inhibitory effects at the
10ꢁ5 M single dose evaluation (Table 1), compounds 15a and 15j
did not exhibit submicromolar GI50 values, except compound 15j
with GI50¼0.33 mM against leukaemia SR cell line. Notably, both
causes a dramatical loss of inhibitory properties. In series D
(11i–l), the 3,4-dimethoxyphenyl ring appears to be the only one
to confer selective inhibitory properties against the above men-
tioned cancer cell lines.
Showing the most significant growth inhibition, compounds compounds showed considerable cytotoxic activity against all
11a, 15a, and 15j were selected for evaluation against 60 cell colon cancer, CNS cancer, renal cancer, and melanoma cell lines,

1590
M.-C. SARDARU ET AL.
but also on some cell lines from non-small cell lung cancer, ovar- contacts upon accommodation at the tubulin binding site could
explain the low biological properties exhibited by this compound.
Docking experiments for compounds 11e, 11f, 11i, 11j, and
11k did not reveal any conformations in which the methoxy-sub-
stituted cycle would overlap with the one in the colchicine bind-
ing site as to permit a polar interaction with bCys241. Instead, this
aromatic moiety was oriented towards a subunit of the binding
pocket, being stabilised by H-bonds with bLys254 (11e, 11i) or
aAsn101 (11j), as well as weak hydrogen bonding interactions
with aSer178, bGln247 and the nonexchangeable GTP molecule.
The indolizine moiety is stabilised by extensive hydrophobic con-
tacts and, in the case of 11f, 11k, by additional amide stacking
with the backbone of bLys254. The pyridyl ring is positioned deep
ian cancer, breast cancer, and prostate cancer, respectively. This
different behaviour of compounds 15a and 15j in comparison
with compounds 11a and Phenstatin, suggests different mecha-
nisms of action.
In vitro tubulin polymerisation inhibition activity
In order to confirm if the observed anticancer activity of the
above mentioned compounds is conferred by a microtubule-tar-
geting mechanism, we evaluated the effect of the active com-
pounds 11a, 11b, 11k, 15a, and 15j, but also of the inactive
compound 14a (for comparison) on the assembly of tubulin. To
confirm their influence on microtubule dynamics, two positive in the colchicine binding site, away from the a/b interface, and is
stabilised through hydrophobic interactions with residues in the b
subunit. Since all these compounds have good binding energies,
yet lack activity, it could be postulated that a polar interaction
with bCys241, or at least the positioning of possible polar inter-
action partners in the proximity of this residue is crucial for the
observed anticancer activity, as has been seen for other colchicine
binding site inhibitors44–46. However, an exception can be seen at
compound 11k, which showed selective activity against nine can-
cer cell lines (GI > 70%), and also inhibited tubulin polymerisation
in vitro, but did not form a favourable contact with this residue in
our docking experiments. Further mutagenesis experiments could
be performed in order to describe the impact of this residue on
the binding properties of the tested compounds. Despite its
potent anticancer activity, the low theoretical binding score of
11a compared to 11e–k suggests that its cytotoxicity may involve
other cellular targets or pathways other than the ab-tubulin heter-
odimer. At the same time, since cancer cells preferentially express
different b-tubulin isoforms, it would be possible that this com-
pound binds with greater affinity to other isoforms. This aspect
could be further studied in silico, as has been done for DAMA-col-
chicine47 and other colchicine binding site microtubule depoly-
merising agents48.
Compound 14a was accommodated in a similar fashion to
compounds 11a,b,d, preserving the interactions of the methoxy-
substituted moiety with the hydrophobic pocket formed by
bCys241, bLeu248, bLeu255, bAla250, bAla316, bIle318, bAla354,
and bIle378, but its indolizine ring rotated as to permit the inter-
action between the pyrid-2-yl ring and bMet259 through pi-sul-
phur stacking. This rotation also led to the formation of an H-
bond between the carboxylate moiety of this compound and
bGln247. While the in vitro tubulin polymerisation assay results are
in agreement with the docking observations, the lack of anti-
cancer activity in the case of compound 14a remains to
be elucidated.
Compound 15a occupied the colchicine binding site similar to
compounds 11a,b,d and Phenstatin, engaging in H-bonds with
bCys241 and bAsn258, and forming hydrophobic contacts with
