Synthesis, characterization and structure-activity relationship of novel N-phosphorylated E,E-3,5-bis(thienylidene)piperid-4-ones

Article abstract of DOI:10.1016/j.ejmech.2009.11.041

In order to design the agents with improved antitumor activity of 3,5-bis(thienylidene)piperid-4-one type, E,E-N-phosphoryl-3,5-bis(thienylidene)piperid-4-ones 6a-c and E,E-N-ω-phosphorylalkyl-3,5-bis-(thienylidene)piperid-4-ones 7a-c were obtained via the direct phosphorylation of the parent NH-3,5-bis(thienylidene)piperid-4-one and by condensation of preformed N-phosphorylalkyl substituted piperidones with thiophene 2-carbaldehyde, respectively. The structures of the compounds were elucidated by ¹H, ³¹P, ¹³C NMR along with a single crystal X-ray diffraction analysis. Under the action of visible light thermodynamically more stable E,E-isomers slowly undergo photochemical conversion in CDCl₃ solution to the corresponding E,Z-isomers and E,Z-N-methyl-3,5-bis(thienylidene)piperid-4-one 5 was isolated in individual state. The importance of phosphorylation for cytotoxic properties of 3,5-bis(thienylidene)piperid-4-ones towards human carcinoma cell lines Caov3, Scov3, and A549 and influence of olefin configuration on antitumor activity were demonstrated.

Full text of DOI:10.1016/j.ejmech.2009.11.041

European Journal of Medicinal Chemistry 45 (2010) 992–1000
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and structure–activity relationship of novel
N-phosphorylated E,E-3,5-bis(thienylidene)piperid-4-ones
Michael V. Makarova, Evgeniya S. Leonova a, Ekaterina Yu. Rybalkina b, Paul Tongwa c,
,
,
*
Victor N. Khrustaleva 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, Russia
b Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 24, 115478 Moscow, Russia
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 design the agents with improved antitumor activity of 3,5-bis(thienylidene)piperid-4-one
Received 26 June 2009
Received in revised form
13 November 2009
Accepted 20 November 2009
Available online 26 November 2009
type, E,E-N-phosphoryl-3,5-bis(thienylidene)piperid-4-ones 6a-c and E,E-N-u-phosphorylalkyl-3,5-
bis-(thienylidene)piperid-4-ones 7a-c were obtained via the direct phosphorylation of the parent NH-
3,5-bis(thienylidene)piperid-4-one and by condensation of preformed N-phosphorylalkyl substituted
piperidones with thiophene 2-carbaldehyde, respectively. The structures of the compounds were
elucidated by 1H, 31P, 13C NMR along with a single crystal X-ray diffraction analysis. Under the action of
visible light thermodynamically more stable E,E-isomers slowly undergo photochemical conversion in
CDCl3 solution to the corresponding E,Z-isomers and E,Z-N-methyl-3,5-bis(thienylidene)piperid-4-one 5
was isolated in individual state. The importance of phosphorylation for cytotoxic properties of 3,5-
bis(thienylidene)piperid-4-ones towards human carcinoma cell lines Caov3, Scov3, and A549 and
influence of olefin configuration on antitumor activity were demonstrated.
Keywords:
Aminophosphonates
N-Phosphoryl-3,5-bis(thienylidene)piperid-
4-ones
4-Piperidones
N-Phosphorylalkylene-3,5-
bis(thienylidene)piperid-4-ones
Synthesis
Ó 2009 Elsevier Masson SAS. All rights reserved.
X-ray structure
Cytotoxicity–structure relationship
Photoisomerization
1. Introduction
inflammatory [18], antibacterial [19], antihepatoxic [20], and anti-
oxidative [21] ways of action. Similar properties are possessed by
Bis(arylidene)cyclohexanones and bis(arylidene)piperidones
(Scheme 1, 1) were synthesized in the beginning of 20th century
and from this time their bioactivity and pharmaceutical applica-
tions attracted significant attention [1–4]. Asymmetrically
substituted derivatives having structure similar to cocaine were
studied and their properties, that might be suitable for applications
as local anesthetics, were considered. Starting from early 1970th
arilidenecyclohexanones and arylidenepiperidones have been
studied in relation to their cytotoxic properties and antitumor
activities [5–12].
curcuminoid structures containing the conjugated a,b-unsaturated
ketone moieties (Scheme 1, 3) which similarity to bis(ar-
ylidene)cyclohexanones and bis(arylidene)piperidones is quite
obvious.
It was found that cytotoxic properties of synthetic curcumin
analogs such as cycloalkan-1-ones [22–24] and 3,5-bis(arylidene)-
4-piperidones [11,13,25–29] are significantly stronger than those
for curcumine. On the other hand, it is remarkable that these
compounds (generally screened as free bases) are non-lethal (as
curcumin) and mice tolerated doses up to and including 300 mg/kg
[30] and even five daily doses up to 240 mg/kg of hydrochloride salt
of 3,5-bis(benzylidene)-4-piperidone did not cause mortalities [8].
Curcumin and other enones are supposed to have multiple
targets and interacting macromolecules within the cell [16,17,28]. It
was suggested that enones display anticancer properties via the
supposed mechanism of action comprising interaction with cellular
thiols with little or no affinity for hydroxyl and amino groups in
nucleic acids [31,32]. The 1,5-diaryl-3-oxo-1,4-pentadienyl
Relatively recently [13–15] antitumor activity of these materials
started to attribute to their structural similarity to Indian spice
curcumine (Scheme 1, 2) that possesses chemotherapeutic and
some chemopreventative properties [16,17], including anti-
* Corresponding author. Tel./Fax: þ7 495 1359356.
E-mail address: odinets@ineos.ac.ru (I.L. Odinets).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.041

M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
993
O
Y
piperidones, we performed the synthesis of two series of
compounds. For comparison we used known NH- (4 [38]) and N-
methyl-bis(thienylidene)-4-piperidones (5 [12,39]) which were
obtained via the literature procedures by aldol condensation of
thiophene 2-carbaldehyde with piperid-4-one and N-methyl-
piperid-4-one, respectively, under basic conditions (10% NaOH/
EtOH).
R
R
1: Arylidenecyclohexanones - Y=CH2; Arylidenepiperidones -Y = NH
O
O
O
R
R
In the first series of phosphorylated bis(thienylidene)-4-piper-
idones 6a-c the phosphorus atom is directly bonded with the
nitrogen atom of the piperidone moiety and possessed substituents
of different nature. The second series comprises compounds 7a-c,
in which the diethoxyphosphoryl group is attached to the N-atom
using an alkylene linker of different length. Scheme 2 below
demonstrates the general structure of 3,5-bis(thienylidene)-4-
piperidones of interest.
