Discovery of indolizines containing triazine moiety as new leads for the development of antitumoral agents targeting mitotic events

Article abstract of DOI:10.1016/j.bmcl.2015.07.025

Abstract A new family of 3-aroylindolizines bearing a dimethoxytriazine unit in their position 1 was designed, synthesized and evaluated for their ability to inhibit tubulin polymerization and cellular growth in vitro. Compound 39 was the best candidate i

Full text of DOI:10.1016/j.bmcl.2015.07.025

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3975–3979
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of indolizines containing triazine moiety as new leads
for the development of antitumoral agents targeting mitotic events
Liliana Lucescu a, Alina Ghinet a,b,c, Dalila Belei a, Benoît Rigo b,c, Joëlle Dubois d, Elena Bîcu a,
⇑
a Department of Organic Chemistry, Faculty of Chemistry, ‘Al. I. Cuza’ University of Iasi, B-dul Carol I, Nr. 11, Corp A, 700506 Iasi, Romania
b Inserm, LIRIC-U995, Université Lille 2, CHRU de Lille, Faculté de Médecine—Pôle Recherche, Place Verdun, F-59045 Lille Cedex, France
c Hautes Etudes d’Ingénieur (HEI), UCLille, Laboratoire de pharmacochimie, 13 rue de Toul, BP 41290, F-59014 Lille Cedex, France
d Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new family of 3-aroylindolizines bearing a dimethoxytriazine unit in their position 1 was designed,
synthesized and evaluated for their ability to inhibit tubulin polymerization and cellular growth
in vitro. Compound 39 was the best candidate in the current study with a GI50 value of 870 nM on
SNB-75 CNS cancer cells and of 920 nM on MDA-MB-231/ATCC breast cancer cells. The standard NCI
Compare results indicated that indolizine 39 may target PLK1 (polo-like kinase 1).
Ó 2015 Elsevier Ltd. All rights reserved.
Received 8 June 2015
Revised 9 July 2015
Accepted 11 July 2015
Available online 17 July 2015
Keywords:
Indolizine
Dimethoxytriazine
Antitumor agent
PLK1
Polo-like kinase 1 inhibitor
O-demethylation
Hilbert–Johnson transposition
Indolizines are important chemical structures having an impor-
tant place both in organic synthesis and in biochemistry as they
represent promising molecules with potential applications in the
fields of pharmaceuticals,1–3 total synthesis,1 organic materials as
novel fluorescent sensors4 and as key intermediates in organic syn-
thesis.1–3 In particular, 3-aroylindolizine systems have attracted
considerable interest from our research group in recent years for
their antitumoral potential.5,6 The reported biological activity for
similar indolizines also highlights efficiency on cardiovascular dis-
orders7–10 or on degenerative joints diseases11 as osteoarthritis or
spondyloses. However, the existing literature on 3-aroylindolizines
bearing a heterocyclic unit in their position 1 is extremely scarce
and mainly concerns the synthetic procedures.7,8,12–18 Only three
of such indolizines have been reported and patented for their bio-
logical activity, particularly as potential antagonists of the binding
of FGFs (fibroblast growth factors) to their receptors (compounds
I–III, Fig. 1).7,8
propose in this manuscript the development of a new family of
3-aroylindolizines substituted by a dimethoxytriazine unit target-
ing mitotic events (target compounds 27–45, Fig. 1). Tubulin is the
structural protein of microtubules, a major component of the
cytoskeleton. Since cancer cells divide more frequently than nor-
mal cells, and since mitotic microtubule dynamics is 4–100 times
greater than that of microtubules during interphase, tubulin is a
target for antitumor compounds.19 Inhibitors of tubulin polymer-
ization will therefore alter the cytoskeleton and inhibit the mitotic
spindle formation, resulting in a high cytotoxic effect. Phenstatin
(compound IV, Fig. 1) is one of the most potent tubulin polymer-
ization inhibitor that binds to the colchicine site of the protein.20
We have already obtained microtubule-interacting agents, phen-
statin analogs, with indolizine skeleton as B-ring that showed
excellent in vitro antiproliferative effect in the nanomolar range
on MDA-MB-435 melanoma cell lines (GI50 = 30 nM) (e.g., com-
pound VI, Fig. 1).5 In order to develop new anti-cancer agents
and enrich the structure–activity relationships in this field, we
designed and synthesized new phenstatin analogs with
dimethoxytriazine–indolizine core as B-ring and diversely substi-
tuted phenyl unit as A-ring (Fig. 1).
