Antiproliferative activity of 2-alkyl-4-halopiperidines and 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines in solid tumor cell lines

Article abstract of DOI:10.1016/j.bmcl.2007.03.010

A series of trans-2-alkyl-4-halopiperidines and 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines were prepared by means of an iron(III) catalyzed process. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780 (ovarian

Full text of DOI:10.1016/j.bmcl.2007.03.010

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2681–2684
Antiproliferative activity of 2-alkyl-4-halopiperidines and
2-alkyl-4-halo-1,2,5,6-tetrahydropyridines in solid tumor cell lines
a,b
a
a,b
´
´
´
´
Leticia G. Leon, Ruben M. Carballo, Marıa C. Vega-Hernandez,
a
a
a,b,
*
´
´
´
´
´
Vıctor S. Martın, Juan I. Padron and Jose M. Padron
aInstituto Universitario de Bio-Orga´nica ‘‘Antonio Gonza´lez’’ (IUBO-AG), Universidad de La Laguna,
C/ Astrofı´sico Francisco Sa´nchez 2, 38206 La Laguna, Spain
bBioLab, Instituto Canario de Investigacio´n del Ca´ncer (ICIC), C/ Astrofı´sico Francisco Sa´nchez 2, 38206 La Laguna, Spain
Received 28 February 2007; accepted 3 March 2007
Available online 7 March 2007
Abstract—A series of trans-2-alkyl-4-halopiperidines and 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines were prepared by means of an
iron(III) catalyzed process. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780 (ovarian
cancer), SW1573 (non-small cell lung cancer), and WiDr (colon cancer). The results on the biological activity revealed that, in
general, the 2-alkyl-4-halo-1,2,5,6-tetrahydropyridine analogs are more potent than the trans-2-alkyl-4-halopiperidine derivatives.
A remarkable selectivity of the aza compound 5f for the resistant cell line WiDr was observed. Cell cycle studies revealed a
G2/M phase arrest for 5f.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Marine organisms have proven to be an endless source
of novel biologically active natural products.1 In partic-
ular, marine secondary metabolites appear as promising
molecules for the development of anticancer drugs.2
Within our program directed at the development of
novel antitumor drugs based on heterocyclic scaffolds
from marine origin (Fig. 1), this type of compounds
has attracted our interest.3
Cl
O
Cl
O
Y
Y
X
X
OAllyl
15—37 μM
R
17
R
R'
O
6
R
O
16
R = Alkyl R = Ph,
R' = -Bu
—28 μM
—
39 μM
—38 μM
c
-Hex
X = OTBS, Y = H
X = H, Y = OTBS
R = CO2Me, CH2OH
i
Figure 1. Heterocyclic marine scaffolds and antiproliferative activity
against the human solid tumor cell lines A2780 (ovarian), SW1573
(non-small cell lung), and WiDr (colon).
A major drawback of marine compounds is the very low
amounts in which they are present in their natural
sources. This fact forbids the direct use of the marine
reservoir to isolate drugs to be used in chemotherapy.
Therefore, chemists are encouraged to develop high
yield synthetic methods to produce these compounds
in a limited number of chemical steps and in large quan-
tities. In our group, we have developed diverse method-
ologies for the synthesis of cyclic ethers.4 Of particular
interest is our approach to the synthesis of oxacycles
based on a one-pot Prins-type cyclization, which is pro-
moted by the inexpensive and environmentally friendly
iron(III) chloride.5
In an effort to find more active analogs, we considered
azacyclic bioisosteres of the previously reported oxacy-
clic derivatives. Herein we report on the antiproliferative
activity of a set of 12 trans-2-alkyl-4-halopiperidines 3
and twelve 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines 5
against a panel of three representative human solid tu-
mor cells: A2780 (ovarian cancer), SW1573 (non-small
cell lung cancer, NSCLC), and WiDr (colon cancer).
We performed cell cycle studies to assess the effect of
the most potent compound 5f in SW1573 and WiDr
cells.