HO
OH
OCH3
OCH3
2
3
Scheme 1. Structures of bioactive bis(arylidene)cyclohexanones and the related
bis(arylidene)piperidones 1, curcumine 2 and linear -unsaturated ketones 3.
a,b
pharmacophore group observed both in alicyclic unsaturated
enones and cyclic piperidone derivatives is speculated to interact
with cellular constituents, while the nature of the group at the
heterocyclic nitrogen atom in the piperidone derivatives could
influence the biological activity of the compounds. In other words,
it can contribute to solubility and bioavailability and either
increase, for example, the cytotoxic properties by facilitating
approach of the cytotoxin to a specific binding site or reduce them
preventing this interaction. These groups may possess inherent
bioactivity, and it would be beneficial if they could enhance the
therapeutic effect resulting in additional impact on cancer cells due
to the different mechanisms of action. Therefore, the attempts to
improve the antitumor properties of 3,5-bis(arylidene)-4-piper-
idones and affect the capacity of their transportation via the cellular
membrane were mostly concentrated on modification of substitu-
ents at the nitrogen atom via N-alkylation [33], N-acylation [10,26],
N-phosphorylation [34,35] or introduction of phosphorylalkylidene
moieties [36].
At the same time, electron properties of the aryl substituents
can affect both these parameters. In fact, piperidinone derivatives
bearing different substituents at the aryl rings were tested for
antitumor properties, where the cytotoxic activity drastically
increases with the increase of electron withdrawing properties of
these substituents [8,24,33,35–37]. According to the Dimmock’s
data [37], a Hammett sP value of the substituents in the aryl rings as
well as conformation of the central core affects the fractional
positive change on the adjacent carbon atoms at the double bond
and, as a consequence, the possibility of the compound to interact
with cellular thiols.
Compounds 7a-c may be considered as substituted
u-amino-
phosphonates bearing additional pharmacofore 3,5-bis(thienyli-
dene)-4-piperidone moiety. It should be mentioned that natural
and synthetic aminophosphonates and aminophosphonic acids
being analogues of amino acids and competing with the latter for
active sites of enzymes, possess different biological activity which
type is defined by the nature of linker and surroundings of the
phosphorus and nitrogen atoms [40]. Therefore, in this case one
may expect that combination of the aminophosphonate moiety and
cytotoxic skeleton may result in improved activity.
The first series of N-phosphorylated compounds, namely ami-
dophosphates 6a,b and amidophosphonate 6c, was obtained by
direct phosphorylation of the parent NH-bis(thienylidene)-4-
piperidone 4 [38] using the appropriate phosphorus(V) acid chlo-
rides in the presence of triethylamine as a base (Scheme 3). The
reaction readily proceeds in THF under ambient conditions to afford
the desired products in ca. 57–66% yield.
Previously we demonstrated [33] that reaction of NH-3,5-
bis(arylidene)piperid-4-ones 3 (R1 ¼ H) with short-chain halogen-
oalkanes proceeded predominately as quaternization to afford N,N-
dialkylated products. To avoid such side reaction in the synthesis of
target
E,E-N-u-phosphorylalkyl-3,5-bis(thienylidene)piperid-4-
ones 7a-c, the condensation of thiophene 2-carbaldehyde with
aminophosphonates 8, 9a,b was the method of choice (Scheme 4).
The
u-aminophosphonates 8, 9a,b differing in the length of an
alkylene linker, were obtained starting either from O-protected 4-
piperidone, namely 1,4-dioxa-8-azaspiro[4.5]decane, or 4-piper-
idone itself, according the procedures elaborated by us earlier [36].
Briefly, N-phosphorylmethyl substituted piperidone 8 (n ¼ 1) was
obtained in the dioxolane protected form via the three-component
Kabachnik–Fields reaction of diethylphosphite, commercially
available 1,4-dioxa-8-azaspiro[4.5]decane and formaldehyde in
Therefore, it seems reasonable to estimate the bioactivity of
relative compounds having heterocyclic rings instead of aryls. To
the best of our knowledge, two such attempts were undertaken by
Dimmock et al. [37] and El-Subbagh et al. [12] who revealed the
diminished antitumor activity of N,N-dimethyl-3,5-bis(4-piper-
idylidene)-4-piperidonium iodide [37] and a broad spectrum of
antitumor activity for N-methyl-3,5-bis(thienylidene)-4-piper-
aqueous media. For the synthesis of
(n ¼ 2) we used the aza–Michael reaction of piperidone and
vinylphosphonate in water while -aminophosphonate 9b was
b-aminophosphonate 9a
idone [12] having IC50 values in the range of 1–9ꢀ10ꢁ5 m
M
g
depending on the cell line.
obtained by alkylation of 4-piperidone hydrochloride with diethyl
Moreover, recently we demonstrated the positive effect of
phosphorus groups attached to the nitrogen atom of 3,5-bis(ar-
ylidene)-4-piperidones [34,35] on their cytotoxic properties. Taking
these results into account, in this paper we described the synthesis,
crystal structure, photoisomerization, and in vitro evaluation of the
antitumor activity of phosphoryl substituted 3,5-bis(thienylidene)-
4-piperidones.
O
7
'
7
'
9
9
8
'
8
4
5
6
3
'
10
10
N 2
R
S
S
11
'
11
2. Results and discussion
4: R=H; 6a: R= P(O)(OEt)2
5: R=Me; 6b: R= P(O)(OPh)2
7a: R= CH2P(O)(OEt)2
7b: R= (CH2)2P(O)(OEt)2
7c: R= (CH2)3P(O)(OEt)2
2.1. Chemistry
6c: R= P(O)(OPh)Me
In order to understand better an influence of the phosphorus
moiety on cytotoxic properties of 3,5-bis(thienylidene)-4-
Scheme 2. General structure of phosphorylated 3,5-bis(thienylidene)-4-piperidones.

994
M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
O
into the extensive planar system of conjugation with the central
piperidone fragment, four possible conformers A, B, C and D
(Scheme 5) are mainly expected for those molecules.
O
O
R1R2P
Cl
S
S
S
S
N
H
It should be noted that, apart from R substituent, the conformers
B and C are converted into each other by the inversion center. In
general, the outstretched structure of conjugated bonds (conformer
A) is more favorable, however, the energy differences between
conformers A, B and D for molecule 5 with R]Me are very small
(their relative energies according to quantum–chemical calcula-
tions are 0.0 for A, 0.68 B and 1.34 kcal/mol for D [39]). Moreover,
the 1H NMR spectra both of piperidone 5 and all other relative
compounds obtained demonstrated only one set of the corre-
sponding protons. Apparently, free rotation over C6–C7 (and C11–
C12) ordinary bond resulted in the quick transformations of these
conformers in solutions to give the averaged chemical shifts. The
insignificant differences in energies indicate a high probability of all
three conformers to be formed over the crystallization, and they
can statistically occupy same positions in crystals. Thus, in crystal 5,
conformers A and C were found in ratio 9:1. Superposition of two
conformers found in crystal 5 is shown in Fig. 1.
