Structure-cytotoxicity relationship in a series of N-phosphorus substituted E,E-3,5-bis(3-pyridinylmethylene)- and E,E-3,5-bis(4-pyridinylmethylene) piperid-4-ones

Article abstract of DOI:10.1016/j.ejmech.2010.09.058

In order to give further insight on the influence of the aromatic ring nature and the presence of the phosphorus substituent at the piperidone nitrogen atom of E,E-3,5-bis((hetero)arylidene)piperid-4-ones on their antitumor properties, a series of phosphorus substituted E,E-3,5-bis(pyridinylmethylene) piperid-4-ones bearing either 3-pyridine or 4-pyridine rings was obtained. Novel NH-3,5-bis(pyridinylmethylene)piperid-4-ones 1a,b were converted into the corresponding N-phosphorylated derivatives 3a-c, 4a-c differing in the substitution at the phosphorus atom (amidophosphates and amidophosphonates), via direct phosphorylation while N-(ω-phosphorylalkyl)-substituted compounds 8a-c were obtained via aldol-crotonic condensation of preformed N-phosphorylalkyl substituted piperidones with the corresponding pyridinecarboxaldehyde. The cytotoxicity screen has revealed that phosphorylated compounds based on E,E-3,5-bis(4-pyridinylmethylene)piperid-4-one framework displayed higher inhibitory properties toward Caov3, A549, KB 3-1 and KB 8-5 human carcinoma cell lines comparing with their analogues with 3-pyridine rings. Introduction of the phosphorus moiety substantially increased the antitumor properties in the case of E,E-3,5-bis(3-pyridinylmethylene)piperid-4-ones derivatives but this influence less pronounced for more active analogues bearing 4-pyridinyl rings. Most of the compounds tested are potent against multi-drug resistant cell line KB 8-5 affording some guidelines for the search of perspective drug-candidates among phosphorus substituted E,E-3,5-bis((hetero) arylidene)piperid-4-ones.

Full text of DOI:10.1016/j.ejmech.2010.09.058

European Journal of Medicinal Chemistry 45 (2010) 5926e5934
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureecytotoxicity relationship in a series of N-phosphorus substituted
E,E-3,5-bis(3-pyridinylmethylene)- and E,E-3,5-bis(4-pyridinylmethylene)
piperid-4-ones
Evgeniya S. Leonova a, Michael V. Makarova, Ekaterina Yu. Rybalkina b, Shravana L. Nayani c,
,
Paul Tongwa c, Alexander Fonari c, Tatiana V. Timofeeva c, Irina L. Odinets a
*
a A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilova str., 28, 119991 Moscow, Russian Federation
b Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Centre, Russian Academy of Medical Sciences, Kashirskoe sh., 24, 115478 Moscow, Russian Federation
c New Mexico Highlands University, Las Vegas, NM 87701, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to give further insight on the influence of the aromatic ring nature and the presence of the
phosphorus substituent at the piperidone nitrogen atom of E,E-3,5-bis((hetero)arylidene)piperid-4-ones
on their antitumor properties, a series of phosphorus substituted E,E-3,5-bis(pyridinylmethylene)
piperid-4-ones bearing either 3-pyridine or 4-pyridine rings was obtained. Novel NH-3,5-bis(pyr-
idinylmethylene)piperid-4-ones 1a,b were converted into the corresponding N-phosphorylated deriva-
tives 3aec, 4aec differing in the substitution at the phosphorus atom (amidophosphates and
Received 28 July 2010
Received in revised form
23 September 2010
Accepted 27 September 2010
Available online 28 October 2010
amidophosphonates), via direct phosphorylation while N-(u-phosphorylalkyl)-substituted compounds
Keywords:
8aec were obtained via aldol-crotonic condensation of preformed N-phosphorylalkyl substituted
piperidones with the corresponding pyridinecarboxaldehyde. The cytotoxicity screen has revealed that
phosphorylated compounds based on E,E-3,5-bis(4-pyridinylmethylene)piperid-4-one framework dis-
played higher inhibitory properties toward Caov3, A549, KB 3-1 and KB 8-5 human carcinoma cell lines
comparing with their analogues with 3-pyridine rings. Introduction of the phosphorus moiety
substantially increased the antitumor properties in the case of E,E-3,5-bis(3-pyridinylmethylene)piperid-
4-ones derivatives but this influence less pronounced for more active analogues bearing 4-pyridinyl
rings. Most of the compounds tested are potent against multi-drug resistant cell line KB 8-5 affording
some guidelines for the search of perspective drug-candidates among phosphorus substituted E,E-3,5-bis
((hetero)arylidene)piperid-4-ones.
3,5-bis((hetero)arylidene)piperid-4-ones
N-Phosphoryl-3,5-bis(pyridinylmethylene)
piperid-4-ones
4-Piperidones
N-Phosphorylalkylene-3,5-bis
(pyridinylmethylene)piperid-4-ones
Synthesis
X-ray structure
Cytotoxicityestructure relationship
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
fact allowsto consider3,5-bis(arylidene)-piperid-4-ones as potential
drug-candidates which may be free of mutagenic properties of
Starting from early 1970s bis(arylidene)piperid-4-ones and the
related bis(arylidene)cycloalkan-4-ones were intensively studied in
relation to their cytotoxic properties and antitumor activities [1e7].
The excellentcytotoxicpropertiesofdifferentcompoundspossessing
the general structure I is believed to be attributed to the presence of
1,5-diaryl-3-oxo-1,4-pentadienyl (dienone) pharmacophore moiety
observed both in alicyclic unsaturated enones and cyclic piperidone
derivatives [4]. The proposed mechanism of action of such
compounds includes their interaction with biogenic thiols (i.e. their
alkylation), the nitrogen-containing intracellular nucleophiles such
as proteins and nucleic acids not being affected by them [8,9]. This
known alkylating anticancer agents used in current medicine prac-
tice [10]. Moreover, these compounds (generally screened as free
bases) are non-lethal and mice tolerated doses up to 300 mg/kg
[11] and even five daily doses upto 240 mg/kgofhydrochloride saltof
3,5-bis(benzylidene)piperid-4-one did not cause mortalities [12].
Many investigations indicate that the presence of strong electron-
withdrawing substituents in aryl moieties results in significant
increase of antitumor activity of 3,5-bis(arylidene)-piperid-4-ones. In
other words, a Hammett sP value of the substituents in the aryl rings
(as well as conformation of the central core) and hence that of
the substituted aryl ring affects the fractional positive change on the
adjacent carbon atoms at the double bond and, as a consequence, the
possibilityof thecompoundtointeractwithcellularthiols[13]. Aswell
known, sP value of 3-pyridinyl group is close to that for 4-NO2eC6H4
* Corresponding author. Tel.: þ7 495 1359356; fax: þ7 495 1359356.
E-mail address: odinets@ineos.ac.ru (I.L. Odinets).
(sP ¼ 0.25 and 0.26, respectively) while the Hammett constant of
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.058

E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
5927
A: Ar = substituted phenyl,
R = H, Alk, C(O)Alk, C(O)OAlk
O
B: Ar = 4-C6H4NMe2,
R = P(O)(OCH2CF3)2, P(O)(OPh)2, P(O)(OPh)Me
Ar
Ar
Ar = 4-C6H4NO2, 3-C6H4NO2, 2-C6H4NO2
R = P(O)(OEt)2, P(O)(OH)2
N
C: Ar = 4-C6H4R1, R1 = NMe2, F, NO2,
R
R = (CH2)nP(O)(OEt)2 or (CH2)nP(O)(ONa)2, n = 1-4
IA-D
D: Ar = 2-thienyl,
R = P(O)(OEt)2, P(O)(OPh)2, P(O)(OEt)Me, or
(CH2)nP(O)(OEt)2, (n = 1-3)
Scheme 1. Modified structures of 3,5-bis(arylidene)-piperid-4-ones.
4-pyridinyl is much higher (sP ¼ 0.44) [14]. Therefore, one may expect
high cytotoxicity for 3,5-bis(pyridinylmethylene)-piperid-4-ones.
Nevertheless, only one compound belonging to pyridine substituted
3,5-bis(arylidene)-piperid-4-ones (namely N,N-dimethyl-3,5-bis
(4-pyridinylmethylene)-4-piperidonium iodide) was tested for this
type of activity [15] and reported to possess the diminished anti-
tumor properties comparing with a wide range of 3,5-bis(aryli-
dene)-piperid-4-ones in the screens used an average of 54 human
tumor cell lines from eight neoplastic diseases such as leukemia,
melanoma, colon, non-small-cell lung, small-cell lung, central
nervous system, ovarian, and renal cancers.
On the other hand, there has been a reasonable suggestion that
the groups at the piperidone nitrogen atom may influence the
cytotoxic properties of 3,5-bis(arylidene)piperid-4-ones via their
interactions with additional binding sites of a biological target.