Keywords: Marine drugs; Anticancer drugs; Halogenated piperidines;
Halogenated tetrahydropyridines; Solid tumors; Structure–activity
relationship.
*
Corresponding author. Tel.: +34 922 318 580; fax: +34 922 318
571; e-mail: jmpadron@ull.es
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.010

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L. G. Leon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2681–2684
2682
The compounds were prepared by means of the so-
called aza-Prins-type cyclization.6 This fast, simple,
and versatile method is based on the consecutive gener-
ation of a c-unsaturated-iminium ion and further nucle-
ophilic attack by the unsaturated carbon–carbon bond.
In this process, homoallyl tosylamine (1) leads to the
corresponding trans-2-alkyl-4-halo-1-tosylpiperidine 3
as the major isomer, while homopropargyl tosylamine
(4) gives 2-alkyl-4-halo-1-tosyl-1,2,5,6-tetrahydropyri-
dine 5 as only product (Scheme 1). The choice of the ir-
on(III) salt-solvent system FeCl3/CH2Cl2 or FeBr3/
CH2Br2 leads to the 4-chloro or 4-bromo derivatives,
respectively.7 Thus, we have prepared the set of trans-
2-alkyl-4-halopiperidines 3a–l (Table 1) and their corre-
sponding analogs 2-alkyl-4-halo-1,2,5,6-tetrahydropyri-
dines 5a–l (Table 2).
X
FeX3
+
RCHO
The in vitro antiproliferative activity was evaluated
using the National Cancer Institute (NCI) protocol8
after 48 h of drug exposure using the sulforhodamine
B (SRB) assay.3b For trans-2-alkyl-4-halopiperidines
3a–l, the results showed that the majority of the com-
pounds were able to induce growth inhibition in the
resistant cancer cell line WiDr (Table 1). With the excep-
tion of compound 3f (R = Bn), A2780 and SW1573 cells
were less sensitive to the derivatives than WiDr cells.
This is a remarkable effect, since the general observation
for conventional antitumor drugs is that WiDr colon
cancer cells are more drug resistant than A2780 ovarian
TsHN
TsHN
N
R
CH2X2
Ts
3a—l
1
4
2
X
FeX3
+
RCHO
N
R
CH2X2
Ts
5a—l
2
Scheme 1. Synthesis of substituted 4-halopiperidines and 4-halo-
1,2,5,6-tetrahydropyridines.
Table 1. Lipophilicity and in vitro antiproliferative activity of trans-2-alkyl-4-halopiperidines against human solid tumor cellsa
Compound
ClogPb
Substituent
A2780
TGI
SW1573
TGI
WiDr
TGI
X
R
GI50
LC50
GI50
LC50
GI50
LC50
3a
3b
3c
3d
3e
3f
3.16
5.14
6.86
5.71
4.92
5.25
3.30
5.28
7.00
5.85
5.06
5.39
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
H
>100
36 ( 6.7)
>100
>100
19 ( 6.9)
>100
>100
i-Bu
n-Hep
c-Hex
Ph
22 ( 7.8)
54 ( 28)
15 ( 6.8)
37 ( 10)
20 ( 1.6)
>100
72 ( 16)
>100
53 ( 25)
>100
Bn
H
19 ( 1.7)
>100
50 ( 1.0)
12 ( 0.3)
>100
66 ( 47)
3g
3h
3i
i-Bu
n-Hep
c-Hex
Ph
39 ( 11)
>100
>100
22 ( 0.8)
>100
14 ( 1.3)
>100
3j
82 ( 20)
>100
73 ( 2.4)
14 ( 5.0)
36 ( 2.4)
37 ( 15)
3k
3l
>100
63 ( 17)
Bn
a Values representing GI50 are given in lM and are means of two to four experiments, standard deviation is given in parentheses. TGI and LC50
values are given only if they are less than 100 lM, which is the maximum concentration tested.
b Ref. 10.