Because of this limited number, the general development of
new methodologies that can target these heterocycles and espe-
cially when they allow their diversification and discovery of new
bioactive agents, is a valuable ambition. In this perspective, we
The chemical strategy started with the synthesis of the key
dipolarophile 3. In this light, propiolic acid 1, after activation as
⇑
Corresponding author. Tel.: +40 232 20 13 47; fax: +40 232 20 13 13.
E-mail address: elena@uaic.ro (E. Bîcu).
acid
chloride
2,
then
treatment
with
zinc
http://dx.doi.org/10.1016/j.bmcl.2015.07.025
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3976
L. Lucescu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3975–3979
O
O
O
O
O
O
O
O
Me
N
Me
N
N
N
N
H
N
H
.
.
.
F
O
N
Me
N
Me
N
Me
HCl
HCl
HCl
Me
Me
Me
N
N
N
III7,8
I8
II8
R2
O
R1
Me
N
R
O
O
O
3
O
2
MeO
MeO
OH
OH
MeO
MeO
N
N
1
N
N
A
OMe
B
N
S
S
N
OMe
OMe
OMe
COOEt
COOEt
N
OMe
MeO
27-45
Target compounds
IV20
V21
VI5
VII22
Phenstatin
Phenstatin analogues
Figure 1. Structure of some biologically active 3-aroylindolizines I–III,7,8 of phenstatin IV,20 phenstatin analogs V–VII5,21,22 and of target compounds 27–45.
OMe
pyridinium salts 6–24, with 2-ethynyl-4,6-dimethoxy-1,3,5-tri-
N
N
O
O
(i)
(ii)
azine 3, followed by spontaneous aromatization of intermediates
26a–r (Scheme 2). The cycloimonium salts 6–24 have been easily
obtained in good yields by reacting commercially available pyri-
N
OH
Cl
OMe
3
1
2
dine derivatives 5a–h with
x-bromoacetophenones 4A–H.
Scheme 1. Reagents and conditions: (i) 1 equiv PCl5, dichloromethane, N2, rt, 1 h,
90% yield; (ii) 0.7 equiv zinc dimethylimidodicarbonimidate, molecular sieves 4 Å,
dichloromethane, N2, rt, 24 h, 55% yield.23
In order to investigate the importance of the dimethoxytriazine
ring of compound 39 on antitumoral activity, additional modula-
tions have been envisaged and realized. In this light, we decided
to use the chemical strategy discovered previously on N-substi-
tuted pyrrolidinones linked to a 4,6-dimethoxy-1,3,5-triazine24
and transpose it to indolizines containing triazine developed in
the current manuscript. Thus, refluxing indolizine 39 in 2% aque-
ous HCl for 24 h resulted in a total O-demethylation of methoxy
groups and furnished triazinedione 46 in 94% yield. A mono
dimethylimidodicarbonimidate salt,23 furnished acetylenic deriva-
tive 3 (Scheme 1), used further for all the cycloaddition reactions.