These groups can contribute to solubility and bioavailability of the
final compound and increase the cytotoxic properties, for example,
by facilitating approach of the cytotoxin to a specific binding site.
However, the above groups can also reduce cytotoxicity preventing
this interaction. Moreover, according to the hypothesis of sequential
cytotoxicity, if these additional groups are also able to alkylate
intracellular nucleophiles, the malignant cells may suffer greater
injury than normal ones. That means that structureeactivity rela-
tionship of compounds of such type presents a multifactor problem.
Therefore, a great number of publications dealing with modifi-
cation of structures of 3,5-bis(arylidene)-piperid-4-ones in order to
elucidate the structureeactivity relationship, enhance antitumor
activity and selectivity to different types of cancer, improve the
capacity of their transportation via the cellular membrane and
bioavailability, and to impart them such properties as ability to
reverse multi-drug resistance can be found in the literature. Mostly,
these modifications include variation of the substituents at the aryl
rings, which differ in electronic, hydrophobic, and steric properties,
and alkylation or acylation of the piperidone nitrogen atom of the
ring (Scheme 1, compounds A) [4,5,16].
More recently, it was revealed that introduction of phosphonate
(phosphinate) group [17e19] or
-phosphonoalkyl moiety [19,20]
u
at the piperidone nitrogen atom significantly increase cytotoxic
properties of 3,5-bis(arylidene)-piperid-4-ones (compounds BeC,
Scheme 1). This phenomenon was more pronounced in the case of
3,5-bis(2-thienylidene)-piperid-4-ones D. Thus, while NH-3,5-bis
(2-thienylidene)-piperid-4-one and 1-methyl-3,5-bis(2-thienyli-
dene)-piperid-4-one did not show significant cytotoxicity towards
human carcinoma cell lines Scov3, Caov3 and A549 (IC50 values ꢀ
80
mM), their phosphorylated analogues were more active (IC50
values in the range of 4e60
m
M), with the methylphosphinate (R ¼
P(O)(OEt)Me) having the lowest IC50 values among all investigated
thiophene derivatives D. Furthermore, the above mentioned
general tendency of more prominent cytotoxicity of derivates
bearing electron-withdrawing substituents as compared to those
having electron-donating ones was also observed for phosphorus-
containing 3,5-bis(arylidene)-piperid-4-ones B and C.
In view of these results, in this paper we report on the synthesis,
crystal structure, and antitumor activity in vitro of phosphoryl
substituted 3,5-bis(pyridinylmethylene)-piperid-4-ones differing
in the position of the nitrogen atom in pyridine ring of the dienone
system.
2. Results and discussion
2.1. Chemistry
In order to give insight into the influence of the phosphorus
moiety on cytotoxic properties of 3,5-bis(pyridinylmethylene)
piperid-4-ones, we performed the synthesis of the corresponding
3-pyridinyl and 4-pyridinyl derivatives bearing either phosphorus
atom in different surrounding directly bonded with the nitrogen
atom of the piperidone cycle or those having an alkylene linker
between the phosphorus and the nitrogen atoms.
O
R=H
R1R2P(O)Cl
O
O
'
7
7
.
O
Py
BF Et O/CH CN
Py
1)
3
2
3
4
Py
5
6
3
2
Py
Py
C
+
2) NaHCO3
N
P
Et3N/THF
N
R
H
N
R
R1
R2
O
3a-c
4a-c
R=H (1a,b)
R=Me (2a,b)
9
8
9
8
10
10 (1b, 2b, 4a-c)
Py =
(1a, 2a, 3a-c),
N
13
11
13
N
12
R1=R2=OEt (3a,4a); R1= Me, R2=OEt (3b,4b), R1=Me, R2=OPh (3c,4c)
Scheme 2. Synthesis of 3,5-bis(pyridinylmethylene)piperid-4-ones 1a,b,2a,b and phosphorylation of compounds 1a,b.

5928
E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
For the synthesis of the first series of the target compounds the
direct phosphorylation of the preformed parent NH-3,5-bis(pyr-
idinylmethylene)piperid-4-ones 1a,b by the appropriate phos-
phorus(V) acid chlorides in the presence of triethylamine as a base
was used (Scheme 2).
O
N
O
N
2
N
CHO
piperidine/EtOH
cat. AcOH
N
N
The starting NH-precursors 1a,b were obtained via the aldol-
crotonic condensation of the corresponding pyridinecarbox-
aldehyde and piperid-4-one in the presence of boron trifluoride
etherate (r.t., CH3CN as a solvent) as the corresponding tris(tet-
rafluoroborate) salts followed by neutralization according to
procedure elaborated by us recently [21]. The similar approach
was used for the synthesis of known N-methyl-3,5-bis(pyridinyl-
methylene)piperid-4-ones 2a,b [22] used for comparison
in cytotoxicity screen (see below). Note, that the above aldol-
crotonic condensation provided high yields in the reaction of
NH-piperid-4-one with 3-pyridinecarboxaldehyde and those
of N-methyl-piperid-4-one with both aldehydes (e90% isolated
yield) while the compound 1b$HBF4 was obtained in 20%
yield only. Moreover, the condensation of 4-piperidone with
2-pyridinecarboxaldehyde afforded 1,2-di(2-pyridinyl)-1,2-ethane
-dione along with traces of (3E)-3-(pyridin-2-ylmethylene)piper-
idin-4-one (monocondensation product) rather than the related
NH-3,5-bis(2-pyridinylmethylene)piperid-4-one 1d.
As regards the further phosphorylation of 1a,b, it should be
emphasized that the starting substrates 1a,b require careful
drying over P2O5 in vacuum for successful reaction as they are
inclined to form stable hydrates. The presence of water results in
domination of side reactions dealing with the hydrolysis of
phosphorylating agent and further formation of pirophosphates
(pirophosphonates) hence reducing significantly the yields of the
desired amidophosphates (phosphonates). The higher yields of
derivatives 3aec bearing 3-pyridinyl fragment comparing with
the products 4aec over phosphorylation may be attributed to the
lesser stability of hydrate formed by NH-3,5-bis(3-pyridinyl-
methylene)piperid-4-one 1a which hence contained less amount
of water after drying (the drying conditions were the same for
both substrates).
P(O)(OEt)2
P(O)(OEt)2
8b
6a
Scheme 4. Synthesis of N-(2-phosphorylethyl)-3,5-bis(3-pyridinyl’methylene)-4-piper-
idone 8b.
2a,b, the final tetrafluoroborate salts 7aec precipitated from reac-
tion mixtures and were isolated via filtration (Scheme 3).
However, using further neutralization with aq. NaHCO3 of tet-
rafluoroborate salts 7aec we succeeded to isolate in a pure form
only N-(4-phosphorylbutyl)-3,5-bis(pyridinylmethylene)piperid-
4-one 8c, while we failed to purify free bases 8a,b with shorter
alkylene chains from unidentified side products using column
chromatography and/or crystallization. Therefore, we performed
the alternative synthesis of N-(2-phosphorylethyl)-substituted
b-aminophosphonate 6a with
3-pyridinecarboxaldehyde under basic conditions which resulted
in 8b in ca. 28% (Scheme 4).
The structures of the phosphorylated products were elucidated
by 1H, 31P, 13C NMR and IR spectral data along with a single crystal
X-ray diffraction analysis. The 31P NMR spectra of the correspond-
ing products displayed the singlet signals observed at ca.7.2 ppm
for amidophosphates 3a,4a and at ca. 30 ppm for phosphonates
3b,c, 4d,c, and 8b,c. Such disposition of the signals is typical for the
mentioned surrounding at the particular phosphorus atom. In the
1H NMR spectra of all compounds the singlet resonances assigned
to vinyl hydrogen atoms are observed at ca. 8.0 ppm for tetra-
fluoroborate salts 7aec while in the spectra of the corresponding
free bases 8b,c these resonances are upfield schifted for ca. 7.7 ppm.
The related tendency was observed in the corresponding pattern of
the 13C spectra: the signals of methyne C7(70)-carbon atom
observed at 143e146 ppm in the spectra tetrafluoroborate salts
7aec shifted up to 133e135 ppm in the spectra of the corre-
sponding free bases 8b,c.
derivative 8b via condensation of
As introduction of the u-phosphorylalkyl group to the nitrogen
atom of the preformed NH-precursors 1a,b is accompanied by
quaternization [16], the aldol-crotonic condensation of 3-pyr-
idinecarboxaldehyde with preformed aminophosphonates 5,6a,b
was the method of choice for the synthesis of
u-amino-
phosphonates 8aec differing in the length of an alkylene linker.