Table 2. Lipophilicity and in vitro antiproliferative activity of 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines against human solid tumor cellsa
Compound ClogPb Substituent
A2780
TGI
SW1573
TGI
WiDr
TGI
X
R
GI50
LC50
GI50
LC50
GI50
LC50
5a
5b
5c
5d
5e
5f
3.42
5.39
7.11
5.97
5.18
5.51
3.51
5.48
7.20
6.06
5.27
5.60
Cl
H
>100
>100
>100
Cl i-Bu
35 ( 8.3)
31 ( 8.5)
49 ( 12)
62 ( 0.6)
21 ( 6.9)
50 ( 9.7)
24 ( 7.1)
Cl n-Hep 92 ( 11)
Cl c-Hex
Cl Ph
39 ( 17)
17 ( 3.8)
4.8 ( 1.1)
>100
42 ( 14) 86 ( 25) 16 ( 5.4)
37 ( 8.1) 87 ( 8.3) 18 ( 5.6)
77 ( 41)
91 ( 16)
Cl Bn
6.6 ( 4.5)
>100
3.3 ( 0.4) 14 ( 0.3) 79 ( 30)
5g
5h
5i
Br
H
>100
Br i-Bu
36 ( 14)
26 ( 7.5)
49 ( 30)
65 ( 3.9)
54 ( 9.4)
16 ( 2.5)
40 ( 26)
18 ( 3.4)
29 ( 13)
Br n-Hep >100
5j
Br c-Hex
Br Ph
44 ( 10)
5k
5l
49 ( 8.3)
27 ( 8.2)
Br Bn
77 ( 31)
21 ( 9.5)
78 ( 38)
24 ( 5.7)
71 ( 25)
a Values representing GI50 are given in lM and are means of two to four experiments, standard deviation is given in parentheses. TGI and LC50
values are given only if they are less than 100 lM, which is the maximum concentration tested.
b Ref. 10.

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L. G. Leon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2681–2684
2683
the trans-2-alkyl-4-halopiperidine derivatives, WiDr
was the most sensitive cell line. The most potent 2-al-
kyl-4-halo-1,2,5,6-tetrahydropyridine derivative was
compound 5f (R = Bn), which produced GI50 values
against the three cell lines in the range 3.3–6.6 lM.
When considering TGI and LC50 values (Table 2) com-
pounds 5e, 5f, and 5l appeared as the most antiprolifer-
ative products of the series.
Cl
O
Cl
O
6d
6f
Figure 2. Structures of antiproliferative six-membered oxacycles.
Lipophilicity is a major determinant of pharmacokinetic
and pharmacodynamic properties of drug molecules.
The general trend of lipophilicity expressed as ClogP
values was found to be ClogP < 4 for inactive deriva-
tives and 4.92 < ClogP < 7.20 for active compounds.10
In view of these results, it appears that the biological
activity does not correlate with the calculated ClogP
values. Differences in activity between chlorinated and
brominated derivatives did not follow a clear trend.
Finally, the endocyclic insaturation at C3 appears as
responsible for the larger activity of 2-alkyl-4-halo-
1,2,5,6-tetrahydropyridine analogs. This result is consis-
tent with our previous findings in 2-alkyl-4-chloro-5,
6-dihydro-2H-pyrans (Fig. 2), in which the chlorovinyl
group was established as the pharmacophore. The new
azacycles only showed a significant selectivity for the
resistant colon cancer cell line when compared to the
corresponding oxacyclic derivatives (Table 3).
Table 3. Comparison of in vitro antiproliferative activity between oxa-
and azacycles against human solid tumor cellsa
Compound
Substituent
R
Cell line
SW1573
A2780
WiDr
5d
6d
5f
c-Hex
c-Hex
Bn
39
62
24
>100b
3.3
>100b
20b
4.8
36b
26b
6.6
58b
6f
Bn
a Values representing GI50 are given in lM.
b Ref. 3.
cancer cells.9 Analog 3f was the most potent compound
of the series with GI50 values in the range 12–20 lM
against the three cell lines. This compound was the only
piperidine analog able to induce total growth inhibition
(TGI) in A2780 and SW1573 cells. However, cytotoxic-
ity (expressed as LC50) was not observed for any of the
trans-2-alkyl-4-halopiperidine derivatives.