Compound 6a crystallizes in monoclinic space group P21/c, with
the two independent molecules in the unit cell (Fig. 2). According to
the disorder analysis of the thiophene rings (see Experimental
part), compound 6a exists as the major conformer A and the two
minor conformers B and C in ratio of 7:2:1. The geometrical
parameters of the two independent molecules in terms of bond
lengths and bond angles are similar. In both molecules of 6a, the
central piperidone cycles adopt a sofa conformation, with the
deviations of the nitrogen atoms from the planes via five other
atoms of 0.619 and 0.626 Å, respectively. Both molecules contain
three planar fragments; the first includes the planar fragment of
the piperidone cycle (PA), while the planar fragments PB and PC
include a thiophene ring and adjacent bridge atoms. The dihedral
angles PA/PB and PA/PC between these fragments are equal to 5.5(1),
9.1(1) and 2.3(3), 5.2(3)ꢂ, respectively. The largest difference
between the two molecules is the rotation angles of the disordered
phosphonate substituents relative to the central heterocycle (see
Supplementary Materials, Fig. S1). The corresponding dihedral
O2]P1–N1–C3 and O6]P2–N2–C23 angles are equal to ꢁ5.1(3)
and ꢁ33.2(3)ꢂ.
N
P
R2
R1
O
4
6a-c
R1=R2=OEt (6a), OPh (6b);
R1=Me, R2=OEt (6c)
Scheme 3. Direct phosphorylation of NH-3,5-bis(thienylidene)-4-piperidone 4.
u
-bromopropylphosphonate under phase transfer conditions
(Scheme 4).
As mentioned above, thiophene 2-carbaldehyde gave higher
yields of the final products (up to 90%) of aldol-crotonic conden-
sation with piperidin-4-one under basic conditions (10% NaOH/
EtOH [12,38,39]). However, we found that N-phosphorylalkyl
substituted piperidones readily undergo dealkylation of ester
groups along with the rupture of P–C bond under basic conditions
and afforded to a complex mixture of products. Moreover, the
dioxalane protected group could be removed only under acidic
conditions. Therefore, we performed the condensation of (1,4-
dioxa-8-azaspiro[4.5]dec-8-ylmethyl)phosphonate
8 with thio-
phene 2-carbaldehyde under acidic conditions (g. HCl/AcOH) which
allowed concurrent formation of C]C bond and removing of
protection group. Nevertheless, acidic conditions were unsuitable
for condensation thiophene 2-carbaldehyde with unprotected
N-phosphorylalkylpiperidones 9a,b. Thus, the synthesis of 7b,c was
accomplished under milder conditions, namely a combination of
Lewis acid MgBr2 and TEA similar to the known synthesis of relative
NH- and N-Me derivatives 4,5 [41]. It should be mentioned that
despite the good yield of the crude desired products estimated by
the NMR spectroscopy, compounds 7a and 7c easily undergo
decomposition under column chromatography purification on silica
gel, after which the yields of isolated products decreased drastically.
2.2. Molecular geometry and crystal structure of phosphorylated
3,5-bis(thienylidene)-4-piperidones
The molecular structure of 5 was studied by our group recently
[39]. From series of compounds presented in this paper we were
able to crystallize compounds 6a, 6c and 7b by slow diffusion of
pentane in solution of the compound in CH2Cl2 (6a, 6c) or EtOAc
(7b) and their molecular and crystal structures were characterized
by a single crystal X-ray diffraction analysis. Taking into account the
fact that there is a clear trend for all such molecules to be involved
In the crystal, the molecules of 6a are bound by the weak C–
H.O hydrogen bonds (see Supplementary Materials, Table S1).
Moreover, there are the weak stacking interactions between the
pairs of molecules connected by the inversion center, with the
interplane distances of 3.40 Å (C1.C5A 3.492(5) Å, C2.C11A
3.544(5) Å, C5.C1A 3.492(5) Å, C11.C2A 3.544(5) Å; label A
denotes the symmetrically equivalent atom by the inversion center
2
O
O
O
O
CHO
S
(EtO)2P(O)H/CH2O
HCl/AcOH
O
N
N
H
N
P(O)(OEt)2
2
8
S
S
(EtO)2(O)P
O
O
(CH2)nP(O)(OEt)2
7a-c
n=2
(EtO)2P(O)(CH2)3Br
n=3
CHO
S
n=1 (7a), 2 (7b), 3 (7c)
MgBr2.Et2O/Et3N
N
H
N
(CH2)nP(O)(OEt)2
9a,b
Scheme 4. Synthetic approach to N-(
u
-phosphorylalkyl)-3,5-bis(thienylidene)-4-piperidones 7a-c.

M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
995
O
N
O
nitrogen atom from the plane via five other atoms of 0.710 Å. The
molecule of 7b also contains the same three planar fragments PA, PB
and PC, however, the dihedral angles PA/PB and PA/PC between them
(9.0(2) and 31.2(2)ꢂ, respectively) are larger than those in 6a and 6c.
The rotation angle of the phosphonate substituent relative to the
central heterocycle in 7b defined as pseudo-torsion O2]P1.N1–
C3 angle (ꢁ166.0(2)ꢂ) is significantly larger than those in 6a and 6c.
Similar to 6c, the molecules of 7b form in the crystal the
centrosymmetric dimers by the stacking interactions, with the
interplane distance of 3.18 Å (C6.C11A 3.318(4) Å, C1.C1A
3.260(4) Å, C11.C6A 3.318(4) Å; label A denotes the symmetrically
equivalent atom by the inversion center (symmetry code [ꢁx, 1ꢁy,
2ꢁz])). Furthermore, the dimers are bound by the weak C–H.O
hydrogen bonds (see Supplementary Materials, Table S1).
S
S
S
S
N
R
R
Conformer A
Conformer B
O
O
S
S
S
S
N
N
R
R
Conformer D
Conformer C
The most striking differences between the molecules of 5, 6a, 6c
and 7b are the geometry of the nitrogen atoms and the arrange-
ment of the phosphonate ligands (Table 1). In contrast to 5 and 7b,
where the nitrogen atoms have trigonal–pyramidal configurations
and the phosphonate ligands occupy the more sterically favored
equatorial positions, in molecules 6a and 6c the nitrogen atoms
have almost planar coordination and ligands at the N atoms occupy
sterically unfavorable pseudo-axial positions. The flattened coor-
dination of the nitrogen atoms was found to be quite common
characteristic of the related molecules containing the pP]O and
pC]O substituents at the nitrogen atoms [25,34,42–45]. Such
arrangement of the substituents at the nitrogen atom has been
already observed in the analogous compounds before [46,47].
