3,5-Bis(Arylidene)-4-Piperidones Modified by Bisphosphonate Groups as Novel Anticancer Agents

Article abstract of DOI:10.1080/10426507.2014.976338

Synthetic approaches for conjugating 3,5-bis(arylidene)-4-piperidones with bisphosphonate moiety were elaborated. These approaches are based either on reaction of Grignard reagent containing dioxolane protected 4-piperidone with tetraethyl ethylidenbisphosphonate followed by crotonic condensation with aromatic aldehydes or on Cu(i) catalyzed 1,3-cycloaddition of tetraethyl but-3-yne-1,1-diylbisphosphonate to N-(2-azidoethyl)-3,5-bis(arylidene)-4-piperidones resulting in corresponding 1,2,3-triazole derivatives. Cytotoxic activity of the synthesized conjugates was dependent on the length of linker connecting piperidone nitrogen atom and bisphosphonate residue. Triazole derivatives of 3,5-bis(arylidene)-4-piperidone series displayed moderate in vitro inhibitory properties towards HCT116 and MCF7 human cancer cell lines with IC₅₀ values in the range of 5.0-7.5 M, whereas conjugates with butylene linker between piperidone nitrogen atom and bisphosphonate moiety were significantly less active.

Full text of DOI:10.1080/10426507.2014.976338

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Phosphorus, Sulfur, and Silicon and the Related
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3,5-Bis(arylidene)-4-piperidones Modified by
Bisphosphonate Groups as Novel Anticancer Agents
Mikhail V. Makarova, Ekaterina Yu. Rybalkinab & Valery K. Brela
a A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Vavilova str., 28, Moscow, 119991, Russian Federation
b Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Russian
Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478, Russian Federation
Accepted author version posted online: 03 Nov 2014.
To cite this article: Mikhail V. Makarov, Ekaterina Yu. Rybalkina & Valery K. Brel (2014): 3,5-Bis(arylidene)-4-piperidones
Modified by Bisphosphonate Groups as Novel Anticancer Agents, Phosphorus, Sulfur, and Silicon and the Related Elements,
DOI: 10.1080/10426507.2014.976338
To link to this article: http://dx.doi.org/10.1080/10426507.2014.976338
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3,5-Bis(arylidene)-4-piperidones Modified by Bisphosphonate Groups as Novel Anticancer
Agents
Mikhail V. Makarova,*, Ekaterina Yu. Rybalkinab and Valery K. Brela
a A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Vavilova str., 28, Moscow, 119991, Russian Federation
b Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Russian Academy
of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478, Russian Federation
e-mail: mmak78@yandex.ru
Abstract. Synthetic approaches for conjugating 3,5-bis(arylidene)-4-piperidones with
bisphosphonate moiety were elaborated. These approaches are based either on reaction of
Grignard reagent containing dioxolane protected 4-piperidone with tetraethyl
ethylidenbisphosphonate followed by crotonic condensation with aromatic aldehydes or
on Cu(I) catalyzed 1,3-cycloaddition of tetraethyl but-3-yne-1,1-diylbisphosphonate to N-
(2-azidoethyl)-3,5-bis(arylidene)-4-piperidones resulting in corresponding 1,2,3-triazole
derivatives. Cytotoxic activity of the synthesized conjugates was dependent on the length
of linker connecting piperidone nitrogen atom and bisphosphonate residue. Triazole
derivatives of 3,5-bis(arylidene)-4-piperidone series displayed moderate in vitro
inhibitory properties towards HCT116 and MCF7 human cancer cell lines with IC50
values in the range of 5.0–7.5 μM, whereas conjugates with butylene linker between
piperidone nitrogen atom and bisphosphonate moiety were significantly less active.