Firstly, the reactions were performed in the presence of BF3$Et2O
both as a catalyst and a solvent. Application of additional co-solvent
such as CH3CN decreased the reaction rate and accelerated side
reactions (hydrolysis of ester groups). Note that the longer linker
2.2. Molecular and crystal structures of 3,5-bis
(pyridinilmethylene)-4-piperidones
has the starting
rate (ca. 1 week for
-aminophosphonate 6b). Similar to the above mentioned
synthesis of 3,5-bis(pyridinylmethylene)piperid-4-ones 1a,b and
u
-aminophosphonate the higher was the reaction
We succeeded to grow crystals of three derivatives 2a,3c and 8b
that were satisfactory for a single crystal X-ray structural charac-
terization. All compounds have 3-pyridyl rings and N-methyl, N-
methyl(phenoxy)phosphoryl, and N-(2-diethoxyphosphoryl)ethyl
a-aminophosphonate 5 versus about 3 h for
d
2
N
CHO
O
N
O
2
N
CHO
O
O
BF3 Et2O
BF3 Et2O
BF4
N
N
N
N
(CH2)nP(O)(OEt)2
H
(CH2)nP(O)(OEt)2
7a-c
P(O)(OEt)2
6a,b
5
n = 1 (5, 7a), 2 (6a, 7b), 3 (6b, 7c)
Scheme 3. Synthesis of N-(u-phosphorylalkyl)-3,5-bis(3-pyridinylmethylene)-4-piperidone tetrafluoroborates 7aec.

E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
5929
Fig. 1. Molecular structure of 2a drawn at 50% probability of thermal ellipsoids.
groups, respectively. Structural features of molecules 2a,3c and 8b
(Figs. 1e3) do not differ significantly from relative molecules that
belong to groups AeD mentioned in introduction. Note that
numeration of the atoms in the NMR spectra of the compounds and
X-ray data is independent.
Fig. 3. Molecular structure of 8b drawn at 50% probability of thermal ellipsoids.
To the best of our knowledge, onlyone example of structural studies
of pyridine substituted 3,5-bis(arylidene)-piperid-4-ones (namely
N,N-dimethyl-3,5-bis(4-pyridinylmethylene)-4-piperidonium iodide)
was presented in literature [15]. It was found that for this compound
deviation of aryl cycles from the plane of central fragment does not
exceed 30ꢁ. Studies of N-phosphorylated compounds have shown that
introduction of even large substituents at the N atom of piperidone ring
do not cause significant rotation of planes of phenyl or thiophene rings
from the plane of central fragment [17,19]. However, for 3,5-bis(2-
nitrobenzylidene)-4-oxo-1-phosphonopiperidine molecule having
nitro group in the ortho-position, increase of the torsion angle around
single bond between an aryl ring and adjusted unsaturated group, that
can be explained by steric hindrances, was found [18].
In molecules of 2a, 3c, and 8b under investigation two types of
orientation of pyridine nitrogen atoms exist: towards carbonyl
oxygen (2a, two nitrogens up) and towards piperidone nitrogen (3c
and 8b, two nitrogens down). This fact demonstrates that in the
other crystalline samples and/or in solutions of 3,5-bis(hetero-
arylidene)-piperid-4-ones upeup, downedown and up-down
positions of heteroatom in heteroaromatic substituents can be
expected, as it was found for thiophene-substituted molecules [19].
As in the previously studied molecules belonging to compounds
of this class, the structures presented in this paper are character-
ized with three planar fragments shown on Scheme 5. Dihedral
angles between these planes, mean atomic deviations from these
planes, and deviation of the nitrogen atom from the central frag-
ment are presented in Table 1. These data show that central frag-
ments in all molecules have envelope conformation with significant
deviation of the N atom from the plane of the central fragment.
Values of dihedral angles show that conjugated pharmacophore
fragment (see, for instance [18]) in molecule 2a is more close to
planarity than in molecules 3c and 8b. Deviation from planarity in
two latter molecules might be caused by larger size of substituent
at the piperidone N atom as well as by unusual conformation of the
substituent in the molecule 3c.
Previously, it was pointed out [19] that the higher cytotoxicity of
a particular bis(aryliden)piperid-4-one might be connected with
the planarity of the piperidone nitrogen atom. Presented data on
molecular geometrical characteristics for compounds 2a, 3c, and 8b
(Table 2) support this supposition as cytotoxic properties of ami-
dophosphonate 3c are markedly higher comparing with those for
N-methyl- and N-phosphorylethyl-substituted analogues 2a and
8b, respectively (see Table 3 below). On the other hand, is seems
that planarity of conjugated pharmacophore fragment is less
significant for cytotoxicity of a compound. Here we are considering
just small number of factors responsible for sample bioactivity and
more data should be acquired before proper structureeproperties
relationship will be established.
2.3. Cytotoxic properties
The cytotoxic activity of the compounds synthesized was tested
against human cancer cell lines, namely Caov3 (ovarian carcinoma),
A549 (lung carcinoma), KB 3-1 (human oral epidermoid carcinoma)
and drug resistant subclone of the latter one, i.e. KB 8-5, with MDR1
hyperexpression. The results are summarized in Table 3 showing
the corresponding IC50 values (IC50 is the concentration of
compound required to inhibit the growth of the cells by 50%).
Plane 1
O
Plane 2
Plane 3
N
N
N
Scheme 5. Planar fragments in the molecules of 3,5-bis(pyridinylmethylene)-piperid-
Fig. 2. Molecular structure of 3c drawn at 30% probability of thermal ellipsoids.
4-ones.

5930
E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
Table 1
Dihedral angles between planar fragments in molecules 2a,3c and 8b shown in Scheme 4, mean atomic deviations from these planes, and deviation of the nitrogen atom from
mean plane of the piperidone fragment.
Compound
Dihedral angles between planes, deg.
Mean atomic deviations from planes, Å
Deviation of
N from Plane 1, Å
1/2
1/3
2/3
2.71(6)
14.26(20)
18.52(10)
Plane 1
Plane 2
Plane 3
2a
3c
8b
14.13(6)
30.32(13)
27.56(8)
12.67(6)
27.60(16)
36.64(7)
0.045
0.015
0.023
0.008
0.005
0.009
0.011
0.010
0.010
0.719
0.701
0.639
Anticancer agent Melphalan (Sarcolysin, alkylation agent) was used
as a positive control similar to assays of cytotoxic properties of
other 3,5-bis(arylidene)-4-piperidone derivatives described in
literature (see, e.g., Ref.[1e5,16e20]). As the second positive
control, we use Doxorubicin (anthracycline antibiotic having
different mechanism of action). For comparison, the activities of
parent NH-piperidones 1a,b and the related N-Me substituted
analogues 2a,b were estimated in the same assay in order to esti-
mate the influence of N-alkylation on cytotoxic properties.
In general, cytotoxicity of all 3,5-bis(pyridinylmethylene)
piperid-4-ones tested in this assay towards ovarian and lung
carcinoma cell lines, CaoV3 and A549, was significantly higher
comparing with that of Melphalan. As one can see, compounds
bearing 4-pyridinyl moieties on the whole are more active than
their analogues having 3-pyridinyl groups. This fact is in agreement
with higher electron-withdrawing properties of 4-pyridinyl
independently on the nature of the substituent at the piperidone
nitrogen atom.
It is known that expression of transmembrane transporting P-
glycoprotein (permeability glycoprotein abbreviated as Pgp170)
being responsible for elimination of hydrophobic compounds
including the cytostatics, from tumor cells, is a plausible reason of
the classical multi-drug resistances in the treatment of cancers [23].
Usually, to overcome such resistance the inhibitors of Pgp170 should
be added to cytostatic compounds but till now the suitable and
multi-purpose inhibitors were not found out. Therefore, resistant
tumors are still difficult to treat. The cell lines KB 3-1 and KB 8-5
differ from each other byexpression of the above Pgp170. Mostly, the
compounds under investigation are less active to the drug resistant
line KB 8-5 comparing KB 3-1 similar to Doxorubicin. However,
compounds 3a, 3c, and 4c have shown the similar toxicity towards
these cell lines. Such data allowed us to suggest that piperidones 3a,
3c, and 4c are not the substrates for Pgp170 and hence can be of
interest for treatment of resistant tumors. However, this problem
requires the additional detailed studies.
compared with that of 3-pyridinyl
respectively).
(
sP
¼
0.44 and 0.25,
Introduction of methyl group at the nitrogen atom of 3,5-bis
(pyridinylmethylene)piperid-4-ones resulted in significant decrease
of antitumor properties towards ovarian and carcinoma cell lines for
compound 2a with 3-pyridine ring while its 4-pyridine containing
analogue 2b possesses even higher activity comparing with that of
NH-piperidone 1b.