Comparison of relative orientations of thiophene rings for
molecules in crystals studied shows superposition of conformers A
and C in structure 5 and A, B and C in 6a. Pure conformers A and B
were found in 6c and 7b, respectively. It gives us reason to suggest
that these conformers resulted in different proportions over the
crystallization and samples 5 and 6a give us the examples of co-
crystallization of two or more conformers.
Scheme 5. Possible conformers of 3,5-bis(thienylidene)-4-piperidones.
(symmetry code [2ꢁx, 2ꢁy, 1ꢁz])) and 3.44 Å (C20.C31A 3.398(5)
Å, C24.C30A 3.530(5) Å, C30.C24A 3.530(5) Å, C31.C20A
3.398(5) Å; label A denotes the symmetrically equivalent atom by
the inversion center (symmetry code [1ꢁx, ꢁy, 1ꢁz])).
Compound 6c crystallizes in triclinic space group Pꢁ1, Z ¼ 2,
and, contrary to 6a, contains in crystal one independent molecule
that represents the only conformer A (Fig. 3). General features of
the molecular structures of 6a and 6c are very similar. As in 6a, the
central piperidone cycle in 6c adopts a sofa conformation, with the
deviation of the nitrogen atom from the plane via five other
piperidone atoms of 0.592 Å. Also, the molecule of 6c contains the
same three planar fragments PA, PB and PC. The dihedral angles
PA/PB and PA/PC between these fragments in 6c (4.5(1) and 9.4(1)ꢂ,
respectively) are very close to those in 6a. The rotation angle of the
phosphonate substituent relative to the central heterocycle in 6c
(the O2]P1–N1–C3 torsion angle is ꢁ1.1(2)ꢂ) is only slightly
smaller than that in one of the two independent molecules of 6a
representing the most populated conformer A.
The molecules of 6c form in crystal centrosymmetric dimers by
both the stacking interactions (the interplane distance of 3.27 Å,
C6.C11A 3.385(2) Å, C1.C1A 3.325(2) Å, C11.C6A 3.385(2) Å;
label A denotes the symmetrically equivalent atom by the inversion
center (symmetry code [1ꢁx, 1ꢁy, 1ꢁz])) and the weak C–H.O
hydrogen bonds (see Supplementary Materials, Table S1). Further-
more, the dimers are bound by both the attractive S2.O3 [2ꢁx, ꢁy,
1ꢁz] interactions (3.183(2) Å) and the weak C–H.O hydrogen
bonds (see Supplementary Materials, Table S1).
Compound 7b crystallizes in monoclinic space group P21/c,
Z ¼ 4, and contains one independent molecule that represents the
only conformer B (Fig. 4). As for 6a and 6c, the central piperidone
cycle in 7b adopts a sofa conformation, with the deviation of the
2.3. Photochemical transformations
It should be mentioned that, independently on reaction condi-
tions of aldol-crotonic condensation, all known types of 3,5-
bis(arylidene)piperidones and the relative cycloalkanone
analogues were isolated as thermodynamically more stable E,E-
isomers. Guilford et al. [48] demonstrated that E,E-3,5-bis(ar-
ylidene)cycloalkanones bearing para-amidine moieties in aryl
rings along with the relative E,E-N-carboethoxy-3,5-bis(ar-
ylidene)piperid-4-one undergo facile photochemical isomerization
to the mixture of the corresponding Z,Z and Z,E olefin isomers.
These compounds are inhibitors of Factor Xa (FXa) involved in
Fig. 1. Superposition of molecular conformers A (solid line) and C (dashed line) in 5 drawn at 50% probability of thermal ellipsoids.

996
M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
Fig. 4. Molecular structure of 7b drawn at 40% probability of thermal ellipsoids. The
alternative positions of the disordered ethyl groups are not shown. All hydrogen atoms
are omitted for clarity.
were irradiated until a constant products ratio was reached as
determined by 1H NMR technique (the product ratio could be
determined by integration of vinyl resonanses in the 1H NMR
spectrum of crude mixtures). Over 6 h no transformation was
observed in DMSO solutions and very slow isomerization pro-
ceeded in CD3CN accompanied by photodegradation (the yield of
the second isomer was 5% and 20% for 5 and 7b respectively). Better
results were obtained in CDCl3 media, where the yields of the
corresponding second isomers reached 45% and 25% for 5 and 7b,
respectively (Scheme 6).
Then, isomerization of 5 was performed at preparative scale. The
second isomer was isolated via preparative TLC. Note that reverse
transformation was observed under acidic conditions, therefore,
the silica gel was pre-neutralized with a mixture of dioxane/
NH4OH: 4/1. According to the 1H and 13C NMR data, the product
represented the corresponding Z,E-isomer (compound 5-Z,E, see
Supplementary Materials, Figs. S2 and S3 respectively; for
comparison, Figs. S5–S7 demonstrate the NMR and IR spectra of the
parent isomer, namely compound 5-E,E). Due to unsymmetrical
Fig. 2. Structure of two independent molecules of 6a drawn at 40% probability of
thermal ellipsoids. Only major conformers A are shown. All hydrogen atoms are
omitted for clarity.
blood coagulation cascade and the order of their potency was
Z,Z > Z,E > E,E. Possibility of similar photochemical rearrangement
was mentioned also by Vatsadze et al. [49] for 3,5-bis(thienyli-
dene)-4-cyclohexanone which molecular and crystal structure is
very similar to structure of 5 but was found for only bis(pyr-
idylidene) analogue. Therefore, it seems reasonable to estimate the
possibility of photochemical isomerization in a series of thienyli-
dene substituted compounds obtained and in case of success to test
the biological activity of the isomers formed.
Firstly, solutions of E,E-isomers of two compounds, namely 5-E,E
and 7b-E,E, in three different solvents such as DMSO-d6, CDCl3, and
CD3CN were irradiated with a white light (175 W standard mercury
lamp) to produce an equilibrium mixture of isomers. The solutions
Table 1
Geometries of the nitrogen atoms in molecules 5, 6a, 6c and 7b.
Compound
Bond angles
at nitrogen
atom, deg.
Configuration of
nitrogen atom
Position of
phosphonate
ligand
5
C3–N1–C4, 110.3(2)
C3–N1–C16, 111.3(2)
C4–N1–C16, 111.0(2)
Sum 332.6(6)
Trigonal–pyramidal
Equatorial
6a
P1–N1–C3, 123.4(2)
P1–N1–C4, 122.2(2)
C3–N1–C4, 114.1(3)
Sum 359.7(7)
P2–N2–C22, 119.2(2)
P2–N2–C23, 120.2(2)
C22–N2–C23, 113.5(2)
Sum 352.9(6)
P1–N1–C3, 122.5(1)
P1–N1–C4, 121.1(1)
C3–N1–C4, 115.4(1)
Sum 359.0(3)
C3–N1–C4, 110.7(2)
C3–N1–C16, 113.5(2)
C4–N1–C16, 114.5(2)
Sum 338.7(6)
Planar
Pseudo-axial
Pseudo-axial
Pseudo-axial
Equatorial
Flattened
6c
Planar
7b
Trigonal–pyramidal
Fig. 3. Molecular structure of 6c drawn at 40% probability of thermal ellipsoids. All
hydrogen atoms are omitted for clarity.