bLeu248, bLeu255, bAla250, bAla316, bIle318, and bAla354. The
additional 3,4,5-trimethoxybenzoyl group reached towards the
H10 helix of the b-tubulin subunit to form H-bonds with bThr353
and bGln336, as well as pi-anion stacking with the sidechain of
bAsp329. The indolizine moiety was stabilised by amide-pi stack-
ing with the backbone of aThr179. This compound had the one of
the lowest binding energies of all tested molecules (–10.2 kcal/
mol), being surpassed only by compound 15j (–10.7 kcal/mol).
Interestingly, compound 15j forms a hydrophobic interaction with
bCys241 and maintains many of the polar and hydrophobic con-
tacts observed at 15a, being accommodated in the same
controls were used: paclitaxel (a tubulin stabiliser) and Phenstatin
(a tubulin polymerisation inhibitor). As presented in Figure 2,
Paclitaxel was found to stimulate tubulin polymerisation, while
Phenstatin and all six tested compounds appeared to inhibit tubu-
lin polymerisation.
Four compounds, namely 11k, 14a, 15a, and 15j showed a
similar strong inhibitory behaviour on tubulin polymerisation,
superior to the one obtained for compounds 11a and 11b. The
obtained data clearly indicated that all tested target compounds
effectively inhibit tubulin polymerisation in vitro.
Molecular modelling
Molecular docking experiments revealed similar docking confor-
mations of the tested compounds to previously reported anti-
cancer pyrrolo[1,2-b]pyridazines (compounds type B, Figure 1),
which are also thought to achieve anticancer activity by binding
to the colchicine binding site of tubulin24. The used docking
protocol was validated by computing the RMSD between re-
docked colchicine and its co-crystallised conformation, which was
0.16 Å in our case. Generally, an RMSD value below 2 Å (the aver-
age resolution of a crystal structure) is considered acceptable43.
Compound 11a is stabilised in the colchicine binding site of
tubulin through H-bonding between its carbonyl moiety and
bAsn258 and through extensive hydrophobic contacts with the
accommodating pocket formed by bCys241, bLeu248, bLeu255,
bAla250, bAla316, bIle318, bAla354, and bIle378, similar to
Phenstatin. Moreover, the indolizine heterocycle extends towards
the a subunit with the help of two polar interaction partners:
bLys254 and aAsn101, and is further stabilised by the nonpolar
chain of bLys352, which is engaged in an H-bond with aThr179
þ
through its e-NH3
group. The 4-methoxy substituent does not
engage in an H-bond with bCys241, as seen in the case of
Phenstatin, but the binding orientation of this compound sug-
gests that this bond could form if the binding site would be opti-
mised. Further molecular dynamics simulations could be
performed in order to investigate the formation and stability of
this H-bond. Removal of the 4-methoxy group leads to a slight
shift of the methoxy-substituted ring compared to 11a, possibly
to optimise hydrophobic contacts, as reflected by the similar the-
oretical binding energy (Table 3), but the formed H-bonds with
bAsn258, bLys254, and aAsn101 are enough to maintain a con-
formation roughly overlapping with 11a, which could account for
the observed biological activity exhibited by this compound. The
bromo-substituted compound 11d was accommodated more
deeply in the colchicine binding pocket, exclusively through
hydrophobic interactions, having one of the lowest theoretical
binding energies of all docked compounds. The absence of polar extended binding site-spanning conformation. Additional

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1591
Table 3. Binding orientation, energy, and amino acid contacts for tested compounds, as predicted by molecular docking experiments.
(continued)

1592
M.-C. SARDARU ET AL.
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1593
For binding orientation, the a,b-tubulin heterodimer is shown as ribbons; aminoacids and ligands are represented as sticks; for 2D interaction diagrams, colours are
as follows: conventional hydrogen bonds – green, carbon–hydrogen bonds – pale green; hydrophobic interactions – light pink; amide–p stacking – dark pink;
anion–p stacking: orange; p–sulphur stacking: dark yellow.

1594
M.-C. SARDARU ET AL.
molecular dynamics experiments should be performed in order to
confirm the stability of the observed interactions, especially
with bCys241.
ORCID
Roxana Maria Amarandi
Ionel I. Mangalagiu
http://orcid.org/0000-0002-9937-6539
http://orcid.org/0000-0002-4632-5076
Ramona Danac
http://orcid.org/0000-0003-4370-5353
Conclusions
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