As for introduction of a phosphorus moiety, in general its influ-
ence was more pronounced for the first series of compounds bearing
3-pyridinyl moieties. Among the derivatives bearing the direct NeP
bond, the activities of amidophosphate 3a and aminophosphonates
3b, 3c towards CaoV3 and A549 cell lines were comparable with
those for the parent NH-piperidone 1a while compounds 3a, 3b
possess higher activity towards KB lines including drug resistant
3. Conclusion
This study has demonstrated that introduction of pyridine rings
into the molecule of 3,5-bis((hetero)arilidene)piperid-4-ones
resulted in compounds possessing excellent antitumor properties
towards both ordinary carcinoma cell lines and drug resistant
subclone KB 8-5. The antitumor properties significantly exceeded
those for the analogues of these compounds bearing thienylidene
rings and those having E,E-3,5-bis(4-pyridinylmethylene)piperid-
4-one framework displayed higher growth inhibitory properties
comparing with those with 3-pyridine rings. Introduction of the
phosphorus moiety substantially increased the antitumor proper-
ties in the case of E,E-3,5-bis(3-pyridinylmethylene)piperid-4-one
derivatives but it is less pronounced for more active analogues
bearing 4-pyridinyl rings. That means the high potential 3,5-bis
(heteroarylidene)-4-piperidones especially phosphorylated ones as
potential drug-candidates for further development to improve
activity and bioavailability of 3,5-bis(arylidene)piperid-4-ones.
KB 8-5. In a series of
longer alkylene chain demonstrated excellent cytotoxic properties
comparable with that for 3a, 3b while -aminophosphonate 8b
u-aminophosphonates, compound 8c with
b
was less active comparing with the parent NH-piperidone 1a. At
the same time, compounds having more electron-withdrawing
4-pyridinyl rings possess in general the same order of activity
Table 2
Configuration of the nitrogen atom and positions of the substituents in the
compounds 2a, 3c and 8b.
4. Experimental protocols
Compound Bond angles at nitrogen
atom, deg.
Configuration Position of
4.1. General remarks
of nitrogen
atom
phosphorus
group
NMR spectra were recorded with a Bruker AMX-400 spec-
trometer (1H, 400.13; 19F, 376.3; 31P, 161.97 and 13C, 100.61 MHz)
using residual proton signals (1H) and that of carbon atom (13C) of
a deuterated solvent as an internal standard relative TMS, and
2a
3c
8b
C(8)eN(2)eC(9)
C(9)eN(2)eC(18)
C(8)eN(2)eC(18)
Sum
109.73(9)
111.05(9)
110.57(10)
331.35(28)
Trigonal-
pyramidal
Equatorial
Pseudo-axial
Equatorial
CF3COOH (19F) and H3PO4 31P) as an external standard. The 13C
(
C(2)eN(1)eC(6)
C(2)eN(1)eP(1)
C(6)eN(1)eP(1)
Sum
113.35(13) Planar
121.83(11)
123.24(11)
NMR spectra were registered using the JMODECHO mode; the
signals for the C atom bearing odd and even numbers of protons
have opposite polarities. The numeration for carbon atoms is
shown in Scheme 2.
Column chromatography was carried out using Merck silica gel
60 (230e400 mesh ASTM). Analytical TLCs were performed with
Merck silica gel 60 F254 plates. Visualization was accomplished by
358.42(35)
C(14)eN(3)eC(15) 110.21(19) Trigonal-
C(14)eN(3)eC(18) 111.87(18) pyramidal
C(15)eN(3)eC(15) 109.18(18)
Sum
331.26(55)

E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
5931
Table 3
Cytotoxicity of phosphorylated 3,5-bis(pyridinylmethylene)-4-piperidones 3aec, 4a,c, 8b,c and the related NH- and NeMe piperidones 1a,b and 2a,b against human Caov3,
A549, KB 3-1, and KB 8-5 cell lines.
Compound
Het
R
CaoV3
A549
KB 3-1
KB 8-5
1a
2a
3a
3b
3c
8b
8c
1b
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
4-Py
H
Me
9.2 ꢃ 0.1
>20
8.1 ꢃ 0.3
10.8 ꢃ 0.6
9.3 ꢃ 0.3
>40
6.2 ꢃ 0.1
6.0 ꢃ 0.1
5.1 ꢃ 0.3
4.3 ꢃ 0.2
7.0 ꢃ 0.3
50 ꢃ 10
e
11.1 ꢃ 0.6
>20
10.4 ꢃ 0.2
11.3 ꢃ 0.4
7.5 ꢃ 0.3
>40
15.4 ꢃ 0.2
10.3 ꢃ 0.1
4.2 ꢃ 0.1
8.2 ꢃ 0.1
8.3 ꢃ 0.2
50 ꢃ 10
0.8
24.4 ꢃ 0.7
20.2 ꢃ 0.4
5.0 ꢃ 0.2
20.3 ꢃ 0.6
7.8 ꢃ 0.1
>40
6.1 ꢃ 0.2
5.0 ꢃ 0.2
3.0 ꢃ 0.2
2.1 ꢃ 0.1
7.1 ꢃ 0.1
e
30.0 ꢃ 2.3
40.4 ꢃ 1.7
5.3 ꢃ 0.4
45.8 ꢃ 5.1
7.6 ꢃ 0.2
>40
9.5 ꢃ 0.5
8.5 ꢃ 0.1
7.4 ꢃ 0.1
5.1 ꢃ 0.4
7.1 ꢃ 0.2
e
P(O)(EtO)2
P(O)(EtO)Me
P(O)(PhO)Me
(CH2)2P(O)(OEt)2
(CH2)4P(O)(OEt)2
H
2b
4a
4c
Me
P(O)(EtO)2
P(O)(PhO)Me
Melphalan
Doxorubicin
1.2 ꢃ 0.1
3.3 ꢃ 0.1
UV light. IR spectra were recorded in KBr pellets on a Fourier-
spectrometer “Magna-IR750” (Nicolet), resolution 2 cmꢂ1, 128
scans. Melting points were determined with MPA 120 EZ-Melt
Automated Melting Point Apparatus and were uncorrected.
The reaction mixtures were stirred magnetically in round-
system CH2Cl2 (15 ml)/aqueous Na2CO3 (0.72 g, 0.0068 mol, 10 ml
H2O). Reaction mixture was stirred for 1.5 h and then solid NaCl was
added. After stirring for 15 min, the layers were separated and the
aqueous phase was extracted with CH2Cl2. The combined organic
layer was dried over Na2SO4, filtered off and the filtrate was
evaporated at reduced pressure to give 0.51 g (100%) of 1a as
bottom flasks, and the reaction course was monitored by 19F or 31
P
NMR technique or by thin-layer chromatography as appropriate.
a yellow powder, mp 166e175 ꢁC (decomp.). 1H (CDCl3),
d, ppm:
All commercial reagents were used as purchased without further
purification, all solvents were reagent grade. Ethyl methyl-
phosphonochloridate [24], phenyl methylphosphonochloridate [25],
1.80 (s, 1H, NH), 4.14 (s, 4H, cyclic NCH2), 7.34 (dd, 2H, 3JHH ¼ 5.0 Hz,
0
0
3JHH ¼ 8.0 Hz, C10-H,0 C10 -H), 7.66 (d, 2H, 3JHH ¼ 8.0 Hz, C9-H, C9 -H),
0
7.74 (s, 2H, C7-H, C7 -H), 8.57 (d, 2H, JHH ¼ 5.0 Hz, C11-H, C11 -H),
3
0
diethyl (1,4-dioxa-8-azaspiro[4.5]dec-8-yl)methylphosphonate
5
8.63 (s, 2H, C13-H, C13 -H). Anal. calcd. for C17H15N3O (%): C 73.63, H
[20], diethyl 2-(4-oxopiperidin-1-yl)ethylphosphonate 6a [20],
diethyl 4-(4-oxopiperidin-1-yl)butylphosphonate 6b [20] were
obtained by the known procedures. (3E,5E)-1-Methyl-4-oxo-3,5-bis
(3-pyridinylmethylene)piperidinone 2a and (3E,5E)-1-methyl-4-
oxo-3,5-bis(4-pyridinylmethylene)piperidinone 2b were obtained
via aldol-crotonic condensation of N-methyl-piperid-4-one with
appropriate pyridinecarboxaldehyde in the presence of BF3$Et2O
followed by neutralization of the corresponding tris(tetra-
fluoroborate) salts [21] and their physicalechemical constants and
spectral data fit well the literature data for the same compounds
obtained via the aldol-crotonic condensation performed under basic
conditions [22].
5.45, N 15.15. Found (%): C 73.38, H 5.44, N 14.95.
4.3. (3E,5E)-4-Oxo-3,5-bis(4-pyridinylmethylene)piperidine (1b)
The compound was obtained according to the procedure similar
to that for the synthesis of its 3-pyridinyl analogue 1a$excluding
the reaction time for condensation being 7 days (r.t.) to afford
(3E,5E)-4-oxo-3,5-bis(4-pyridinylmethylene)piperidinium tris(tetra-
fluoroborate) (1b$3HBF4) as an yellow solid in 21% yield. Mp 249 ꢁC
(decomp.). IR (KBr, n
, cmꢂ1): 3287, 3187, 3111, 1700 (C]O), 1634
(C]C), 1615, 1502, 1322, 1305, 1239, 1190, 1084, 1064, 925, 760, 746,
524. 1H (DMSO-d6),
d
, ppm: 4.57 (s, 4H, cyclic NCH2), 8.02 (0s, 2H, C7-
0
0
H, C7 -H), 8.13 (d, 4H, 3JHH ¼ 6.0 Hz, C9-H, C9 -H, C13-H, C13 -H), 9.01
0
0
3
(d, 4H, JHH ¼ 6.2 Hz, C10-H, C10 -H, C12-H, C12 -H), 12.43 (s, HBF4).