M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
997
Comparing the activities of compounds 6a, 7a-c having the
S
O
similar surroundings at the phosphorus atom one may mention
O
N
that aminophosphate 6 and
similar order of cytotoxicity,
toxic while -aminophosphonate 7b with odd number of methy-
a
-aminophosphonate 7a possess the
hν
g-aminophosphonate 7c is slightly less
S
S
b
N
R
S
lene units in the alkylene chain demonstrated the lowest cytotox-
icity among all phosphorylated bis(thienylidene)-4-piperidones.
R
Z,E
E,E
R=Me (5), (CH2)2P(O)(OEt)2 (7b)
3. Conclusion
Scheme 6. Photochemical isomerization of 3,5-bis(thienylidene)-4-piperidones 5, 7b.
This study has demonstrated that introduction of phosphoryl
moiety to the nitrogen atom of the 3,5-bis(thienylidene)-4-piper-
idones resulted in increased cytotoxic properties comparing with
relative non-phosphorylated analogues. In contrast, the presence of
phosphorylalkylene chain decreased the rate of photochemical
transformations. This study has revealed the high potential 3,5-
bis(heteroarylidene)-4-piperidones especially phosphorylated
ones for further development to improve activity and bioavail-
ability of 3,5-bis(arylidene)-4-piperidones.
nature of the isomer, the 1H NMR and 13C spectra of 5-Z,E are more
complex showing separate resonances of many hydrogen and
carbon atoms, respectively.
2.4. Cytotoxic properties
The cytotoxic activity of the compounds synthesized was tested
against human cancer cell lines, namely Scov3 and Caov3 (ovarian
carcinoma), and A549 (lung carcinoma). The results are summa-
rized in Table 2 showing the corresponding IC50 values (IC50 is the
concentration of compound required to inhibit the growth of the
cells by 50%). Anticancer agent Melphalan (Sarcolysin) was used as
a positive control similar to assays of cytotoxic properties of other
3,5-bis(arylidene)-4-piperidone derivatives described in literature
[24,25,30–33,36]. For comparison, the activities of parent NH-
piperidone 4 and relative N-Me substituted analogue 5 were esti-
mated in the same assay.
The NH-piperidone 4 did not demonstrate cytotoxic properties
towards the cell lines under investigation while introduction of
methyl group to the nitrogen atom (compound 5-E,E) resulted in
reasonable increase of cytotoxicity towards two cell lines (Scov3
and A549) but this compound still had higher IC50 figures than
melphalan. Interestingly, the other isomer of 5, namely 5-Z,E did
not possess cytotoxicity and we failed to achieve 50% cell death
under the experimental conditions.
At the same time all bis(thienylidene)-4-piperidones bearing
phosphoryl moieties had lower IC50 figures than melphalan
towards the mentioned cell lines. Therefore, the introduction of the
phosphoryl group to the nitrogen atom clearly resulted in a drastic
increase of cytotoxic activity among 3,5-bis(thienylidene)-4-
piperidones. In general, compounds 6a-c having almost planar
configuration of nitrogen atom in piperidone ring are more active
than 7a-c, in which the phosphorus atom and the nitrogen one are
separated by alkylene linker and where nitrogen atom has trigonal-
pyramidal configuration. At that, amidophosphinate 6c was the
most active among compounds tested.
4. Experimental protocols
4.1. General remarks
NMR spectra were recorded with a Bruker AMX-400 spec-
trometer (1H, 400.13; 31P, 161.97 and 13C, 100.61 MHz) using
residual proton signals of deuterated solvent as an internal stan-
dard (1H, 13C), and H3PO4 (31P) as an external standard. The 13C 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 numbering of carbon atoms used for description of
13C NMR spectra is shown in Scheme 2. Melting points are uncor-
rected. HRMS were obtained on a Varian MAT CH7A instrument at
70 eV. Analytical TLCs were performed with Merck silica gel 60 F254
plates. Visualization was accomplished by UV light. IR spectra were
recorded in KBr pellets on a Fourier-spectrometer ‘‘Magna-IR750’’
(Nicolet), resolution 2 cmꢁ1, 128 scans. The assignment of the
absorption bands in IR spectra was made according to ref. [50,51].
The starting NH-bis(thienylidene)-4-piperidone 4 [38], its N-
methyl derivative 5 [12] and aminophosphonates on the piper-
idone base [36] were obtained by the known procedures. Other
reactants were purchased from Aldrich and used without further
purification.
4.2. Diethyl (3E,5E)-4-oxo-3,5-bis(2-thienylydene)-1-
piperidinylphosphonate 6a
A solution of diethyl chlorophosphate (0.26 g, 0.0015 mol) in
THF (5 mL) was added to a stirred mixture of NH-3,5-bis(2-thie-
nylydene)-4-piperidinone 4 (0.36 g, 0.00125 mol) and Et3N (0.18 g,
0.00175 mol) in THF (5 mL). After stirring at r.t. over 24 h, the
mixture was evaporated at reduced pressure to dryness and the
residue was purified by column chromatography (SiO2, eluent –
CH2Cl2). Evaporation of the appropriate fractions afforded crude 6a
as a yellow solid. The latter was dissolved in CH2Cl2 and precipi-
tated with pentane to give 6a (0.35 g, 66%) as yellow long needles,
Table 2
Cytotoxicity of phosphorylated 3,5-bis(thienylidene)-4-piperidones 6a-c, 7a-c and
comparison NH- and N-Me piperidons 4,5 against human Scov3, Caov3 and A549
cell lines.