Target indolizines containing triazine 27–45 were next
obtained by [3+2] cycloaddition reaction of the corresponding
ylide 25a–r, generated in situ by triethylamine treatment of
Y
6 R1 = R2 = R3 = OMe, X = Y = Z = W = H, 70%
X
Z
O
7 R1 = R2 = R3 = OMe, X = Me, Y = Z = W = H, 96%
O
R3
R2
Br
N
X
Y
R3
R2
N
8 R1 = R2 = R3 = OMe, X = Y = W = H, Z = Me, 65%
9 R1 = R2 = R3 = OMe, X = Ph, Y = Z = W = H, 20%
10 R1 = R3 = H, R2 = OMe, X = Y = Z = W = H, 93%
11 R1 = R3 = H, R2 = OMe, X = Y = W = H, Z = CN, 23%
12 R1 = R3 = H, R2 = Me, X = Y = Z = W = H, 94%
13 R1 = R3 = H, R2 = Me, X = Y = W = H, Z = CN, 84%
14 R1 = R3 = H, R2 = Ph, X = Y = Z = W = H, 70%
15 R1 = R3 = H, R2 = CN, X = Y = Z = W = H, 93%
16 R1 = R3 = H, R2 = F, X = Y = Z = W = H, 70%
17 R1 = R3 = H, R2 = Cl, X = Y = Z = W = H, 92%
18 R1 = R3 = H, R2 = Br, X = Y = Z = W = H, 99%
19 R1 = R3 = H, R2 = Br, X = Me, Y = Z = W = H, 89%
20 R1 = R3 = H, R2 = Br, X = Y = W = H, Z = Me, 97%
21 R1 = R3= H, R2 = Br, X = Z = H, Y = W =Me, 96%
22 R1 = R3 = H, R2 = Br, X = Y = W = H, Z = CN, 56%
23 R1 = R3 = H, R2 = Br, X-Y = (CH)4, Z = W = H, 97%
24 R1 = R3 = H, R2 = Br, X = Y = H, Z-W = (CH)4, 89%
W
(i)
+
Br
W
R1
Z
R1
4A-H
5a-h
6-24
(ii)
OMe
N
N
N
Y
H
Y
X
X
Z
O
OMe
O
3
Z
R3
R2
R3
R2
N
N
W
H
W
H
R1
N
R1
N
OMe
N
25a-r
MeO
26a-r
4A R1 = R2 = R3 = OMe
4B R1 = R3 = H, R2 = OMe
4C R1 = R3 = H, R2 = Me
4D R1 = R3 = H, R2 = Ph
4E R1 = R3 = H, R2 = CN
4F R1 = R3 = H, R2 = F
4G R1 = R3 = H, R2 = Cl
4H R1 = R3 = H, R2 = Br
27 R1 = R2 = R3 = OMe, X = Y = Z = W = H, 58%
28 R1 = R2 = R3 = OMe, X = Me, Y = Z = W = H, 51%
29 R1 = R2 = R3 = OMe, X = Y = W = H, Z = Me, 59%
30 R1 = R2 = R3 = OMe, X = Ph, Y = Z = W = H, 55%
31 R1 = R3 = H, R2 = OMe, X = Y = Z = W = H, 45%
32 R1 = R3 = H, R2 = OMe, X = Y = W = H, Z = CN, 52%
33 R1 = R3 = H, R2 = Me, X = Y = Z = W = H, 53%
34 R1 = R3 = H, R2 = Me, X = Y = W = H, Z = CN, 44%
35 R1 = R3 = H, R2 = Ph, X = Y = Z = W = H, 40%
36 R1 = R3 = H, R2 = CN, X = Y = Z = W = H, 52%
37 R1 = R3 = H, R2 = F, X = Y = Z = W = H, 47%
38 R1 = R3 = H, R2 = Cl, X = Y = Z = W = H, 52%
39 R1 = R3 = H, R2 = Br, X = Y = Z = W = H, 44%
40 R1 = R3 = H, R2 = Br, X = Me, Y = Z = W = H, 49%
41 R1 = R3 = H, R2 = Br, X = Y = W = H, Z = Me, 58%
42 R1 = R3 = H, R2 = Br, X = Z = H, Y = W =Me, 63%
43 R1 = R3 = H, R2 = Br, X = Y = W = H, Z = CN, 55%
44 R1 = R3 = H, R2 = Br, X-Y = (CH)4, Z = W = H, 55%
45 R1 = R3 = H, R2 = Br, X = Y = H, Z-W = (CH)4, 52%
- H2
Y
N
X
O
Z
R3
R2
N
W
5a
X = Y = Z = W = H
5b X = Me, Y = Z = W = H
5c X = Y = W = H, Z = Me
5d X = Ph, Y = Z = W = H
5e X = Z = H, Y = W =Me
5f X = Y = W = H, Z = CN
5g X-Y = (CH)4, Z = W = H
5h X = Y = H, Z-W = (CH)4
R1
N
OMe
N
MeO
27-45
Scheme 2. Reagents and conditions: (i) EtOAc, reflux, 24 h; (ii) 1.5 equiv TEA, acetonitrile, rt, 24 h.