4.2. (3E,5E)-4-Oxo-3,5-bis(3-pyridinylmethylene)piperidine (1a)
90
130
13C (DMSO-d6), , ppm: 43.97 (C2, C6), 127.27 (C9, C13, C , C ),
d
0 0 0
134.22 (C3, C5),134.79 (C7, C7 ),143.05 (C10, C12, C10 , C12 ),149.95 (C8,
Boron trifluoride etherate (4 mmol) was added to a solution of 4-
piperidone monohydrate hydrochloride (1 mmol) and the 3-pyr-
idinecarboxaldehyde (2 mmol) in CH3CN (2 ml). The mixture was
stirred at room temperature for 2 days (until consumption of the
starting compounds as monitored by TLC). Then the mixture was
dissolved in hot EtOH. This solution was cooled to room temperature
resulting in formation of crystalline precipitate. The precipitate was
filtered off and air-dried to give (3E,5E)-4-oxo-3,5-bis(3-pyridinyl-
methylene)piperidinium tris(tetrafluoroborate) (1a$3HBF4) as a yellow
0
C8 ), 181.68 (C4). Anal. calcd. for C17H15N3O$3HBF4 (%): C 37.76, H
3.36, N 7.77. Found (%): C 37.76, H 3.83, N 7.79.
Further neutralization provided 1b as a free base in 94% yield
based on the intermediate tris(tetrafluoroborate) salt. 1H (CDCl3),
d
,
3
ppm: 1.83 (s, 1H, NH), 4.12 (s, 4H, cyclic NCH2), 7.20 (d, 4H, JHH
¼
0
0
0
5.0 Hz, C9-H, C9 -H, C13-H, C103 -H), 7.65 (s, 2H, C7-H, C7 -H), 8.65 (d,
0
4H, 3JHH ¼ 5.0 Hz, C10-H, C10 -H, C12-H, C12 -H). 13C (CDCl3),
d, ppm:
0
0
47.61 (C2, C6), 123.82 (C9, C13, C9 , C13 ), 132.98 (C3,0 C5), 137.72 (C7,
0
0
0
C7 ), 141.97 (C10, C12, C10 , C12 ), 149.97 (C8, C8 ), 186.82 (C4).
Analytically pure sample was obtained by column chromatography
using CH2Cl2/EtOH ¼ 100:15 as an eluent. Anal. calcd. for C17H15N3O
(%): C 73.63, H 5.45, N 15.15. Found (%): C 73.66, H 5.28, N 15.19.
solid, mp ca. 160 ꢁC (decomp.). Yield 84%. IR (KBr, , cmꢂ1): 3584,
n
3194, 3103, 2872, 1687 (C]O), 1629 (C]C), 1593, 1561, 1467, 1305,
1195, 1084,1059, 980, 817, 679, 544, 523. 1H (DMSO-d6),
d
, ppm: 4.59
0
(s, 4H, cyclic NCH2), 8.01 (s, 2H, C7-H, C7 -H), 8.12 (dd, 2H, 3JHH ¼ 5.6
0
0
Hz, 3JHH ¼ 8.0 Hz, C10-H, C10 -H), 8.61 (d, 2H, 3JHH ¼ 8.3 Hz, C9-H, C9 -
0
0
H), 8.94 (d, 2H, 3JHH ¼ 5.4 Hz, C11-H, C11 -H), 9.10 (s, 2H, C13-H, C13 -H),
4.4. Phosphorylation of 4-oxo-3,5-bis(pyridinylmethylene)-1-
piperidinones 1a,b (general procedure)
0
9.50 (s, HBF4). 13C (DMSO-d6),
d
, ppm: 43.93 (C2, C6),127.01 (C10, C10 ),
0
0
0
131.95 (C3, C5),132.58 (C8, C8 ),133.82 (C9, C9 ),143.55 (C7, C7 ),144.46
0
0
and 145.22 (C13, C13 and C11, C11 ),181.81 (C4). 19F (DMSO-d6),
d, ppm:
To a mixture of the corresponding NH-3,5-bis(pyridinyl-
ꢂ147.98,-148.04.Anal. calcd. for C17H15N3O$3HBF4 (%): C 37.76, H
methylene)piperidone 1a,b (1.8 mmol) and triethylamine (2.3
mmol) in dry tetrahydrofuran (9 ml) a solution of corresponding
chlorophosphonate or chlorophosphate (2.2 mmol) in dry tetra-
hydrofuran (4 ml) was added. The reaction mixture was stirred at
3.36, N 7.77. Found (%): C 37.72, H 2.98, N 7.42.
For isolation of 1a as a free base, the corresponding tris(tetra-
fluoroborate) salt (1.00 g, 0.00185 mol) was taken up into biphasic

5932
E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
room temperature over 24 h. The reaction was monitored by TLC
and the 31P NMR technique. After completion of the reaction, the
mixture was evaporated at reduced pressure, dissolved in CH2Cl2
and washed with water. The organic layer was separated, evapo-
rated at reduced pressure and purified by column chromatography
(SiO2, THF or mixture of CH2Cl2/EtOH ¼ 100:5 as an eluent). The
appropriate fractions were evaporated under reduced pressure to
give the solid product. If desired, the product can be additionally
purified by precipitation with pentane from solution in CH2Cl2.
31P (CDCl3),
d, ppm: 29.80. Anal. calcd. for C24H22N3O3P$0.75EtOH (%):
C 65.73, H 5.73, N 9.02. Found (%): C 65.56, H 5.64, N 8.83.
4.4.4. Diethyl (3E,5E)-4-oxo-3,5-bis(4-pyridinylmethylene)-1-
piperidinylphosphonate 4a
Yellow solid, mp 97.5e99.5 ꢁC. Yield 62%. IR (KBr, , cmꢂ1):
n
2982, 1672 (C]O), 1616 (C]C), 1593, 1585, 1544, 1414, 1391, 1365,
1325, 1264, 1253, 1233, 1186, 1150, 1101, 1050, 1021, 965, 940, 858,
3
821, 754, 690, 626, 537. 1H (CDCl3),
d
, ppm: 1.17 (t, 6H, JHH ¼ 7.0
Hz, P(OCH2CH3)2), 3.84e4.04 (m, 4H, P(OCH2CH3)2), 4.41 (d, 4H,
0
4.4.1. Diethyl (3E,5E)-4-oxo-3,5-bis(3-pyridinylmethylene)-1-
3JPH ¼ 8.4 Hz, cyclic NCH2), 7.23 (d, 4H, 3JHH ¼ 4.4 Hz, C9-H, C9 -H,
0
0
3
piperidinylphosphonate 3a
C
C
13-H, C130 -H), 7.68 (s, 2H, C7-H, C7 -H), 8.68 (d, 4H, JHH ¼ 4.6 Hz,
0
Yellow solid, mp 122e124 ꢁC. Yield 52%. IR (KBr,
n
, cmꢂ1): 2984,
10-H, C10 -H, C12-H, C12 -H). 13C (CDCl3),
d
, ppm: 15.88 (d, 3JPC ¼ 7.0
2
2
1669 (C]O), 1614(C]C), 1587, 1565, 1480, 1415, 1269, 1256, 1241,
Hz, P(OCH2CH3)2), 45.92 (d, JPC ¼ 3.7 Hz, C2, C6), 62.83 (d, JPC
¼
0
0
0
1212, 1154, 1047, 1023, 970, 960, 807, 769, 702, 628, 531. 1H (CDCl3),
5.9 Hz, P(OCH2CH3)2), 123.71 (C9, C13, C9 , C130 ), 134.11 (C7, C7 ),
3
d
, ppm: 1.15 (t, 6H, 3JHH ¼ 7.0 Hz, P(OCH2CH3)2), 3.81e4.03 (m, 4H, P
135.48 (d, JPC ¼ 6.1 Hz, C3, C5), 141.66 (C8, C8 ), 150.26 (C10, C12
,
0
0
3
(OCH2CH3)2), 4.43 (d, 4H, JHP ¼ 8.2 Hz, cyclic NCH2), 7.36 (dd, 2H,
C
10 , C12 ), 185.86 (C4). 31P (CDCl3),
d
, ppm: 7.20. Anal. calcd. for
0
3JHH ¼ 4.9 Hz, 3JHH ¼ 7.9 Hz, C10-H, C10 -H), 7.69 (d, 2H, 3JHH ¼ 7.9 Hz,
C21H24N3O4P (%): C 61.01, H 5.85, N 10.16. Found (%): C 60.88, H
5.67, N 9.94.