Compound
R
Scov3
Caov3
A549
IC50
NT
(
m
M)
IC50
(m
M)
IC50
NT
(mM)
4-E,E
5-E,E
5-Z,E
H
Me
Me
NT
NT
NT
mp 121.5–123.5 ꢂC. IR (KBr,
n
cmꢁ1): 1659 (C]O), 1595 (C]C),
85 ꢃ 5
80 ꢃ 5
NT
NT
1560, 1420 and 1367 (thiophene ring vibration), 1330, 1264 (P]O),
6a-E,E
6b-E,E
6c-E,E
7a-E,E
7b-E,E
7c-E,E
Melphalan
P(O)(OEt)2
P(O)(OPh)2
P(O)(OPh)Me
CH2P(O)(OEt)2
(CH2)2P(O)(OEt)2
(CH2)3P(O)(OEt)2
30 ꢃ 3
60 ꢃ 5
8 ꢃ 1
26 ꢃ 6
44 ꢃ 4
10 ꢃ 2
20 ꢃ 1
27 ꢃ 5
35 ꢃ 4
4 ꢃ 2
1211 (thiophene ring vibration), 1192, 1164, 1150, 1046 (P–O–C),
1023, 963, 855, 699. 1H (CDCl3)
d, ppm: 1.23 (t, 6H, P(OCH2CH3)2,
3JHH ¼ 7.1 Hz), 3.93–4.05 (m, 4H, P(OCH2CH3)2), 4.47 (d, 2H, cyclic
38 ꢃ 3
64 ꢃ 4
40 ꢃ 5
60 ꢃ 7
15 ꢃ 1
35 ꢃ 1
30 ꢃ 3
2
3
3
N(CH2)2, JPH ¼ 9.2 Hz,), 7.13 (dd, 2H, JHH ¼ 3.8 Hz, JHH ¼ 4.9 Hz),
70 ꢃ 10
40 ꢃ 4
3
3
7.32 (d, 2H, JHH ¼ 3.6 Hz), 7.55 (d, 2H, JHH ¼ 5.0 Hz), 7.92 (s, 2H).
13C (CDCl3)
d
, ppm: 16.09 (d, POCH2CH3, 3JCP ¼ 7.3 Hz), 46.02 (d, C2,
50 ꢃ 10
50 ꢃ 10
3
2
NT, non toxic.
C6, JCP ¼ 4.4 Hz), 62.77 (d, POCH2CH3, JCP ¼ 5.8 Hz), 128.15 (C10
,

998
M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
0
0
0
3
3
C10 ), 128.72 (C90, C9 ), 129.62 (d, 0C3, C5, JCP ¼ 5 Hz), 130.84 (C7, C7 ),
8H P(OCH2CH3)2 þ cyclic N(CH2)2), 7.13 (dd, 2H, JHH ¼ 3.8 Hz,
133.36 (C11, C11 ), 138.35 (C8, C8 ), 185.83 (C4). 31P (CDCl3)
7.68. Anal. calcd. for C19H22NO4PS2 (%): C 53.89, H 5.24, N 3.31.
Found (%): C 53.64, H 5.09, N 3.20.
Compounds 6b,c were obtained similarly using the appropriate
chlorophosphate or chlorophosphinate respectively.
d
, ppm:
3JHH ¼ 4.9 Hz), 7.33 (d, 2H, 3JHH ¼ 3.2 Hz), 7.55 (d, 2H, 3JHH ¼ 5 Hz),
7.94 (s, 2H). 13C (CDCl3),
d, ppm: 16.14 (d, POCH2CH3,
3JCP ¼ 5.7 Hz), 52.38 (d, NCH2P, JCP ¼ 162.2 Hz), 55.47 (d, C2, C6,
0
3JCP ¼ 9.7 Hz), 61.99 (d, POCH2CH3, JCP ¼ 6.7 Hz), 127.7 (C10, C100),
2
0
0
0
128.60 (C7, C7 ), 129.49 (C8, C8 ), 130.44 (C9, C9 ), 132.92 (C11, C11 ),
138.19 (C3, C5), 185.53 (C4). 31P (CDCl3),
d, ppm: 23.46. Anal. calcd.
4.3. Diphenyl (3E,5E)-4-oxo-3,5-bis(2-thienylydene)-1-
piperidinylphosphonate 6b
for C20H24NO4PS2 (%): C 54.91, H 5.53, N 3.20. Found (%):C 54.99,
H 5.65, N 3.06.
Yellow crystalline powder, mp 172–173 ꢂC. Yield 57%. IR (KBr,
n
4.6. Diethyl 2-[(3E,5E)-4-oxo-3,5-bis(2-thienylmethylene)-1-
piperidinyl]ethyl-phosphonate 7b
cmꢁ1): 1662 (C]O), 1593 (C]C), 1564 1490, 1486, 1419 and 1365
(thiophene ring vibration), 1320, 1271 (P]O), 1251, 1236 (thio-
phene ring vibration), 1204, 1186, 1069 (P–O–C), 965, 936, 928, 781,
Diethyl 2-(4-oxo-1-piperidinyl)ethylphosphonate (1.00 g,
0.0038 mol), 2-thiophene carboxaldehyde (0.85 g, 0.0076 mol),
Et3N (0.77 g, 0.0076 mol), MeOH (0.12 g, 0.0038 mol) were placed
in a flask. Magnesium bromide ethyl etherate (1.96 g, 0.0076 mol)
was added in portions to the mixture of reagents at good stirring.
To ensure satisfactory stirring, small amounts of abs. THF were
periodically added to a reaction mixture (9 ml of THF in total).
Next day water was added followed by extraction with CH2Cl2.
Evaporation of CH2Cl2 afforded yellow oil which was purified by
column chromatography (Al2O3, l 25 cm, d 2 cm; eluent – EtOAc).
Elutes were evaporated at reduced pressure to give yellow solid.
The product obtained was dissolved in EtOAc, and pentane was
added on the top of solution obtained. Diffusion of pentane at
room temperature resulted in the formation of nice yellow nee-
dles of the desired compound (0.79 g, 46%), mp 111–113 ꢂC. IR
761, 734. 1H (CDCl3),
d, ppm: 4.62 (d, 2H, cyclic N(CH2)2,
2JPH ¼ 9.1 Hz), 7.12–7.18 (m, 10H), 7.28 (t, 4H, 3JHH ¼ 7.9 Hz), 7.34 (d,
3
3
2H, JHH ¼ 3.6 Hz), 7.59 (d, 2H, JHH ¼ 5.0 Hz), 7.87 (s, 2H). 13C
2
(CDCl3),
d
, ppm: 45.96 (d, C2, C6, JCP ¼ 4.2 Hz), 119.96 (d, CPh in o-
0
3
position to O, JCP ¼ 4.9 Hz), 125.03 (C10, C10 ), 128.08 (CPh in p-
0
position to O), 128.49 (d, C3, C5, JCP ¼ 4.6 Hz), 128.94 (C9, C9 ),
3
0
0
129.63 (CPh in m-position to O), 130.86 (C7, C7 ), 133.42 (C11, C11 ),
0
138.04 (C8, C8 ), 150.34 (d, CPh–O–P, JCP ¼ 6.5 Hz), 184.75 (C4). 31P
2
(CDCl3),
d
, ppm: ꢁ1.76. Anal. calcd. for C27H22NO4PS2 (%): C 62.41, H
4.27, N 2.70. Found (%): C 62.55, H 4.23, N 2.57.