Cell cycle control is the major regulatory mechanism of
cell growth. Many cytotoxic agents and/or DNA dam-
aging agents arrest the cell cycle at the G0/G1, S, or
G2/M phase and then induce apoptotic cell death. We
examined cell cycle phase distribution by flow cytometry
to determine if cell growth inhibition involved cell cycle
The results on the biological activity of the 2-alkyl-4-
halo-1,2,5,6-tetrahydropyridine analogs are shown in
Table 2. Majority of the compounds induced antiprolif-
erative effect on the three cell lines tested. Similarly to
Figure 3. Effects of compound 5f on cell cycle distribution of SW1573 NSCLC cells (a–c) and WiDr colon cancer cells (d–f). Cells were cultured for
24 h in the absence (control, a and d) or presence of 5f at 10 lM (b and e) and 20 lM (c and f).

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L. G. Leon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2681–2684
2684
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(d) Padron, J. M.; Donadel, O. J.; Leon, L. G.; Martın, T.;
Martın, V. S. Lett. Drug Des. Discovery 2006, 3, 29; (e)
´
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changes. Therefore, SW1573 and WiDr cells were
exposed for 24 h to the most active derivative 5f at drug
doses of 10 and 20 lM.11 The results are shown in Fig-
ure 3. For SW1573 cells exposed to 10 lM of derivative
5f, it was possible to observe a slight increase in the per-
centage of cells in the G2/M phase (25%) with respect to
control cells (11%). At the higher dose, the amount of
NSCLC cells in G2/M phase augmented to 38%. The rise
was concomitant with a decrease in the G0/G1 phase
compartment. Interestingly, the cell cycle arrest was
more evident for WiDr cells exposed to compound 5f.
Thus, the percentage of cells at G2/M was 53% and
81% after exposure to 10 and 20 lM, respectively. These
values are much larger than that of non-treated WiDr
cells, which was 11%. The results are in agreement with
those obtained in the antiproliferative study, showing a
remarkable selectivity of the new compound for the
more resistant colon cancer cell line WiDr.
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Crisostomo, F. R. P.; Padron, J. M.; Martın, T.; Villar, J.;
Martın, V. S. Eur. J. Org. Chem. 2006, 1910; (f) Carrillo,
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R.; Leon, L. G.; Martın, T.; Martın, V. S.; Padron, J. M.
Bioorg. Med. Chem. Lett. 2006, 16, 6135; (g) Carrillo, R.;
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Leon, L. G.; Martın, T.; Martın, V. S.; Padron, J. M.
Bioorg. Med. Chem. Lett. 2007, 17, 780.
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4. For recent examples, see: (a) Donadel, O. J.; Martın, T.;
Martın, V. S.; Padron, J. M. Bioorg. Med. Chem. Lett.
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2007, 17, 18; (b) Ortega, N.; Martın, T.; Martın, V. S.
Org. Lett. 2006, 8, 871; (c) Crisostomo, F. R. P.;
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Martın, T.; Martın, V. S. Org. Lett. 2004, 6, 565; (d)
Pinacho Crisostomo, F. R.; Carrillo, R.; Leon, L. G.;
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Martın, T.; Padron, J. M.; Martın, V. S. J. Org. Chem.
2006, 71, 2339.
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5. (a) Miranda, P. O.; Dıaz, D. D.; Padron, J. I.; Ramırez,
M. A.; Martın, V. S. J. Org. Chem. 2005, 70, 57; (b)
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Miranda, P. O.; Ramırez, M. A.; Martın, V. S.; Padron, J.
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6. Carballo, R. M.; Ramırez, M. A.; Rodrıguez, M. L.;
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In summary, we have determined the antiproliferative
activity of a series of trans-2-alkyl-4-halopiperidines
and 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines. Active
compounds showed a significant selectivity for the more
resistant cancer cell line WiDr. The halovinyl group
seems responsible for the enhanced activity of the pyri-
dine analogs when compared to piperidine derivatives.