0
0
C9-H, C9 -H), 7.76 (s, 2H, C7-H, C7 -H), 8.58 (d, 2H, 3JHH ¼ 4.7 Hz, C11
-
0
0
H, C11 -H), 8.64 (s, 2H, C13-H, C13 -H). 13C (CDCl3),
d
, ppm: 15.85 (d,
3JPC ¼ 6.9 Hz, P(OCH2CH3)2), 46.02 (d, JPC ¼ 3.6 Hz, C2, C6), 62.72
4.4.5. Ethyl methyl[(3E,5E)-4-oxo-3,5-bis(4-pyridinylmethylene)-1-
piperidinyl] phosphinate 4b
2
0
0
(d, JPC ¼ 5.5 Hz, P(OCH2CH3)2), 123.48 (C10, C10 ), 130.36 (C8, C8 ),
2
0
0
133.30 (C7, C7 ), 133.95 (d, JPC ¼ 4.9 Hz, C3, C5), 136.83 (C9, C9 ),
Yellow solid, mp 60e62 ꢁC. Yield 12%, purity according to the 1H
3
0
0
149.89 and 151.00 (C13, C13 and C11, C11 ), 185.75 (C4). 31P (CDCl3),
d
,
NMR data w89%. IR (KBr, n
, cmꢂ1): 3030, 2982, 1661 (C]O), 1644,
ppm: 7.21. Anal. calcd. for C21H24N3O4P: C, 61.01; H, 5.85; N, 10.16%.
Found: C, 60.68; H, 6.13; N, 9.98%.
1594, 1547, 1528, 1506, 1443, 1415, 1390, 1362, 1327, 1308, 1280,
1267, 1223, 1183, 1164, 1102, 1040, 995, 958, 903, 788, 754, 533, 494.
1H (CDCl3),
d
, ppm: 1.20 (t, 3H, 3JHH ¼ 7.0 Hz, POCH2CH3),1.41 (d, 3H,
4.4.2. Ethyl methyl[(3E,5E)-4-oxo-3,5-bis(3-pyridinylmethylene)-1-
2JHP ¼ 16.3 Hz, PCH3), 3.79e3.85 (m, 1H, OCH2), 3.97e4.18 (m, 1H,
OCH2), 4.48 (d, 4H, 3JHP ¼ 8.0 Hz, cyclic NCH2), 7.31 (d, 4H, 3JHH ¼ 5.8
piperidinyl]-phosphinate 3b
0
0
0
Yellow solid, mp 131e139 ꢁC (decomp.). Yield 60%. IR (KBr,
n
,
Hz, C9-H, C9 -H, C13-H, C13 -H), 7.76 (s, 2H, C7-H, C7 -H), 8.74 (d, 4H,
0
0
cmꢂ1): 3085, 2925, 1726, 1665 (C]O), 1612 (C]C), 1579, 1560, 1477,
3JHH ¼ 5.7 Hz, C10-H, C10 -H, C12-H, C12 -H). 13C (CDCl3),
d, ppm: 11.89
1420, 1309, 1269, 1240, 1197, 1186, 1155, 1072, 1034, 987, 959, 939,
(d, 1JPC ¼ 132.8 Hz, PCH3), 15.91 (d, 3JPC ¼ 6.6 Hz, POCH2CH3), 45.16
902, 810, 755, 709, 695, 632b 545. 1H (CDCl3),
d
, ppm: 1.07 (t, 3H,
(d, 2JPC ¼ 3.8 Hz, C2, C6), 59.89 (d, 2JPC ¼ 6.6 Hz, POCH2CH3), 123.65
0
0
0
2
3
3JHH ¼ 7.1 Hz, POCH2CH3), 1.29 (d, 3H, JHP ¼ 18.4 Hz, PCH3),
(C9, C13, C9 , C13 ), 134.21 (C7, C7 ), 135.35 (d, JPC ¼ 3.3 Hz, C3, C5),
0
0
0
3.67e3.73 (m, 1H, OCH2), 3.86e3.92 (m, 1H, OCH2), 4.40 (d, 4H, 3JHP
141.44 (C8, C8 ), 150.22 (C10, C12, C10 , C12 ), 185.77 (C4). 31P (CDCl3),
ppm: 31.68.
d,
¼ 8.0 Hz, cyclic NCH2), 7.32 (dd, 2H, 3JHH ¼ 4.9 Hz, 3JHH ¼ 8.0 Hz, C10
-
0
0
H,0C10 -H), 7.66 (d, 2H, 3JHH ¼ 8.0 Hz, C9-H, C9 -H), 7.73 (s, 2H, C7-H,
0
C7 -H), 8.54 (d, 2H, JHH ¼ 4.6 Hz, C11-H, C11 -H), 8.60 (s, 2H, C13-H,
4.4.6. Phenyl methyl[(3E,5E)-4-oxo-3,5-bis(4-pyridinylmethylene)-
3
0
1
C
13 -H). 13C (CDCl3),
d
, ppm: 11.91 (d, JPC ¼ 133 Hz, PCH3), 15.86
1-piperidinyl]phosphinate 4c
(d, 3JPC ¼ 6.6 Hz, POCH2CH3), 45.29 (d, 2JPC ¼ 4.4 Hz, C2, C6), 59.77 (d,
Yellow solid, mp 52e57 ꢁC. Yield 43%. IR (KBr, , cmꢂ1): 3420,
n
0
0
2JPC ¼ 6.6 Hz, POCH2CH3), 123.45 (C10, C10 ), 130.18 (C80 , C8 ), 133.37
3038, 2921, 2853, 1660 (C]O), 1643 (C]C), 1593, 1554, 1527, 1490,
0
(C7, C7 ), 133.83 (d, 3JPC ¼ 3.7 Hz, C3, C5), 136.78 (C9, C9 ), 149.89 and
1414, 1309, 1264, 1234, 1205, 1187, 1164, 1110, 1064, 1025, 995, 984,
0
0
150.91 (C13, C13 and C11, C11 ), 185.65 (C4). 31P (CDCl3),
d, ppm: 31.52.
917, 898, 813, 775, 766, 692, 531, 504, 474. 1H (CDCl3),
d, ppm: 1.55
Anal. calcd. for C20H22N3O3P$0.8H2O (%): C 60.39, H 5.98, N 10.56, P
7.79. Found (%): C 60.69, H 5.48, N 10.07, P 7.72.
(d, 3H, 2JHP ¼ 16.5 Hz, PCH3), 4.49 (d, 4H, 3JHP ¼ 8.4 Hz, cyclic NCH2),
7.01 (d, 2H, 3JHH ¼ 7.4 Hz, o-H in C6H5O), 7.17 (t, 1H, 3JHH ¼ 7.3 Hz, p-
0
H in C6H5O), 7.25e7.28 (m, 4H, m-H in C6H5O and C9-H, C9 -H, C13
-
0
0
4.4.3. Phenyl methyl[(3E,5E)-4-oxo-3,5-bis(3-pyridinylmethylene)-
1-piperidinyl]-phosphinate 3c
The compound was purified by column chromatography using
gradientelutionfrom100% CH2Cl2 to CH2Cl2/EtOH ¼ 20/1to give3c as
a strong solvate with ethanol which was observed in the 1H NMR
H, C13 -H), 7.65 (s, 2H, C7-H, C7 -H), 8.73 (d, 4H, 3JHH ¼ 5.5 Hz, C10-H,
0
0
C
10 -H, C12-H, C12 -H). 13C (CDCl3),
d
, ppm: 12.27 (d, 1JPC ¼ 135.6 Hz,
PCH3), 45.00 (d, 2JPC ¼ 3.8 Hz, C2, C6), 119.73 (d, 3JPC ¼ 4.9 Hz, o-C in
0
0
C6H5O), 123.63 (C9, C13, C9 , C13 ), 124.77 (p-C in C6H5O), 129.64 (m-C
0
in C6H5O), 134.20 (C7, C7 ), 134.85 (d, 3JPC ¼ 3.3 Hz, C3, C5),141.44 (C8,
0
0
0
spectra. Yellow solid, mp. >99 ꢁC (decomp.). Yield 51%. IR (KBr,
n
,
C8 ), 150.13 (C10, C12, C10 , C12 ), 185.10 (C4). 31P (CDCl3),
d, ppm:
cmꢂ1): 3429, 3054, 2916, 1733, 1670 (C]O), 1613 (C]C), 1585, 1564,
1495, 1482, 1415, 1329, 1310, 1274, 1240, 1200, 1187, 1167, 1159, 1070,
1026, 987, 917, 907, 893, 814, 774, 764, 715, 693, 566, 550, 514, 475. 1H
29.79. Anal. calcd. for C24H22N3O3P$H2O (%): C 64.14, H 5.39, N 9.35.
Found (%): C 63.96, H 5.03, N 9.11.