4.4. O-Phenyl methyl[(3E,5E)-4-oxo-3,5-bis(2-thienylmethylene)-
1-piperidinyl]phosphinate 6c
Yellow crystalline powder, mp 159–161 ꢂC. Yield 65%. IR (KBr,
n
(KBr, n
cmꢁ1): 1659 (C]O), 1598 (C]C), 1561, 1419 and 1359
(thiophene ring vibration), 1336, 1316, 1261 (P]O), 1251, 1234,
1220 (thiophene ring vibration), 1190, 1173, 1167, 1055 (P–O–C),
cmꢁ1): 1656 (C]O), 1592 (C]C), 1554, 1494, 1421 and 1376 (thio-
phene ring vibration), 1332, 1302, 1261 (P]O), 1249 (thiophene
ring vibration), 1203, 1194, 1169, 1141, 1061 (P–O–C), 969, 927, 900,
1027, 1012, 972, 958, 948, 722. 1H (CDCl3),
d
, ppm: 1.27 (t, 6H,
778, 724, 703. 1H (CDCl3),
d
, ppm: 1.60 (d, 3H, CH3-P, 2JPH ¼ 16.6 Hz),
P(OCH2CH3)2, JHH ¼ 7.1 Hz), 2.03 (ABX-system, 2H, NCH2CH2P,
3
2
4.45–4.58 (m, 4H, cyclic N(CH2)2), 7.11–7.18 (m, 5H), 7.28 (t, 2H,
2JHH ¼ 16.0 Hz, JPH(A) ¼ 2JPH(B) ¼ 7.9 Hz), 2.89–2.95 (apparent q,
3JHH ¼ 7.8 Hz), 7.34 (d, 2H, 3JHH ¼ 3.4 Hz), 7.58 (d, 2H, 3JHH ¼ 5.0 Hz),
2H, NCH2CH2P), 3.81 (s, 4H, cyclic N(CH2)2), 4.02–4.10 (m, 4H,
7.89 (s, 2H). 13C (CDCl3),
d, ppm: 12.33 (d, P-CH3, JCP ¼ 134.0 Hz),
P(OCH2CH3)2), 7.10 (apparent t, 2H, JHH ¼ 4.3 Hz), 7.28 (d, 2H,
1
3
2
3
45.02 (d, C2, C6, JCP ¼ 4.2 Hz), 120.08 (d, CPh in o-position to O,
3JHH ¼ 3.2 Hz), 7.52 (d, 2H, JHH ¼ 5.0 Hz), 7.88 (s, 2H). 13C (CDCl3),
0
3JCP ¼ 4.9 Hz), 124.56 (C10, C10 ), 128.02 (CPh in p-position to O),
d
, ppm: 16.23 (d, POCH2CH3, JCP ¼ 6.6 Hz), 24.33 (d, NCH2CH2P,
3
0
128.72 (C9, C9 ), 128.86 (d C3,0 C5, JCP ¼ 3.5 Hz), 129.56 (CPh in m-
JCP ¼ 139 Hz), 50.69 (NCH2CH2P), 53.79 (C2, C6), 61.50 (d,
3
0
0
0
0
position to O), 130.74 (C7, C7 ), 133.37 (C11, C11 ), 137.92 (C8, C8 ),
POCH2CH3, JCP ¼ 6.6 Hz), 127.8 (C10, C100), 128.38 (C9, C9 ), 129.81
2
0
0
2
150.29 (CPh–O–P, JCP ¼ 8.4 Hz), 185.05 (C4). 31P (CDCl3),
d, ppm:
(C3, C5), 130.34 (C7, C7 ), 132.99 (C11, C11 ), 138.34 (C8, C8 ), 185.83
30.40. Anal. calcd. for C22H20NO3PS2 (%): C 59.85, H 4.57, N 3.17.
Found (%): C 59.85, H 4.61, N 3.09.
(C4). 31P (CDCl3), , ppm: 31.08. HRMS: found (Mþ) 451.10358,
d
calcd. (Mþ) 451.10409.
4.5. Diethyl 2-[(3E,5E)-4-oxo-3,5-bis(2-thienylmethylene)-1-
4.7. Diethyl 2-[(3E,5E)-4-oxo-3,5-bis(2-thienylmethylene)-1-
piperidinyl]methyl-phosphonate 7a
piperidinyl]propyl-phosphonate 7c
Dry hydrochloric acid was bubbled through a suspension of
diethyl (1,4-dioxa-8-azaspiro[4.5]dec-8-ylmethyl)phosphonate
(1.47 g, 0.005 mol) and 2-thiophene carboxaldehyde (1.4 g,
0.0125 mol) in glacial acetic acid (10 mL) over 2–4 h. The reaction
mixture was kept at room temperature for 4 days. The volatiles
were removed under reduced pressure, and the residue was
treated with CH2Cl2 and an aqueous solution of potassium
carbonate (pH w 8–9). After stirring over 10 min the organic
phase was separated, dried over Na2SO4 and evaporated to
dryness. The residue was purified by column chromatography on
SiO2 (eluent: mixture of CH2Cl2/CH3CN ¼ 100:10) to give desired
phosphonate in a yield of ca. 10% as yellow oily product, gradually
Using the above procedure for the synthesis of compound 7b,
propylphosphonate 7c was obtained in 10% yield as yellow
powder, mp 80–83 ꢂC. IR (KBr,
n
cmꢁ1): 1660 (C]O), 1597 (C]C),
1561, 1420 and 1366 (thiophene ring vibration), 1339, 1302, 1264
(P]O), 1241, 1227 (thiophene ring vibration), 1198, 1185, 1163,
1053 (P–O–C), 1027, 1005, 964, 905, 706. 1H (CDCl3),
d, ppm: 1.24
(t, 6H, P(OCH2CH3)2, 3JHH ¼ 7 Hz), 1.75–1.90 (m, 4H,
NCH2CH2CH2P), 2.72 (t, 2H, NCH2, 3JHH ¼ 6.3 Hz), 3.84 (s, 4H, cyclic
3
3
N(CH2)2), 7.14 (dd, 2H, JHH ¼ 3.8 Hz, JHH ¼ 4.9 Hz), 7.32 (d, 2H,
3JHH ¼ 3.6 Hz), 7.54 (d, 2H, JHH ¼ 5.0 Hz), 7.92 (s, 2H). 13C (CDCl3),
3
d,
ppm: 16.26 (d, POCH2CH3, 3JCP ¼ 6.0 Hz), 20.26 (d,
NCH2CH2CH2P,
2JCP ¼ 4.5 Hz),
22.93
(d,
NCH2CH2CH2P,
transforming into semi-solid compound, mp 78–81 ꢂC. IR (KBr,
n
JCP ¼ 141.1 Hz), 54.13 (C2, C6), 57.16 (d, NCH2CH2CH2P,
0
cmꢁ1): 1652 (C]O), 1598 (C]C), 1564, 1414 and 1392 (thiophene
ring vibration), 1331, 1313, 1293, 1262 (P]O), 1253, 1239 (thio-
phene ring vibration), 1206, 1186, 1165, 1051 (P–O–C), 1024, 972,
3JCP ¼ 16.5 Hz), 61.35 (d, POCH2CH3, JCP ¼ 6.8 Hz), 127.9 (C10, C100),
2
0
0
128.35 (C9, C90 ), 130.15 (C3, C5), 130.33 (C7, C7 ), 133.03 (C11, C11 ),
138.51 (C8, C8 ), 186.28 (C4). 31P (CDCl3),
d, ppm: 31.96. Anal. calcd.