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O
N
O
Ar
Ar
Ar
Ar
N
N
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
N
N
2a
3a,c
Ar = 4-O2NC6H4 (a), 4-NCC6H4 (c)
IC50 = 5.0 - 7.0 μM (MCF7 breast cancer)
IC50 = 40 μM (MCF7 breast cancer)
Key words: 3,5-bis(arylidene)-4-piperidones, aminobisphosphonates, bisphosphonates,
anticancer properties
Shortened title of the article: Bisphosphonates of bis(arylidene)piperidone series
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Bisphosphonates (BPs) occupy a prominent position among biologically active
organophosphorus compounds due to high efficacy of some their representatives in treatment of
osteoporosis and bone metastases.1 They are usually classified in two types: non-nitrogen BPs
(first generation as exemplified by etidronate in Fig. 1) and nitrogen-containing BPs or amino-
BPs (second and third generations as exemplified by aledronate and risedronate, respectively, in
Fig. 1). Compounds belonging to the second generation contain nitrogen atom in a short alkyl
chain attached to the carbon atom of bisphosphonate (P–C–P) moiety. In the case of the third
generation BPs, nitrogen atom is a part of a heterocyclic system connected with the BP moiety
through a short methylene linker. The third generation amino-BPs demonstrate the highest
antiresorptive activity that is several thousand times higher than that of the first generation BPs,
and some of the former, for example, risedronate and zoledronate, find wide application in
clinical practice. Structure-activity relationship studies revealed that the proximity of nitrogen
atom to the P–C–P moiety in the structure of amino-BPs plays a crucial role for the biological
activity of these compounds. Indeed, in the case of the most active representatives the P–C–P
residue and nitrogen atom are spaced from each other by only one to three carbon atoms.1
Moreover, due to high affinity of BPs to osseous tissue, these compounds may be used
for targeted delivery of other pharmacophores to the affected bone. The idea of conjugation of
BP moieties with various anticancer drugs was implemented in a number of publications
describing BP conjugates of gemcitabine2, methotrexate3, doxorubicin4, and nucleoside
antimetabolites5. Further examples of pharmacologically active compounds conjugated with BPs
are provided elsewhere.6
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In our research we are trying to modify the structure of anticancer active 3,5-
bis(arylidene)-4-piperidones using organophosphorus groups, including BP residues, to improve
their pharmacological properties and to enhance cytotoxic activity. Recently, we have shown that
NH-3,5-bis(arylidene)-4-piperidones
may
be
easily
reacted
with
tetraethyl
ethylidenbisphosphonate in an aza-Michael type addition reaction resulting in a series of
corresponding β-amino-BPs 1.7 Some of these β-amino-BPs showed high in vitro cytotoxicity
(with inhibition concentration IC50 values in the range of 0.5–4 μM) towards human cancer cell
lines CaoV3 (ovarian cancer), A549 (lung cancer), PC3 (prostate cancer) and KB3-1 (oral
epidermoid cancer). That study demonstrated that combining in one molecule two
pharmacophores – 3,5-bis(arylidene)-4-piperidone moiety and BP group – may be an effective
approach to increase the cytotoxicity because, in some instances, BP-modified piperidones were
more active than the parent NH-compounds.
The aim of this study was to evaluate the influence of type and length of a linker
connecting the BP moiety with the piperidone scaffold on the cytotoxic activity of 3,5-
bis(arylidene)-4-piperidones. In this regard, synthetic approaches to novel bisphosphonates 2a,b
and 3a-d containing 3,5-bis(arylidene)-4-piperidone pharmacophore were elaborated (Scheme 1)
and in vitro cytotoxicity of these compounds towards some human cancer cell lines was
evaluated and compared with that of compounds 1.
Detailed synthetic procedures for preparing compounds 2a,b and 3a-d have been
described in our recent publications;8 therefore, only short description of their synthesis will be
provided now. Commercially available 1,4-dioxa-8-azaspiro[4.5]decane (4) was used as a
starting compound and was converted to chlorides 5 and 6 using alkylation with 2-chlroethanol
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or 3-chloropropanol followed by reaction of resulting alcohols with thionyl chloride.9 In the first
approach, chloride 6 was treated with a sodium hydrogen carbonate aqueous solution to remove
intramolecular HCl which resulted in corresponding free base subsequently converted to a
Grignard reagent; the latter was reacted with tetraethyl ethylidenbisphosphonate resulting in
aminobisphosphonate 7 isolated in the yield of 55% after column chromatography. Crotonic
condensation of compound 7 with aromatic aldehydes under acidic conditions provided desired
piperidones 2a,b in the yields of ca. 30% due to partial hydrolysis of ethoxy groups at
phosphorus atoms.
In the second approach, chloride 5 was reacted with sodium azide in water to afford
corresponding azide from which dioxolane protection was removed to liberate ketone function.
Resulting compound 8 was reacted with aromatic aldehydes in the presence of lithium
perchlorate and diethylamine according to known procedure10 to give N-(2-azidoethyl)-3,5-
bis(arylidene)-4-piperidones 9a-d in the yields of 31–56%. The final step in the synthesis of
compounds 3a-d was copper(I) catalyzed 1,3-cycloaddition reaction between azides 9a-d and
terminal acetylene containing BP moiety. This reaction afforded desired conjugates 3a-d in
regioselective manner (only 1,4-regioisomers of 1,2,3-triazoles were formed) in good yields (57–
89%).
The cytotoxic activity of BPs 2a,b was tested in vitro against following human cancer
cell lines: ovarian (Scov3, Caov3), lung (A549) and breast (MCF7) carcinoma using MTT
method and compared with that of analogues 1a,b containing shorter alkylene chain. The results
are summarized in Table 1 showing the corresponding IC50 values (IC50 is the concentration of
5
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compound required to inhibit the growth of the cells by 50%). Anticancer antibiotic Doxorubicin
was used as a positive control.
Two basic conclusions may be drawn form the data in Table 1. First, BPs 2a and 1a
containing electron-withdrawing nitro substituents in aromatic rings have higher anticancer
activity as compared to analogues 2a and 1a, respectively, having electron-releasing
dimethylamino groups. Second, as it follows from comparison of activity of compounds 2a and
1a, bisphosphonate 2a having longer alkylene chain of four carbon atoms between piperidone
and BP moieties revealed significantly lower activity than counterpart 1a with one CH2 unit
between those moieties.