A clear structure–activity relationship for chlorinated
and brominated derivatives was not observed. Cell cycle
arrest at G2/M was confirmed for the most active deriv-
ative. Ongoing studies on the mechanism of action will
be reported elsewhere.
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Martın, V. S.; Padron, J. I. Org. Lett. 2006, 8, 3837.
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7. Miranda, P. O.; Dıaz, D. D.; Padron, J. I.; Bermejo, J.;
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8. In this method, for each drug a dose–response curve is
generated and three levels of effect can be calculated, when
possible. The effect is defined as percentage of growth
(PG), where 50% growth inhibition (GI50), total growth
inhibition (TGI), and 50% cell killing (LC50) represent the
drug concentration at which PG is +50, 0, and –50,
respectively. Skehan, P.; Storeng, P.; Scudeiro, D.; Monks,
A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
9. Pizao, P. E.; Peters, G. J.; van Ark-Otte, J.; Smets, L. A.;
Smitskamp-Wilms, E.; Winograd, B.; Pinedo, H. M.;
Giaccone, G. Eur. J. Cancer 1993, 29A, 1566.
Acknowledgments
10. Software-predicted lipophilicity of the compounds was
calculated with the program New ClogP accessible via
internet (http://intro.bio.umb.edu/111-112/OLLM/111F98/
newclogp.html).
This research was supported by the EU INTERREG
IIIB-MAC initiative (05/MAC/2.5/C14 BIOPOLIS),
´
the Ministerio de Educacion y Ciencia of Spain, co-fi-
11. Cells were seeded in six-well plates at a density of 0.7–1 ·
106 cells/well. After 24 h, the products were added to the
respective well and incubated for an additional period of
24 h. Cells were trypsinized, harvested, transferred to test
tubes (12.75 mm), and centrifuged at 1500 rpm for 10 min.
The supernatant was discarded and the cell pellets were
resuspended in 200 ll of cold PBS and fixed by the addition
of 3 mL ice-cold 70% ethanol. Fixed cells were incubated
overnight at À20 ꢁC after which time were centrifuged at
1500 rpm for 10 min. The cell pellets were resuspended in
500 ll PBS. Then, 20 ll of DNAse-free RNAse (200 ll/ml)
and 10 ll of propidium iodide (40 ll/ml) were sequentially
added. The mixture was incubated in the dark at 37 ꢁC
for 30 min. Flow cytometric determination of DNA content
(25,000 cells/sample) was analyzed by FACSCalibur
nanced by the European Regional Development Fund
(CTQ2005-09074-C02-01/BQU) and the Canary Islands
Government. R.M.C. thanks the Spanish MEC for an
FPU fellowship. J.I.P. and J.M.P. thank the Spanish
´
MEC-FSE for Ramon y Cajal contracts.
References and notes
1. Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1, and earlier
reviews in the same series.
2. Mayer, A. M. S.; Gustafson, K. R. Eur. J. Cancer 2006,
42, 2241, and references therein.
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3. (a) Donadel, O. J.; Martın, T.; Martın, V. S.; Villar, J.;
Padron, J. M. Bioorg. Med. Chem. Lett. 2005, 15, 3536; (b)
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Flow Cytometer (Becton Dickinson, San Jose, CA, USA).
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Miranda, P. O.; Padron, J. M.; Padron, J. I.; Villar, J.;
Martın, V. S. Chem. Med. Chem. 2006, 1, 323; (c)
´
The fractions of the cells in G0/G1, S, and G2/M phase
in addition to apoptosis were analyzed using cell cycle
analysis software, ModFit LT 3.0 (Verity Software House,
Topsham, ME, USA).
´
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Miranda, P. O.; Leon, L. G.; Martın, V. S.; Padron, J.
I.; Padron, J. M. Bioorg. Med. Chem. Lett. 2006, 16, 3135;
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