927, 705. 1H (CDCl3),
d
,
ppm: 1.27 (t, 6H, P(OCH2CH3)2,
for C22H28NO4PS2 (%): C 56.76, H 6.06, N 3.01. Found (%): C 56.54, H
6.04, N 2.88.
2
3JHH ¼ 7.1 Hz), 3.09 (d, 2H, NCH2P, JPH ¼ 11.6 Hz), 4.09–4.17 (m,

M.V. Makarov et al. / European Journal of Medicinal Chemistry 45 (2010) 992–1000
999
4.8. (3Z,5E)-1-methyl-3,5-bis(2-thienylmethylene)piperidin-4-one
5-Z,E
isotropic displacement parameters (Uiso(H) ¼ 1.5Ueq(C) for the CH3-
groups and Uiso(H) ¼ 1.2Ueq(C) for the other groups). All calcula-
tions were carried out using the SHELXTL program [53]. Crystallo-
graphic data for 6a, 6c and 7b have been deposited with the
Cambridge Crystallographic Data Center. CCDC 735 987–735 989
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).
A solution of 5-E,E isomer (20 mg, 0.666 mmol) in CHCl3 (2 ml)
was irradiated with standard mercury lamp (175W) for a total of
6 h. The mixture was concentrated under vacuum without heating
and separated by thin-layer preparative chromatography on SiO2-
plate using CH2Cl2:CH3CN 5:1 as a developing eluent. Previously,
the silica gel was neutralized with a mixture of dioxane/NH4OH: 4/
1. The area on a plate containing the product was brushed and
extracted with Et2O. Insoluble product was filtered off, the residue
evaporated to dryness without heating to give 2 mg (10%) of 5-E,Z-
4.10. Biological evaluations
isomer as yellow oil. IR (thin film,
n
cmꢁ1): 3078, 2921, 2850, 2772,
Cell lines used for estimation of toxicity of compounds 4,5, 6a-c
and 7a-c were Scov3 and CaoV3 (human ovarian carcinoma), and
A549 (human lung carcinoma). Cells were grown in RPMI-1640
medium (Sigma–Aldrich, UK) supplemented with 10% fetal bovine
serum (FBS, HyClone, USA), 2 mM L-glutamine and gentamicin.
Cytotoxicity 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
1733, 1656 (C]O), 1591 (C]C), 1403 and 1331 (thiophene ring
vibration), 1276 (thiophene ring vibration), 1180, 1112, 1053, 991,
857, 709. 1H NMR (CDCl3)
d, ppm: 2.54 (s, 3H, CH3), 3.57 (s, 2H, CH2),
3.79 (s, 2H, CH2), 6.98 (s, 1H, CH), 7.09 (t, 1H, 3JHH ¼ 4.4 Hz), 7.15 (t,
3
3
3
1H, JHH ¼ 4.4 Hz), 7.33 (d, 1H, JHH ¼ 3.1 Hz), 7.41 (d, 1H, JHH ¼ 3.1
Hz), 7.51 (d, 1H, 3JHH ¼ 5.1 Hz), 7.55 (d, 1H, 3JHH ¼ 5.1 Hz), 7.99 (s, 1H,
CH). 13C NMR (CDCl3), , ppm: 45.1 (NCH3), 57.33 (C2, CH2N), 61.45
d
0
0
(C6 ), 125.88 (C3, C5 ) 126.44, 127.78, 128.83 (C7), 130.37, 132.15,
132.92, 133.29, 136.85, 138.31, 138.71, 186.02 (C4).
4.9. X-ray structure determination
seeded into 96-well plates at a concentration of 1 ꢀ104 cells/100
ml/
well. The cells were allowed to attach overnight at 37 ꢂC 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 concentra-
Data were collected on a Bruker SMART APEX II CCD diffrac-
tometer (l(MoKa)-radiation, graphite monochromator, u and 4
scan mode) and corrected for absorption using the SADABS program
(version 2.03 [52]). For details, see Table 3. 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 unit cell of 6a contains the two independent
molecules. One of the two thiophene rings of each of the inde-
pendent molecules in 6a are disordered over two sites connected
by rotation on 180ꢂ around the ordinary C11–C12 and C30–C31
bonds, respectively, with the occupancies 0.8:0.2 and 0.6:0.4. Also,
one of the two ethyl groups of the phosphonate fragments in both
independent molecules are disordered over two sites each, with the
equal occupancies. The ethyl groups of the phosphonate fragment
in 7b are disordered over two sites each, with the occupancies of
0.75:0.25. The hydrogen atoms in all compounds were placed in
calculated positions and refined within the riding model with fixed
tions. After 72 h incubation, 20 ml of MTT reagent (5 mg/ml) were
added and cell cultures were incubated for 3 h at 37 ꢂC. After
removal of the culture medium formazan crystals were dissolved in
dimethylsulfoxide (Sigma–Aldrich) to determine the amount of
formazan 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 ꢀ10ꢁ5, 10ꢁ5, 10ꢁ6, 10ꢁ7 M. Commercially available
Melphalan (Sarcolysin) purchased from Arkelan-Glaxo was used as
a positive control in the assay.
Table 3
Acknowledgements
Crystallographic data for 6a, 6c and 7b.
Compound
6a
6c
7b
This work was supported by the Deutsche For-
Empirical formula
fw
T, K
Crystal system
Space group
a, Å
b, Å
c, Å
C19H22NO4PS2 C22H20NO3PS2 C21H26NO4PS2
ꢂ
schungsgemeinschaft (DFG N 436 RUS 113/905/0-1; RO 362/35-
423.47
150(2)
Monoclinic
P21/c
11.5370(10)
14.9382(13)
23.614(2)
90
96.148(1)
90
4046.2(6)
8
441.48
100(2)
Triclinic
P-1
9.0893(11)
9.3757(11)
12.7390(15)
72.773(2)
87.761(2)
85.524(2)
1033.6(2)
2
451.52
100(2)
Monoclinic
P21/c
15.5351(14)
5.9751(5)
23.339(2)
90
90.352(1)
90
2166.4(3)
4
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 and NIH via the RIMI program
(grant 1P20MD001104-01).
a
, deg.
, deg.
Appendix. Supplementary data
b
g
, deg.
V, Å3
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.11.041.
Z
dc, g cm-3
1.390
0.367
1776
54
1.419
0.359
460
54
1.384
0.347
952
56
m
, mmꢁ1
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