L. Lucescu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3975–3979
3977
O
(i)
(iii)
N
Total O-demethylation
Hilbert-Johnson transposition
Br
O
O
O
N
39
MeO
N
N
N
N
N
N
OMe
N
Br
Me
Br
Me
Br
NH
N
N
O
N
O
46
(ii)
O
Partial O-demethylation
49
48
NH
N
NH
O
O
O
Me
O
N
Br
NH
N
47
O
N
MeO
Scheme 3. Reagents and conditions: (i) HCl aq 2%, dioxane, reflux, 24 h, 94% yield; (ii) 9 equiv KOH, dioxane, reflux, 48 h, 85% yield; (iii) 3.75 equiv Me2SO4, toluene, reflux,
72 h, 34% yield.
O-demethylation of indolizine–triazine 39 was next accomplished
in basic conditions and provided indolizine 47. Finally, the Hilbert–
Johnson oxygen to nitrogen transposition of the methyl groups24
was tested by refluxing dimethoxytriazine 39 in toluene in pres-
ence of dimethyl sulfate for 72 h. Unexpectedly, only the transpo-
sition of one methyl group has been observed and we were able to
isolate indolizine 48 as unique reaction product. No traces of
expected transposition product 49 have been detected. This under-
lines a different chemical behavior for the dimethoxytriazine unit
when linked to a sp2 carbon compared to dimethoxytriazines
linked to a sp3 carbon in previously described pyrrolidin-2-ones24
(Scheme 3).
Table 1
In vitro percentage of growth inhibition (GI%) caused by the compounds 27–34, 38–41, and 43–45 against some tumor cell lines in the single-dose assaya
Panel
Cell line
Compound
27
28
29
30
31
32
33
34
38
39
40
41
43
44
45
b
Leukemia
CCRF-CEM
HL-60(TB)
K-562
RPMI-8226
SR
—
—
10
33
45
10
67
26
60
56
30
74
62
—
29
52
29
—
—
—
—
19
—
—
—
—
13
13
—
—
26
22
—
—
—
—
—
11
—
—
13
26
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12
22
26
36
10
—
20
Non-small cell lung cancer
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H460
NCI-H522
—
—
—
—
—
—
17
34
—
—
—
36
28
—
—
16
43
28
—
—
—
14
11
21
97
—
12
—
—
25
—
—
—
—
—
—
—
—
—
—
11
60
11
—
—
21
21
77
—
—
20
47
62
96
59
75
70
40
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
55
—
—
28
71
—
49
79
—
35
68
—
56
60
Colon cancer
CNS cancer
HCT-116
—
15
27
—
36
—
12
—
26
65
—
—
—
—
—
SF-295
SF-539
SNB-75
U251
—
—
—
—
15
14
67
—
—
40
—
23
—
47
32
—
—
46
—
—
—
18
—
47
42
33
39
À3
44
87
76
À5
61
22
—
75
—
20
—
—
—
—
27
—
76
—
75
—
59
—
69
—
29
58
15
À17
À14
56
56
—
Melanoma
MALME-3M
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
11
35
—
—
—
37
86
14
41
—
61
100
40
63
38
63
—
—
—
67
—
—
—
—
—
—
11
—
—
—
—
—
—
—
—
—
19
—
—
17
—
—
—
16
14
30
—
12
—
—
14
37
45
63
27
62
30
18
—
14
—
—
—
73
—
30
—
31
—
—
—
—
—
11
—
67
—
52
—
32
—
58
—
43
—
30
—
—
30
39
Ovarian cancer
Renal cancer
OVCAR-3
OVCAR-4
OVCAR-8
NCI/ADR-RES
SK-OV-3
—
—
—
—
—
19
—
12
15
18
75
21
15
24
29
28
18
41
38
—
—
—
16
19
54
—
—
—
—
—
—
—
—
—
—
—
—
14
—
—
24
—
55
44
88
52
50
15
18
29
14
20
—
95
—
—
—
—
—
—
—
—
51
33
50
46
—
31
30
50
43
—
42
À11
786-0
ACHN
CAKI-1