(CDCl3),
d
, ppm:1.50(d,3H, 2JHP ¼ 16.6 Hz, PCH3), 4.47 (d, 4H, 3JHP ¼ 8.3
4.5. (3E,5E)-1-[(Diethoxyphosphoryl)methyl]-4-oxo-3,5-bis(3-
pyridinylmethylene)-piperidinium tetrafluoroborate 7a
Hz, cyclic NCH2), 6.97 (d, 2H, 3JHH ¼ 8.4 Hz, o-H in C6H5O), 7.12 (t, 1H,
3JHH ¼ 7.3 Hz, p-H in C6H5O), 7.25 (d, 2H, 3JHH ¼ 7.8 Hz, m-H in C6H5O),
0
7.38(dd, 2H, 3JHH ¼ 5.0Hz,3JHH ¼ 7.8Hz,C10-H, C10 -H),7.68(d, 2H, 3JHH
Boron trifluoride etherate (4 ml) was added to a mixture of
diethyl (1,4-dioxa-8-azaspiro[4.5]dec-8-yl)methyl-phosphonate 6a
(0.28 g, 1 mmol) and 3-pyridinaldehyde (0.20 g, 2 mmol) cooled on
ice-bath. The resulting mixture was stirred at room temperature for
7 days. Addition of Et2O (15 ml) resulted in the formation of the oily
residue. The ether layer was discarded and the crude product was
recrystallized from MeOH followed by trituration in Et2O to afford
0
0
¼ 7.8 Hz, C9-H, C9 -H), 7.70 (s, 2H, C7-H, C7 -H), 8.61 (dd, 2H, 3JHH ¼ 5.3
0
0
Hz, C11-H, C11 -H), 8.62 (s, 2H, C13-H, C13 -H).13C (CDCl3),
d, ppm: 12.33
(d,1JPC ¼ 136 Hz, PCH3), 45.20 (d, 2JPC ¼ 4.4 Hz, C2, C6),119.80 (d, 3JPC
¼
0
4.9 Hz, o-C in C6H5O), 123.52 0(C10, C10 ), 124.75 (p-C in C6H5O), 129.66
(m-C in C6H5O),130.23 (C8, C8 ),133.43 (C3, C5),133.45 (d, 4JPC ¼ 4.9 Hz,
0
0
0
0
C7, C7 ), 136.89 (C9, C9 ),149.95 (C13, C13 ), 150.88 (C11, C11 ),185.16 (C4).

E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
5933
7a (0.2 g, 34%) as a yellow powder. Mp. >156 ꢁC (decomp.). IR (KBr,
, cmꢂ1): 3434, 3129, 2983, 2928, 2599, 1676, 1632, 1598, 1550,
1465, 1265, 1235, 1205, 1124, 1084, 1063, 992, 814, 792, 766, 681,
reduced pressure, the oily residue was purified by column chro-
matography on silica gel. Elution was started with acetone
(w150 ml) and continued with THF. The appropriate THF fractions
were evaporated under reduced pressure, and the residue was
taken up in THF. After addition of Et2O to this solution, its slow
diffusion resulted in precipitation of the product as a yellow crys-
talline solid, mp 137e144 ꢁC (decomp.). Yield 0.78 g (28%). 1H
n
522. 1H (DMSO-d6),
d
, ppm: 1.11 (t, 3JHH ¼ 7.0 Hz, 6H, P(OCH2CH3)2),
3.16 (d, 2JHP ¼ 11.5 Hz, 2H, NCH2P), 3.86e3.99 (m, 4H, P(OCH2CH3)2),
0
4.18 (s, 4H, cyclic NCH2), 5.07 (s, HBF4), 7.78 (s, 2H, C7-H, C7 -H), 7.93
0
(dd, 2H, 3JHH ¼ 5.6 Hz, 3JHH ¼ 8.0 Hz, C10-H, C10 -H), 8.37 (d, 2H, 3JHH
0
0
¼ 7.5 Hz, C9-H, C9 -H), 8.83 (d, 2H, 3JHH ¼ 5.2 Hz, C11-H, C11 -H), 8.97
(CDCl3),
d
, ppm: 1.25 (t, 6H, 3JHH ¼ 7.0 Hz, P(OCH2CH3)2), 1.80e1.98
0
(s, 2H, C13-H, C13 -H). 13C (DMSO-d6),
d
, ppm: 16.49 (d, 3JPC ¼ 5.5 Hz,
(m, 2H, CH2P), 2.78e2.91 (m, 2H, CH2CH2P), 3.82 (s, 4H, cyclic N
P(OCH2CH3)2), 51.06 (d, 1JPC ¼ 149 Hz, NCH2P), 54.28 (d, JPC ¼ 7.5
(CH2)2), 3.95e4.10 (m, 4H, 0P(OCH2CH3)2), 7.37 (dd, 2H, JHH ¼ 5.00
2
3
Hz, cyclic NCH2), 61.33 (d, 2JPC ¼ 5.8 Hz, P(OCH2CH3)2), 126.16 (C10
,
Hz, 3JHH ¼ 8.0 Hz, C10-H, C10 -H), 7.68 (d, 2H, 3JHH ¼ 8.0 Hz, C9-H, C9 -
0
0
0
0
0
0
C
10 ), 132.17 (C3, C5), 132.89 (C8, C8 ), 133.73 (C9, C9 ), 143.07 (C7, C7 ),
H), 7.76 (s, 2H, C7-H, C7 -H), 8.59 (d, 2H, 3JHH ¼ 5.0 Hz, C11-H, C11 -H),
0
0
0
145.20 and 146.37 (C13, C13 and C11, C11 ), 183.38 (C4). 19F (DMSO-
8.64 (s, 2H, C13-H, C13 -H). 13C (CDCl3),
d
, ppm: 16.10 (d, 3JPC ¼ 6 Hz, P
1
d6),
(DMSO-d6),
d
, ppm: ꢂ147.98, ꢂ148.03. 31P (DMSO-d6),
d
, ppm: 22.82. 19F
(OCH2CH3)2), 23.95 (d, JPC ¼ 140 Hz, CH2P), 50.42 (s, CH2CH2P),
0
d,
ppm: ꢂ147.98, ꢂ148.03. Anal. calcd. for
53.90 (C2, C6), 61.37 (d, 2JPC ¼ 6 Hz, P(OCH2CH3)2), 123.21 (C10, C100),
0
0
C22H26N3O4P$2HBF4$H2O (%): C 42.54, H 4.87, N 6.77. Found (%): C
130.49 (C8, C8 ), 132.37 (C7, C7 ), 134.11 (C3, C5), 136.70 (C9, C9 ),
0 0
42.46, H 4.68, N 6.74.
149.53 and 150.72 (C13, C13 and C11, C11 ), 185.66 (C4). 31P (CDCl3),
d,
ppm: 30.66. HRMS: found (Mþ) 441.18120, calcd. (Mþ) 441.18175.
4.5.1. (3E,5E)-1-[4-(Diethoxyphosphoryl)butyl]-4-oxo-3,5-bis(3-
pyridinylmethylene)piperidinium tris(tetrafluoroborate) 7c
4.6. X-ray structure determination
Boron trifluoride etherate (10 ml) was added to a cooled (ice-
bath) mixture of diethyl 3-(4-oxopiperidin-1-yl)butylphosphonate
6c (0.88 g, 3 mmol) and 3-pyridinecarboxaldehyde (0.64 g, 6
mmol). Formation of oily precipitate was immediately observed.
The mixture was stirred at room temperature for 18 h. After addi-
tion of Et2O (15 ml), the upper ether layer was decanted from oily
residue, which was recrystallized from a mixture of CH2Cl2/MeOH
¼ 10:1 to give 1.27 g (56%) of 7c as a yellow powder, mp > 160 ꢁC
The single crystals of 2a suitable for X-ray experiments were
obtained by recrystallization from DCM, by slow diffusion of diethyl
ether into the solution in DMSO (3c) or in THF (8b). Data were
collected on a Bruker SMART APEX II CCD diffractometer (l(Mo Ka)-
radiation, graphite monochromator, omega and phi scan mode) and
corrected for absorption using the SADABS program (version 2.03
[26]). The structures were solved by direct methods and refined by
a full-matrix least squares technique on F2 with anisotropic
displacement parameters for non-hydrogen atoms. The hydrogen
atoms in all compounds were placed in calculated positions and
refined within the riding model with fixed isotropic displacement
parameters (Uiso(H) ¼ 1.5 Ueq(C) for the CH3-groups and Uiso(H)
¼ 1.2 Ueq(C) for the other groups). All calculations were carried out
using the SHELXTL program [27]. For more details of data collection
and structure solution see Table 3. Crystallographic data for 2a, 3c
and 8b have been deposited with the CambridgeCrystallographic
Data Center. CCDC 776801e776803 contain the supplementary
crystallographic data for this paper. These data can be obtained free
of charge from the Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (Fax: þ44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk or
www.ccdc.cam.ac.uk) Table 4.