[Insert Table 1]
The cytotoxic activity of triazoles 3a-d was tested in vitro against human colon cancer
(HCT116) and human breast cancer (MCF7) cell lines as well as normal human embryonic
fibroblasts (HEF) using MTT method. Cytotoxic activity of nitro derivative 3a was also
compared with the activity of analogues 1a and 2a containing different linkers between
piperidone and bisphosphonate moieties. The results are summarized in Table 2, with anticancer
agent Doxorubicin being used again as a positive control.
As is seen from the data in Table 2, compounds 3a,c with triazole linker between BP and
piperidone moieties and having electron-withdrawing substituents demonstrate moderate activity
towards used cell lines. At the same time, pyridine derivative 3d and bisphosphonate 3b with
electron-releasing dimethylamino groups were non-active. Among nitro-derivatives 1a, 2a and
3a, compound 1a with shortest alkylene chain is the most active towards breast cancer cell line.
6
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Therefore, we believe that further studies may include synthesis of 3,5-bis(arylidene)-4-
piperidones with BP moiety attached directly to piperidone nitrogen atom.
[Insert Table 2]
In conclusion, this study has demonstrated the suitable approaches to the modification of
3,5-bis(arylidene)-4-piperidone structure with BP moiety using different linkers connecting these
two pharmacologically active units. The cytotoxicity screening revealed that the highest
anticancer activity is revealed by BP conjugates of 3,5-bis(arylidene)-4-piperidones having the
shortest linker between BP moiety and piperidone nitrogen atom. Comparison of IC50 values for
triazoles 3a,c (in the range of 5.0–7.5 μM) with those for related BP 1a (in the range of 2.6–5.2
μM) showed that 1,2,3-triazole ring did not have strong negative impact on the antitumor activity
of compounds and, therefore, the approach based on conjugation of 3,5-bis(arylidene)-4-
piperidone pharmacophore with other biologically active moieties using 1,2,3-triazole linker may
be effectively used for the synthesis of novel cytostatics.
This work was supported by the Russian Foundation for Basic Research (project no. 14-03-
00687).
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[7] (a) Makarov, M. V.; Leonova, E. S.; Matveeva, E. V.; Rybalkina, E. Yu.;
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O
N
Ar
Ar
N
O
O
P(O)(OEt)2
P(O)(OEt)2
N
N
N
H
4
3a-d
1) Cl(CH2)nOH
K2CO3 / MeCN
P(O)(OEt)2
P(O)(OEt)2
Cu(I)
DMF - H2O
2) SOCl2/ C6H6
O
N
O
N
O
O
1) NaN3
H2O, 80oC
ArCHO
Ar
Ar
LiClO
2) AcOH - HCl
75oC
4 / Et2NH
N
HCl
(CH2)nCl
5 (n=2), 6 (n=3)
n=2
(CH2)2N3
(CH2)2N3
8
9a-d
1) NaHCO3
P(O)(OEt)2
P(O)(OEt)2
n=3
O
2) Mg / THF then
O
N
O
ArCHO
AcOH - HCl
Ar
Ar
N
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
7
2a,b
Ar = 4-O2NC6H4 (a), 4-Me2NC6H4 (b), 4-NCC6H4 (c), 3-Py (d)
Scheme 1. Synthesis of aminobisphosphonates containing 3,5-bis(arylidene)-4-piperidone
pharmacophore
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HO
O
N
HO
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
Alendronate
Me
H2N
Ar
Ar
P(O)(OH)2
Etidronate
P(O)(OEt)2
P(O)(OEt)2
HO
P(O)(OH)2
1a,b
P(O)(OH)2
Residronate
N
Ar = 4-O2NC6H4 (a), 4-Me2NC6H4 (b)
Fig. 1. Structures of known aminobisphosphonates and compounds of series 1
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Table 1. In vitro cytotoxicity of BPs 2a,b and 1a,b towards human carcinoma cell lines
Cell lines/IC50, μM
Compound
Scov3
38±10
Caov3
31±4
A549
58±2
MCF7
40±5
2a
2b
IC50 > 70 μM for all cell lines
1a
1b
4.0±0.8
56±7
3.6±1.0
25±11
5.2±0.6
70±10
2.6±0.5
32±5
Doxorubicin
0.8±0.15
0.38±0.12
0.34±0.12
0.27±0.07
Table 2. In vitro cytotoxicity of BPs 3a-d towards HCT116, MCF7, and HEF cells
Cell lines/IC50, μM
Compound
HCT116
MCF7
HEF
3a
7.5±0.5
n/d
5.0±0.4
2.6±0.5
40±5
6.0±0.4
n/d
1a
2a
n/d
n/d
3c
3b
7.5±0.6
7.0±0.5
5.0±0.3
>20 for all cell lines
0.3±0.1
3d
Doxorubicin
1.6±0.3
2.1±0.4
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