RXF 393
TK-10
—
—
—
—
—
—
20
47
25
41
—
—
27
54
26
59
18
—
—
—
14
—
—
—
43
—
—
40
15
—
52
—
31
56
—
37
12
59
70
56
75
78
14
30
—
36
—
—
—
—
—
—
—
14
—
81
29
44
81
—
45
22
33
61
—
30
11
31
—
30
43
15
—
—
—
Prostate cancer
Breast cancer
PC-3
—
19
36
37
18
—
13
—
19
55
—
—
58
37
MCF7
MDA-MB-231/ATCC
HS 578T
—
—
—
—
55
17
17
—
73
25
35
22
23
14
32
13
50
25
17
—
10
35
—
—
19
43
46
19
18
48
96
52
24
27
22
19
38
—
—
—
11
—
60
96
65
38
44
80
56
31
18
16
—
39
42
—
T-47D
15
—
—
The bold values highlight the best activities (the most representative results).
a
Data obtained from NCI’s in vitro disease-oriented human tumor cell screen at 10
No inhibitory activity.
lM concentration.
b

3978
L. Lucescu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3975–3979
Table 2
National Cancer Institute (NCI) for initial biological screening on
the NCI-60 cell lines panel at a single dose of 10
M.26 The repre-
Results of the 5-dose in vitro 60-cell-lines screena for compounds 39 and 44
l
sentative results are shown in Table 1. Most indolizines containing
triazine showed the best cellular growth inhibition on SNB-75 CNS
cancer cell line. Additionally, indolizines 31, 34, 38 and 39
O
O
N
N
exhibited
a
cytotoxic effect at this concentration. 3,4,5-
Br
Br
Trimethoxyphenylindolizines 27–29 displayed the best cellular
inhibitory activity on MDA-MB-435 melanoma cell line in the same
way as reference compound VI (GI% (MDA-MB-435) = 100)5
(Fig. 1). However, these derivatives showed the best inhibitory
potential on leukemia cancer cell lines among the tested com-
pounds. The replacement of the trimethoxyphenyl unit by a
para-bromophenyl resulted in superior biological potential and,
particularly, in a different inhibition profile (indolizines 27 vs 39,
Table 1), suggesting a different biological mode of action. Thus,
an interesting cytotoxic potential on SK-OV-3 ovarian cancer cells,
overexpressing Pgp (permeability glycoprotein) has been regis-
tered for indolizine–triazine 39 (Table 1). On non-small cell lung
cancer cells, indolizines 31 and 39 were the most active,
para-methoxyphenyl substituted indolizine 31 having similar
inhibitory percentage as para-bromophenyl derivative 39. Again,
compound 39 was the most active, inhibiting the growth of 65%
of HCT-116 colon cancer cells. The growth of renal cancer cell lines
(786-0 and RXF 393) was mainly inhibited by compound 44, a ster-
ically hindered analog of indolizine 39 bearing a pyrrolo[2,1-a]iso-
quinolinyl unit. The same compound was the best inhibitor of the
growth of PC-3 prostate cancer cells. Finally, on breast cancer cells,
the most active molecules were again indolizines 39 (96% inhibi-
tion of HS 578T cells) and 44 (96% inhibition of MDA-MB-
231/ATCC cells). Compound 45, the position isomer of indolizine
44, showed decreased biological potency.