(decomp.). IR (KBr, n
, cmꢂ1): 3429, 3044, 2979, 2933, 2866, 2804,
1669 (C]O), 1649 (C]C), 1619, 1583, 1563, 1479, 1415, 1391, 1276,
1267,1235,1203,1166,1097,1052,1023, 987, 961, 942, 809, 789, 707,
546. 1H (DMSO-d6),
d
, ppm: 1.20 (t, 6H, 3JHH ¼ 7.0 Hz, P(OCH2CH3)2),
1.45e1.56 (m, 2H, CH2CH2CH2P), 1.69e1.82 (m, 4H, CH2CH2CH2P),
3.33 (t, 2H, 3JHH ¼ 6.9 Hz, NCH2CH2CH2CH2P), 3.94e4.03 (m, 4H, P
(OCH2CH3)2), 4.72 (s, 4H, cyclic N(CH2)2), 7.97 (m 4H), 8.43 (s, 2H),
8.89 (s, 2H), 9.03 (s, 2H). 13C (DMSO-d6),
d
, ppm: 16.48 (d, 3JPC ¼ 5.4
2
Hz, P(OCH2CH3)2), 19.41 (d, JPC ¼ 4.3 Hz, CH2CH2CH2CH2P), 24.00
1
3
(d, JPC ¼ 138 Hz, CH2CH2CH2CH2P), 24.03 (d, JPC ¼ 16.2 Hz,
CH2CH2CH2CH2P), 51.65 (C2, C6), 55.63 (CH2CH2CH2CH2P), 061.20 (d,
0
2JPC ¼ 6.5 Hz, P(OCH2CH3)2), 127.08 (C10, C10 ), 131.15 (C8, C8 ), 132.37
0
(C3, C5), 134.40 (C7, C7 ), 143.53, 144.68, 145.39, 181.21 (C4). 31P
(DMSO-d6), d, ppm: 31.10. Anal. calcd. for C25H32N3O4P$3HBF4$H2O
(%): C 39.98, H 4.97, N 5.60. Found (%): C 40.27, H 4.68, N 5.64.
Further neutralization of 7c provided free base 8c as a viscous
Table 4
semisolid substance. 1H (CDCl3),
d
, ppm: 1.20 (t, 6H, 3JHH ¼ 7.0 Hz, P
Crystallographic data for 2a,3c,and 8b.
(OCH2CH3)2), 1.45e1.65 (m, 6H, CH2CH2CH2P), 2.50 (t, 2H, 3JHH ¼ 6.7
Hz, NCH2CH2CH2CH2P), 3.74 (s, 4H, cyclic N(CH2)2), 3.94e4.05 (m,
4H, P(OCH2CH3)2), 7.33 (dd, 2H, 3JHH ¼ 4.9 Hz, 3JHH ¼ 7.9 Hz, C10-H,
Compound
2a
3c
8b
Empirical formula
FW
T, K
Crystal system
Space group
a, Å
b, Å
c, Å
C18H17N3O
291.35
100(2)
Triclinic
P-1
5.8194(8)
9.0874(12)
14.3210(19)
102.490(2)
96.681(2)
98.004(2)
723.76(17)
2
C23H28N3O4P
441.45
C24H22N3O3P
431.42
0
0
0
C
10 -H), 7.65 (d, 2H, 3JHH ¼ 8.0 Hz, C9-H, C9 -H), 7.69 (s, 2H, C7-H, C7 -
298(2)
136(2)
0
0
H), 8.54 (d, 2H, 3JHH ¼ 3.6 Hz, C11-H, C11 -H), 8.60 (s, 2H, C13-H, C13
-
Triclinic
P-1
Triclinic
P-1
H). 13C (CDCl3),
d
, ppm: 16.22 (d, 3JPC ¼ 6.0 Hz, P(OCH2CH3)2), 20.03
8.529(5)
10.865(6)
12.991(7)
95.126(8)
100.524(8)
93.928(8)
1174.5(11)
2
11.0617(13)
11.3621(12)
11.5682(14)
83.128(3)
62.021(3)
62.263(2)
1127.4(2)
2
2
1
(d, JPC ¼ 5.2 Hz, CH2CH2CH2CH2P), 25.14 (d, JPC ¼ 140 Hz,
3
CH2CH2CH2CH2P), 27.59 (d, JPC ¼ 16.3 Hz, CH2CH2CH2CH2P), 54.47
(C2, C6), 56.52 (CH2CH2CH2CH2P), 61.23 (d, JPC ¼ 6.4 Hz, P
2
a
, deg.
, deg.
0
0
0
(OCH2CH3)2),123.32 (C10, C10 ), 130.77 (C8, C8 ), 132.67 0(C7, C7 ),134.63
b
0
0
g
, deg.
(C3, C5), 136.92 (C9, C9 ), 149.52 and 150.79 (C13, C13 and C11, C11 ),
V, Å3
186.19 (C4). 31P (CDCl3),
d
, ppm: 31.72. Anal. calcd. for C25H32N3O4P
Z
(%): C 63.95, H 6.87, N 8.95. Found (%): C 63.44, H 7.08, N 8.57.
dc, mg/m3
F(000)
1.337
308
0.085
55.98
1.248
1.271
468
452
m
, mmꢂ1
0.150
0.152
4.5.2. Diethyl 2-[(3E,5E)-4-oxo-3,5-bis(3-pyridinylmethylene)
piperidin-1-yl]ethylphosphonate 8b
2
qmax, deg.
52
49.98
Reflections
7297/3457
13682/4616
3962/3962
A solution of diethyl 2-(4-oxopiperidin-1-yl)ethylphosphonate
6b (1.65 g, 0.0063 mol), 3-pyridinecarboxaldehyde (1.34 g, 0.0125
mol) in EtOH (5 ml), 8 drops of piperidine and 8 drops glacial acetic
acid was refluxed for 4 days. After removing of volatiles under
collected/unique
R1; wR2 (I > 2
s(I))
0.0390; 0.0982
0.0497; 0.1041
1.009
0.0588; 0.1816
0.0770; 0.1980
1.374
0.0368; 0.0911
0.0435; 0.0942
1.019
R1; wR2 (all data)
GOF on F2

5934
E.S. Leonova et al. / European Journal of Medicinal Chemistry 45 (2010) 5926e5934
4.7. Biological evaluations
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Cell lines used for estimation of toxicity of compounds were
CaoV3 (human ovarian carcinoma), A549 (human lung carcinoma),
KB 3-1 (human oral epidermoid carcinoma cells), and drug resis-
tant subclone of the latter one, i.e. KB 8-5, with MDR1 hyper-
expression. Cells were grown in RPMI-1640 medium
(SigmaeAldrich, UK) supplemented with 10% fetal bovine serum
(FBS, HyClone, USA), 2 mM L-glutamine and gentamicin. Cytotox-
icity of the individual compounds was measured for each cell line
after 72 of cultivation by the MTT (3-(4,5-dimethyldiazolyl-2)-2,5-
diphenyl tetrazolium-bromide) colorimetric assay. The test is based
on the ability of mitochondrial dehydrogenase in viable cells to
convert MTT reagent (ICN Biomedicals, Germany) into a soluble
blue formazan dye. Briefly, the different cell lines were seeded into
96-well plates at a concentration of 1 ꢄ 104 cells/100
ml/well. The
cells were allowed to attach overnight at 37 ꢁC in a humidified
atmosphere containing 5%CO2. The tested compounds were
initially dissolved in dimethylsulfoxide (DMSO) and the working
solutions were added to FBS free culture medium. The compounds
were added to wells with increasing drug concentrations. After 72 h
incubation, 20 ml of MTT reagent (5 mg/ml) were added and cell
cultures were incubated for 3 h at 37 ꢁC. After removal of the
culture medium formazan crystals were dissolved in dime-
thylsulfoxide (SigmaeAldrich) to determine the amount of for-
mazan product. The optic density (OD) was determined by the
multi-well plate reader (Uniplan, Picon, Russia) at 590 nm The
results were expressed as percent decrease of cell viability as
compared to untreated controls. Each concentration of the
compound tested was examined in triplicate and the IC50 values
were determined graphically. The concentrations of compounds
used were 5 ꢄ 10ꢂ5, 10ꢂ5, 10ꢂ6, 10ꢂ7 M. Commercially available
Melphalan (Sarcolysin) and Doxorubicin purchased from Arkelan-
Glaxo were used as a positive control in the assay.
Acknowledgements
Thisworkwas supported by the DeutscheForschungsgemeinschaft
ꢁ
(DFG N 436 RUS 113/905/0-1; RO 362/35-1), program of President of
Russian Federation “For Young PhD scientists” (No MK-2464.2008.3),
Program of Chemistry and Material Science Division of RAS, NIH via the
RIMI program (grant 1P20MD001104-01), and NSF/DMR grant
0934212.