N
N
39
44
N
N
OMe
OMe
N
N
MeO
MeO
b
Cell type
Cell line
GI50
39
(l
M)
GI50 (lM)
44
Breast cancer
Melanoma
Prostate cancer
CNS cancer
MDA-MB-231/ATCC
SK-MEL-28
DU-145
0.92
83.8
29.1
0.87
1.87
3.32
2.6
4.74
2.18
1.08
ndc
nd
nd
SNB-75
U251
TK-10
786-0
RXF 393
HOP-62
HOP-92
2.58
nd
Renal cancer
nd
4.06
3.57
22.8
13.2
Non-small cell lung cancer
The bold values highlight the best activities (the most representative results).
Data obtained from NCI’s in vitro 60 cell 5-dose screening.26
a
b
GI50 is the molar concentration of indolizine–triazine causing 50% growth
inhibition of tumor cells.
c
Not determined.
All synthesized indolizines containing triazine 27–48 have
been tested in vitro for their ability to inhibit tubulin polymeriza-
tion.25 Unpredictably, no active molecule was detected at a
100 lM concentration. The absence of inhibitory potential of ana-
logs 27–30 bearing the classical 3,4,5-trimethoxyphenyl unit of
parent phenstatin IV as A ring was the most astonishing and con-
firms that the biological target of this series is different than
tubulin.
The biological evaluation continued on fifteen indolizines con-
taining triazine 27–34, 38–41, and 43–45, selected by the
The best candidates issued from this single-dose assay, indoli-
zines 39 and 44, progressed in the 5-dose in vitro 60-cell-lines
screen in order to evaluate their GI50 values. Results are reported
in Table 2 and confirm that compound 39 is the best candidate
in the current study with a GI50 value of 870 nM on SNB-75 CNS
cancer cells and of 920 nM on MDA-MB-231/ATCC breast cancer
Table 3
Standard NCI COMPARE results obtained for indolizine 39
Compound ID
Structure
NSC number
S763415
Rank
Correlation
Count common cell lines
O
N
N
Br
39
—
1
42
N
OMe
N
MeO
O
N
S
N
OMe
O
GSK1030058A
S756135
1
0.942
42
MeO
CF3
OMe
N
N
N
+
63875
S63875
2
3
0.865
0.859
42
42
N
N
N
O -
OMe
OMe
O
EtO
EtO
O
OEt
OEt
492247
S625157
O
O

L. Lucescu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3975–3979
3979
cells. The bulky derivative 44 was globally less active with a GI50
value in the low micromolar range on SNB-75 cells.
Treatment and Diagnosis (the URL to the Program’s website:
http://dtp.cancer.-gov) and for NCI COMPARE program.
In our search for the biological target of the newly synthesized
indolizines containing triazine, we decided to use the NCI
COMPARE program. The NCI COMPARE is an online database and
a comparison tool that analyzes the profile of cellular growth inhi-
bition from the NCI-60 cell line panel of tested compounds to assist
in the identification of molecules with similar activity profiles, pre-
viously screened by the DTP (Developmental Therapeutics
Program).27 Thus, strong correlation with drugs from the DTP data-
base may provide guidance on the biological target. Interestingly,
the standard compare results for the most active molecule in the
current study (indolizine 39), indicated a correlation of 0.942 with
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
025.
References and notes
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Acknowledgments
This work was supported by the strategic Grant
POSDRU/159/1.5/S/137750, Project ‘Doctoral and Postdoctoral pro-
grams support for increased competitiveness in Exact Sciences
research’ cofinanced by the European Social Fund within the
Sectorial Operational Program Human Resources Development
2007–2013 (PhD scholarship L.L.). The authors acknowledge the
National Cancer Institute (NCI) for biological evaluation of com-
pounds on their 60-cell panel: the testing was performed by the
Developmental Therapeutics Program, Division of Cancer